US20050125161A1 - Differentially-expressed conifer cDNAs, and their use in improving somatic embryogenesis - Google Patents

Differentially-expressed conifer cDNAs, and their use in improving somatic embryogenesis Download PDF

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US20050125161A1
US20050125161A1 US10/651,991 US65199103A US2005125161A1 US 20050125161 A1 US20050125161 A1 US 20050125161A1 US 65199103 A US65199103 A US 65199103A US 2005125161 A1 US2005125161 A1 US 2005125161A1
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expression
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John Cairney
Nanfei Xu
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Georgia Tech Research Corp
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    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16BBIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00ICT programming tools or database systems specially adapted for bioinformatics
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to a relational database of cDNA molecules, including those corresponding to Loblolly Pine Major Intrinsic Protein (MIP), which are differentially expressed during plant embryogenesis.
  • MIP Loblolly Pine Major Intrinsic Protein
  • the present invention further relates to the use of DNA arrays for evaluating gene expression in somatic and zygotic embryos.
  • the invention encompasses related nucleic acids, proteins, antigens, and antibodies derived from these cDNAs as well as the use of such molecules for the staging, characterization, and manipulation of plant embryogenesis, in particular conifer embryogenesis.
  • the cDNAs and related nucleic acids, proteins, antigens, and antibodies derived from these cDNAs are useful in the design, selection, and cultivation of improved crops, specifically including coniferous trees, which provide raw materials for paper and wood products.
  • Asexual propagation permits the application of very high selection intensity, resulting in the propagation of only those progeny showing a high genetic gain potential. These highly desirable progeny can have unique genetic combinations that result in superior growth and performance characteristics. Thus, with asexual propagation it is possible to genetically select individuals while avoiding a concomitant reduction of genetic gain due to intra-familial variation.
  • Asexual propagation of trees can be accomplished currently by grafting, vegetative propagation, and micropropagation. Grafting, widely used to propagate select individuals in limited quantities for seed orchard establishment, is not applicable to large-scale production for reforestation. Vegetative propagation, achieved by the rooting of cuttings, and micropropagation by somatic embryogenesis, currently hold the most potential for reforestation of conifers. Although vegetative propagation by rooted cuttings can be achieved in many coniferous species, large-scale production via this method is extremely costly due to difficulties in automating and mechanizing the process, not to mention the need for tremendous quantities of stock tissue. This propagation method is still further limited by the fact that the rooting potential of stock plants decrease with time, making it difficult to serially propagate from select genotypes over extended periods of time.
  • Micropropagation is the most promising method of asexual propagation for mass plantings. This process involves the production of somatic embryos in vitro from minute pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Both vegetative propagation and micropropagation have the potential to capture all genetic gain of highly desirable genotypes. However, unlike conventional vegetative propagation methods, somatic embryogenesis is amenable to automation and mechanization, making it highly desirable for large-scale production of planting stock for reforestation. Moreover, somatic embryogenesis is particularly amenable to high intensity selection of a large number of clones.
  • somatic embryogenesis has great potential for clonal production of conifer embryos to meet the increased demands of the pulp and paper industry.
  • Assessment of embryo quality needs improvement.
  • the process of creating better tree stock begins with understanding the process of tree development from embryogenesis through full maturation.
  • plant tissue culture is the broad science of growing plant tissues on or in a nutrient medium containing minerals, sugars, vitamins and plant hormones. By adjusting the composition of the media, cultured tissues can be induced to grow or differentiate into specific cell types or organs.
  • Somatic embryogenesis is a type of plant tissue culture where a piece of a donor plant is excised, cultured and induced to form multiple embryos. An embryo is a discrete mass of cells with a well-defined structure that is capable of growing into a whole plant.
  • somatic embryogenesis involves several steps. Prior to the tissue culture process, a suitable “explant” is harvested.
  • a typical explant in conifer somatic embryogenesis is the “megagametophyte”, a haploid nutritive tissue of the conifer seed, which is extracted from the ovule of a pollinated female cone. This ovule contains single or multiple zygotic seed embryos.
  • cleavage polyembryony where a zygotic embryo grows and divides to form a small clones of embryos.
  • the first step in somatic embryogenesis is the initiation step.
  • the explant is placed on a suitable media.
  • a process called extrusion occurs. Extrusion involves the emergence or expulsion of a zygotic embryo or multiple embryos and embryogenic tissue out of the megagametophyte. If culture conditions are suitable, initiation proceeds and the extruded embryo or embryos undergo the process of cleavage polyembryony. This results in the formation of early stage somatic embryos in a glossy, mucilaginous mass.
  • somatic embryos are transferred to a second medium with an appropriate composition of plant hormones and other factors to induce the somatic embryos to multiply.
  • cultures can double up to 2-6 times in one week.
  • the embryos are moved to a development and maturation medium.
  • the correct balance of plant hormones and other factors will induce the early-stage embryos to mature into late stage embryos.
  • embryos are germinated to form small seedlings. These seedlings are then acclimated for survival outside of the culture vessel. After acclimation, the seedlings are ready for planting.
  • the current invention will allow one to stage embryos based on a relational database system profiling gene expression patterns instead of physical morphological differences, thereby permitting one less skilled in the art of visual staging to biologically determine the stages of embryogenesis.
  • the traditional morphological staging method provides only a crude indication of the underlying biochemical condition or state of an embryo. This level of information is insufficient for refining culture conditions, including media formulations, or for selecting potentially advantageous embryo clones for further development.
  • the current invention will allow definitive staging significantly beyond that currently practiced in the art, and provides a detailed analysis of the biochemical state and potential fitness of an embryo by comparison to developed relational database profiles.
  • Visual staging methods depend on morphological markers to assign a numerical stage of 1-9 to an embryo. Nevertheless, it is well accepted that visually undetectable developmental changes occur in an embryo after it reaches stage 9.
  • the current invention is particularly useful in providing means for monitoring and evaluating the developmental state of these older embryos, as genetic responses occur and are detectable up to and through an adult tree's life.
  • the relational database system provides a platform for which to monitor individual gene expression levels during embryo development while directly correlating expression with, for example, environmental conditions, age, and embryo fitness, as well as the protein identification achieved by BLAST searches of publicly available databases (i.e., GenBank) for desirable genes. Accordingly, the present invention therefore provides the additional ability to correlate the direct, global gene expression response within the embryo system to a typically non-expressing gene driven by a stage-specific promoter.
  • the present invention addresses these needs by providing in a relational database format nucleic acid and protein sequences that are differentially expressed during various stages of plant embryogenesis.
  • the invention encompasses a set of isolated nucleic acid molecules comprising the DNA sequence of any one of SEQ ID NOS: 1-334 and nucleic acid molecules related or complementary to any one of SEQ ID NOS: 1-334. (See Table I)
  • the invention includes both single-stranded and double-stranded RNA and DNA nucleic acids, including variants thereof.
  • the nucleic acids of the invention can be used as an expression template in the form of DNA arrays, including for example, gene arrays, DNA chips, and dot array Southerns, for which to compare and evaluate expression in test samples.
  • the nucleic acids of the invention can be further used as probes to detect the presence or level of both single-stranded and double-stranded RNA and DNA encoding variants of polypeptides or fragments of polypeptides encompassed by the invention.
  • the nucleic acids of the invention can be further used as promoters for the expression of sense and antisense molecules at specific stages of embryo development. Data acquired through the use of the present invention can in turn be provided to the relational database for further development.
  • Isolated nucleic acid molecules that hybridize to a denatured, double-stranded DNA comprising the DNA sequence of any one of SEQ ID NOS: 1-334 under conditions of moderate or high stringency are also encompassed by the invention.
  • the invention further encompasses synthetic and naturally-occurring variants of the nucleic acids described in SEQ ID NOS: 1-334, for example, isolated nucleic acid molecules derived by in vitro mutagenesis from SEQ ID NOS: 1-334.
  • In vitro mutagenesis would include numerous techniques known in the art including, but not limited to, site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis.
  • the invention also encompasses related molecules (variants) including isolated nucleic acid molecules degenerate from SEQ ID NOS: 1-334 as a result of the genetic code, for example, naturally-occurring or synthetic allelic variants of the genes encoding SEQ ID NOS: 1-334.
  • related molecules also encompass both smaller and larger nucleic acids that contain sufficient sequence to support hybridization to any of SEQ ID NOS: 1-334 under conditions of moderate or high stringency. Consequently, recombinant vectors, including those that direct the expression of these nucleic acid molecules and host cells transformed or transfected with these vectors are herein defined as variants and are encompassed by the invention.
  • transgenic vectors and transgenic plants comprising vectors or other nucleic acids comprising any one of SEQ ID NOS: 1-334 and related molecules. Particularly preferred are those capable of expressing polypeptides or peptides encoded by any of SEQ ID NOS: 1-327.
  • the transgene comprises SEQ ID NO: 327, or a variant thereof.
  • SEQ ID NO: 327 encodes a protein which corresponds to a novel Loblolly pine homolog of the plant Major Intrinsic Protein (MIP) family.
  • MIPs comprise a large family of related proteins that function as membrane channels for the transport of water and possibly ions across cellular membranes.
  • the encoded protein of SEQ ID NO: 327 may be referred to as Loblolly MIP.
  • Variants including naturally-occurring and artifactually-programmed allelic variants, vectors, and other nucleic acids which hybridize to SEQ ID NO: 327 under conditions of moderate or high stringency are encompassed by the invention. Also encompassed are plant cells, seeds, embryos and trees, transgenic for loblolly pine MIP, and variants thereof.
  • the invention also encompasses isolated polypeptides, or fragments thereof, encoded by any one of the nucleic acid molecules of SEQ ID NOS: 1-327, including variants thereof.
  • the invention further encompasses the use of these peptide sequences as markers for staging, monitoring, and selecting embryos and embryo cultures.
  • the invention also encompasses methods for the production of these polypeptides or fragments thereof including culturing a host cell under conditions promoting expression and recovering the polypeptide or peptide from the culture medium.
  • the expression of polypeptides or peptides encoded by SEQ ID NOS: 1-327 in viral vectors, bacteria, yeast, plant, and animal cells is encompassed by the invention.
  • Isolated polyclonal or monoclonal antibodies that bind to peptides encoded by SEQ ID NOS: 1-327 are also encompassed by the invention.
  • nucleic acid molecules of any one of SEQ ID NOS: 1-327 to obtain full length cDNA and genomic sequences of the corresponding genes, including cognate, homologous, or otherwise related genetic sequences, which hybridize to any of SEQ ID NOS: 1-327 under conditions of moderate or high stringency.
  • oligonucleotides derived from any one of SEQ ID NOS: 1-334 that can be used as probes and/or as primers in PCR, RT-PCR, and other assays to detect the presence or level of the nucleic acids of SEQ ID NOS: 1-334 and related molecules.
  • the primers and other probes of the invention may be used to monitor and characterize the development of plant embryos, in particular, pine tree embryos. Characterization of embryonic gene expression provides means for correlating gene expression with current and potential plant phenotypes. Consequently, the present invention encompasses means for monitoring and adjusting growth conditions (see FIG. 6 ), as well as means for selecting genetically superior embryonic clones for further propagation and expansion (see FIG. 8 ).
  • the present invention encompasses the use of DNA or RNA probes derived from the nucleic acid molecules of SEQ ID NOS: 1-334 in any form, e.g., in DNA arrays, and antibodies raised against polypeptides or peptide fragments encoded by SEQ ID NOS: 1-327, to determine relative or absolute levels of expression of the genes or proteins encoded by SEQ ID NOS: 1-327.
  • these nucleic acid and antibody probes may be used for staging, monitoring, characterizing, or selecting plant embryos or embryo cultures, particularly pine tree embryos.
  • the relational database of the present invention allows expression information pertaining to embryo stages to be viewed as sequence data generated in accordance with the present invention.
  • the invention includes a database for storing a plurality of sequence records for which to correlate embryo stages to sequence records.
  • the method further involves providing an interface which allows a user to select one or more expression categories contained within the database.
  • the relational database is designed to include separate parts or cells for information storage.
  • One cell or part may contain a gene expression database which contains nucleic acid molecules of SEQ ID NOS: 1-327.
  • Other cells or parts may contain descriptive information pertaining to each nucleic acid molecules of SEQ ID NOS: 1-327, additional sequence data related to the gene expression database, protein encoded by nucleic acids disclosed herein, similarity values to known proteins of other systems, and to conditions under which expression data was obtained.
  • the database system described in the present invention will allow identification or selection of particular genes of interest for further use with DNA arrays. Identification or selection of particular genes may include, for example, those related to patterns of expression, those identified with homology to known genes from other studies, and those sequences considered novel.
  • FIG. 1 depicts differential display of loblolly pine zygotic and somatic embryos at different stages of development.
  • FIG. 2 displays embryo gene expression observed by high-density array Southern hybridization.
  • FIG. 3 provides a general schematic for gene regulation studies arising from the cDNA cloning of genes expressed in embryos.
  • FIG. 4 depicts graphical representation of hybridization of ‘dehydrin’ and LPZ216 cDNA probes to total RNA isolated from zygotic embryos of loblolly pine.
  • FIG. 5 displays ABA concentration of loblolly pine embryos.
  • FIG. 6 shows schematic of gene study for improved somatic embryogenesis.
  • FIG. 7 shows detection of gene expression by high-density array Southern hybridization for loblolly pine genotype 333 after 12 weeks on two maturation media.
  • FIG. 8 depicts the application of embryogenic gene expression studies.
  • FIG. 9 displays slot blots and expression levels for three embryogenesis-related genes.
  • FIG. 10 depicts clone LPS-097 sequence (LP2-3 differential display fragment.)
  • FIG. 11 displays a northern blot for the LP2-3 gene during stages 1-3.
  • FIG. 12 displays a slot blot of total RNA from somatic embryo tissue probed with an LP2-3-specific probe.
  • FIG. 13 displays a slot blot of total RNA from zygotic embryo tissue probed with an LP2-3-specific probe.
  • FIG. 14 depicts the quantified expression of early zygotic embryos compared to early somatic embryos.
  • the three hundred and twenty-seven differentially expressed cDNAs isolated from plant specimens of known developmental ages are disclosed in SEQ ID NOS: 1-327.
  • the seven stage-specific promoters isolated from plant specimens are disclosed in SEQ ID NOS: 328-334.
  • the discovery of these cDNAs and promoters enables the design, isolation, and construction of related nucleic acids, proteins, antigens, antibodies other heterologous genes. Both the cDNAs and promoters facilitate the staging, characterization, and manipulation of plant embyrogenesis, in particular, conifer embryogenesis.
  • These molecules, and related nucleic acids, peptides, proteins, antigens, and antibodies are particularly useful when compiled into a relational database for the monitoring, design, selection, and cultivation of improved crop plants.
  • the cDNAs of SEQ ID NOS: 1-327, in addition to the promoters of SEQ ID NOS: 328-334, were originally derived from Pinus taeda embryos, commonly known as the Loblolly Pine. Nevertheless, it is understood that the invention is applicable to and encompasses all plants, including all dicotyledonous plants, including all conifers, including all species of Pinus, Picea , and Pseudotsuga . Exemplary conifers may include Picea abies , and Psedotsuga menziesii , and Pinus taeda.
  • nucleic acid or “nucleic acid molecule” refer to a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • a “nucleotide sequence” also refers to a polynucleotide molecule or oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid.
  • the nucleotide sequence or molecule may also be referred to as a “nucleotide probe.”
  • the nucleic acid molecules of the invention are derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequence by standard biochemical methods. Examples of such methods, including methods for PCR protocols that may be used herein, are disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), Current Protocols in Molecular Biology edited by F. A. Ausubel et al., John Wiley and Sons, inc. (1987), and Innis, M. et al., eds., PCR Protocols: A Guide to Methods and Applications , Academic Press (1990), each of which are herein incorporated by reference in their entirety.
  • a “nucleotide probe” is defined as an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, through complementary base pairing, or through hydrogen bond formation.
  • the oligonucleotide probe may include natural (ie. A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
  • bases in a oligonucleotide probe may be joined by a linkage other than a phosphodiester bond, so long as it does not prevent hybridization.
  • oligonucleotide probes may have constituent bases joined by peptide bonds rather than phosphodiester linkages.
  • a “target nucleic acid” herein refers to a nucleic acid to which the nucleotide probe or molecule can specifically hybridize.
  • the probe is designed to determine the presence or absence of the target nucleic acid, and the amount of target nucleic acid.
  • the target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target.
  • the probe may also contain additional nucleic acids or other moieties, such as labels, which may not specifically hybridize to the target.
  • target nucleic acid may refer to the specific nucleotide sequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect.
  • the overall sequence e.g., gene or mRNA
  • the nucleic acid molecules of the invention include DNA in both single-stranded and double-stranded form, as well as the RNA complement thereof.
  • DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof.
  • Genomic DNA, including translated, non-translated and control regions, may be isolated by conventional techniques, e.g., using any one of the cDNAs of SEQ ID NO: 1 through SEQ ID NO: 327, or suitable fragments thereof, as a probe, to identify a piece of genomic DNA which can then be cloned using methods commonly known in the art.
  • nucleic acid molecules within the scope of the invention include sequences that hybridize to sequences of SEQ ID NOS: 1-334 under hybridization and wash conditions of 5°, 10°, 15°, 20°, 25°, or 30° below the melting temperature of the DNA duplex of sequences of SEQ ID NOS: 1-334, including any range of conditions subsumed within these ranges.
  • DNA arrays are used to identify hybridizing sequences from test samples.
  • the term “DNA array” refers to “gene arrays,” “DNA chips,” “dot array Southerns,” etc.
  • the DNA array will typically include one or a multiplicity of nucleic acid molecules derived from SEQ ID NO: 1 through SEQ ID NO: 327 that specifically hybridize to the nucleic acid expression of which is to be detected.
  • the array may include one or more control probes to monitor the expression system. Control probes refer to known expression products present at each stage of expression, e.g., ribosomal gene products or the transcripts of other housekeeping genes.
  • this invention provides an isolated nucleic acid molecule selected from the group consisting of:
  • moderate stringency is herein defined as a nucleic acid having 10, 11, 12, 13, 14, 15, 16, or 17, contiguous nucleotides identical to any of the sequences of SEQ ID NOS: 1-334, or a complement thereof.
  • high stringency is hereby defined as a nucleic acid having 18, 19, 20, 21, 22, or more contiguous identical nucleotides, or a longer nucleic acid having at least 80, 85, 90, 95, or 99 percent identity with any of the sequences of SEQ ID NOS: 1-334; for sequences of at least 50, 100, 150, 200, or 250 nucleotides, high stringency may comprise an overall identity of at least 60, 65, 70 or 75 percent.
  • nucleic acid hybridization simply involves providing a denatured nucleotide molecule or probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. The nucleic acids that do not substantially form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is further generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under lower stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed sequences are not perfectly complementary.
  • lower stringency conditions e.g., low temperature and/or high salt
  • hybridization conditions may be selected to provide any degree of stringency.
  • the percent identity between an amino acid sequence encoded by any of SEQ ID NOS: 1-334 and a potential hybridizing variant can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. ( Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch ( J. Mol. Biol, 48:443, 1970), as revised by Smith and Waterman ( Adv. Appl. Math 2:482, 1981).
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess ( Nuci. Acids Res. 14:6745, 1986), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure , National Biomedical Research Foundation, pp. 353-358, 1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • DNA sequences can code for the same polypeptide.
  • variant DNA sequences can result from genetic drift or artificial manipulation (e.g., occurring during PCR amplification or as the product of deliberate mutagenesis of a native sequence).
  • the present invention thus encompasses any nucleic acid capable of encoding a protein derived from SEQ ID NOS: 1-327, or variants thereof.
  • Deliberate mutagenesis of a native sequence can be carried out using numerous techniques well known in the art. For example, oligonucleotide-directed site-specific mutagenesis procedures can be employed, particularly where it is desired to mutate a gene such that predetermined restriction nucleotides or codons are altered by substitution, deletion or insertion. Exemplary methods of making such alterations are disclosed by Walder et al. ( Gene 42:133, 1986); Bauer et al. ( Gene 37:73, 1985); Craik ( BioTechniques , Jan. 12-19, 1985); Smith et al. ( Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel ( Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. ( Methods in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462, all of which are incorporated by reference.
  • the invention further provides an isolated nucleic acid molecule selected from the group comprising of (1), (2), and (3) above and further consisting of:
  • the cDNAs isolated and cloned through the differential display procedure will often only represent a partial sequence (generally the 3′ end) of the mRNA from which it was derived due to the nature of the arbitrary primer used in the differential display PCR reaction. Consequently, the cDNA sequences of SEQ ID NOS: 1-327 provide powerful tools for obtaining the sequences of full-length cDNAs. This can be accomplished by using a partial cDNA as a probe to identify and isolate the full length cDNA from a population of full length cDNAs or from a full length cDNA library. As is well known in the art, similar procedures can be used to identify corresponding genomic DNA sequences.
  • RACE Rapid Amplification of cDNA Ends
  • Clonetech Laboratories, Inc. (Palo Alto, Calif.) offers a SMARTM cDNA product line that allows one to generate high quality full length cDNAs and cDNA libraries.
  • SMARTM technology can also be used to perform RACE.
  • Full length cDNAs may be sequenced and their sequences compared to sequences in public databases to assess their identities and/or homologies to other known sequences.
  • Cloned full length cDNAs can be used in the construction of expression vectors for the production and purification of pine tree polypeptides which contain the pine tree peptides encoded by the cDNAs of any one of SEQ ID NOS: 1-327.
  • the present invention encompasses oligonucleotide fragments derived from any one of SEQ ID NO: 1 through SEQ ID NO: 327 or from the reverse complement sequence of any one of SEQ ID NO: 1 through SEQ ID NO: 327.
  • oligonucleotides would be useful as primers in the performance of RT-PCR assays to detect, or even quantify, pine embryo stage-specific transcripts.
  • Such oligonucleotide primers will generally comprise from 10 to 25 nucleotides substantially complementary to the ends of the target sequence and may contain additional non-complementary nucleotides, for example, nucleotides that generate a restriction endonuclease site or cloning junction.
  • Programs useful in selecting PCR primers may be used to design the oligonucleotides of this invention, but use of such programs is not necessary.
  • the Wisconsin PackageTM software available from the Genetic Computer Group includes a program called Prime that can aid in selecting primers from a given template sequence.
  • Protocols for the design and optimization of PCR reactions are commonly known in the art and are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology , Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications , Innis et al., eds., Academic Press, Inc. (1990).
  • antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences.
  • Antisense or sense oligonucleotides comprise a fragment of DNA from any one of SEQ ID NO: 1 through SEQ ID NO: 327. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 To about 30 nucleotides.
  • Antisense or sense oligonucleotides thus may be used to block expression of proteins or the function of RNA.
  • Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases.
  • Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sufficient sequence specificity to be able to bind to target nucleotide sequences.
  • sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine).
  • intercalating agents such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides. Such modifications may modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, lipofection, CaPO 4 -mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus or adenovirus.
  • Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
  • Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448.
  • the sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
  • the cDNAs of SEQ ID NOS: 1-327 can be translated into amino acid sequences potentially corresponding to portions of developmentally-regulated plant proteins. These amino acid sequences can be identified from sequences listed in Table I, below.
  • the cDNAs encoding these predicted polypeptides are grouped into early, middle, and late transcripts according to the staged embryo population from which they were derived.
  • peptide is generally understood to reference synthetic sequences, or fragments of larger proteins, and includes short amino acid sequences of between 2 and 10 amino acids
  • polypeptide refers to larger sequences and full-length proteins
  • the terms are used interchangeably herein to indicate that the invention applies to peptides and polypeptides of any length and variants thereof.
  • the discovery of presumptive open reading frames in SEQ ID NOS: 1-327, and the ability to isolate additional cDNA sequence enables the construction of expression vectors comprising nucleic acid sequences encoding those polypeptides.
  • the cDNAs of the invention also enable cells transfected or transformed with expression vectors driving the expression of the encoded polypeptides and antibodies reactive with the polypeptides.
  • the invention provides for isolated polypeptides, preferably, pine tree polypeptides.
  • polypeptides refers to a genus of polypeptide or peptide fragments that encompass the amino acid sequences identified from Table I, as well as smaller fragments. Consequently, the invention encompasses any polypeptide fragment comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous amino acids encoded by the cDNAs of any of SEQ ID NOS: 1-327, or comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous amino acids of any of amino acid sequence derived from Table I.
  • a polypeptide may be defined in terms of its antigenic relatedness to any peptide encoded by SEQ ID NOS:-1-327.
  • a polypeptide within the scope of the invention is defined as an amino acid sequence comprising a linear or 3-dimensional epitope shared with any peptide encoded by the cDNAs of SEQ ID NOS: 1-327.
  • a polypeptide within the scope of the invention is recognized by an antibody that specifically recognizes any peptide encoded by SEQ ID NOS: 1-327.
  • Antibodies are defined to be specifically binding if they bind pine tree polypeptides with a K a of greater than or equal to about 10 7 M ⁇ , and preferably greater than or equal to 10 8 M ⁇ 1 .
  • a polypeptide “variant” as referred to herein means a polypeptide substantially homologous to a native polypeptide, but which has an amino acid sequence different from that encoded by any of SEQ ID NOS: 1-327 because of one or more deletions, insertions or substitutions.
  • the variant amino acid sequence preferably is at least 80% identical to a native polypeptide amino acid sequence, preferably at least 90%, more preferably, at least 95% identical over at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, or 26-30 contiguous amino acids.
  • the percent identity between an amino acid sequence encoded by any of SEQ ID NOS: 1-327 and a potential variant can be determined manually, or, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. ( Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program described above, utilizes the alignment method of Needleman and Wunsch ( J. Mol. Biol. 48:443,1970), as revised by Smith and Waterman ( Adv. Appl. Math 2:482, 1981).
  • Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.
  • conservative substitutions include substitution of one aliphatic residue for another, such as lie, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. See Zubay, Biochemistry, Addison-Wesley Pub. Co., (1983) incorporated by reference in its entirety.
  • substitutions can be calculated using substitution score matrices such a PAM-120, PAM-200, and PAM-250 as discussed in Altschul, ( J. Mol. Biol. 219:555-65, 1991).
  • Other such conservative substitutions for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Naturally-occurring peptide variants are also encompassed by the invention.
  • examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides of Table I.
  • Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides encoded by the sequences of Table I (generally from 1-5 terminal amino acids).
  • the invention provides recombinant and non-recombinant, isolated and purified polypeptides, preferably pine tree polypeptides.
  • Variants and derivatives of native polypeptides can be obtained by isolating naturally-occurring variants, or the nucleotide sequence of variants, of other plant lines or species, or by artificially programming mutations of nucleotide sequences coding for native pine tree polypeptides. Alterations of the native amino acid sequence can be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence.
  • the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion. Exemplary methods of making such alterations are discussed supra.
  • Recombinant expression vectors containing a nucleic acid sequence encoding the polypeptides of the invention can be prepared using well known methods.
  • the expression vectors include a cDNA sequence encoding the polypeptide operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
  • suitable transcriptional or translational regulatory nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
  • regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation initiation and termination.
  • Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the cDNA sequence of the invention.
  • a promoter nucleotide sequence is operably linked to a cDNA sequence if the promoter nucleotide sequence controls the transcription of the cDNA sequence.
  • the ability to replicate in the desired host cells, usually conferred by an origin of replication, and a selection gene by which transformants are identified can additionally be incorporated into the expression vector.
  • sequences encoding appropriate signal peptides that are not naturally associated with the polypeptides of the invention can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide secretory leader
  • a signal peptide that is functional in the intended host cells enhances extracellular secretion of the expressed polypeptide.
  • the signal peptide can be cleaved from the polypeptide upon secretion from the cell.
  • Fusions of additional peptide sequences at the amino and carboxyl terminal ends of the polypeptides of the invention can be used to enhance expression of the polypeptides or aid in the purification of the protein.
  • Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al., ( Bio/Technology 6:1204, 1988).
  • Suitable host cells for expression of polypeptides of the invention include prokaryotes, yeast or higher eukaryotic cells.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual , Elsevier, N.Y., (1985).
  • Cell-free translation systems could also be employed to the disclosed polypeptides using RNAs derived from DNA constructs disclosed herein.
  • Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli .
  • Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium , and various other species within the genera Pseudomonas, Streptomyces , and Staphylococcus .
  • the disclosed polypeptides can include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal methionine can be cleaved from the expressed recombinant polypeptide.
  • Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes.
  • a phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement.
  • useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
  • an appropriate promoter and a DNA sequence encoding one or more of the polypeptides of the invention are inserted into the pBR322 vector.
  • vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM-1 (Promega Biotec, Madison, Wis., USA).
  • Other commercially available vectors include those that are specifically designed for the expression of proteins; these would include pMAL-p2 and pMAL-c2 vectors that are used for the expression of proteins fused to maltose binding protein (New England Biolabs, Beverly, Mass., USA).
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include P-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EP-A-36776), and tac promoter ( Maniatis, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, p. 412, 1982).
  • P-lactamase penicillinase
  • lactose promoter system Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979
  • tryptophan (trp) promoter system Goeddel et al., Nucl. Acids Res. 8:4057
  • a particularly useful prokaryotic host cell expression system employs a phage ⁇ P L promoter and a c1857ts thermolabile repressor sequence.
  • Plasmid vectors available from the American Type Culture Collection (“ATCC”) which incorporate derivatives of the PL promoter, include plasmid pHUB2 (resident in E. Coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).
  • DNA encoding one or more of the polypeptides of the invention may be cloned in-frame into the multiple cloning site of an ordinary bacterial expression vector.
  • the vector would contain an inducible promoter upstream of the cloning site, such that addition of an inducer leads to high-level production of the recombinant protein at a time of the investigator's choosing.
  • expression levels may be boosted by incorporation of codons encoding a fusion partner (such as hexahistidine) between the promoter and the gene of interest.
  • the resulting “expression plasmid” may be propagated in a variety of strains of E. coli.
  • the bacterial cells are propagated in growth medium until reaching a pre-determined optical density. Expression of the recombinant protein is then induced, e.g., by addition of IPTG (isopropyl-b-D-thiogalactopyranoside), which activates expression of proteins from plasmids containing a lac operator/promoter. After induction (typically for 1-4 hours), the cells are harvested by pelleting in a centrifuge, e.g., at 5,000 ⁇ G for 20 minutes at 4° C.
  • IPTG isopropyl-b-D-thiogalactopyranoside
  • the pelleted cells may be resuspended in ten volumes of 50 mM Tris-HCl (pH 8)/1 M NaCl and then passed two or three times through a French press. Most highly expressed recombinant proteins forms insoluble aggregates known as inclusion bodies. Inclusion bodies can be purified away from the soluble proteins by pelleting in a centrifuge at 5,000 ⁇ G for 20 minutes, 4° C. The inclusion body pellet is washed with 50 mM Tris-HCl (pH 8)/1% Triton X-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTT.
  • any material that cannot be dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTT may be removed by centrifugation (10,000 ⁇ G for 20 minutes, 20° C.).
  • the protein of interest will, in most cases, be the most abundant protein in the resulting clarified supernatant.
  • This protein may be “refolded” into the active conformation by dialysis against 50 mM Tris-HCl (pH 8)/5 mM CaCl 2 /5 mM Zn(OAc) 2 /1 mM GSSG/0.1 mM GSH.
  • purification can be carried out by a variety of chromatographic methods such as ion exchange or gel filtration. In some protocols, initial purification may be carried out before refolding.
  • hexahistidine-tagged fusion proteins may be partially purified on immobilized Nickel.
  • Polypeptides of the invention can also be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae ). Other genera of yeast, such as Pichia or Kluyveromyces (e.g. K. lactis ), can also be employed.
  • yeast vectors will often contain an origin of replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionine, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.
  • glycolytic enzymes Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978
  • enolase glyceraldehyde-3-phosphate dehydrogenase
  • hexokinase hexokinase
  • pyruvate decarboxylase phosphofructokinase
  • glucose-6-phosphate isomerase 3-phosphoglycerate mutase
  • pyruvate kinase triosephosphate isomerase
  • phosphoglucose isomerase phosphoglucose isomerase
  • glucokinase glucokinase
  • yeast vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van den Berg et. al., Bio/Technology, 8:135-139 (1990).
  • Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. ( J. Biol. Chem. 258:2674, 1982) and Beier et al. ( Nature 300:724, 1982).
  • Shuttle vectors replicable in both yeast and E. coli can be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp gene and origin of replication) into the above-described yeast vectors.
  • the yeast ⁇ -factor leader sequence can be employed to direct secretion of one or more of the disclosed polypeptides.
  • the ⁇ -factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274.
  • Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art.
  • a leader sequence can be modified near its 3′ end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
  • Yeast transformation protocols are known to those of skill in the art.
  • One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978.
  • the Hinnen et al. protocol selects for Trp+transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine, and 20 ⁇ g/ml uracil.
  • Yeast host cells transformed by vectors containing ADH2 promoter sequence can be grown for inducing expression in a “rich” medium.
  • a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ⁇ g/ml adenine and 80 ⁇ g/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
  • Mammalian or insect host cell culture systems could also be employed to express recombinant polypeptides of the invention.
  • Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also can be employed.
  • suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line (ATCC CRL 10478) derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. ( EMBO J. 10: 2821, 1991).
  • DHFR dihydrofolate reductase
  • a suitable host strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).
  • a plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media.
  • selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotcs, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
  • Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes.
  • Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and later promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell.
  • Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. in Enzymology, 1990). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
  • Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475-13491,1982).
  • EASE expression augmenting sequence element
  • TPL tripartite leader
  • VA gene RNAs from Adenovirus 2
  • the internal ribosome entry site (IRES) sequences of viral origin allows dicistronic mRNAs to be translated efficiently (Oh and Sarnow, Current Opinion in Genetics and Development 3:295-300, 1993; Ramesh et al., Nucleic Acids Research 24:2697-2700, 1996).
  • IRS internal ribosome entry site
  • a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (eg. DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman, Meth. in Enzymology, 1990).
  • Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by Mosser et al., Biotechniques 22:150-161, 1997, and p2A51 described by Morris et al., Animal Cell Technology, 1997, pp. 529-534.
  • a useful high expression vector, pCAVNOT has been described by Mosley et al., Cell 59:335-348,1989.
  • Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg ( Mol. Cell. Biol. 3:280, 1983).
  • a useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. ( Mol. Immunol. 23:935, 1986).
  • a useful high expression vector, PMLSV N1/N4 described by Cosman et al., Nature 312:768, 1984, has been deposited as ATCC 39890.
  • Additional useful mammalian expression vectors are described in EP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filed May 16, 1991, incorporated by reference herein.
  • the vectors can be derived from retroviruses.
  • a heterologous signal sequence can be added, such as the signal sequence for IL-7 described in U.S. Pat. No. 4,965,195; the signal sequence for IL-2 receptor described in Cosman et al., Nature 312:768 (1984); the IL4 signal peptide described in EP 367,566; the type I IL-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type H IL-1 receptor signal peptide described in EP 460,846.
  • the polypeptides of the invention and the nucleic acid molecules encoding them can also be used as reagents to identify (a) proteins that the disclosed polypeptides or their constituent proteins regulate, and (b) other proteins with which it might interact.
  • the disclosed polypeptides can be coupled to a recombinant protein, to an affinity matrix, or by using them as a bait in the yeast two-hybrid system.
  • the use of the yeast two-hybrid system developed by Stanley Fields and coworkers is well known in the art and described in Golemis, E., et al Section 20.1 in: Current Protocols in Molecular Biology , ed. Ausubel, F. M., et al., John Wiley & Sons, NY, 1997 and in The Yeast Two - Hybrid System ., ed. P. L. Bartel and S. Fields, Oxford University Press, 1997.
  • Purified polypeptides of the invention can be used to generate antibodies that bind to one or more epitopes of the disclosed polypeptide.
  • anti-polypeptide antibodies includes polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab′) 2 , and Fab fragments, as well as any recombinantly produced binding partners.
  • Antibodies are defined to be specifically binding if they bind pine tree polypeptides with a K a of greater than or equal to about 10 7 M ⁇ 1 . Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).
  • Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice, hamsters, guinea pigs, or rats, using procedures that are well-known in the art, for example, as described for example, U.S. Pat. No. 5,585,100, incorporated by reference herein.
  • a composition comprising at least one of the polypeptides of the invention is administered to the host animal, typically through intra-peritoneal or subcutaneous injection.
  • a peptide is used as the immunogen, it is preferable to conjugated it to a suitable carrier molecule, such as a T-dependent antigen (Bovine Serum Albumin, cholera toxin, and the like).
  • a suitable carrier molecule such as a T-dependent antigen (Bovine Serum Albumin, cholera toxin, and the like).
  • the immunogenicity of the disclosed polypeptides can also be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant or alum. Following booster immunizations, small samples of serum are collected and tested for reactivity to the disclosed polypeptides or their constituent epitopes.
  • Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and 4,486,530, each of which is incorporated by reference in their entirety.
  • CIEP countercurrent immuno-electrophoresis
  • ELISA enzyme-linked immuno-sorbent assays
  • sandwich assays see U.S. Pat. Nos. 4,376,110 and 4,486,530, each of which is incorporated by reference in their entirety.
  • Monoclonal antibodies (or fragments thereof), directed against epitopes of the disclosed polypeptides can also be readily prepared using well-known procedures, such as, for example, the procedures described in U.S. Patent No. RE 32,011, U.S. Pat. Nos. 4,902,614, 4,543,439, and 4,411,993 ; Monoclonal Antibodies, Hybrddomas: A New Dimension in Biological Analyses , Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, each of which is incorporated by reference.
  • the host animals such as mice
  • the host animals are injected intraperitoneally at least once, and preferably at least twice at about 3 week intervals with isolated and purified polypeptides optionally in the presence of adjuvant.
  • peptide fragments may need to be conjugated to a suitable carrier protein.
  • Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse.
  • ABSC antibody capture
  • mice are given an intravenous boost of pine tree polypeptides.
  • Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols.
  • ATCC Ag8.653
  • the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell.
  • the fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as, 125 I-pine tree polypeptides is added to each well followed by incubation. Positive wells can be subsequently detected by autoradiography. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).
  • Monoclonal antibodies and specific-binding fragments of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., “Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference.
  • binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody Such a technique is described in Larrick et al., Biotechnology, 7:394 (1989).
  • the disclosed nucleic acids, polypeptides, and antibodies directed against the disclosed polypeptides can be used in a variety of research protocols, such as in DNA arrays or as reagents.
  • a sample of such research protocols are given in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, (1989), incorporated by reference.
  • the compiled sequences, polypeptides, etc. can serve as markers for cell specific or tissue specific expression of RNA or proteins.
  • this system can be used to investigate constitutive and transient expression of the genes encoding the cDNAs of SEQ ID NOS: 1-327 and the proteins encoded by these genes.
  • the disclosed cDNA sequences can be used to determine the chromosomal location of the genomic DNA and to map genes in relation to this chromosomal location.
  • the disclosed nucleotide sequence can be further used to identify additional genes related to the nucleotides of SEQ ID NOS: 1-334 and to establish evolutionary relatedness among species based on the comparison of sequences.
  • the disclosed nucleotide and polypeptide sequences can be used to select for those genes or proteins that are homologous to the disclosed cDNAs or polypeptides, using well-established positive screening procedures such as Southern blotting and immunoblotting and negative screening procedures such as subtractive hybridization.
  • cDNAs and related molecules of this invention can be used as markers for different stages of tree embryogenesis, thereby eliminating the need for a subjective eye to assess maturity and potentially allowing for more accurate staging of tree embryos.
  • the relational database of this invention aids the ability to monitor expression levels and tailor research approaches, such as the use of DNA arrays, to the specific needs of the objective, i.e., staging.
  • the information provided in this invention can be used in whole or in part to stage embryos.
  • one or a multiplicity of nucleic acid molecules from SEQ ID NOS: 1-327 having an expression profile consistent with a particular embryo stage can be used in this invention.
  • a researcher may find it beneficial to use oligonucleotide probes or antibodies, for example, that specifically recognize proteins derived from genes expressed during middle embryonic stages, or that specifically monitor expression levels for embryos that have reached maturity associated with late developmental stages.
  • a researcher can quickly determine that an embryo subset has progressed to or through an embryonic stage with the use of this invention and make appropriate changes in conditions if necessary, e.g. alter growth media or other environmental conditions.
  • Expression patterns of SEQ ID NOS: 1-327 indicate that gene activation can be classified as stage-specific, such as in the case of SEQ ID NO: 327, otherwise known as “LP2-3.”
  • the promoter that drives such a gene can perform valuable functions.
  • a promoter from LP2-3 operatively linked to a reporter gene presented within an embryo system is expected to produce the reporter product under the conditions for expression of gene LP2-3.
  • the system allows a rapid determination of stage specific embryos by a simple phenotypic reporter screen, perhaps by visualization of green fluorescent protein (GFP) or by loss of fluorescent protein product.
  • GFP green fluorescent protein
  • a set of promoters from known, differently staged genes operatively linked to reporter genes will be effective for monitoring developmental changes within the system as the embryos mature.
  • the LP2-3 promoter is identified as SEQ ID NOS: 328-334 in Table I.
  • the promoter expression pattern is that of the natively linked gene, LP2-3.
  • reporter genes are genes typically not present in the recipient organism or tissue and which encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes and assays are provided by Schenborn, E. and Groskreutz, D., Mol. Biotechnol., 13:29, 1999; Helfand, S. L. and Rogina, B., Results Probl. Cell Differ., 29:67, 2000; Kricka, L. J., Methods Enzymol., 305:333, 2000; Himes, S. R. and Shannon, M. F., Methods Mol.
  • the reporter used is GFP, or any ariant of the fluorescent protein.
  • a promoter like that from LP2-3, has potential to stimulate production of products not ordinarily observed at a particular stage.
  • a promoter derived from a gene that expresses during a known stage for example an early stage, can be operatively linked to a gene that does not normally express during that stage, yielding controlled expression of any targeted gene. It may be shown that earlier or later expression, or prolonged expression of a particular gene may give a desirable genotype or phenotype in a mature plant, may result in increased vigor in culture, or may be sufficient to alter the normal maturation process of the embryo. Prolonged expression of any desired gene also may be achieved from linking a constitutively expressed promoter to the targeted gene. Further, the ability to manipulate gene expression during embryogenesis allows for a detailed study of the effects of an individual gene or multiple genes on embryogenesis, leading to a better understanding of the developmental processes involved in embryogenesis.
  • the cDNAs and related molecules of the invention can also be used as markers to examine genetic heterogeneity and heredity through the use of techniques such as genetic fingerprinting. These markers can also be correlated with improved agronomic traits including good initiation frequency, embryonic maturation, high frequency of germination, rapid growth rates, herbicide tolerance, insect resistance, pathogen resistance, climate and environmental adaptability wood quality, and wood fiber quality and content, to name a few. Additionally, the expression of these developmentally regulated genes can be compared among genetically identical clones grown under different culture conditions to determine the best protocols and media for somatic embryogenesis.
  • Cryogenic storage of pine tree embryos is effective for maintaining stocks of embryos determined by this invention to have the desired fitness traits or exist at the appropriate developmental stage. With such storage, one can specifically target desirable embryos for expansion many years after they are frozen. For example, a culture of somatic embryos can be divided into at least three portions, one of which is cryogenically stored, one which is used to study gene embryonic gene (and protein) expression, and one that is used to produce seedlings for field testing. Clones producing valuable mature plants could be selected and expanded from frozen stocks. Additional clones exhibiting similar expression patterns could be selected for future expansion and cultivation.
  • nucleic acids, polypeptides and antibodies of this invention might be used to characterize the gene expression of embryos.
  • stage-specific gene expression of embryos of several different genotypes and at several different stages of embryogenesis are characterized.
  • sets of oligonucleotide primers designed using any one of SEQ ID NOS: 1-327 may be used in RT-PCR assays to characterize expression of a gene product.
  • In situ hybridization assays or antibody staining protocols may also be used to characterize RNA and/or protein expression and localization.
  • Embryos of the same genotype in which gene expression has been characterized may also used be to generate plantlets that are used in field testing. Once the embryos have developed into mature trees, the various genotype trees can be evaluated for important traits such as growth rates, herbicide tolerance, insect resistance, pathogen resistance, climate and environmental adaptability, wood quality, and wood fiber quality and content, among others. Finally the phenotypic data collected from the field testing can be correlated with gene expression during early embryogenesis to further enhance the database of the present invention. This will allow further identification of gene products which whose expression is correlated, either positively or negatively, with commercially valuable tree characteristics.
  • transgenic plants there are several methods known in the art for the creation of transgenic plants. These include, but are not limited to: electroporation of plant protoplasts, liposome-mediated transformation, polyethylene-glycol-mediated transformation, microinjection of plant cells, and transformation using viruses. Because the invention is especially concerned with the transformation of woody species, the two prevalent methods for transforming forest trees, namely Agrobacteriurm -mediated transfer and direct gene transfer by particle bombardment, will be discussed in more detail, though it is understood that the present invention encompasses generation of transgenic plants via standard methods commonly known in the art.
  • A. tumefaciens and A. rhizogenes are two soil microorganisms that naturally infect a wide variety of plants including dicotyledonous plants, gymnosperms and some monocotyledonous plants. Infection by these organisms results in the growth of crown gall tumors or in hairy root disease, respectively. Each of these organisms carries a large plasmid, the tumor inducing (Ti) plasmid, in the case of A. tumefaciens and the root-inducing (Ri) plasmid in the case of A. rhizogenes .
  • T-DNA a segment of DNA that is delimited by conserved regions of approximately 25 base pairs known as the left and right borders.
  • T-DNA is transferred to the plant cell where it is able to stably integrate in single copy in the plant genome. Transfer of T-DNA requires the function of the virulence genes.
  • T-DNA contains genes that mediate progression of disease such as growth hormones or genes controlling root morphogenesis.
  • T-DNA may be modified to contain an expression cassette encoding a foreign gene of interest.
  • T-DNA vector systems commonly in use for the transformation of plants. Several of these vector systems are reviewed in Hansen et al., Current Topics in Microbiology and Immunology 240: 21-57 (1999) which is hereby incorporated by reference.
  • T-DNA vectors must include the left and right borders. In addition they must either be capable of replication in Agrobacterium or be designed so as to recombine with a plasmid that does so. The latter type of vector is known as a co-integrate vector.
  • vir virulence genes
  • the vir genes may be on the same plasmid with the T-DNA or more likely supplied by a helper plasmid.
  • binary T-DNA vector systems are comprised of two plasmids, one containing the vir genes and the other containing T-DNA.
  • a selectable marker that will allow identification and selection of transformed plant cells.
  • the selectable marker should be one that works in both Agrobacterium and the target plant.
  • the genes encoding chloramphenicol acetyltransferase and neomycin phosphotransferase are suitable marker genes that confer resistance to chloramphenicol and kanamycin, respectively.
  • a selectable marker may be provided on a separate T-DNA from the T-DNA encoding the gene of interest. Co-transformed T-DNAs can integrate at separate sites in the plant genome. This can be useful because it will later allow segregation of the marker gene in progeny enabling the generation of transgenic trees expressing the gene of interest but not the marker gene.
  • the gene of interest and the selectable marker genes must also be under the control of promoters that function in the transformed plant cell.
  • suitable promoters include, but are not limited to: the abscisic acid (ABA)-inducible promoter from the early methionine (Em) gene from wheat (Marcotte et al., Plant Cell 1:976-979 (1989); the cauliflower mosaic virus (CaMV) 35S promoter (Odell et al., Nature 313:810-812 (1985); and the nopaline synthase (nos) promoter (Sanders et al., Nucl. Acids Res. 15(4):1543-58 (1987). Tissue-specific plant promoters or plant promoters responsive to chemical, hormone, heat or light treatments may be used. Additionally, the gene of interest may be expressed under the control of its endogenous promoter to ensure proper regulation.
  • ABA abscisic acid
  • Em early methionine
  • CaMV cauliflower mosaic virus
  • nos nopaline syntha
  • the process of transformation requires plant cells that are competent and that are either embryogenic or organogenic.
  • the plant cells to be transformed are then co-cultivated with Agrobacterium containing an engineered T-DNA vector system for 1-5 days. Following the co-cultivation period, the cells are incubated with the antibiotic against which the selectable marker confers resistance, and transformed lines are selected for further cultivation.
  • Agrobacterium mediated transfer in woody trees is described in Loopstra et al., Plant Molecular Biology 15:1-9 (1990), Gallardo et al., Planta 210:19-26 (1999) and Wenck et al., Plant Molecular Biology 39:407-419 (1999), each of which is hereby incorporated by reference.
  • Direct gene transfer by particle bombardment provides another method for transforming plant tissue. This method can be especially useful when plant species are recalcitrant to transformation by other means.
  • a particle, or microprojectile, coated with DNA is shot through the physical barriers of the cell.
  • Particle bombardment can be used to introduce DNA into any target tissue that is penetrable by DNA coated particles, but for stable transformation, it is imperative that regenerable cells be used.
  • the particles are made of gold or tungsten.
  • the particles are coated with DNA using either CaCl 2 or ethanol precipitation methods which are commonly known in the art.
  • DNA coated particles are shot out of a particle gun.
  • a suitable particle gun can be purchased from Bio-Rad Laboratories (Hercules, Calif.). Particle penetration is controlled by varying parameters such as the intensity of the explosive burst, the size of the particles, or the distance particles must travel to reach the target tissue.
  • the DNA used for coating the particles should comprise an expression cassette suitable for driving the expression of the gene of interest. Minimally this will comprise a promoter operably linked to the gene of interest.
  • Suitable promoters include, but are not limted to, the the abscisic acid (ABA)-inducible Em promoter from wheat (Marcotte et al., Plant Cell 1:976-979 (1989), the CaMV35S promoter (Odell, et al., Nature 313:810-812 (1985), and the NOS:promoter (Sanders et., Nucl. Acids Res. 15(4):1543-58 (1987).
  • ABA abscisic acid
  • Em promoter from wheat
  • CaMV35S promoter Odell, et al., Nature 313:810-812 (1985
  • NOS:promoter anders et., Nucl. Acids Res. 15(4):1543-58 (1987).
  • the cDNAs of the invention may be expressed in such a way as to produce either sense or antisense RNA.
  • Antisense RNA is RNA that has a sequence which is the reverse complement of the mRNA (sense RNA) encoded by a gene.
  • a vector that will drive the expression of antisense RNA is one in which the cDNA is placed in “reverse orientation” with respect to the promoter such that the non-coding strand (rather than the coding strand) is transcribed.
  • the expression of antisense RNA can be used to down-modulate the expression of the protein encoded by the mRNA to which the antisense RNA is complementary.
  • antisense suppression This phenomenon is also known as “antisense suppression.” It is believed that down-regulation of protein expression following antisense RNA is caused by the binding of the antisense RNA to the endogenous mRNA molecule to which it is complementary, thereby, inhibiting or preventing translation of the endogenous mRNA.
  • the antisense RNA expressed need not be the full-length cDNA and need not be exactly homologous to the target mRNA. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the endogenous mRNA will be needed for effective antisense suppression.
  • the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases.
  • the length of the antisense sequence in the vector may be greater than 100 nucleotides.
  • Vectors producing antisense RNA's could be used to make transgenic plants, as described above, in situations when desirable tree characteristics are produced when the expression of a particular gene is reduced or inhibited.
  • the cDNAs of the current invention can be derived from any sets of plant tissue.
  • the cDNAs of SEQ ID NOS: 1-334 were originally derived from embryonic tissues of pine tree embryos staged 1-9.9 as classified in Pullman and Webb TAPPI R&D Division 1994 Biological Sciences Symposium, pages 31-34, which is hereby incorporated by reference.
  • LPS and LPZ clones are derived from somatic and zygotic embryos, respectively.
  • embryos may be of either somatic or zygotic derivation, and the embryos may be grown in either semi-solid or liquid tissue culture systems. Applicable methods for growing embryos in semi-solid or liquid tissue culture systems are disclosed in U.S. Pat.
  • RNA isolated from staged cell populations provides the starting material for reverse transcription, differential display, and cloning of amplified cDNA.
  • Methods and kits for isolating total RNA from cellular populations, or for generating poly(A)+ RNA are commonly known in the art. For example, several procedures for isolating RNA are disclosed in Chapter 4 of Current Protocols in Molecular Biology edited by F. A. Ausubel et al., John Wiley and Sons, Inc. (1987) (incorporated herein by reference).
  • the TRI Reagent7 available from Molecular Research Center, Inc. (Cincinnati, Ohio) is a suitable reagent (used according to the manufacturer's instructions) for isolation of RNA from plant tissues.
  • Differential display provides a method to identify individual messenger RNAs that are differentially expressed among two or more cell populations.
  • these cell populations may be provided by pine tree or other plant embryos of different developmental stages.
  • the differential display procedure is taught in Liang et al., Science, 257:967-71 (1992) and in U.S. Pat. No. 5,262,311, which are hereby incorporated by reference. Briefly, mRNA sequences are PCR-amplified using two types of oligonucleotide primers known as “anchor” and “arbitrary” primers. Anchor primers are designed to recognize the polyadenylate tail of messenger RNAs.
  • Arbitrary primers are short and arbitrary in sequence ard anneal to complementary sequences in various mRNAs. Products amplified with these primers will vary in size and can be differentiated on an agarose or sequencing gel based on their size. If different cell populations are amplified with the same anchor and arbitrary primers, one can compare the amplification products to identify differentially expressed RNA sequences.
  • PCR-amplified bands representing differentially expressed RNA samples are excised from the gel, transferred to tubes and reamplified using the same primer pairs and PCR conditions as used in the differential display procedure.
  • Methods for the cloning of PCR products are commonly known in the art and there are several commercially available reagents and kits for cloning PCR products.
  • the pCR-SciptTM Cloning kit from Stratagene, La Jolla, Calif.
  • E. coli transformants containing plasmids with PCR fragment inserts can rapidly be identified using blue/white color selection followed by plasmid purification and restriction digests.
  • the pCR-Script vector contains T3 and T7 polymerase recognition sites allowing for in vitro transcription of the inserted fragment.
  • sequence of a cloned cDNA insert obtained may be compared against public databases such as Genbank to discern any identity or homology to known sequences.
  • Programs, such BLAST, for performing such a search are available on the National Center for Biotechnology Information's web page located at hftp://www.ncbi.nim.nih.qov.
  • Genbank search may reveal the potential function of a polypeptide or RNA molecule encoded by the cDNA.
  • search may reveal the potential function of a polypeptide or RNA molecule encoded by the cDNA.
  • FRAMES displays open reading frames for the six DNA translation frames, allowing one to quickly assess the presence or absence of stretches of open-reading frames that are likely to be protein encoding regions.
  • CodonPreference is a more sophisticated program that identifies and displays possible protein coding regions based on similarity of the codon usage in the sequence to a codon frequency table (Gribskov et al., 1984).
  • cDNA libraries were prepared from staged pine tree embryos, as described above.
  • the differential display technique was used to identify 327 novel cDNAs that were preferentially-expressed during early, middle, or late stages of pine tree embryogenesis, as set forth below. Clone nomenclature is divided into subsets based on tissue type; a clone is designated LPS to indicate somatic origins and LPZ for zygotic origins.
  • Somatic embryos were collected at different stages of development. Cultures of somatic embryos of were initiated from Loblolly pine immature zygotic embryos as described by Becwar et al., Forestry Science 44:287-301 (1994) (incorporated by reference) or with minor modifications in media mineral composition. Somatic embryos were grown in cell suspension culture medium 16 (Pullman and Webb, Tappi R&D Division 1994 Biological Sciences Symposium) and a maturation medium similar to that of a standard maturation media. Resulting somatic embryos were selected and classified as stages 1-9 according to morphological development following the teachings of Pullman and Webb, Tappi R&D Division 1994 Biological Sciences Symposium pp.31-34. Somatic embryos were sorted into tubes containing the same stages and stored at ⁇ 70° C.
  • the early phase is represented by a liquid suspension culture containing embryos of stages 1 through stage 3.
  • Middle phase contains embryos of stages 4 through stage 6, while stages 7 through 9 formed the late phase.
  • 60-100 mg aliquots of staged frozen embryos were ground in 1.0 ml of TRI Reagent® Isolation Reagent (Molecular Research Center, Inc.), a commercial product that includes phenol and guideline thiocyanate in a monophase solution and extracted according to the manufacturer's instructions.
  • the total RNA was used as a template to synthesize single stranded DNA mediated by MMLV reverse transcriptase (100 U/ ⁇ l).
  • the method involves the reverse transcription by PCR of the mRNA with an oligo-dT primer (H-T 11 G: 5′ B AAGCTTTTTTTTTTTG 3′) anchored to the beginning of the poly(A) tail, followed by a PCR reaction in the presence of a second short (13-mer) primer which is arbitrary in sequence [AP 1 (5′ B AAGCTTGATTGCC-3′) or AP 2 (5′ B AAGCTTCGACTGT-3′)].
  • Reverse transcription and Differential Display were conducted using the GenHunter RNAimage Kit 1.
  • a 19 ⁇ l reverse transcription reaction (10 ⁇ l sterile water, 2.0 ⁇ l 5 ⁇ RT buffer, 1.6 ⁇ l dNTP (250 ⁇ M), 2.0 ⁇ l anchored primer (2.0 ⁇ M), 2.0 ⁇ l RNA template at 100 ng/ ⁇ l) was prepared for each embryo phase sample.
  • the reaction mixture was heated to 65° C. for 5 minutes in a thermocycler, cooled to 37° C. and paused after 10 minutes while 1.0 ⁇ l MMLV was added. The program was allowed to resume at 37° C. for 50 minutes.
  • the reaction was then heated to 75° C. for 5 minutes, cooled to 4° C. and stored at ⁇ 20° C.
  • Differential Display PCR was performed in a 20 ⁇ l reaction containing 2 ⁇ l of the reverse-transcribed cDNA template; 10 ⁇ l sterile water 2.0 ⁇ l 10 ⁇ PCR buffer, 1.6 ⁇ l dNTP (25 ⁇ M), 2.0 ⁇ l anchored primer H-T 11G, (2.0 ⁇ M), 2.0 ⁇ l 13 mer arbitrary primer (AP 1 or AP 2 (2.0 ⁇ M), 0.2 ⁇ l Taq DNA polymerase, and 0.2 ⁇ l ⁇ 32 P-dATP (2000 Ci/mmole).
  • the cDNA was amplified by PCR: 94° C. for 3 minutes, 40 cycles of 94° C. for 30 seconds, 40° C. for 2 minutes, and 72° C. for 30 seconds, followed by 72° C. for 5 minutes.
  • the reaction was cooled to 4° C. and stored at ⁇ 20° C.
  • PCR products were separated on a Stratagene (La Jolla, Calif.) pre-cast 6% polyacrylamide sequencing gel at 30 watts constant power for approximately 2.5 to 3 hours.
  • 3.5 ⁇ l of sample was mixed with 2.0 ⁇ l, of loading dye and incubated at 80° C. for 2 minutes immediately before loading onto the gel.
  • the gel was rinsed in water and dried. Dilute 35 P-dATP with loading dye was spotted at the corners as alignment markers and the gels were exposed to Kodak BioMaXTM autoradiography film.
  • An exemplary gel is shown in FIG. 1 .
  • the PCR products from the gel fragments were purified, polished, ligated and cloned into XL 10-Gold Kan ultracompetent cells by heat shock with the Stratagene pCR-Script Amp SK(+) Supercompetent Cell Cloning Kit according to manufacturer's instructions.
  • the transformed cells were spread on LB agar plates containing ampicillin, IPTG, and X-Gal each at 50 ⁇ g/ml. The plates were incubated overnight at 37° C. Plasmids containing PCR inserts were identified using blue-white colony screening. The presence of inserts was confirmed by digesting the clones with restriction endonucleases, Msc I and Nla ll, followed by standard DNA gel electrophoresis. Transformants representing early, middle, and late phase embryos were sequenced using standard dideoxy protocols known in the art with the T3 primer.
  • SEQ ID NO: 327 was first identified through differential display with T 12 MG and AP 1 primers (GeneHunter).
  • the differential display band appeared to be present only in liquid suspension cultures of Loblolly Pine somatic embryos.
  • the conditions for mRNA isolation, reverse-transcription, differential display-PCR, and gel separation/visualization for producing this band were all as described in Example 1.
  • the band containing the original LP2-3 fragment was excised from the differential display gel, amplified, and cloned into pCR-Script AMP SK(+) according to standard protocols known in the art.
  • FIG. 11 Northern analysis demonstrated that the LP2-3 differential display clone hybridized to an approximately 1.2 Kb mRNA from liquid suspension culture embryos but was undetectable in late (6-9) stage embryo RNA.
  • FIG. 11 In general, LP2-3 is most highly expressed in early stage embryos in liquid culture. LP2-3 mRNA is found most abundantly in early stage somatic embryos, especially for embryos grown in liquid multiplication medium.
  • FIG. 12 Further, transcription decreases rapidly as embryos are transferred to maturation medium (stage 3 and stage 4) and begin to mature. LP2-3 transcripts are virtually undetectable at stage 6-9 somatic embryos grown on maturation medium. (See FIG.
  • FIGS. 13 and 14 Additional studies indicate that LP23 mRNA is expressed zygotically, particularly in early stage zygotic embryos, but is undetectable in mature vegetative tissues.
  • FIGS. 13 and 14 Specifically, the signal intensity from liquid suspension somatic embryo RNA was about 3 times greater than the signal from the analogous stage 1 zygotic embryo RNA.
  • FIGS. 13 and 14 LP2-3 transcripts were not detectable in total RNA from needles, stems, or roots of one year old seedlings, including those exposed to cold, ozone, wound stresses, or the hormone jasmonic acid (not shown).
  • a ‘full-length’ cDNA was captured from SMARTTM cDNA made from somatic embryo liquid suspension by using a biotinylated LP2-3 differential display fragment as a capture probe.
  • the “full-length” cDNA was cloned and sequenced according to standard protocols known in the art. This sequence was designated at LP2-3 + .
  • This family of proteins encodes membrane channels for the transport of water and/or ions across cell membranes. They may play a significant role in osmoregulation and may play a role in the cellular responses to water and salt stresses.
  • the MIPs are induced by dessication, flooding, and high levels of the plant hormone ABA.
  • the LP2-3 sequence was not detected in desiccated late-stage embryos which have high levels of ABA and, thus, appears to be regulated by some embryo-specific signal.
  • tissue culture practices arises via hypothesis, evaluation and adoption. Hypotheses arise from observation of size, shape, weight, etc. and physiological measurement of ion or sugar content ( FIG. 6 , box 1). These observations are limited in scope and this limits the improvements that can be made to the tissue culture process.
  • Gene expression is closely linked to metabolic condition, thus the observation of which genes are induced or repressed under a given growth condition, naturally, on the tree, or in a culture vessel, provides insight into the metabolic state of the embryo. This information can be used to create new hypotheses that can be evaluated by modifying tissue culture.
  • mRNA levels of two cDNAs (LPZ-202 and LPZ-216), similar to “Late Embryogenesis Abundant” (LEA) proteins, identified in other plants, were monitored. These genes are induced by the plant hormone ABA. Two peaks of mRNA were observed in these clones rather than the typical single peak in most plants. (See FIG. 4 for clone LPZ-216; clone LPZ-202 is similar but data is not shown.) It was subsequently confirmed that two peaks in ABA activity are observed during development and that these correspond in timing to the elevation in mRNA for LPZ-202 and LPZ-216. Thus mRNA abundance profiles are providing insight into embryo physiology. (See FIG.
  • Loblolly pine cones were collected weekly from a breeding orchard near Lake Charles, La., and shipped on ice for experimentation. Embryos were excised and evaluated for developmental stage (Pullman et al. 1994). Stage 9 embryos were separated by the week they were collected-9.1 (week 1), 9.2 (week 2), etc. Staged zygotic embryos were sorted into vials partially immersed in liquid nitrogen and stored at ⁇ 70° C. Somatic embryos for loblolly pine were initiated as described by Becwar et al. (1995) or with minor modifications. Somatic embryos were grown, selected, and staged as described by Pullman et al. (1994) and stored at ⁇ 70° C.
  • RNA slot blot was pre-hybridized in hybridization buffer (0.5 M sodium-phosphate, pH 7.2, 5% SDS, and 10 mM EDTA) at 65° C. for 2 hours in a hybridization oven (Model 400, Robbins Scientific, Sunnyvale, Calif.) and the hybridized in the same conditions with the cDNA probes. After hybridization, the membranes were washed at 65° C. in 0.2 ⁇ SSC and 0.1% SDS. Each wash was 15 min. The membranes were then exposed to Image Plate.
  • hybridization buffer 0.5 M sodium-phosphate, pH 7.2, 5% SDS, and 10 mM EDTA
  • the probes can be stripped from the RNA slot blot by pouring boiling 0.5% SDS onto the membrane twice and incubating without heating for 30 min. The stripped blot was then exposed to Image Plate for overnight to check the completeness of the de-probing before next round of hybridization.
  • Lea proteins and mRNAs appear in embryos at a stage when ABA is high and the genes can be induced in vegetative tissue by application of ABA.
  • the transcript level of Lea genes LPZ-202 and LPZ-216 showed two peaks, rising from stage 5 and returning to a base line about stage 9.2 then rising again around stage 9.5. (See FIG. 4 for clone LPZ-216).
  • Dehydrins are a class of lea protein, originally identified as water deficit inducible proteins. Since the expression of this class of protein is well characterized, in contrast to our lea genes, the dehydrin expression profile could act as a reference point.
  • blots were stripped and probed with a 26S rDNA probe from Arabidopsis to check the loading of the original gel.
  • the normalized expression pattern of dehydrin in the zygotic embryogenesis is illustrated in the top panel of FIG. 4 .
  • the expression of the dehydrin gene was induced at stage 5 and reached a peak at stage 6. It declined at stage 7-8, just prior to the onset of the desiccation. Then the mRNAs level remained low from stage 9.1 through 9.5.
  • the dehydrin mRNA levels rose again late in development, from stage 9.6 on, apparently dropping in very late development.
  • a similar pattern of expression was observed in a parallel experiment when our lea-like clone, LPZ-216, was used as a probe.
  • the model and goal for somatic embryogenesis is to produce an embryo that in vigor, germinatability, etc., resembles a zygotic embryo. Standard measurements reveal relatively little about the embryos; thus the metabolic state of somatic and zygotic embryos is unknown.
  • the metabolic state of zygotic (natural) embryos can be evaluated by DNA arrays containing the cDNA clones described in this application. A database of mRNA levels for the genes represented on the DNA arrays can then be established. Embryos growing under a new tissue culture protocol ( FIG. 6 , box #2) can be evaluated by DNA array southerns ( FIG. 6 , box #3). The array elucidates patterns of gene activity and reveals whether those patterns are similar to the natural state ( FIG. 6 , box #4). The germination, or further development can be checked ( FIG. 6 , box #5) to confirm the conclusion. Where a link between specific gene activity and embryo performance has been demonstrated, protocols can be modified with efficiency as seen in FIG. 6 , box 6.
  • Loblolly pine cones were collected weekly from a breeding orchard near Lake Charles, La., and shipped on ice for experimentation. Embryos were excised and evaluated for developmental stage (Pullman et al. 1994). Stage 9 embryos were separated by the week they were collected-9.1 (week 1), 9.2 (week 2), etc. Staged zygotic embryos were sorted into vials partially immersed in liquid nitrogen and stored at ⁇ 70° C. Somatic embryos for loblolly pine were initiated as described by Becwar et al. (1995) or with minor modifications. Somatic embryos were grown, selected, and staged as described by Pullman et al. (1994) and stored at ⁇ 70° C.
  • RNA differential display method is sensitive enough to produce banding patterns from one mid- to late-stage embryo or 10-20 early stage embryos.
  • This technique which extracts mRNA directly from tissue using oligo(dt) beads, avoids losses inherent in conventional RNA extraction methods, is fast, reliable, and inexpensive. Differences in gene expression during development, as well as between somatic and zygotic embryos, can be easily detected.
  • 50-100 ⁇ l lysis buffer containing 100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, 1% SDS and 5 mM DTT was added to 10-100 mg of staged embryos in a 1.5 ml tube.
  • the mixture was ground thoroughly with an electric drill containing a plastic pestle bit (VWR, Cat# KT95050-99) that had been sterilized by autoclaving.
  • An additional 50-100 ⁇ l lysis buffer was added and ground briefly.
  • the grinder and vortex was washed with 100 ⁇ l lysis buffer. If multiple samples were processed, each is stored on ice until ready for the next step.
  • the grinding tip was washed with sterile water and dried for the next sample.
  • the mixture was then incubated on ice for 5 min., placed on a magnetic stand (Promega) for 5 min., and partially dried by careful removal of the liquid.
  • 100 ⁇ l of washing buffer with LiDS containing 100 mM Tris-HCl, pH 8.0, 0.15 mM LiCl, 1.0 mM EDTA, and 0.1% SDS was added, (mRNA DIRECT kit.)
  • the mix was transferred to a 200 ⁇ l PCR tube.
  • the beads were washed once with 100 ⁇ l washing buffer with LIDS and once with 50 ⁇ l washing buffer containing 100 mM Tris-HCl, pH 8.0, 0.15 mM LiCl, and 1.0 mM EDTA.
  • PCR products A portion of the PCR products was run on a gel to determine amount and size of PCR products; DNA that did not correspond to the size of the original differential display band was discarded.
  • the remaining PCR fractions were purified using CHROMA SPIN-100 columns (Clontech, Palo Alto, Calif.) according to the manufacturer's instructions.
  • the purified PCR fragments were cloned into the pCR2.1 TA cloning vector (Invitrogen) according to Invitrogen cloning protocols supplied with the vector.
  • the only variation from the standard protocol was an increase in the molar concentration of PCR product to vector (over 100-fold); multiple insertions were not found to be a problem. All ligations were performed at 16° C. overnight, transformed into E. coli strain DH5 ⁇ , and plated onto LB with X-gal/IPTG.
  • PCR products Five colonies were chosen for PCR verification; PCR products of expected size were selected. About 10 ⁇ l of the 30 ⁇ l PCR reaction was simultaneously digested with Nla III and Mse I overnight at 37° C. (a 5 h digestion was used as well.) cDNA clones were selected according to the colony PCR and the restriction enzyme digestion pattern.
  • the differential display protocol for finely staged zygotic embryos of loblolly pine as described above has produced more than 600 differential display patterns and more than 60,000 bands. Within that set of bands, we have identified bands that increased and/or decreased during embryo development. From those bands cDNA clones of this invention were isolated and sequenced.
  • a micro-array assay was developed.
  • the cloned cDNAs were amplified by PCR and adjusted to equal concentrations (0.1 ⁇ g/ ⁇ l).
  • the cDNAs were then dispensed in the wells of a 384-well plate, denatured in 0.3 M NaOH at 65° C. for 30 min. and neutralized with 2 volumes of 20 ⁇ SSPE mixed with 0.00125% bromophenol blue and 0.0125% xylene cyanol FF (5% gel loading dye).
  • the denatured DNAs were then blotted on to Hybond N+membranes (Amersham) as arrays using a VP 386 pin blotter (V&P Scientific, Inc., San Diego, Calif.). Each DNA was dot-blotted four times as a quartet on the membrane. An example of quartet spotting is seen in FIG. 7 . Each dot is about 1.2 mm in diameter and contains about 3 ng of DNA. DNA was then cross-linked to the membrane at 120,000 mJ/cm2 in a CL-1000 UV-linker. (Strategene, Inc., Upland, Calif.) The dot image of each membrane was scanned, numbered and saved in computer for later use in data digitizing.
  • the cDNA array membranes were pre-hybridized in hybridization buffer (0.5 M Na-phosphate, pH 7.2, 5% SDS, and 10 mM EDTA) at 65° C. for 30′ in a hybridization oven (Model 400, Robbins Scientific, Sunnyvale, Calif.) and then hybridized under the same conditions with total cDNA probes made from mRNA.
  • the membranes were washed twice at room temperature in 2 ⁇ SSPE and 0.1% SDS, twice in 0.5 ⁇ SSPE and 0.1% SDS, and twice in 0.1 ⁇ hybridization buffer. Each wash was roughly 20 min. Each membrane was then exposed to Kodak Biomax MR films.
  • the total cDNA probes referred to above were made by initially creating the first strand cDNA. This was accomplished by mixing loblolly pine embryos (0.05-0.1 gm fresh weight) with 100 ⁇ l lysis buffer (containing 100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, 1% SDS and 5 mM DTT) in a 1.5 ml Eppendorf tube. The mix was then ground with an electric drill as described above. Another 100 ⁇ l lysis buffer was added and the lysate was ground again briefly. The drill pestle was washed with 100 ⁇ l lysis buffer that was pooled with the lysate. After centrifugation at 14K at 4° C. for 15 min.
  • the beads were washed twice with 100 ⁇ l of washing buffer with LiDS and once with 50 ⁇ l of washing buffer.
  • the mRNA was eluted from the beads in 6 ⁇ l water at 65° C. for 2′.
  • One ⁇ l T21VN primer (10 ⁇ M) and 1 ⁇ l SCSP oligo (cap switch primer, 5′-ctcttaattaagtacgcggg-3′, 10 ⁇ M) were added to the mRNA eluate. The mixture was incubated at 70° C. for 2′ and cooled on ice.
  • the first strand cDNAs synthesized as described above contains a T21VN sequence at their 5′ ends and the SCSP sequence (see “SMARTTM cDNA, Clontech, Palo Alto, Calif.) at their 3′ terminals.
  • Total cDNA probes were made by PCR amplifying the first strand cDNAs using SMART cDNA PCR (Clontech, Palo Alto, Calif.) in the presence of labeling agent.
  • Table 4 describes several publicly available clones. Lec. Fie, and Pkl, used to provide a representative model for this example. Any clone within Table 1, SEQ ID NOS: 1-327, can be substituted for those in Table 4 to assay increased performance in tissue culture. Any promoter within Table 1, SEQ ID NOS: 328-334, can be incorporated with those in Table 4 or SEQ ID NOS: 1-327 to assay increased performance in tissue culture. In this scenario, Table 5, a representation of the information contained in FIG. 9 , shows performance of selected genotypes (260, 480, 499, and 500) in various media (1133 or 16) determined by the total number of embryos produced per medium as described by Pullman and Webb (1994), incorporated herein.
  • Embryo maturation was determined by the presence of recognized morphology according to methods previously mentioned above. (Pullman and Webb, (1994)) Genotypes that produced high, medium, and low numbers of embryos were selected for RNA extraction. Gene expression assays, such as DNA arrays, Northern blots, slot blots, etc., were used in attempt to correlate embryo performance with mRNA abundance for selected genes. In the example shown in FIG. 9 and Table 5, expression of loblolly pine genes, designated as Lec, Fie, and Pkl, obtained from the Pine Gene Discovery Project, was evaluated. The preliminary correlation appears to be that the high levels of the Lec gene's mRNA correlates with greater number of pine embryos.
  • Somatic embryos were initiated as described by Becwar et al. (1990) or with modifications in media mineral composition.
  • the early stage somatic embryos were grown in cell suspension culture medium 16 and sub-cultured every week (Pullman and Webb, 1994).
  • the embryos collected from the suspension which include stage 1 and stage 2 somatic embryos, are referred to as stage S embryos.
  • stage S embryos At the end of the subculture week, the somatic embryos in the suspension were settled in a cylinder and transferred to maturation medium 240 (Pullman and Webb, 1994). Resulting somatic embryos were selected, staged, sorted into vials containing the same stage, and stored at ⁇ 70° C. until analyses were performed.
  • RNA was isolated from embryos at different stages in development, early stage somatic embryos and late-stage somatic embryos.
  • the cDNA probes used in this example are not contained in the SEQ ID NOS: 1-327, but rather, are generic, publicly available pine sequences obtained from the Pine Gene Discovery project located at (http://www.cbc.med.umn.edu/ResearchProiects/Pine/DOE.pine/index.html). These clones are homologs to the well-studied Arabidopsis genes that have been shown to have significant influence on embryo development in this plant.
  • the pine clone names (first column) and corresponding references for the Arabidopsis homologs are shown in Table 4.
  • Probes were made by preparation of DNA using Wizard Minipreps (Promega, Madison, Wis.) and cDNA inserts isolated by restriction enzyme digestion. For the cDNA probes, 50 ng of the isolated cDNA insert DNA was used to make 32 P-labeled probes with Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech) according to manufacturer's instructions. Blots were prehybridized (7% SDS, 1% BSA, 0.25 M NaPO 4 (pH 7.2), 1.0 mM EDTA) for 3 hours at 65° C. and hybridized in fresh buffer at 65° C. for 12 to 18 hours (4).
  • FIG. 9 shows that Lec is not expressed in late stages of embryogenesis in somatic embryos.
  • the Lec gene is expressed throughout embryogenesis in Arabidopsis .
  • the blot reveals that the Lec gene is a useful early expression marker for embryogenesis.
  • the somatic embryos do not express Lec in the manner that Lec is expressed in zygotic embryos, i.e. the use of Lec expression has highlighted a defect in gene expression in somatic embryos. This defect could be used to identify desirable genotypes, i.e. those likely to progress through development and produce a large number of healthy plantlets compared to undesirable genotypes that will cease development prematurely or produce low numbers of plantlets.
  • PICKLE is a CHD3 chromatin- after germination. over 155aa remodeling factor that Represses lec regulates the transition from expression embryonic to vegetative development in Arabidopsis .
  • PNAS. 96(24): 13839-13844 PC08C06 FIE fertilization-independent Fie mutants initiate 61% ID 92 8e ⁇ 20 (‘FIE’ in endosperm protein. Ohad, et al endosperm development 75% + ve figure.) 1999. Mutations in FIE, a WD w/o fertilization over 67aa polycomb group gene, allow endosperm development without fertilization. Plant Cell 11 (3), 407-416

Abstract

The invention relates to a method for staging embryos of plants. In particular, this invention relates to a method for creating a relational database by determining transcript levels of sets of genes expressed at predetermined stages in embryo development. This approach creates a method by which embryos of unknown stage development can be determined by comparisons between expression levels of those embryos to the expression levels found in the database. This approach further allows rapid identification of transcripts in an embryo to be staged by the utilization of probes corresponding to cDNAs comprising the database. Additionally, this invention relates to a method for selecting advantageous plant clones for future propagation. Specifically, this method relates to an approach to link the biochemical condition of an embryo to current culture conditions and thus provides a method for enhancing conditions to produce embryos with a desired biochemical state.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This patent application claims benefit of priority of provisional application U.S. Ser. No. 60/239,250, filed Oct. 11, 2000, and claims benefit of priority of provisional application U.S. Ser. No. 60/260,882, filed Jan. 12, 2001.
  • FIELD OF THE INVENTION
  • The present invention relates to a relational database of cDNA molecules, including those corresponding to Loblolly Pine Major Intrinsic Protein (MIP), which are differentially expressed during plant embryogenesis. The present invention further relates to the use of DNA arrays for evaluating gene expression in somatic and zygotic embryos. The invention encompasses related nucleic acids, proteins, antigens, and antibodies derived from these cDNAs as well as the use of such molecules for the staging, characterization, and manipulation of plant embryogenesis, in particular conifer embryogenesis. The cDNAs and related nucleic acids, proteins, antigens, and antibodies derived from these cDNAs are useful in the design, selection, and cultivation of improved crops, specifically including coniferous trees, which provide raw materials for paper and wood products.
  • BACKGROUND OF THE INVENTION
  • The world demand for paper is expected to increase nearly 50% by the year 2010 (McNutt and Rennel, Pulp Paper Intern 39: 48 (1997)). The United States' forest products industry faces a great challenge in order to keep pace with the growing demand for paper. This challenge is made greater by the decreasing availability of a forest land-base, resulting from environmental restrictions and urban growth, from which to harvest timber resources. Additionally, valuable wood resources are lost to the environmental stresses and biotic diseases. Consequently, the push to secure a renewable and sustainable source of raw material for paper and other wood related products has become an important priority for the forest products industry.
  • Current forestry related research and development is focused on creating sustainable fiber farms or tree plantations. Farming trees with elite germplasms will increase growth rates and yields of wood per acre. However, creating improved tree stock requires the ability to identify and generate genetically superior trees and a way to propagate such superior trees without diluting their genetic quotient.
  • A. Breeding and Selection
  • Addressing the need to propagate genetically superior trees without genetic diminution demands full research attention. Traditional methods of tree propagation relied on selected breeding programs to achieve genetic gain, i.e., the development of a strain, sub-strain, or line having any heritable and economically valuable characteristic or combination of characteristics not found in the parents. Based on the results of progeny tests, superior maternal trees are selected and used in “seed orchards” for mass production of genetically improved seed. The genetic gain in such an open-pollinated sexual propagation strategy is, however, limited by the breeder's inability to control the paternal parent. Additional gains can also be achieved by control-pollination of the maternal tree with pollen from individual trees whose progeny have demonstrated superior growth characteristics. Nevertheless, even under controlled conditions where both parents of each seed are the same, sexual propagation results in a “family” of seeds, i.e., siblings, comprised of many different genetic combinations. As not all genotype combinations are favorable, the genetic gain in any particular progeny is frequently offset and obscured by the genetic variation among sibling seeds and those seedlings retaining undesirable or previously masked pre-cross traits.
  • In addition to inherent genetic limitations of a traditional breeding programs, large-scale production of control pollinated seeds is also expensive. Consequently, economic and biological limitations of large-scale seed production has lead the industry to turn towards methods of asexual reproduction, such as grafting, vegetative propagation and micropropagation, as more viable alternatives.
  • B. Asexual (Clonal) Propagation
  • Asexual propagation permits the application of very high selection intensity, resulting in the propagation of only those progeny showing a high genetic gain potential. These highly desirable progeny can have unique genetic combinations that result in superior growth and performance characteristics. Thus, with asexual propagation it is possible to genetically select individuals while avoiding a concomitant reduction of genetic gain due to intra-familial variation.
  • Asexual propagation of trees can be accomplished currently by grafting, vegetative propagation, and micropropagation. Grafting, widely used to propagate select individuals in limited quantities for seed orchard establishment, is not applicable to large-scale production for reforestation. Vegetative propagation, achieved by the rooting of cuttings, and micropropagation by somatic embryogenesis, currently hold the most potential for reforestation of conifers. Although vegetative propagation by rooted cuttings can be achieved in many coniferous species, large-scale production via this method is extremely costly due to difficulties in automating and mechanizing the process, not to mention the need for tremendous quantities of stock tissue. This propagation method is still further limited by the fact that the rooting potential of stock plants decrease with time, making it difficult to serially propagate from select genotypes over extended periods of time.
  • Micropropagation is the most promising method of asexual propagation for mass plantings. This process involves the production of somatic embryos in vitro from minute pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Both vegetative propagation and micropropagation have the potential to capture all genetic gain of highly desirable genotypes. However, unlike conventional vegetative propagation methods, somatic embryogenesis is amenable to automation and mechanization, making it highly desirable for large-scale production of planting stock for reforestation. Moreover, somatic embryogenesis is particularly amenable to high intensity selection of a large number of clones. These advantages are compounded by the ability to safely preserve somatic embryogenic cultures in liquid nitrogen for long-term storage. Consequently, long-term cryogenic preservation offers immense advantages over other vegetative propagation systems that attempt to maintain the juvenility of stock plants. Techniques for somatic embryogenesis in a wide variety of plant species are well known in the art; exemplary methods for performing somatic embryogenesis in conifers are taught in U.S. Pat. Nos.: 5,036,007; 5,236,841; 5,294,549; 5,413,930; 5,491,090; 5,506,136; 5,563,061; 5,677,185; 5,731,203; 5,731,204; and 5, 856,191, herein incorporated by reference in their entirety.
  • Thus, somatic embryogenesis has great potential for clonal production of conifer embryos to meet the increased demands of the pulp and paper industry. Assessment of embryo quality, however, needs improvement. The process of creating better tree stock begins with understanding the process of tree development from embryogenesis through full maturation.
  • In general, plant tissue culture is the broad science of growing plant tissues on or in a nutrient medium containing minerals, sugars, vitamins and plant hormones. By adjusting the composition of the media, cultured tissues can be induced to grow or differentiate into specific cell types or organs. “Somatic embryogenesis” is a type of plant tissue culture where a piece of a donor plant is excised, cultured and induced to form multiple embryos. An embryo is a discrete mass of cells with a well-defined structure that is capable of growing into a whole plant.
  • The methods generally in use for somatic embryogenesis today involve several steps. Prior to the tissue culture process, a suitable “explant” is harvested. A typical explant in conifer somatic embryogenesis is the “megagametophyte”, a haploid nutritive tissue of the conifer seed, which is extracted from the ovule of a pollinated female cone. This ovule contains single or multiple zygotic seed embryos. In the seeds of many coniferous species, one or more genetically unique embryos naturally undergo a process called cleavage polyembryony, where a zygotic embryo grows and divides to form a small clones of embryos.
  • The first step in somatic embryogenesis is the initiation step. The explant is placed on a suitable media. When the explant is an ovule, a process called extrusion occurs. Extrusion involves the emergence or expulsion of a zygotic embryo or multiple embryos and embryogenic tissue out of the megagametophyte. If culture conditions are suitable, initiation proceeds and the extruded embryo or embryos undergo the process of cleavage polyembryony. This results in the formation of early stage somatic embryos in a glossy, mucilaginous mass.
  • After embryogenic cultures are initiated, the somatic embryos are transferred to a second medium with an appropriate composition of plant hormones and other factors to induce the somatic embryos to multiply. In the multiplication stage, cultures can double up to 2-6 times in one week. Once large numbers of embryos are obtained in the multiplication stage, the embryos are moved to a development and maturation medium. Here, the correct balance of plant hormones and other factors will induce the early-stage embryos to mature into late stage embryos. Following the maturation and development stage, embryos are germinated to form small seedlings. These seedlings are then acclimated for survival outside of the culture vessel. After acclimation, the seedlings are ready for planting.
  • The relative ability to propagate plants by somatic embryogenesis can vary greatly between species. Among conifers, for example, spruce (Picea) species and Douglas fir are easily propagated, while Pinus species are much more difficult. Many Pinus species, including Loblolly pine (Pinus taeda), do not readily initiate embryonic cultures. Typical initiation frequencies between 1% and 12% are reported for various Pinus species (Becwar et al., For. Sci. p1-18 (1988), Jain et al., Plant Sci. 65:233-241 (1989), Becwar et al., Can. J. For. Res. 20:810 (1990), Li and Huang, J. Tissue Cult. Assoc. 32:129 (1996)). Laine and David, (Plant Sci. 69:215 (1990)), however, were able to obtain high frequencies of initiation (up to 59%) in Pinus caibaea, suggesting that not all Pinus species are recalcitrant. Also, one earlier report described initiation frequencies of 54% in White pine (Pinus strobus). Finer et al., Plant Cell Rep. 8:203 (1989). However, other workers were not able to duplicate this success. Michler et al., Plant Sci. 77:111 (1991). The results in the literature demonstrate the recalcitrance of Pinus species, especially Loblolly pine, in regeneration by somatic embryogenesis.
  • Nevertheless, once this process is understood from the standpoint of developmental genetics, breeders will then have the appropriate tools to monitor, intervene, and improve both the regeneration frequency and the overall quality of tree stock through genetic engineering. For example, both environmental requirements and responsiveness of a developing embryo change as the embryo passes various developmental milestones. Consequently, accurate and timely knowledge of the developmental stage of an embryonic culture would allow the skilled practitioner to beneficially adjust the growth media components and other environmental factors to achieve optimal embryo survival, growth, and maturation. In addition, an understanding of developmentally regulated genes would allow for early selection of advantageous clones and provide tools for developmentally regulated transgenic expression systems.
  • Currently, a reasonable determination of the precise developmental stage of an embryo requires a practiced, physical familiarity with the morphological appearance of embryos at different stages, which is further complicated by the presence of morphological variations between species. Consequently, visual determination is performed best by experts in the field. Thus, there is a need in the art for a staging method which can be reliably practiced by the ordinary practitioner. The current invention will allow one to stage embryos based on a relational database system profiling gene expression patterns instead of physical morphological differences, thereby permitting one less skilled in the art of visual staging to biologically determine the stages of embryogenesis.
  • The traditional morphological staging method provides only a crude indication of the underlying biochemical condition or state of an embryo. This level of information is insufficient for refining culture conditions, including media formulations, or for selecting potentially advantageous embryo clones for further development. Thus, there is a need in the art for a more sensitive staging method that precisely defines the physiological age, health, growth requirements, and potential fitness of a particular embryo. The current invention will allow definitive staging significantly beyond that currently practiced in the art, and provides a detailed analysis of the biochemical state and potential fitness of an embryo by comparison to developed relational database profiles.
  • Visual staging methods depend on morphological markers to assign a numerical stage of 1-9 to an embryo. Nevertheless, it is well accepted that visually undetectable developmental changes occur in an embryo after it reaches stage 9. The current invention is particularly useful in providing means for monitoring and evaluating the developmental state of these older embryos, as genetic responses occur and are detectable up to and through an adult tree's life.
  • There further exists in the art a need for information regarding the proteins, genes, and gene expression patterns in plant embryo development, as well as a more thorough understanding of how this information relates to the physiology, developmental potential, and genetic quotient of a plant embryo. The relational database system provides a platform for which to monitor individual gene expression levels during embryo development while directly correlating expression with, for example, environmental conditions, age, and embryo fitness, as well as the protein identification achieved by BLAST searches of publicly available databases (i.e., GenBank) for desirable genes. Accordingly, the present invention therefore provides the additional ability to correlate the direct, global gene expression response within the embryo system to a typically non-expressing gene driven by a stage-specific promoter.
  • SUMMARY OF THE INVENTION
  • The present invention addresses these needs by providing in a relational database format nucleic acid and protein sequences that are differentially expressed during various stages of plant embryogenesis. The invention encompasses a set of isolated nucleic acid molecules comprising the DNA sequence of any one of SEQ ID NOS: 1-334 and nucleic acid molecules related or complementary to any one of SEQ ID NOS: 1-334. (See Table I) As such, the invention includes both single-stranded and double-stranded RNA and DNA nucleic acids, including variants thereof. The nucleic acids of the invention can be used as an expression template in the form of DNA arrays, including for example, gene arrays, DNA chips, and dot array Southerns, for which to compare and evaluate expression in test samples. (See Table II) The nucleic acids of the invention can be further used as probes to detect the presence or level of both single-stranded and double-stranded RNA and DNA encoding variants of polypeptides or fragments of polypeptides encompassed by the invention. The nucleic acids of the invention can be further used as promoters for the expression of sense and antisense molecules at specific stages of embryo development. Data acquired through the use of the present invention can in turn be provided to the relational database for further development.
  • Isolated nucleic acid molecules that hybridize to a denatured, double-stranded DNA comprising the DNA sequence of any one of SEQ ID NOS: 1-334 under conditions of moderate or high stringency are also encompassed by the invention. The invention further encompasses synthetic and naturally-occurring variants of the nucleic acids described in SEQ ID NOS: 1-334, for example, isolated nucleic acid molecules derived by in vitro mutagenesis from SEQ ID NOS: 1-334. In vitro mutagenesis would include numerous techniques known in the art including, but not limited to, site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis.
  • The invention also encompasses related molecules (variants) including isolated nucleic acid molecules degenerate from SEQ ID NOS: 1-334 as a result of the genetic code, for example, naturally-occurring or synthetic allelic variants of the genes encoding SEQ ID NOS: 1-334. Such related molecules also encompass both smaller and larger nucleic acids that contain sufficient sequence to support hybridization to any of SEQ ID NOS: 1-334 under conditions of moderate or high stringency. Consequently, recombinant vectors, including those that direct the expression of these nucleic acid molecules and host cells transformed or transfected with these vectors are herein defined as variants and are encompassed by the invention.
  • Another embodiment of this invention is the production of transgenic vectors and transgenic plants comprising vectors or other nucleic acids comprising any one of SEQ ID NOS: 1-334 and related molecules. Particularly preferred are those capable of expressing polypeptides or peptides encoded by any of SEQ ID NOS: 1-327. In a preferred embodiment, the transgene comprises SEQ ID NO: 327, or a variant thereof.
  • SEQ ID NO: 327 encodes a protein which corresponds to a novel Loblolly pine homolog of the plant Major Intrinsic Protein (MIP) family. MIPs comprise a large family of related proteins that function as membrane channels for the transport of water and possibly ions across cellular membranes. Henceforth, the encoded protein of SEQ ID NO: 327 may be referred to as Loblolly MIP. Variants, including naturally-occurring and artifactually-programmed allelic variants, vectors, and other nucleic acids which hybridize to SEQ ID NO: 327 under conditions of moderate or high stringency are encompassed by the invention. Also encompassed are plant cells, seeds, embryos and trees, transgenic for loblolly pine MIP, and variants thereof.
  • The invention also encompasses isolated polypeptides, or fragments thereof, encoded by any one of the nucleic acid molecules of SEQ ID NOS: 1-327, including variants thereof. The invention further encompasses the use of these peptide sequences as markers for staging, monitoring, and selecting embryos and embryo cultures. The invention also encompasses methods for the production of these polypeptides or fragments thereof including culturing a host cell under conditions promoting expression and recovering the polypeptide or peptide from the culture medium. In particular, the expression of polypeptides or peptides encoded by SEQ ID NOS: 1-327 in viral vectors, bacteria, yeast, plant, and animal cells is encompassed by the invention. Isolated polyclonal or monoclonal antibodies that bind to peptides encoded by SEQ ID NOS: 1-327 are also encompassed by the invention.
  • Further encompassed by this invention are methods for using the nucleic acid molecules of any one of SEQ ID NOS: 1-327 to obtain full length cDNA and genomic sequences of the corresponding genes, including cognate, homologous, or otherwise related genetic sequences, which hybridize to any of SEQ ID NOS: 1-327 under conditions of moderate or high stringency. Also provided by this invention are oligonucleotides derived from any one of SEQ ID NOS: 1-334 that can be used as probes and/or as primers in PCR, RT-PCR, and other assays to detect the presence or level of the nucleic acids of SEQ ID NOS: 1-334 and related molecules.
  • The primers and other probes of the invention may be used to monitor and characterize the development of plant embryos, in particular, pine tree embryos. Characterization of embryonic gene expression provides means for correlating gene expression with current and potential plant phenotypes. Consequently, the present invention encompasses means for monitoring and adjusting growth conditions (see FIG. 6), as well as means for selecting genetically superior embryonic clones for further propagation and expansion (see FIG. 8). Thus, the present invention encompasses the use of DNA or RNA probes derived from the nucleic acid molecules of SEQ ID NOS: 1-334 in any form, e.g., in DNA arrays, and antibodies raised against polypeptides or peptide fragments encoded by SEQ ID NOS: 1-327, to determine relative or absolute levels of expression of the genes or proteins encoded by SEQ ID NOS: 1-327. In addition, these nucleic acid and antibody probes may be used for staging, monitoring, characterizing, or selecting plant embryos or embryo cultures, particularly pine tree embryos.
  • The relational database of the present invention allows expression information pertaining to embryo stages to be viewed as sequence data generated in accordance with the present invention. The invention includes a database for storing a plurality of sequence records for which to correlate embryo stages to sequence records. The method further involves providing an interface which allows a user to select one or more expression categories contained within the database.
  • The relational database is designed to include separate parts or cells for information storage. One cell or part may contain a gene expression database which contains nucleic acid molecules of SEQ ID NOS: 1-327. Other cells or parts may contain descriptive information pertaining to each nucleic acid molecules of SEQ ID NOS: 1-327, additional sequence data related to the gene expression database, protein encoded by nucleic acids disclosed herein, similarity values to known proteins of other systems, and to conditions under which expression data was obtained.
  • The database system described in the present invention will allow identification or selection of particular genes of interest for further use with DNA arrays. Identification or selection of particular genes may include, for example, those related to patterns of expression, those identified with homology to known genes from other studies, and those sequences considered novel.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts differential display of loblolly pine zygotic and somatic embryos at different stages of development.
  • FIG. 2 displays embryo gene expression observed by high-density array Southern hybridization.
  • FIG. 3 provides a general schematic for gene regulation studies arising from the cDNA cloning of genes expressed in embryos.
  • FIG. 4 depicts graphical representation of hybridization of ‘dehydrin’ and LPZ216 cDNA probes to total RNA isolated from zygotic embryos of loblolly pine.
  • FIG. 5 displays ABA concentration of loblolly pine embryos.
  • FIG. 6 shows schematic of gene study for improved somatic embryogenesis.
  • FIG. 7 shows detection of gene expression by high-density array Southern hybridization for loblolly pine genotype 333 after 12 weeks on two maturation media.
  • FIG. 8 depicts the application of embryogenic gene expression studies.
  • FIG. 9 displays slot blots and expression levels for three embryogenesis-related genes.
  • FIG. 10 depicts clone LPS-097 sequence (LP2-3 differential display fragment.)
  • FIG. 11 displays a northern blot for the LP2-3 gene during stages 1-3.
  • FIG. 12 displays a slot blot of total RNA from somatic embryo tissue probed with an LP2-3-specific probe.
  • FIG. 13 displays a slot blot of total RNA from zygotic embryo tissue probed with an LP2-3-specific probe.
  • FIG. 14 depicts the quantified expression of early zygotic embryos compared to early somatic embryos.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The three hundred and twenty-seven differentially expressed cDNAs isolated from plant specimens of known developmental ages are disclosed in SEQ ID NOS: 1-327. The seven stage-specific promoters isolated from plant specimens are disclosed in SEQ ID NOS: 328-334. The discovery of these cDNAs and promoters enables the design, isolation, and construction of related nucleic acids, proteins, antigens, antibodies other heterologous genes. Both the cDNAs and promoters facilitate the staging, characterization, and manipulation of plant embyrogenesis, in particular, conifer embryogenesis. These molecules, and related nucleic acids, peptides, proteins, antigens, and antibodies are particularly useful when compiled into a relational database for the monitoring, design, selection, and cultivation of improved crop plants.
  • The cDNAs of SEQ ID NOS: 1-327, in addition to the promoters of SEQ ID NOS: 328-334, were originally derived from Pinus taeda embryos, commonly known as the Loblolly Pine. Nevertheless, it is understood that the invention is applicable to and encompasses all plants, including all dicotyledonous plants, including all conifers, including all species of Pinus, Picea, and Pseudotsuga. Exemplary conifers may include Picea abies, and Psedotsuga menziesii, and Pinus taeda.
  • Nucleic Acid Molecules
  • In a particular embodiment, the invention relates to certain isolated nucleotide sequences including those that are substantially free from contaminating endogenous material. The terms “nucleic acid” or “nucleic acid molecule” refer to a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides. A “nucleotide sequence” also refers to a polynucleotide molecule or oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid. The nucleotide sequence or molecule may also be referred to as a “nucleotide probe.” The nucleic acid molecules of the invention are derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequence by standard biochemical methods. Examples of such methods, including methods for PCR protocols that may be used herein, are disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), Current Protocols in Molecular Biology edited by F. A. Ausubel et al., John Wiley and Sons, inc. (1987), and Innis, M. et al., eds., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990), each of which are herein incorporated by reference in their entirety.
  • As used herein a “nucleotide probe” is defined as an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, through complementary base pairing, or through hydrogen bond formation. As described above, the oligonucleotide probe may include natural (ie. A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, bases in a oligonucleotide probe may be joined by a linkage other than a phosphodiester bond, so long as it does not prevent hybridization. Thus, oligonucleotide probes may have constituent bases joined by peptide bonds rather than phosphodiester linkages.
  • A “target nucleic acid” herein refers to a nucleic acid to which the nucleotide probe or molecule can specifically hybridize. The probe is designed to determine the presence or absence of the target nucleic acid, and the amount of target nucleic acid. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target. As recognized by one of skill in the art, the probe may also contain additional nucleic acids or other moieties, such as labels, which may not specifically hybridize to the target. The term target nucleic acid may refer to the specific nucleotide sequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. One skilled in the art will recognize the full utility under various conditions.
  • As described herein, the nucleic acid molecules of the invention include DNA in both single-stranded and double-stranded form, as well as the RNA complement thereof. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA, including translated, non-translated and control regions, may be isolated by conventional techniques, e.g., using any one of the cDNAs of SEQ ID NO: 1 through SEQ ID NO: 327, or suitable fragments thereof, as a probe, to identify a piece of genomic DNA which can then be cloned using methods commonly known in the art. In general, nucleic acid molecules within the scope of the invention include sequences that hybridize to sequences of SEQ ID NOS: 1-334 under hybridization and wash conditions of 5°, 10°, 15°, 20°, 25°, or 30° below the melting temperature of the DNA duplex of sequences of SEQ ID NOS: 1-334, including any range of conditions subsumed within these ranges.
  • DNA Arrays
  • In a further embodiment, DNA arrays are used to identify hybridizing sequences from test samples. The term “DNA array” refers to “gene arrays,” “DNA chips,” “dot array Southerns,” etc. One of skill in the art will appreciate that an enormous number of array designs are suitable for the practice of this invention. The DNA array will typically include one or a multiplicity of nucleic acid molecules derived from SEQ ID NO: 1 through SEQ ID NO: 327 that specifically hybridize to the nucleic acid expression of which is to be detected. In addition, the array may include one or more control probes to monitor the expression system. Control probes refer to known expression products present at each stage of expression, e.g., ribosomal gene products or the transcripts of other housekeeping genes. The organization of the DNA array will be known to facilitate interpretation of results. Examples in the art describing the uses and composition of DNA arrays can be found in U.S. Pat. Nos.: 5,700,637, 5,837,832, 5,843,655, 5,874,219, 6,040,138, 6,045,996, and are incorporated by reference.
  • Molecules That Hybridize to Identified Sequences
  • Thus, in a particular embodiment, this invention provides an isolated nucleic acid molecule selected from the group consisting of:
    • (1) a DNA sequence comprising any one of the sequences presented in SEQ ID NO: 1 through SEQ ID NO: 334;
    • (2) an isolated nucleic acid molecule that hybridizes to either strand of a denatured, double-stranded DNA comprising the nucleic acid sequence of (a) under conditions of moderate stringency; and
    • (3) an isolated nucleic acid molecule that hybridizes to either strand of a denatured, double-stranded DNA comprising the nucleic acid sequence of (a) under conditions of high stringency.
  • As used herein, stringency conditions in nucleic acid hybridizations can be readily determined by those having ordinary skill in the art based on, for example, the length and composition of the nucleic acid. In one embodiment, moderate stringency is herein defined as a nucleic acid having 10, 11, 12, 13, 14, 15, 16, or 17, contiguous nucleotides identical to any of the sequences of SEQ ID NOS: 1-334, or a complement thereof. Similarly, high stringency is hereby defined as a nucleic acid having 18, 19, 20, 21, 22, or more contiguous identical nucleotides, or a longer nucleic acid having at least 80, 85, 90, 95, or 99 percent identity with any of the sequences of SEQ ID NOS: 1-334; for sequences of at least 50, 100, 150, 200, or 250 nucleotides, high stringency may comprise an overall identity of at least 60, 65, 70 or 75 percent.
  • Generally, nucleic acid hybridization simply involves providing a denatured nucleotide molecule or probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. The nucleic acids that do not substantially form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is further generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under lower stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed sequences are not perfectly complementary. Thus specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer mismatches. One of skill in the art will appreciate that hybridization conditions may be selected to provide any degree of stringency.
  • As used herein, the percent identity between an amino acid sequence encoded by any of SEQ ID NOS: 1-334 and a potential hybridizing variant can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol, 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (Nuci. Acids Res. 14:6745, 1986), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • Alternatively, basic protocols for empirically determining hybridization stringency are set forth in section 2.10 of Current Protocols in Molecular Biology edited by F. A. Ausubel et al., John Wiley and Sons, Inc. (1987). Stringency conditions can be determined readily by the skilled artisan. An example of moderate stringency hybridization conditions would be hybridization in 5×SSC, 5× Denhardt's Solution, 50% (w/v) formamide, and 1% SDS at 42° C. with washing conditions of 0.2×SSC and 0.1% SDS at 42° C. An example of high stringency conditions can be defined as hybridization conditions as above, and with washing at approximately 68° C., in 0.1×SSC, and 0.1% SDS. The skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe.
  • Due to the degeneracy of the genetic code wherein more than one codon can encode the same amino acid, multiple DNA sequences can code for the same polypeptide. Such variant DNA sequences can result from genetic drift or artificial manipulation (e.g., occurring during PCR amplification or as the product of deliberate mutagenesis of a native sequence). The present invention thus encompasses any nucleic acid capable of encoding a protein derived from SEQ ID NOS: 1-327, or variants thereof.
  • Deliberate mutagenesis of a native sequence can be carried out using numerous techniques well known in the art. For example, oligonucleotide-directed site-specific mutagenesis procedures can be employed, particularly where it is desired to mutate a gene such that predetermined restriction nucleotides or codons are altered by substitution, deletion or insertion. Exemplary methods of making such alterations are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, Jan. 12-19, 1985); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462, all of which are incorporated by reference.
  • Thus, the invention further provides an isolated nucleic acid molecule selected from the group comprising of (1), (2), and (3) above and further consisting of:
    • (4) an isolated nucleic acid molecule degenerate from SEQ ID NOS: 1-334 as a result of the genetic code; and
    • (5) an isolated nucleic acid molecule selected from the group consisting of an allelic variants and species homologs of SEQ ID NOS: 1-334.
      Obtaining Full Length cDNAs
  • The cDNAs isolated and cloned through the differential display procedure will often only represent a partial sequence (generally the 3′ end) of the mRNA from which it was derived due to the nature of the arbitrary primer used in the differential display PCR reaction. Consequently, the cDNA sequences of SEQ ID NOS: 1-327 provide powerful tools for obtaining the sequences of full-length cDNAs. This can be accomplished by using a partial cDNA as a probe to identify and isolate the full length cDNA from a population of full length cDNAs or from a full length cDNA library. As is well known in the art, similar procedures can be used to identify corresponding genomic DNA sequences.
  • Alternatively, one can obtain the 5′ sequence of a partial cDNA by performing Rapid Amplification of cDNA Ends (RACE) procedures such as those disclosed in Frohman, Methods in Enzymology, 218:340-356 (1993) and Bertling et al., PCR Methods and Applications 3:95-99 (1993) which are hereby incorporated by reference. For example, Clonetech Laboratories, Inc. (Palo Alto, Calif.) offers a SMAR™ cDNA product line that allows one to generate high quality full length cDNAs and cDNA libraries. SMART™ technology can also be used to perform RACE. One skilled in the art will readily recognize that there are other equivalent products and procedures for obtaining full length cDNAs. Full length cDNAs may be sequenced and their sequences compared to sequences in public databases to assess their identities and/or homologies to other known sequences.
  • Cloned full length cDNAs can be used in the construction of expression vectors for the production and purification of pine tree polypeptides which contain the pine tree peptides encoded by the cDNAs of any one of SEQ ID NOS: 1-327.
  • Oligonucleotide Primers for PCR Assays
  • In another embodiment, the present invention encompasses oligonucleotide fragments derived from any one of SEQ ID NO: 1 through SEQ ID NO: 327 or from the reverse complement sequence of any one of SEQ ID NO: 1 through SEQ ID NO: 327. Such oligonucleotides would be useful as primers in the performance of RT-PCR assays to detect, or even quantify, pine embryo stage-specific transcripts. Such oligonucleotide primers will generally comprise from 10 to 25 nucleotides substantially complementary to the ends of the target sequence and may contain additional non-complementary nucleotides, for example, nucleotides that generate a restriction endonuclease site or cloning junction. Programs useful in selecting PCR primers may be used to design the oligonucleotides of this invention, but use of such programs is not necessary. By way of example, the Wisconsin Package™ software available from the Genetic Computer Group (Madison, Wis.) includes a program called Prime that can aid in selecting primers from a given template sequence. Protocols for the design and optimization of PCR reactions are commonly known in the art and are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).
  • Antisense Nucleic Acid Molecules
  • Other useful fragments of the nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of DNA from any one of SEQ ID NO: 1 through SEQ ID NO: 327. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 To about 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (Bio/Techniques 6:958, 1988).
  • Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes or other nucleic acid complexes inimical to efficient production of gene products. The antisense oligonucleotides thus may be used to block expression of proteins or the function of RNA. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sufficient sequence specificity to be able to bind to target nucleotide sequences.
  • Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides. Such modifications may modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
  • Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, lipofection, CaPO4-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus or adenovirus.
  • Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. In one embodiment, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
  • Polypeptides Encoded by Differentially-Expressed cDNAs
  • The cDNAs of SEQ ID NOS: 1-327 can be translated into amino acid sequences potentially corresponding to portions of developmentally-regulated plant proteins. These amino acid sequences can be identified from sequences listed in Table I, below. The cDNAs encoding these predicted polypeptides are grouped into early, middle, and late transcripts according to the staged embryo population from which they were derived.
  • (See Table I)
  • Although the term “peptide” is generally understood to reference synthetic sequences, or fragments of larger proteins, and includes short amino acid sequences of between 2 and 10 amino acids, whereas “polypeptide” refers to larger sequences and full-length proteins, the terms are used interchangeably herein to indicate that the invention applies to peptides and polypeptides of any length and variants thereof. Moreover, the discovery of presumptive open reading frames in SEQ ID NOS: 1-327, and the ability to isolate additional cDNA sequence, enables the construction of expression vectors comprising nucleic acid sequences encoding those polypeptides. The cDNAs of the invention also enable cells transfected or transformed with expression vectors driving the expression of the encoded polypeptides and antibodies reactive with the polypeptides.
  • In one embodiment, the invention provides for isolated polypeptides, preferably, pine tree polypeptides. As used herein, the term “polypeptides” refers to a genus of polypeptide or peptide fragments that encompass the amino acid sequences identified from Table I, as well as smaller fragments. Consequently, the invention encompasses any polypeptide fragment comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous amino acids encoded by the cDNAs of any of SEQ ID NOS: 1-327, or comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous amino acids of any of amino acid sequence derived from Table I.
  • Alternatively, a polypeptide may be defined in terms of its antigenic relatedness to any peptide encoded by SEQ ID NOS:-1-327. Thus, in one embodiment a polypeptide within the scope of the invention is defined as an amino acid sequence comprising a linear or 3-dimensional epitope shared with any peptide encoded by the cDNAs of SEQ ID NOS: 1-327. Alternatively, a polypeptide within the scope of the invention is recognized by an antibody that specifically recognizes any peptide encoded by SEQ ID NOS: 1-327. Antibodies are defined to be specifically binding if they bind pine tree polypeptides with a Ka of greater than or equal to about 107 M, and preferably greater than or equal to 108 M−1.
  • A polypeptide “variant” as referred to herein means a polypeptide substantially homologous to a native polypeptide, but which has an amino acid sequence different from that encoded by any of SEQ ID NOS: 1-327 because of one or more deletions, insertions or substitutions. The variant amino acid sequence preferably is at least 80% identical to a native polypeptide amino acid sequence, preferably at least 90%, more preferably, at least 95% identical over at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-25, or 26-30 contiguous amino acids. The percent identity between an amino acid sequence encoded by any of SEQ ID NOS: 1-327 and a potential variant can be determined manually, or, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program, described above, utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443,1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981).
  • Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as lie, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. See Zubay, Biochemistry, Addison-Wesley Pub. Co., (1983) incorporated by reference in its entirety. The effects of such substitutions can be calculated using substitution score matrices such a PAM-120, PAM-200, and PAM-250 as discussed in Altschul, (J. Mol. Biol. 219:555-65, 1991). Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Naturally-occurring peptide variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides of Table I. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides encoded by the sequences of Table I (generally from 1-5 terminal amino acids).
  • As stated above, the invention provides recombinant and non-recombinant, isolated and purified polypeptides, preferably pine tree polypeptides. Variants and derivatives of native polypeptides can be obtained by isolating naturally-occurring variants, or the nucleotide sequence of variants, of other plant lines or species, or by artificially programming mutations of nucleotide sequences coding for native pine tree polypeptides. Alterations of the native amino acid sequence can be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion. Exemplary methods of making such alterations are discussed supra.
  • The following sections are examples of the various expression vectors, host cells, and protein purification methods that are known in the art. These examples are provided merely as illustrative and should not be construed as the only means to express and purify the polypeptides and polypeptide variants of the invention.
  • Expression Vectors and Purified proteins
  • Recombinant expression vectors containing a nucleic acid sequence encoding the polypeptides of the invention can be prepared using well known methods. In one embodiment, the expression vectors include a cDNA sequence encoding the polypeptide operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the cDNA sequence of the invention. Thus, a promoter nucleotide sequence is operably linked to a cDNA sequence if the promoter nucleotide sequence controls the transcription of the cDNA sequence. The ability to replicate in the desired host cells, usually conferred by an origin of replication, and a selection gene by which transformants are identified can additionally be incorporated into the expression vector.
  • In addition, sequences encoding appropriate signal peptides that are not naturally associated with the polypeptides of the invention can be incorporated into expression vectors. For example, a DNA sequence for a signal peptide (secretory leader) can be fused in-frame to the pine tree nucleotide sequence so that the polypeptides of the invention is initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells enhances extracellular secretion of the expressed polypeptide. The signal peptide can be cleaved from the polypeptide upon secretion from the cell.
  • Fusions of additional peptide sequences at the amino and carboxyl terminal ends of the polypeptides of the invention can be used to enhance expression of the polypeptides or aid in the purification of the protein. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Pat. No. 5,011,912 and in Hopp et al., (Bio/Technology6:1204, 1988).
  • Suitable host cells for expression of polypeptides of the invention include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-free translation systems could also be employed to the disclosed polypeptides using RNAs derived from DNA constructs disclosed herein.
  • Prokaryotic Expression Systems
  • Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, the disclosed polypeptides can include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal methionine can be cleaved from the expressed recombinant polypeptide.
  • Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. To construct an expression vector using pBR322, an appropriate promoter and a DNA sequence encoding one or more of the polypeptides of the invention are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM-1 (Promega Biotec, Madison, Wis., USA). Other commercially available vectors include those that are specifically designed for the expression of proteins; these would include pMAL-p2 and pMAL-c2 vectors that are used for the expression of proteins fused to maltose binding protein (New England Biolabs, Beverly, Mass., USA).
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include P-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EP-A-36776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage λ PL promoter and a c1857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection (“ATCC”), which incorporate derivatives of the PL promoter, include plasmid pHUB2 (resident in E. Coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).
  • DNA encoding one or more of the polypeptides of the invention may be cloned in-frame into the multiple cloning site of an ordinary bacterial expression vector. Ideally the vector would contain an inducible promoter upstream of the cloning site, such that addition of an inducer leads to high-level production of the recombinant protein at a time of the investigator's choosing. For some proteins, expression levels may be boosted by incorporation of codons encoding a fusion partner (such as hexahistidine) between the promoter and the gene of interest. The resulting “expression plasmid” may be propagated in a variety of strains of E. coli.
  • For expression of the recombinant protein, the bacterial cells are propagated in growth medium until reaching a pre-determined optical density. Expression of the recombinant protein is then induced, e.g., by addition of IPTG (isopropyl-b-D-thiogalactopyranoside), which activates expression of proteins from plasmids containing a lac operator/promoter. After induction (typically for 1-4 hours), the cells are harvested by pelleting in a centrifuge, e.g., at 5,000×G for 20 minutes at 4° C.
  • For recovery of the expressed protein, the pelleted cells may be resuspended in ten volumes of 50 mM Tris-HCl (pH 8)/1 M NaCl and then passed two or three times through a French press. Most highly expressed recombinant proteins forms insoluble aggregates known as inclusion bodies. Inclusion bodies can be purified away from the soluble proteins by pelleting in a centrifuge at 5,000×G for 20 minutes, 4° C. The inclusion body pellet is washed with 50 mM Tris-HCl (pH 8)/1% Triton X-100 and then dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTT. Any material that cannot be dissolved in 50 mM Tris-HCl (pH 8)/8 M urea/0.1 M DTT may be removed by centrifugation (10,000×G for 20 minutes, 20° C.). The protein of interest will, in most cases, be the most abundant protein in the resulting clarified supernatant. This protein may be “refolded” into the active conformation by dialysis against 50 mM Tris-HCl (pH 8)/5 mM CaCl2/5 mM Zn(OAc)2/1 mM GSSG/0.1 mM GSH. After refolding, purification can be carried out by a variety of chromatographic methods such as ion exchange or gel filtration. In some protocols, initial purification may be carried out before refolding. As an example, hexahistidine-tagged fusion proteins may be partially purified on immobilized Nickel.
  • While the preceding purification and refolding procedure assumes that the protein is best recovered from inclusion bodies, those skilled in the art of protein purification will appreciate that many recombinant proteins are best purified out of the soluble fraction of cell lysates. In these cases, refolding is often not required, and purification by standard chromatographic methods can be carried out directly.
  • Yeast Expression Systems
  • Polypeptides of the invention can also be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces (e.g. K. lactis), can also be employed. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionine, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980), or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657 or in Fleer et. al., Gene, 107:285-195 (1991); and van den Berg et. al., Bio/Technology, 8:135-139 (1990). Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E. coli can be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp gene and origin of replication) into the above-described yeast vectors.
  • The yeast α-factor leader sequence can be employed to direct secretion of one or more of the disclosed polypeptides. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence can be modified near its 3′ end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
  • Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp+transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg/ml adenine, and 20 μg/ml uracil.
  • Yeast host cells transformed by vectors containing ADH2 promoter sequence can be grown for inducing expression in a “rich” medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
  • Mammalian Expression Systems
  • Mammalian or insect host cell culture systems could also be employed to express recombinant polypeptides of the invention. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also can be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line (ATCC CRL 10478) derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).
  • Established methods for introducing DNA into mammalian cells have been described (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp. 15-69). Additional protocols using commercially available reagents, such as Lipofectamine (Gibco/BRL) or Lipofectamine-Plus, can be used to transfect cells (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). In addition, electroporation can be used to transfect mammalian cells using conventional procedures, such as those in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, 1989). Selection of stable transformants can be performed using resistance to cytotoxic drugs as a selection method. Kaufman et al., Meth. in Enzymology 185:487-511, 1990, describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection can be CHO strain DX-B11, which is deficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980). A plasmid expressing the DHFR cDNA can be introduced into strain DX-B11, and only cells that contain the plasmid can grow in the appropriate selective media. Other examples of selectable markers that can be incorporated into an expression vector include cDNAs conferring resistance to antibiotcs, such as G418 and hygromycin B. Cells harboring the vector can be selected on the basis of resistance to these compounds.
  • Transcriptional and translational control sequences for mammalian host cell expression vectors can be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and later promoter, enhancer, splice, and polyadenylation sites can be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment, which can also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. in Enzymology, 1990). Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
  • Additional control sequences shown to improve expression of heterologous genes from mammalian expression vectors include such elements as the expression augmenting sequence element (EASE) derived from CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534) and the tripartite leader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475-13491,1982). The internal ribosome entry site (IRES) sequences of viral origin allows dicistronic mRNAs to be translated efficiently (Oh and Sarnow, Current Opinion in Genetics and Development 3:295-300, 1993; Ramesh et al., Nucleic Acids Research 24:2697-2700, 1996). Expression of a heterologous cDNA as part of a dicistronic mRNA followed by the gene for a selectable marker (eg. DHFR) has been shown to improve transfectability of the host and expression of the heterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplary expression vectors that employ dicistronic mRNAs are pTR-DC/GFP described by Mosser et al., Biotechniques 22:150-161, 1997, and p2A51 described by Morris et al., Animal Cell Technology, 1997, pp. 529-534.
  • A useful high expression vector, pCAVNOT, has been described by Mosley et al., Cell 59:335-348,1989. Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4, described by Cosman et al., Nature 312:768, 1984, has been deposited as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filed May 16, 1991, incorporated by reference herein. The vectors can be derived from retroviruses. In place of the native signal sequence, a heterologous signal sequence can be added, such as the signal sequence for IL-7 described in U.S. Pat. No. 4,965,195; the signal sequence for IL-2 receptor described in Cosman et al., Nature 312:768 (1984); the IL4 signal peptide described in EP 367,566; the type I IL-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type H IL-1 receptor signal peptide described in EP 460,846.
  • The polypeptides of the invention and the nucleic acid molecules encoding them can also be used as reagents to identify (a) proteins that the disclosed polypeptides or their constituent proteins regulate, and (b) other proteins with which it might interact. The disclosed polypeptides can be coupled to a recombinant protein, to an affinity matrix, or by using them as a bait in the yeast two-hybrid system. The use of the yeast two-hybrid system developed by Stanley Fields and coworkers is well known in the art and described in Golemis, E., et al Section 20.1 in: Current Protocols in Molecular Biology, ed. Ausubel, F. M., et al., John Wiley & Sons, NY, 1997 and in The Yeast Two-Hybrid System., ed. P. L. Bartel and S. Fields, Oxford University Press, 1997.
  • Antibodies and Peptide Binding Proteins
  • Purified polypeptides of the invention can be used to generate antibodies that bind to one or more epitopes of the disclosed polypeptide. Such anti-polypeptide antibodies includes polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab′)2, and Fab fragments, as well as any recombinantly produced binding partners. Antibodies are defined to be specifically binding if they bind pine tree polypeptides with a Ka of greater than or equal to about 107 M−1. Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).
  • Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice, hamsters, guinea pigs, or rats, using procedures that are well-known in the art, for example, as described for example, U.S. Pat. No. 5,585,100, incorporated by reference herein. In general, a composition comprising at least one of the polypeptides of the invention is administered to the host animal, typically through intra-peritoneal or subcutaneous injection. In the case where a peptide is used as the immunogen, it is preferable to conjugated it to a suitable carrier molecule, such as a T-dependent antigen (Bovine Serum Albumin, cholera toxin, and the like). The immunogenicity of the disclosed polypeptides can also be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant or alum. Following booster immunizations, small samples of serum are collected and tested for reactivity to the disclosed polypeptides or their constituent epitopes. Examples of various assays useful for such determination include those described in: Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well as procedures such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA), dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and 4,486,530, each of which is incorporated by reference in their entirety.
  • Monoclonal antibodies (or fragments thereof), directed against epitopes of the disclosed polypeptides can also be readily prepared using well-known procedures, such as, for example, the procedures described in U.S. Patent No. RE 32,011, U.S. Pat. Nos. 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybrddomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, each of which is incorporated by reference. Briefly, the host animals, such as mice, are injected intraperitoneally at least once, and preferably at least twice at about 3 week intervals with isolated and purified polypeptides optionally in the presence of adjuvant. Again, if peptide fragments are used they may need to be conjugated to a suitable carrier protein. Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given an intravenous boost of pine tree polypeptides. Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as, 125I-pine tree polypeptides is added to each well followed by incubation. Positive wells can be subsequently detected by autoradiography. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).
  • Monoclonal antibodies and specific-binding fragments of the invention can be produced using alternative techniques, such as those described by Alting-Mees et al., “Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody Such a technique is described in Larrick et al., Biotechnology, 7:394 (1989).
  • It is understood of course that many techniques could be used to generate antibodies against the polypeptides of the invention and that the above embodiments in no way limits the scope of the invention.
  • Nucleotides. Proteins, Antibodies, and Binding Proteins As Probes and Reagents
  • The disclosed nucleic acids, polypeptides, and antibodies directed against the disclosed polypeptides can be used in a variety of research protocols, such as in DNA arrays or as reagents. A sample of such research protocols are given in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory Press, (1989), incorporated by reference. For example, the compiled sequences, polypeptides, etc., can serve as markers for cell specific or tissue specific expression of RNA or proteins. Similarly, this system can be used to investigate constitutive and transient expression of the genes encoding the cDNAs of SEQ ID NOS: 1-327 and the proteins encoded by these genes.
  • Further, the disclosed cDNA sequences can be used to determine the chromosomal location of the genomic DNA and to map genes in relation to this chromosomal location. The disclosed nucleotide sequence can be further used to identify additional genes related to the nucleotides of SEQ ID NOS: 1-334 and to establish evolutionary relatedness among species based on the comparison of sequences. The disclosed nucleotide and polypeptide sequences can be used to select for those genes or proteins that are homologous to the disclosed cDNAs or polypeptides, using well-established positive screening procedures such as Southern blotting and immunoblotting and negative screening procedures such as subtractive hybridization.
  • Method for Using Nucleic Acid Probes or Antibodies to Stage Embryos
  • Accurate staging of tree embryos is critical. It is known that different stages of tree embryos have different capacities as subjects for genetic transformation and genetic engineering. In addition, environmental requirements exhibited by embryos vary due to increasing physiologic age. Currently, the staging of tree embryogenesis is most accurately performed by an expert in the field who is very familiar with the morphological appearance of embryos at different stages. The cDNAs and related molecules of this invention can be used as markers for different stages of tree embryogenesis, thereby eliminating the need for a subjective eye to assess maturity and potentially allowing for more accurate staging of tree embryos. Moreover, by monitoring the expression of the underlying genes, it is possible to determine when an embryo has reached a certain level of development even if that level does not correspond to a visible difference in embryo morphology. The relational database of this invention aids the ability to monitor expression levels and tailor research approaches, such as the use of DNA arrays, to the specific needs of the objective, i.e., staging.
  • The information provided in this invention can be used in whole or in part to stage embryos. For example, one or a multiplicity of nucleic acid molecules from SEQ ID NOS: 1-327 having an expression profile consistent with a particular embryo stage can be used in this invention. A researcher may find it beneficial to use oligonucleotide probes or antibodies, for example, that specifically recognize proteins derived from genes expressed during middle embryonic stages, or that specifically monitor expression levels for embryos that have reached maturity associated with late developmental stages. A researcher can quickly determine that an embryo subset has progressed to or through an embryonic stage with the use of this invention and make appropriate changes in conditions if necessary, e.g. alter growth media or other environmental conditions.
  • Method for Monitoring, Enhancing, or Determining Expression of Stage-Specific Genes
  • Expression patterns of SEQ ID NOS: 1-327 indicate that gene activation can be classified as stage-specific, such as in the case of SEQ ID NO: 327, otherwise known as “LP2-3.” The promoter that drives such a gene can perform valuable functions. For example, a promoter from LP2-3 operatively linked to a reporter gene presented within an embryo system is expected to produce the reporter product under the conditions for expression of gene LP2-3. Thus, the system allows a rapid determination of stage specific embryos by a simple phenotypic reporter screen, perhaps by visualization of green fluorescent protein (GFP) or by loss of fluorescent protein product. Similarly, a set of promoters from known, differently staged genes operatively linked to reporter genes will be effective for monitoring developmental changes within the system as the embryos mature. The LP2-3 promoter is identified as SEQ ID NOS: 328-334 in Table I. The promoter expression pattern is that of the natively linked gene, LP2-3.
  • Virtually any indicator or reporter gene can be used for this approach or for other methods associated with this invention provided they are compatible with the system studied. Generally, reporter genes are genes typically not present in the recipient organism or tissue and which encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes and assays are provided by Schenborn, E. and Groskreutz, D., Mol. Biotechnol., 13:29, 1999; Helfand, S. L. and Rogina, B., Results Probl. Cell Differ., 29:67, 2000; Kricka, L. J., Methods Enzymol., 305:333, 2000; Himes, S. R. and Shannon, M. F., Methods Mol. Biol., 130:165, 2000; and Leffel, S. M. et al., Biotechniques, 23:912, 1997, which are incorporated in their entirety by reference. In one embodiment of this invention, the reporter used is GFP, or any ariant of the fluorescent protein.
  • Additionally, one skilled in the art would recognize that a promoter, like that from LP2-3, has potential to stimulate production of products not ordinarily observed at a particular stage. A promoter derived from a gene that expresses during a known stage, for example an early stage, can be operatively linked to a gene that does not normally express during that stage, yielding controlled expression of any targeted gene. It may be shown that earlier or later expression, or prolonged expression of a particular gene may give a desirable genotype or phenotype in a mature plant, may result in increased vigor in culture, or may be sufficient to alter the normal maturation process of the embryo. Prolonged expression of any desired gene also may be achieved from linking a constitutively expressed promoter to the targeted gene. Further, the ability to manipulate gene expression during embryogenesis allows for a detailed study of the effects of an individual gene or multiple genes on embryogenesis, leading to a better understanding of the developmental processes involved in embryogenesis.
  • Method of Correlatinq Gene Expression with Improved Tree Stock or Culture Conditions
  • Importantly, the cDNAs and related molecules of the invention can also be used as markers to examine genetic heterogeneity and heredity through the use of techniques such as genetic fingerprinting. These markers can also be correlated with improved agronomic traits including good initiation frequency, embryonic maturation, high frequency of germination, rapid growth rates, herbicide tolerance, insect resistance, pathogen resistance, climate and environmental adaptability wood quality, and wood fiber quality and content, to name a few. Additionally, the expression of these developmentally regulated genes can be compared among genetically identical clones grown under different culture conditions to determine the best protocols and media for somatic embryogenesis.
  • Cryogenic storage of pine tree embryos is effective for maintaining stocks of embryos determined by this invention to have the desired fitness traits or exist at the appropriate developmental stage. With such storage, one can specifically target desirable embryos for expansion many years after they are frozen. For example, a culture of somatic embryos can be divided into at least three portions, one of which is cryogenically stored, one which is used to study gene embryonic gene (and protein) expression, and one that is used to produce seedlings for field testing. Clones producing valuable mature plants could be selected and expanded from frozen stocks. Additional clones exhibiting similar expression patterns could be selected for future expansion and cultivation.
  • As will be evident to the ordinary practitioner, there are numerous ways in which the nucleic acids, polypeptides and antibodies of this invention might be used to characterize the gene expression of embryos. Ideally the stage-specific gene expression of embryos of several different genotypes and at several different stages of embryogenesis are characterized. For example, sets of oligonucleotide primers designed using any one of SEQ ID NOS: 1-327 may be used in RT-PCR assays to characterize expression of a gene product. In situ hybridization assays or antibody staining protocols may also be used to characterize RNA and/or protein expression and localization.
  • Embryos of the same genotype in which gene expression has been characterized may also used be to generate plantlets that are used in field testing. Once the embryos have developed into mature trees, the various genotype trees can be evaluated for important traits such as growth rates, herbicide tolerance, insect resistance, pathogen resistance, climate and environmental adaptability, wood quality, and wood fiber quality and content, among others. Finally the phenotypic data collected from the field testing can be correlated with gene expression during early embryogenesis to further enhance the database of the present invention. This will allow further identification of gene products which whose expression is correlated, either positively or negatively, with commercially valuable tree characteristics.
  • It will be clear to those skilled in the art that identification of such gene products can have several uses. Determining the correlation between a desirable phenotype and a genotype would allow for the “pre-selection” of tree embryos for field testing. It would also be useful in evaluating experimental tissue culture conditions for somatic embryogenesis; in other words, the expression level of a gene known to correlate with the development of trees with desirable characteristics could serve as the criterion on which culture media is evaluated, as opposed to assessing the phenotype of fully matured trees. The ability to evaluate culture conditions without having to develop fully mature trees and do field testing would save a great deal of research time and expense. And of course, the knowledge of the correlation between gene expression and desirable tree phenotypes would serve to identify target genes for genetic engineering.
  • Genetically Engineering Trees and Other Plants
  • There are several methods known in the art for the creation of transgenic plants. These include, but are not limited to: electroporation of plant protoplasts, liposome-mediated transformation, polyethylene-glycol-mediated transformation, microinjection of plant cells, and transformation using viruses. Because the invention is especially concerned with the transformation of woody species, the two prevalent methods for transforming forest trees, namely Agrobacteriurm-mediated transfer and direct gene transfer by particle bombardment, will be discussed in more detail, though it is understood that the present invention encompasses generation of transgenic plants via standard methods commonly known in the art.
  • Agrobacterium Mediated Transfer
  • A. tumefaciens and A. rhizogenes are two soil microorganisms that naturally infect a wide variety of plants including dicotyledonous plants, gymnosperms and some monocotyledonous plants. Infection by these organisms results in the growth of crown gall tumors or in hairy root disease, respectively. Each of these organisms carries a large plasmid, the tumor inducing (Ti) plasmid, in the case of A. tumefaciens and the root-inducing (Ri) plasmid in the case of A. rhizogenes. These plasmids have two critical features, a set of virulence genes and a segment of DNA called T-DNA that is delimited by conserved regions of approximately 25 base pairs known as the left and right borders. During infection, the T-DNA is transferred to the plant cell where it is able to stably integrate in single copy in the plant genome. Transfer of T-DNA requires the function of the virulence genes.
  • In its natural state, T-DNA contains genes that mediate progression of disease such as growth hormones or genes controlling root morphogenesis. Using recombinant DNA technology, however, T-DNA may be modified to contain an expression cassette encoding a foreign gene of interest. There are several T-DNA vector systems commonly in use for the transformation of plants. Several of these vector systems are reviewed in Hansen et al., Current Topics in Microbiology and Immunology 240: 21-57 (1999) which is hereby incorporated by reference. T-DNA vectors must include the left and right borders. In addition they must either be capable of replication in Agrobacterium or be designed so as to recombine with a plasmid that does so. The latter type of vector is known as a co-integrate vector. For transformation to proceed, there must also be a source of virulence (vir) genes. The vir genes may be on the same plasmid with the T-DNA or more likely supplied by a helper plasmid. For example, binary T-DNA vector systems are comprised of two plasmids, one containing the vir genes and the other containing T-DNA. Some plants known to be recalcitrant to Agrobacterium-mediated transformation may be transformed if additional copies of some or all virulence genes are provided. Extra copies of VirG and VirE can be particularly useful.
  • Additionally, it is convenient to include in the T-DNA a selectable marker that will allow identification and selection of transformed plant cells. The selectable marker should be one that works in both Agrobacterium and the target plant. For example, the genes encoding chloramphenicol acetyltransferase and neomycin phosphotransferase are suitable marker genes that confer resistance to chloramphenicol and kanamycin, respectively. Additionally, a selectable marker may be provided on a separate T-DNA from the T-DNA encoding the gene of interest. Co-transformed T-DNAs can integrate at separate sites in the plant genome. This can be useful because it will later allow segregation of the marker gene in progeny enabling the generation of transgenic trees expressing the gene of interest but not the marker gene.
  • The gene of interest and the selectable marker genes must also be under the control of promoters that function in the transformed plant cell. Examples of suitable promoters include, but are not limited to: the abscisic acid (ABA)-inducible promoter from the early methionine (Em) gene from wheat (Marcotte et al., Plant Cell 1:976-979 (1989); the cauliflower mosaic virus (CaMV) 35S promoter (Odell et al., Nature 313:810-812 (1985); and the nopaline synthase (nos) promoter (Sanders et al., Nucl. Acids Res. 15(4):1543-58 (1987). Tissue-specific plant promoters or plant promoters responsive to chemical, hormone, heat or light treatments may be used. Additionally, the gene of interest may be expressed under the control of its endogenous promoter to ensure proper regulation.
  • The process of transformation requires plant cells that are competent and that are either embryogenic or organogenic. The plant cells to be transformed are then co-cultivated with Agrobacterium containing an engineered T-DNA vector system for 1-5 days. Following the co-cultivation period, the cells are incubated with the antibiotic against which the selectable marker confers resistance, and transformed lines are selected for further cultivation. The use of Agrobacterium mediated transfer in woody trees is described in Loopstra et al., Plant Molecular Biology 15:1-9 (1990), Gallardo et al., Planta 210:19-26 (1999) and Wenck et al., Plant Molecular Biology 39:407-419 (1999), each of which is hereby incorporated by reference.
  • Direct Gene Transfer by Particle Bombardment
  • Direct gene transfer by particle bombardment provides another method for transforming plant tissue. This method can be especially useful when plant species are recalcitrant to transformation by other means. In this technique a particle, or microprojectile, coated with DNA is shot through the physical barriers of the cell. Particle bombardment can be used to introduce DNA into any target tissue that is penetrable by DNA coated particles, but for stable transformation, it is imperative that regenerable cells be used. Typically, the particles are made of gold or tungsten. The particles are coated with DNA using either CaCl2 or ethanol precipitation methods which are commonly known in the art.
  • DNA coated particles are shot out of a particle gun. A suitable particle gun can be purchased from Bio-Rad Laboratories (Hercules, Calif.). Particle penetration is controlled by varying parameters such as the intensity of the explosive burst, the size of the particles, or the distance particles must travel to reach the target tissue.
  • The DNA used for coating the particles should comprise an expression cassette suitable for driving the expression of the gene of interest. Minimally this will comprise a promoter operably linked to the gene of interest. As with Agrobacterium mediated transformation. Suitable promoters include, but are not limted to, the the abscisic acid (ABA)-inducible Em promoter from wheat (Marcotte et al., Plant Cell 1:976-979 (1989), the CaMV35S promoter (Odell, et al., Nature 313:810-812 (1985), and the NOS:promoter (Sanders et., Nucl. Acids Res. 15(4):1543-58 (1987).
  • Methods for performing direct gene transfer by particle bombardment are disclosed in U.S. Pat. No. 5,990,387 to Tomes et al. Additionally, Ellis et al. describe the successful use of direct gene transfer to white spruce and larch trees in Bio/Technology 11, 84-89 (1993).
  • Researchers skilled in the area of DNA or gene transformation will recognize that additional procedures, or combination of procedures, may be useful for the successful tranformation of genetic stock.
  • Antisense Expression
  • The cDNAs of the invention may be expressed in such a way as to produce either sense or antisense RNA. Antisense RNA is RNA that has a sequence which is the reverse complement of the mRNA (sense RNA) encoded by a gene. A vector that will drive the expression of antisense RNA is one in which the cDNA is placed in “reverse orientation” with respect to the promoter such that the non-coding strand (rather than the coding strand) is transcribed. The expression of antisense RNA can be used to down-modulate the expression of the protein encoded by the mRNA to which the antisense RNA is complementary. This phenomenon is also known as “antisense suppression.” It is believed that down-regulation of protein expression following antisense RNA is caused by the binding of the antisense RNA to the endogenous mRNA molecule to which it is complementary, thereby, inhibiting or preventing translation of the endogenous mRNA.
  • The antisense RNA expressed need not be the full-length cDNA and need not be exactly homologous to the target mRNA. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the endogenous mRNA will be needed for effective antisense suppression. Preferably, the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases. The length of the antisense sequence in the vector may be greater than 100 nucleotides. Vectors producing antisense RNA's could be used to make transgenic plants, as described above, in situations when desirable tree characteristics are produced when the expression of a particular gene is reduced or inhibited.
  • METHODS
  • Tissue Samples
  • The cDNAs of the current invention can be derived from any sets of plant tissue. The cDNAs of SEQ ID NOS: 1-334, for example, were originally derived from embryonic tissues of pine tree embryos staged 1-9.9 as classified in Pullman and Webb TAPPI R&D Division 1994 Biological Sciences Symposium, pages 31-34, which is hereby incorporated by reference. LPS and LPZ clones are derived from somatic and zygotic embryos, respectively. As noted, embryos may be of either somatic or zygotic derivation, and the embryos may be grown in either semi-solid or liquid tissue culture systems. Applicable methods for growing embryos in semi-solid or liquid tissue culture systems are disclosed in U.S. Pat. Nos.: 5,036,007; 5,236,841; 5,294,549; 5,413,930; 5,491,090; 5,506,136; 5,563,061; 5,677,185; 5,731,203; 5,731,204; and U.S. Patent Application 60/212,651 filed Jun. 19, 2000, which are hereby incorporated by reference.
  • RNA Isolation
  • In one embodiment, RNA isolated from staged cell populations provides the starting material for reverse transcription, differential display, and cloning of amplified cDNA. Methods and kits for isolating total RNA from cellular populations, or for generating poly(A)+ RNA, are commonly known in the art. For example, several procedures for isolating RNA are disclosed in Chapter 4 of Current Protocols in Molecular Biology edited by F. A. Ausubel et al., John Wiley and Sons, Inc. (1987) (incorporated herein by reference). As an example, the TRI Reagent7 available from Molecular Research Center, Inc. (Cincinnati, Ohio) is a suitable reagent (used according to the manufacturer's instructions) for isolation of RNA from plant tissues.
  • Differential Display
  • Differential display provides a method to identify individual messenger RNAs that are differentially expressed among two or more cell populations. In the practice of the present invention, these cell populations may be provided by pine tree or other plant embryos of different developmental stages. The differential display procedure is taught in Liang et al., Science, 257:967-71 (1992) and in U.S. Pat. No. 5,262,311, which are hereby incorporated by reference. Briefly, mRNA sequences are PCR-amplified using two types of oligonucleotide primers known as “anchor” and “arbitrary” primers. Anchor primers are designed to recognize the polyadenylate tail of messenger RNAs. Arbitrary primers are short and arbitrary in sequence ard anneal to complementary sequences in various mRNAs. Products amplified with these primers will vary in size and can be differentiated on an agarose or sequencing gel based on their size. If different cell populations are amplified with the same anchor and arbitrary primers, one can compare the amplification products to identify differentially expressed RNA sequences.
  • Cloning
  • PCR-amplified bands representing differentially expressed RNA samples are excised from the gel, transferred to tubes and reamplified using the same primer pairs and PCR conditions as used in the differential display procedure. Methods for the cloning of PCR products are commonly known in the art and there are several commercially available reagents and kits for cloning PCR products. For instance, the pCR-Scipt™ Cloning kit from Stratagene, La Jolla, Calif.) is suitable for this purpose. Using this kit, E. coli transformants containing plasmids with PCR fragment inserts can rapidly be identified using blue/white color selection followed by plasmid purification and restriction digests. The pCR-Script vector contains T3 and T7 polymerase recognition sites allowing for in vitro transcription of the inserted fragment.
  • Sequence Determination
  • Methods for sequencing DNA, including cloned PCR products, are commonly known in the art. The selection of cloning vectors having M13, T7 or T3 primer annealing sites flanking the PCR-amplified insert can be used in sequencing reactions directly. Most sequencing procedures in use today are modifications of Sanger's dideoxy chain termination sequencing reaction as disclosed in and Sanger et al., Proceedings of the National Academy of Sciences, 74:5463-5467 (1977); which is hereby incorporated by reference.
  • Homology Searching and Identification of Protein Coding Sequences
  • As understood by one of ordinary skill in the art, the sequence of a cloned cDNA insert obtained, may be compared against public databases such as Genbank to discern any identity or homology to known sequences. Programs, such BLAST, for performing such a search are available on the National Center for Biotechnology Information's web page located at hftp://www.ncbi.nim.nih.qov. The results from Genbank search may reveal the potential function of a polypeptide or RNA molecule encoded by the cDNA. In addition to searching gene sequence database, the use of commercially available analysis software is well known in the art. For example, software packages such as the Wisconsin Package™ (Genetic Computer Group, Madison, Wis.) include programs such as FRAMES and CodonPreference that help to identify protein coding sequences in a query nucleotide sequence. FRAMES displays open reading frames for the six DNA translation frames, allowing one to quickly assess the presence or absence of stretches of open-reading frames that are likely to be protein encoding regions. CodonPreference is a more sophisticated program that identifies and displays possible protein coding regions based on similarity of the codon usage in the sequence to a codon frequency table (Gribskov et al., 1984).
  • EXAMPLE 1 Differential Gene Expression Analysis in Pine Tree Embryo enesis
  • cDNA libraries were prepared from staged pine tree embryos, as described above. The differential display technique was used to identify 327 novel cDNAs that were preferentially-expressed during early, middle, or late stages of pine tree embryogenesis, as set forth below. Clone nomenclature is divided into subsets based on tissue type; a clone is designated LPS to indicate somatic origins and LPZ for zygotic origins.
  • Plant Materials
  • Somatic embryos were collected at different stages of development. Cultures of somatic embryos of were initiated from Loblolly pine immature zygotic embryos as described by Becwar et al., Forestry Science 44:287-301 (1994) (incorporated by reference) or with minor modifications in media mineral composition. Somatic embryos were grown in cell suspension culture medium 16 (Pullman and Webb, Tappi R&D Division 1994 Biological Sciences Symposium) and a maturation medium similar to that of a standard maturation media. Resulting somatic embryos were selected and classified as stages 1-9 according to morphological development following the teachings of Pullman and Webb, Tappi R&D Division 1994 Biological Sciences Symposium pp.31-34. Somatic embryos were sorted into tubes containing the same stages and stored at −70° C.
  • RNA Isolation
  • Total RNA was isolated from all stages of somatic embryos of loblolly pine and grouped into early, middle, and late phases of development. The early phase is represented by a liquid suspension culture containing embryos of stages 1 through stage 3. Middle phase contains embryos of stages 4 through stage 6, while stages 7 through 9 formed the late phase. 60-100 mg aliquots of staged frozen embryos were ground in 1.0 ml of TRI Reagent® Isolation Reagent (Molecular Research Center, Inc.), a commercial product that includes phenol and guideline thiocyanate in a monophase solution and extracted according to the manufacturer's instructions.
  • Reverse Transcription of mRNA (RT-PCR)
  • The total RNA was used as a template to synthesize single stranded DNA mediated by MMLV reverse transcriptase (100 U/μl). The method involves the reverse transcription by PCR of the mRNA with an oligo-dT primer (H-T11G: 5′ B AAGCTTTTTTTTTTTG 3′) anchored to the beginning of the poly(A) tail, followed by a PCR reaction in the presence of a second short (13-mer) primer which is arbitrary in sequence [AP1 (5′ B AAGCTTGATTGCC-3′) or AP2 (5′ B AAGCTTCGACTGT-3′)]. Reverse transcription and Differential Display were conducted using the GenHunter RNAimage Kit 1.
  • A 19 μl reverse transcription reaction (10 μl sterile water, 2.0 μl 5×RT buffer, 1.6 μl dNTP (250 μM), 2.0 μl anchored primer (2.0 μM), 2.0 μl RNA template at 100 ng/μl) was prepared for each embryo phase sample. The reaction mixture was heated to 65° C. for 5 minutes in a thermocycler, cooled to 37° C. and paused after 10 minutes while 1.0 μl MMLV was added. The program was allowed to resume at 37° C. for 50 minutes. The reaction was then heated to 75° C. for 5 minutes, cooled to 4° C. and stored at −20° C.
  • Incorporation of Radiolabeled Nucleotides by PCR
  • Differential Display PCR was performed in a 20 μl reaction containing 2 μl of the reverse-transcribed cDNA template; 10 μl sterile water 2.0 μl 10×PCR buffer, 1.6 μl dNTP (25 μM), 2.0 μl anchored primer H-T 11G, (2.0 μM), 2.0 μl 13 mer arbitrary primer (AP1 or AP2 (2.0 μM), 0.2 μl Taq DNA polymerase, and 0.2 μl α32P-dATP (2000 Ci/mmole). The cDNA was amplified by PCR: 94° C. for 3 minutes, 40 cycles of 94° C. for 30 seconds, 40° C. for 2 minutes, and 72° C. for 30 seconds, followed by 72° C. for 5 minutes. The reaction was cooled to 4° C. and stored at −20° C.
  • Differential Display
  • The PCR products were separated on a Stratagene (La Jolla, Calif.) pre-cast 6% polyacrylamide sequencing gel at 30 watts constant power for approximately 2.5 to 3 hours. 3.5 μl of sample was mixed with 2.0 μl, of loading dye and incubated at 80° C. for 2 minutes immediately before loading onto the gel. The gel was rinsed in water and dried. Dilute 35P-dATP with loading dye was spotted at the corners as alignment markers and the gels were exposed to Kodak BioMaX™ autoradiography film. An exemplary gel is shown in FIG. 1.
  • Bands that appeared to be possible markers for phase specific gene expression were marked on the film and aligned over the gel. The bands were excised by cutting through the film. The gel pieces were scraped from the gel and transferred to tubes and re-amplified using the same primer pairs and PCR conditions as used for incorporation of radiolabeled nucleotides.
  • Cloning of DNA Fragments from Differential Display
  • The PCR products from the gel fragments were purified, polished, ligated and cloned into XL 10-Gold Kan ultracompetent cells by heat shock with the Stratagene pCR-Script Amp SK(+) Supercompetent Cell Cloning Kit according to manufacturer's instructions. The transformed cells were spread on LB agar plates containing ampicillin, IPTG, and X-Gal each at 50 μg/ml. The plates were incubated overnight at 37° C. Plasmids containing PCR inserts were identified using blue-white colony screening. The presence of inserts was confirmed by digesting the clones with restriction endonucleases, Msc I and Nla ll, followed by standard DNA gel electrophoresis. Transformants representing early, middle, and late phase embryos were sequenced using standard dideoxy protocols known in the art with the T3 primer.
  • Sequence Analysis
  • All sequences were analyzed using a program-database pair search of the NCBI BLAST 2.0 server, blastn-nr, blastn-others ests, and blastx-nr. In each case, the query sequence was filtered for low complexity regions by default and entered in FASTA format. Other formatting options were set by default; alignment view-pairwise, descriptions-100, and alignments-50. Using these parameter settings, significant similarity to known DNA, RNA, or protein sequences was found for several of the nucleic acid molecules of SEQ ID NOS: 1-334, for example, those described herein. (Alignment data not shown).
  • EXAMPLE 2 Characterization of Full Length LP2-3 cDNA Sequence
  • SEQ ID NO: 327, designated LP2-3, was first identified through differential display with T12MG and AP1 primers (GeneHunter). The differential display band appeared to be present only in liquid suspension cultures of Loblolly Pine somatic embryos. The conditions for mRNA isolation, reverse-transcription, differential display-PCR, and gel separation/visualization for producing this band were all as described in Example 1. Likewise, the band containing the original LP2-3 fragment was excised from the differential display gel, amplified, and cloned into pCR-Script AMP SK(+) according to standard protocols known in the art.
  • Northern Hybridizations Demonstrating Early-Specific Expression
  • Northern analysis demonstrated that the LP2-3 differential display clone hybridized to an approximately 1.2 Kb mRNA from liquid suspension culture embryos but was undetectable in late (6-9) stage embryo RNA. (FIG. 11) In general, LP2-3 is most highly expressed in early stage embryos in liquid culture. LP2-3 mRNA is found most abundantly in early stage somatic embryos, especially for embryos grown in liquid multiplication medium. (FIG. 12) Further, transcription decreases rapidly as embryos are transferred to maturation medium (stage 3 and stage 4) and begin to mature. LP2-3 transcripts are virtually undetectable at stage 6-9 somatic embryos grown on maturation medium. (See FIG. 12) Additional studies indicate that LP23 mRNA is expressed zygotically, particularly in early stage zygotic embryos, but is undetectable in mature vegetative tissues. (FIGS. 13 and 14) Specifically, the signal intensity from liquid suspension somatic embryo RNA was about 3 times greater than the signal from the analogous stage 1 zygotic embryo RNA. (FIGS. 13 and 14) LP2-3 transcripts were not detectable in total RNA from needles, stems, or roots of one year old seedlings, including those exposed to cold, ozone, wound stresses, or the hormone jasmonic acid (not shown).
  • LP2-3 Differential Display and ‘Full-Length’ cDNA Sequences
  • A ‘full-length’ cDNA was captured from SMART™ cDNA made from somatic embryo liquid suspension by using a biotinylated LP2-3 differential display fragment as a capture probe. The “full-length” cDNA was cloned and sequenced according to standard protocols known in the art. This sequence was designated at LP2-3+.
  • GenBank blastx searches conducted with the above sequence translated in all 6 reading frames indicated that LP2-3+likely encodes a member of the major intrinsic protein family. This family of proteins encodes membrane channels for the transport of water and/or ions across cell membranes. They may play a significant role in osmoregulation and may play a role in the cellular responses to water and salt stresses. As is known in the art, the MIPs are induced by dessication, flooding, and high levels of the plant hormone ABA. In contrast, the LP2-3 sequence was not detected in desiccated late-stage embryos which have high levels of ABA and, thus, appears to be regulated by some embryo-specific signal.
  • EXAMPLE 3: Hypothesis Development for Improved Protocols
  • Currently the improvement of tissue culture practices arises via hypothesis, evaluation and adoption. Hypotheses arise from observation of size, shape, weight, etc. and physiological measurement of ion or sugar content (FIG. 6, box 1). These observations are limited in scope and this limits the improvements that can be made to the tissue culture process. Gene expression is closely linked to metabolic condition, thus the observation of which genes are induced or repressed under a given growth condition, naturally, on the tree, or in a culture vessel, provides insight into the metabolic state of the embryo. This information can be used to create new hypotheses that can be evaluated by modifying tissue culture.
  • To this end, mRNA levels of two cDNAs (LPZ-202 and LPZ-216), similar to “Late Embryogenesis Abundant” (LEA) proteins, identified in other plants, were monitored. These genes are induced by the plant hormone ABA. Two peaks of mRNA were observed in these clones rather than the typical single peak in most plants. (See FIG. 4 for clone LPZ-216; clone LPZ-202 is similar but data is not shown.) It was subsequently confirmed that two peaks in ABA activity are observed during development and that these correspond in timing to the elevation in mRNA for LPZ-202 and LPZ-216. Thus mRNA abundance profiles are providing insight into embryo physiology. (See FIG. 7) The effect of giving two pulses of ABA to our somatic embryos is assessed; a tissue culture modification that we might not have considered as important had the gene expression data been unavailable. Internal data shows fluctuations in the abundance of mRNA for cDNAs listed in this collection (data not shown.)
  • Zygotic and Somatic Loblolly Pine Embryos
  • Loblolly pine cones were collected weekly from a breeding orchard near Lake Charles, La., and shipped on ice for experimentation. Embryos were excised and evaluated for developmental stage (Pullman et al. 1994). Stage 9 embryos were separated by the week they were collected-9.1 (week 1), 9.2 (week 2), etc. Staged zygotic embryos were sorted into vials partially immersed in liquid nitrogen and stored at −70° C. Somatic embryos for loblolly pine were initiated as described by Becwar et al. (1995) or with minor modifications. Somatic embryos were grown, selected, and staged as described by Pullman et al. (1994) and stored at −70° C.
  • cDNA Probe Preparation and Hybridization
  • 30 ng of purified Lea protein cDNA fragments was labeled with 32P dCTP using the Ready-To-Go cDNA Random Labeling kit (Pharmacia). The labeled cDNAs were purified using NICK Column (Pharmacia) and heat denatured for hybridization. The RNA slot blot was pre-hybridized in hybridization buffer (0.5 M sodium-phosphate, pH 7.2, 5% SDS, and 10 mM EDTA) at 65° C. for 2 hours in a hybridization oven (Model 400, Robbins Scientific, Sunnyvale, Calif.) and the hybridized in the same conditions with the cDNA probes. After hybridization, the membranes were washed at 65° C. in 0.2×SSC and 0.1% SDS. Each wash was 15 min. The membranes were then exposed to Image Plate.
  • The probes can be stripped from the RNA slot blot by pouring boiling 0.5% SDS onto the membrane twice and incubating without heating for 30 min. The stripped blot was then exposed to Image Plate for overnight to check the completeness of the de-probing before next round of hybridization.
  • To ensure the equal loading of the each RNA sample, the same membranes were stripped and hybridized with a 32P-dCTP labeled 26S ribosomal rDNA fragment. These results were used as controls to normalize the Lea protein gene expression levels.
  • As a means of evaluating the usefulness of these arrays, we followed the expression of three cDNAs that have strong sequence similarity to late embryo-abundant proteins, (Lea) proteins from cotton (Baker et al 1988). Lea proteins and mRNAs appear in embryos at a stage when ABA is high and the genes can be induced in vegetative tissue by application of ABA. The transcript level of Lea genes LPZ-202 and LPZ-216 showed two peaks, rising from stage 5 and returning to a base line about stage 9.2 then rising again around stage 9.5. (See FIG. 4 for clone LPZ-216).
  • To confirm the fluctuation in lea transcript levels by Northern analysis. RNA was extracted from zygotic embryos at different stages of development A in ‘dehydrin’ cDNA from the North Carolina State University cDNA collection (hftp://www.cbc.med.umn.edu/ResearchProiects/Pine/DOE.pine/index.html) was used as probe for some experiments. Dehydrins are a class of lea protein, originally identified as water deficit inducible proteins. Since the expression of this class of protein is well characterized, in contrast to our lea genes, the dehydrin expression profile could act as a reference point. After probing with dehydrin, blots were stripped and probed with a 26S rDNA probe from Arabidopsis to check the loading of the original gel. The normalized expression pattern of dehydrin in the zygotic embryogenesis is illustrated in the top panel of FIG. 4. The expression of the dehydrin gene was induced at stage 5 and reached a peak at stage 6. It declined at stage 7-8, just prior to the onset of the desiccation. Then the mRNAs level remained low from stage 9.1 through 9.5. The dehydrin mRNA levels rose again late in development, from stage 9.6 on, apparently dropping in very late development. A similar pattern of expression was observed in a parallel experiment when our lea-like clone, LPZ-216, was used as a probe.
  • This pattern reveals two significant peaks at the early development of the embryos and high expression levels for the stage 9.6 and beyond. The expression pattern of these two lea genes in loblolly pine embryos is consistent with the changes in ABA concentration observed in pine during embryogenesis. (See FIG. 5)
  • EXAMPLE 4 Evaluation of Metabolic State of Somatic
  • Embryos as Compared to Zygotic Embryos for Fitness Determination
  • The model and goal for somatic embryogenesis is to produce an embryo that in vigor, germinatability, etc., resembles a zygotic embryo. Standard measurements reveal relatively little about the embryos; thus the metabolic state of somatic and zygotic embryos is unknown. The metabolic state of zygotic (natural) embryos can be evaluated by DNA arrays containing the cDNA clones described in this application. A database of mRNA levels for the genes represented on the DNA arrays can then be established. Embryos growing under a new tissue culture protocol (FIG. 6, box #2) can be evaluated by DNA array southerns (FIG. 6, box #3). The array elucidates patterns of gene activity and reveals whether those patterns are similar to the natural state (FIG. 6, box #4). The germination, or further development can be checked (FIG. 6, box #5) to confirm the conclusion. Where a link between specific gene activity and embryo performance has been demonstrated, protocols can be modified with efficiency as seen in FIG. 6, box 6.
  • To illustrate this process, elevation of plant hormone ABA in maturation medium was evaluated as a protocol modification, as described below. This modification proved beneficial, elevating the number and quality of the embryos produced. The mRNA abundance for cDNAs was assessed by DNA array using RNA isolated from control and elevated ABA conditions; several differences were observed in the mRNA levels of specific genes. Further, abundance of mRNA in the elevated ABA condition, more closely resembled the mRNA abundance observed for the these same genes in zygotic embryos. Thus a protocol which produces higher quality embryos produces, in these embryos, a mRNA profile that more closely resembles that observed in natural embryos.
  • Zygotic and Somatic Loblolly Pine Embryos
  • Loblolly pine cones were collected weekly from a breeding orchard near Lake Charles, La., and shipped on ice for experimentation. Embryos were excised and evaluated for developmental stage (Pullman et al. 1994). Stage 9 embryos were separated by the week they were collected-9.1 (week 1), 9.2 (week 2), etc. Staged zygotic embryos were sorted into vials partially immersed in liquid nitrogen and stored at −70° C. Somatic embryos for loblolly pine were initiated as described by Becwar et al. (1995) or with minor modifications. Somatic embryos were grown, selected, and staged as described by Pullman et al. (1994) and stored at −70° C.
  • Mass Isolation of Genes Differentially Expressed in Loblolly Pine Zygotic Embryos
  • The following RNA differential display method is sensitive enough to produce banding patterns from one mid- to late-stage embryo or 10-20 early stage embryos. This technique, which extracts mRNA directly from tissue using oligo(dt) beads, avoids losses inherent in conventional RNA extraction methods, is fast, reliable, and inexpensive. Differences in gene expression during development, as well as between somatic and zygotic embryos, can be easily detected.
  • To achieve these results, 50-100 μl lysis buffer containing 100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, 1% SDS and 5 mM DTT was added to 10-100 mg of staged embryos in a 1.5 ml tube. The mixture was ground thoroughly with an electric drill containing a plastic pestle bit (VWR, Cat# KT95050-99) that had been sterilized by autoclaving. An additional 50-100 μl lysis buffer was added and ground briefly. The grinder and vortex was washed with 100 μl lysis buffer. If multiple samples were processed, each is stored on ice until ready for the next step. The grinding tip was washed with sterile water and dried for the next sample.
  • After all the samples were ground, they were spun at 4° C. for 15 minutes in a bench top centrifuge at 14,000 rpm. 8 μl oligo(dT) coated Dynal beads (mRNA DIRECT Kit, Dynal, N.Y.) was placed in a 1.5 ml tube. The Dynal beads were washed twice with a 100 μl of the above mentioned lysis buffer and suspended in an equal volume of the lysis buffer used in tissue grinding. If more than one sample is handled, the beads for all the samples can be washed together and dispensed in several 1.5-ml tubes. The cleared embryo lysate (after centrifugation) was added to the beads and mixed well.
  • The mixture was then incubated on ice for 5 min., placed on a magnetic stand (Promega) for 5 min., and partially dried by careful removal of the liquid. To this, 100 μl of washing buffer with LiDS containing 100 mM Tris-HCl, pH 8.0, 0.15 mM LiCl, 1.0 mM EDTA, and 0.1% SDS was added, (mRNA DIRECT kit.) The mix was transferred to a 200 μl PCR tube. The beads were washed once with 100 μl washing buffer with LIDS and once with 50 μl washing buffer containing 100 mM Tris-HCl, pH 8.0, 0.15 mM LiCl, and 1.0 mM EDTA. (mRNA DIRECT kit.) The beads were then washed quickly with 20 μl 1×RT Buffer (25 mM Tris-HCl, pH 8.3, 37.6 mM KCl, 2.5 mM MgCl2, and 5 mM DTT) and 20 μl RT Mix containing 1×RT Buffer and 20 μM dNTP was added. The tube was heated at 65° C. for 5 min. and cooled to 37° C. 1 μl MMLV reverse transcriptase (Promega) was added and the mixture was incubated at 37° C. for 1 h. with occasional shaking. Next, 20 μl of water was added to the RT reaction, mixed and a 1.0 μl to 20 μl aliquot of the PCR mix containing 1×Perkin-Elmer PCR buffer, 2.0 μM dNTP, 1.0 μM T12VN, 0.2 μM arbitrary 10-mer, 1 unit AmpliTaq (Perkin-Elmer), 50 μCi α35S-dATP (Amersham) was taken. PCR using temperature settings of 94° C. 30″, 40° C. 1′, 72° C. 2′, 40 cycles, and 72° C. 10′ extension was performed with the Perkin Elmer 9600 Thermal Cycler. All PCR product was run on appropriate gels for band visualization.
  • cDNA cloning of Differential Display Bands
  • All dried gels were marked with radioactive ink prior to film exposure for proper alignment between the X-ray film and the dried gel plate. Appropriate bands were marked by puncturing. A scalpel blade was used to score the gel around each band to be excised. The excised gel pieces were placed into a PCR tube containing 2 μl water. PCR was performed using a 50 μl PCR mix (same as for differential display with the following modifications: the primer concentration was 1 μM, and the dNTP concentration was 200 μM; no α35S-dATP is added.) The cycle settings were the same as above.
  • A portion of the PCR products was run on a gel to determine amount and size of PCR products; DNA that did not correspond to the size of the original differential display band was discarded. The remaining PCR fractions were purified using CHROMA SPIN-100 columns (Clontech, Palo Alto, Calif.) according to the manufacturer's instructions. The purified PCR fragments were cloned into the pCR2.1 TA cloning vector (Invitrogen) according to Invitrogen cloning protocols supplied with the vector. The only variation from the standard protocol was an increase in the molar concentration of PCR product to vector (over 100-fold); multiple insertions were not found to be a problem. All ligations were performed at 16° C. overnight, transformed into E. coli strain DH5α, and plated onto LB with X-gal/IPTG.
  • Five colonies were chosen for PCR verification; PCR products of expected size were selected. About 10 μl of the 30 μl PCR reaction was simultaneously digested with Nla III and Mse I overnight at 37° C. (a 5 h digestion was used as well.) cDNA clones were selected according to the colony PCR and the restriction enzyme digestion pattern.
  • The differential display protocol for finely staged zygotic embryos of loblolly pine as described above, has produced more than 600 differential display patterns and more than 60,000 bands. Within that set of bands, we have identified bands that increased and/or decreased during embryo development. From those bands cDNA clones of this invention were isolated and sequenced.
  • Detection of Gene Expression by Micro-Array Assay
  • In order to verify expression patterns of the cloned DNA in loblolly pine embryos a micro-array assay was developed. The cloned cDNAs were amplified by PCR and adjusted to equal concentrations (0.1 μg/μl). The cDNAs were then dispensed in the wells of a 384-well plate, denatured in 0.3 M NaOH at 65° C. for 30 min. and neutralized with 2 volumes of 20×SSPE mixed with 0.00125% bromophenol blue and 0.0125% xylene cyanol FF (5% gel loading dye). The denatured DNAs were then blotted on to Hybond N+membranes (Amersham) as arrays using a VP 386 pin blotter (V&P Scientific, Inc., San Diego, Calif.). Each DNA was dot-blotted four times as a quartet on the membrane. An example of quartet spotting is seen in FIG. 7. Each dot is about 1.2 mm in diameter and contains about 3 ng of DNA. DNA was then cross-linked to the membrane at 120,000 mJ/cm2 in a CL-1000 UV-linker. (Strategene, Inc., Upland, Calif.) The dot image of each membrane was scanned, numbered and saved in computer for later use in data digitizing.
  • The cDNA array membranes were pre-hybridized in hybridization buffer (0.5 M Na-phosphate, pH 7.2, 5% SDS, and 10 mM EDTA) at 65° C. for 30′ in a hybridization oven (Model 400, Robbins Scientific, Sunnyvale, Calif.) and then hybridized under the same conditions with total cDNA probes made from mRNA. The membranes were washed twice at room temperature in 2×SSPE and 0.1% SDS, twice in 0.5×SSPE and 0.1% SDS, and twice in 0.1× hybridization buffer. Each wash was roughly 20 min. Each membrane was then exposed to Kodak Biomax MR films.
  • The total cDNA probes referred to above were made by initially creating the first strand cDNA. This was accomplished by mixing loblolly pine embryos (0.05-0.1 gm fresh weight) with 100 μl lysis buffer (containing 100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 10 mM EDTA, 1% SDS and 5 mM DTT) in a 1.5 ml Eppendorf tube. The mix was then ground with an electric drill as described above. Another 100 μl lysis buffer was added and the lysate was ground again briefly. The drill pestle was washed with 100 μl lysis buffer that was pooled with the lysate. After centrifugation at 14K at 4° C. for 15 min. in a Beckman bench top centrifuge, the clear embryo lysate was mixed with 10 μl Dynal beads washed twice with lysis buffer. The suspension was incubated on ice for 5 min., with occasional mixing to allow binding of Poly (A) RNA to the oligo (dT) on the beads, and then left on a magnetic stand at room temperature for another 5 min. The liquid was removed and the beads were moved to a 0.2 ml PCR tube by suspending in 100 μl lysis buffer.
  • The beads were washed twice with 100 μl of washing buffer with LiDS and once with 50 μl of washing buffer. The mRNA was eluted from the beads in 6 μl water at 65° C. for 2′. One μl T21VN primer (10 μM) and 1 μl SCSP oligo (cap switch primer, 5′-ctcttaattaagtacgcggg-3′, 10 μM) were added to the mRNA eluate. The mixture was incubated at 70° C. for 2′ and cooled on ice. Three μl 5×First Strand Buffer, 1.5 μl DTT (20 mM), 1.5 μl dNTP (10 mM each) and 1 μl MMLV Superscript II (Gibco BRL) were added to the mRNA-primer mixture followed by incubation at 42° C. for 1 h to synthesis first strand cDNAs. The cDNA was heated to 72° C. for 1 min. to degrade RNA and then diluted to 100 μl with water. The lysis buffer, washing buffer and Dynal beads are components of the mRNA DIRECT kit (Dynal, N.Y.). The first strand buffer (5×), 20 mM DTT and 10 mM dNTP are components of the SMART PCR cDNA synthesis kit (Clontech, Palo Alto, Calif.).
  • The first strand cDNAs synthesized as described above contains a T21VN sequence at their 5′ ends and the SCSP sequence (see “SMARTTM cDNA, Clontech, Palo Alto, Calif.) at their 3′ terminals. Total cDNA probes were made by PCR amplifying the first strand cDNAs using SMART cDNA PCR (Clontech, Palo Alto, Calif.) in the presence of labeling agent. Five 5 μl first strand cDNA solution was mixed with 5 μl 10×KlenTaq PCR buffer (Clonetech), 5 μl dATP+dGTP+dUTP (5 μM each), 1 μl T21VN primer, 1 μl SCSP oligo, 1 μl KlenTaq Mix, 5 μl 32P-dCTP (10 mCi/ml, Amersham) and 27 μl water. The PCR was performed using the setting of 94° C. 2′, 15 cycles of 95° C. 15″, 52° C. 30″, 68° C. 6′. The PCR products were purified using NICK column (Pharmacia) according to the manufacture's instructions.
  • Currently, high-density array Southerns for both somatic and zygotic embryos at all the developmental stages have been performed. The dot array Southern data indicate that gene expression of late stage somatic embryos resembles middle stage zygotic embryos; many transcripts present during late zygotic embryogenesis (ZE) are absent in somatic embryos and late stage somatic embryo gene expression patterns resemble the patterns of middle stage zygotic embryos.
  • Cairney et al. (In Vitro Cell. & Devel. Biol.-Plant. 36:155-162 (2000); Appl. Biochem. Biotech. 77-79:5-17 (1999)) have discussed how this gene expression information may be used to improve the process of somatic embryogenesis; the rare incorporated in their entirety. As shown in FIG. 2, the high-density array Southerns allows rapid evaluation of embryos subjected to protocol changes. Following the expression of a known gene permits inferences about metabolism and is very valuable in developing media-improvement hypotheses. Further, detailed gene expression studies may help by providing an understanding of the timing and location of gene expression (e.g., in situ hybridization). The isolation of key genes also provides the ability to monitor the expression of these genes as stage specific markers and allows protocol variations to be quickly evaluated.
  • EXAMPLE 5 Identification of Markers for Superior Performance in Tissue Culture”
  • The evaluation of tissue culture modifications for pine somatic embryogenesis, depicted in FIG. 8, is typically a lengthy process. However, where molecular tools are available, potentially improved media or genotypes can be discerned more rapidly, thereby avoiding the months of costly evaluation. (See FIG. 8) Table 5 illustrates this proposition.
  • Table 4 describes several publicly available clones. Lec. Fie, and Pkl, used to provide a representative model for this example. Any clone within Table 1, SEQ ID NOS: 1-327, can be substituted for those in Table 4 to assay increased performance in tissue culture. Any promoter within Table 1, SEQ ID NOS: 328-334, can be incorporated with those in Table 4 or SEQ ID NOS: 1-327 to assay increased performance in tissue culture. In this scenario, Table 5, a representation of the information contained in FIG. 9, shows performance of selected genotypes (260, 480, 499, and 500) in various media (1133 or 16) determined by the total number of embryos produced per medium as described by Pullman and Webb (1994), incorporated herein. Embryo maturation was determined by the presence of recognized morphology according to methods previously mentioned above. (Pullman and Webb, (1994)) Genotypes that produced high, medium, and low numbers of embryos were selected for RNA extraction. Gene expression assays, such as DNA arrays, Northern blots, slot blots, etc., were used in attempt to correlate embryo performance with mRNA abundance for selected genes. In the example shown in FIG. 9 and Table 5, expression of loblolly pine genes, designated as Lec, Fie, and Pkl, obtained from the Pine Gene Discovery Project, was evaluated. The preliminary correlation appears to be that the high levels of the Lec gene's mRNA correlates with greater number of pine embryos. (See table 5.) These experiments can be further expanded to incorporate additional or alternative genotypes with the prospect of identifying a large collection of gene indicators of good or poor performance in tissue culture based on high or low mRNA levels. It is clear from the above that this approach, using the sequences disclosed in this application, can evaluate a genotype entering tissue culture, saving both time and expense.
  • Somatic Embryos
  • Immature zygotic seeds were collected from loblolly pine genotype 260 (mother tree BC-3, Boise Cascade). Somatic embryos were initiated as described by Becwar et al. (1990) or with modifications in media mineral composition. The early stage somatic embryos were grown in cell suspension culture medium 16 and sub-cultured every week (Pullman and Webb, 1994). The embryos collected from the suspension, which include stage 1 and stage 2 somatic embryos, are referred to as stage S embryos. At the end of the subculture week, the somatic embryos in the suspension were settled in a cylinder and transferred to maturation medium 240 (Pullman and Webb, 1994). Resulting somatic embryos were selected, staged, sorted into vials containing the same stage, and stored at −70° C. until analyses were performed.
  • Probes
  • For the following example analysis RNA was isolated from embryos at different stages in development, early stage somatic embryos and late-stage somatic embryos. The cDNA probes used in this example are not contained in the SEQ ID NOS: 1-327, but rather, are generic, publicly available pine sequences obtained from the Pine Gene Discovery project located at (http://www.cbc.med.umn.edu/ResearchProiects/Pine/DOE.pine/index.html). These clones are homologs to the well-studied Arabidopsis genes that have been shown to have significant influence on embryo development in this plant. The pine clone names (first column) and corresponding references for the Arabidopsis homologs are shown in Table 4. The three clones listed, Lec, Lie, and Pkl, are for representative purposes within this example and it will be clear to one skilled in the art that any of the SEQ ID NOS: 1-327 could be substituted for those here as all will help identify conditions for improved performance in culture.
  • Probes were made by preparation of DNA using Wizard Minipreps (Promega, Madison, Wis.) and cDNA inserts isolated by restriction enzyme digestion. For the cDNA probes, 50 ng of the isolated cDNA insert DNA was used to make 32P-labeled probes with Ready-To-Go DNA labeling beads (Amersham Pharmacia Biotech) according to manufacturer's instructions. Blots were prehybridized (7% SDS, 1% BSA, 0.25 M NaPO4 (pH 7.2), 1.0 mM EDTA) for 3 hours at 65° C. and hybridized in fresh buffer at 65° C. for 12 to 18 hours (4). Each blot was washed 6 times with the following conditions: 1) RT, 2×SSC, 0.1% SDS, 15 min; 2) RT, 2×SSC, 0.1% SDS, 30 min; 3) 42° C., 0.2×SSC, 0.1% SDS, 15 min; 4) 42° C., 0.2×SSC, 0.1% SDS, 30 min; 5) 60° C., 0.2×SSC, 0.1% SDS, 30 min; 6) 60° C., 0.2×SSC, 0.1% SDS, 30 min. Blots were exposed to a phosphorimaging plate for 10 minutes. Screens were read with a BAS1800 (software v1.0) and images were manipulated with ImageGauge (v2.54) (Fuji Photo Film Co., Ltd., Kanagawa, Japan).
  • The hypothesis tested within this example is that genotypes that produce large numbers of embryos have high Lec expression and low Pkl expression, poor genotypes have the opposite pattern, and that Lec and Pkl expression act as indicators of embryogenic potential. FIG. 9 shows that Lec is not expressed in late stages of embryogenesis in somatic embryos. The Lec gene is expressed throughout embryogenesis in Arabidopsis. The blot reveals that the Lec gene is a useful early expression marker for embryogenesis. One interpretation of these results is that the somatic embryos do not express Lec in the manner that Lec is expressed in zygotic embryos, i.e. the use of Lec expression has highlighted a defect in gene expression in somatic embryos. This defect could be used to identify desirable genotypes, i.e. those likely to progress through development and produce a large number of healthy plantlets compared to undesirable genotypes that will cease development prematurely or produce low numbers of plantlets. This is an example of the principle described pictorially in FIG. 8.
  • The results described in the previous section of Example 5 reveal ways in which gene expression analyses can be used to improve somatic embryogenesis based on several genes. However, this principle applies as well when the assay is expanded to determine the expression of hundreds or thousands of genes simultaneously (e.g. by DNA arrays). We can create hypotheses which state that expression of a single specific gene can be used to determine the potential of a culture, or hypotheses that state that the expression of a group of genes (e.g., hypothetical genes A, B, C, D, E, F) acts as an indicator of high embryogenic potential. For example, all these genes may be expressed at a high level in cell lines that produce large numbers of embryos, thus we would select cell lines which exhibited this characteristic. Alternatively specific levels of expression for genes A, B, C, D, E and F may be required and a combination of high and low expression of particular genes will identify desirable cultures. Alternatively, experience will determine that certain exceptions can be tolerated.
  • While the previous paragraphs discuss numbers of embryos produced, the principle applies to ANY desired characteristic: by establishing a correlation of gene expression with e.g., germination potential, embryo size, growth of plantlets in their first year, disease resistance of mature plants, environmental hardiness or wood quality. Any trait where could be evaluated by these gene expression assays and correlations with gene expression established, resulting in a molecular tool which could be used to predict desirable characteristics. Explicitly, we could use these gene expression tools to select cell lines which will produce high quality plantlets months before they grow into plantlets, or cell lines or juvenile plantlets which will produce hardy trees with desirable wood quality, years before these traits are expressed.
    TABLE I
    Embryo
    cDNA Phase Clone Nucleotide Sequence
    SEQ ID NO:1 Late LPS-001 GGTACTCCACCGTAATAACCCTTGGGAAATAGCCTATGATCCAGGGGAGGCAACC
    ACCTATATCATTGACAACAGCGAAAAATGTGGCGCAAGAAGTTTCACATACAATTCA
    TGGTTACAAAGATCACATACCAGGTGTTGGAGCAGATTCGATAGATATTGAAGATAT
    GAAGCCAAGGAGTGGAGCAGTTATTGAAAAGGGCACAAAAAAATTTGCCATTTACA
    AAGATGAAAATGGGCTGATTCACAAATACTCGGCAATATGCCCACACATGAACTGT
    ATTGTGAAATGGAATCCTATAGACTCAACTTTCGATTGCCCCTGCCATGGTTCAATG
    TTTGATAATCTGGGTCGATGCATCAATGGACCTGCCAAGGCGGACCTATTTCCCGA
    AGATTAACGATAGTTGTTTGTACATGTAATTATCTTGATATTGTATATATATGTATTTA
    AATTATACAGTACAATAAATCCATGTTGCAGGCTATTTCTGCTTGATAATTTAGCTC
    CAGATTATACATAACCAGTTATTGGCTGTTTTCCCCTGGCAAAAAAAAAAAA
    SEQ ID NO:2 Late LPS-003 003GGTACTCCACAGAAAGAAATGATTTGACAGAAAAAGAGAGCTGTAGGATTGGG
    AAACCCTGCAGTGGATATATACAATGTATATGTACTCTGTCTGTTTTTCTGTTATTTG
    ACGGAAATAAAAACGCCATAGCGACGGATGACTGTAAATCCTTAGGGACGGATGAC
    TGTAAATCCTTAGGTTGGAAGATTACAAACGACATATGGGTCTTTCAATTTTCAGAT
    TTCTGTAAGACTTACATTTCAAAGACTGTTTGGATGGGCAAAAAAAAAAAA
    SEQ ID NO:3 Middle LPS-004 GGTACTCCACCAGAATGCCGCAGTTTAGTTCTCTAAAGCAAGCAGTAAATTAATTTT
    GTCAAAATCTAAAGAGTGTATAGTATCAGTGGGTTTGTATTTCCTAGTTTGCCTACA
    ATAACGATGGGGATTCACCAGTTTTTGTAGAATTTGCAATCATCGGATGACAATTTC
    AAAGTTTTCTCTAAGTCACCCGCATTGATATCGAGAAGCCTTCCATTTTCAATTATTT
    AATATCAGAAAATCTTTTCAGTTGGCAAAAAAAAAAAA
    SEQ ID NO:4 Middle LPS-006 AGCCCAGCTGCGAAGGGGATGTGCTGCAAGCGATAAGTGGTAACGCCAGGTTTCC
    AGTCAGACGTGTAAACGACGCCAGTGATGTATACGAATCACTATAGGCGATGGCCT
    TCTAGATGCATGCTCGAGCGCCGCAGTGTGATGAATTGCAGAATCGGCTGGTACT
    CACGGGCTAGAGAAAGGCACAAGCACTTTTTGTCATTTTAGGATCAGAGGCATTCA
    GGTATAGGAAGGGTGGCTCAGATAGGCAGATGGATCGGCATTTTGCCCAGTCATG
    AAACATTTTATGCATGTTATTGCCTCCCAAGGACGAAATCAGTTCTTTGTGCCTTCT
    GGTGATATCACTTCAAACAAAAGGCAACAGTTCTGTGATTTCATATGGTTTGTCACT
    GAATATTTTGTTGCAGATGTTCTCTACTATTTTTTATCTGCTTTCAAGTGATTATTTG
    TTGATTCCCCATGGATAGTTATGCTAATCAGTTGCATTTCTCTTGTACCAGTCAACA
    AACAAAAATGCTTGTAGGAATCCATTACTATTTATTTTCAGACAGGTAAACGTGTAG
    CTAATTGTTCTGGCAAAAAAAAAAAA
    SEQ ID NO:5 Middle LPS-007 TCCAAAATACAAAGGCTTTATTTGCATCATGATATAATACAAAGTAAGAAATTTACCC
    AACTGTTTAACCTAATAATAATACAAAGGAAGCATTTTACCCAACTCTTTAACGTAAT
    AATACCAAAGAGTGGAATGCTTTATTGACCAGCAAGACCTTGAAATTTTTATAACCA
    ATGCCCATCAACAGAGCCTTTCTTAAAAAACGCAAAGCCCAGCTCTGTCACCTTATT
    AGTTAGTATAAACTGACATTCTTCCAAGCTTGTGTGCGCAGAAACAATAAAGAACT
    CACCTTGGTTTAAAGAACGTGCCATGAAGAAAACGTCCCAAGAAAAATGAAATGGC
    TCCTTCGACCATTCAGTCCTCCCTAGAAAAATCAAAAGACTCCTTCGACCATTAGGT
    CCTCCAATTGGGCATCTAACTACAAGCGGTC
    SEQ ID NO:6 Middle LPS-008 GGTACTCCACGGGCTAGAGAAAAGGCACAAGCACTTCTTCGTCATTTTAGGGATCA
    GAGGCATTCAGGTATAGGAAGGGGTGGCTCAGATAGGCAGATGGATCGGCATTTT
    GCCCAGTCATGAAACATTTTATGCATGTTATTGCCTCCCAAGGACGAAATCAGTTCT
    TTGTGCCTTCTGGTGATATCACTTCAAACAAAGGCAACAGTTCTGTGATTTCATAT
    GGTTTGTCACTGAATATTTTGTTGCAGATGTTCTCTACTATTTTTTATCTGCTTTCAA
    GTGATTATTTGTTGATTCCCCATGGATAGTTATGCTAATCAGTTGCATTTCTCTTGTA
    CCAGTCAACAAACAAAAATGCTTGTAGGAATCCATTACTATTTATTTTCAGACAGGT
    AAACGTGTAGCTAATTGTCTGGCAAAAAAAAAAAA
    SEQ ID NO:7 Middle LPS-010 ACGACGTGTAAACGACGGCCAGTGATTGTATACGACTCACTATAGGGCGATTGGC
    CTTCTAGATGCATGCTCGAGCGGCCGCAGGTGATGGATATCTGCAGAATTCGCTT
    GGTACTCCACGGCTAGAGAAAAGGCACAAGCACTTCTTCGTCATTTTAGGATCAGA
    GGCATTCAGGTATAGGAAGGGTGGTCAGATAGGCAGATGGATCGGCATTTTGCCC
    AGTCATGAAACATTTTATGCATGTTATTGCCTCCCAAGGACGAAATCAGTTCTTTGT
    GCCTTCTGGTGATATCACTTCAAACAAAAGGCAACAGTTCTGTGATTTCATATGGTT
    TGTCACTGAATATTTTGTTGCAGATGTTCTCTACTATTTTTTATCTGCTTTCAAGTGA
    TTATTTGTTGATTCCCCATGGATAGTTATGCTAATCAGTTGCATTTCTCTTGTACCAG
    TCAACAAACAAAAATGCTTGTAGGAATCCATTACTATTTATTTTCAGACAGGTAAAC
    GTGTAGCTAATTGTTCTGGCAAAAAAAAAAA
    SEQ ID NO:8 Middle LPS-011 GGTACTCCACGAAGCAAAAAGAGTCAGGGGAATGAAGATGGGGGGCTCCGACAAG
    AAGCGGATCAGAGAAGAGCAGGAAATGAGTCCACCTGAGGAATCCTGGAGACAGA
    AACAGGGGCGTTTTAATGGAGTTTGAGGCAGGGATGGCCTATGATAAACCTGAAAAT
    GCCGGTGCAGGTAATGAGAATTTGCCAGAGTTTTGCTCTCTTTCAAATGAGTACTC
    GATGTTATTGAAAGATCCATGGAGTTGGGAGGATAGCACTGGTTTCGGAATCCGAA
    GCTTAGCTGCTGTCAGGAAGCAGTCTTGTATATTGGACTATCTCCATGATTCTGCT
    GTAGATAATCGCTGTGAAAAGGATTTTGCCGAGCAGCACAAGGTACAGGAAGAGG
    AGGATTGTTTGAGAAGGTCTCTTTTTGAAGCCACAGATGATCAGCTCTGGAGGCTT
    CAGAGTCTTTGCAGGATACAGAAGGTCTGTTTCCTCTGGATTCCGTGGGTAGCCAT
    GATTGCACGACCTTGTTGCAGGATGAGAGCATTGTTCAGGGCGCTGCTCTTACTT
    CAGAATTTGGGAACAGGATGATGGTCACAAGGATGCCAAAATTCATGAAGATGGCA
    TTGGTTTTGTGTATGGGAGTGGGATCTCGGATTGGATTCGGAGGGCTCCCTCGAA
    TCAATCTGAGTTTTCTGAATCTGTTGAATTTGAAAGCTCTATGTTTTCACTGTAATTT
    GGGTCTTTTTAATTTCTTCCTATGTAATTTGGGTGTTTCTAATTTCTTCCTTCAGCAA
    AAAAAAAAAA
    SEQ ID NO:9 Middle LPS-012 GGTACTCCACCATATCCAGGTAACAAGGGAAAACAGAGTCAGCTTCTAGTATGTT
    GTATGCCTTGCTCTGTCTGTTTTCTTTGATCTTTGATGCCAAGCAAGTTGAATGTGA
    TCACTAAATGTTGCTGGCAGTAGAGCTGGAGATGTGCTGTCTCTTTGGTGTCATTA
    GCACAGAAGCTATTGGAGAAATGATTATTATCTGTTTGATAACTTCTAGAGCATTTT
    TCTGCTTCCAATTCCACAAGGTGGAAAGTGCAAGGATGTTTACTTTCTTAAACTGTA
    CTTGCCTTGTATTTGATGATGTAAGGTTGTGTGGCAAAAAAAAAAAA
    SEQ ID NO:10 Middle LPS-013 GGTACTCACCATATCCGGTAACAAGGGAACAAGTCAGTTTTAGAAAGTGGACCCCC
    GGTTCCGTCGTTTTCTTGATCTCGGAGCCAAGCAAGTGGATGTGATCACTAAATGT
    TGCTGGCAGTAGAGGTGGAGATGTGCTGTCTCTTTGGGTCATTAGCACAGAAGCTA
    TTGGAGAAATGATTATGGTATTCCACCATATCCAGGTAAACAAGGGAAAACAGAGC
    TCAGCTTCTAGTATGTTGTATGCCCTGCTCTGTCTGTTTTCTTTGATCTTTGATGCC
    AAGCAAGTTGAATGTGATCACTAAATGTTGCTGGCAGTAGAGCTGGAGATGTGCTG
    TCTCTTTGGTGTCATTAGCACAGAAGCTATTGGAGAAATGATTATTATCTGTTTGAT
    AACTTCTAGAGCATTTTTCTGCTTCCAATCCACAAGGTGGAAAGTGCAAGGATGTT
    TACTTTCTTAAACTGTACTTGCCTTGTATTTGATGATGTAAGGTGTGTGGCAAAAA
    AAAAAAA
    SEQ ID NO:11 Middle LPS-014 GGTACTCCACCATATCCATGTAAACAAGGGAAAACAGAGCTCAGCTTCTAGTATGT
    AGTATGCCCTGCTCTGTCTGTTTTCTTGATCTTTGATGCCAAGCAAGTTGAATGTG
    ATCACTAAATGTTGCTGGCAGTAGAGCTGGAGATGTGCTGTCTCTTTGGTGTCATT
    AGCACAGAAGCTATTGGAGAAATGATTATTATCTGTTACATAACTTATAGAGCATTTT
    TCTGCTTCCAATTCCACAAGGTGGAAAGTGCAAGGATGTTACTTTCTTAAACTGTA
    CTTGCCTTGTATTTGATGATGTAAGGTTGTGTGGCAAAAAAAAAAAA
    SEQ ID NO:12 Late LPS-015 GGTACTCCACTAGACCGGGTAGGGTCTCTCCATGGTTTTGCGACTAGGTTAGGTG
    TCCTGTTCTGTTAATGATTTTGAGGTTTTGTTAATTGTGAGTATGTTTCCAGGGTTTT
    GAACCTGGGTACTCGGCCTTTGTTGGAATGTAGTCTGGTTAATTTATATGTATATGT
    AACCTTGGGGTTTCGAGCCCAGTTCTCTGTTCTTCTTGAAATGAAATGCGATTTGTT
    CTAAAAAAAAAAAA
    SEQ ID NO:13 Late LPS-019 ATATATACGTATGGTATTCCACAGCATGAACTCTCGACATTATATGCTTGTTATAGT
    TTTTAAGAGAGGAGACTTACCTCACACATGTACAGCTTTTTATTGTCGTGCTTTCAG
    TTGATGGATGATTGTTGTAGTCCTGTCATTGGTTGGACAATTTCATCATCCTAAAG
    ATCCAAGAATTCATGTGGCAAGAAACTTTAATAAAGTCAAATATAATCCGATGACGT
    AACCCTAAAAAAAAAAAA
    SEQ ID NO:14 Late LPS-020 GGTACTCCACTAGTGATCGATTCTCTGTATGTGACGCTGCGCGGCGGCTATAGC
    GCTTCACTGAGAATGTACGGTATATTATGATTGATGTGATGGATTTGCTCCGCAGC
    TTCGGCTGTTGTATCTGCTCACTTCGGCGTATATATGTAATATGTTGCTTCTTCAGA
    GAGATGAACTTCCCCCTAAAAAAAAAAAA
    SEQ ID NO:15 Middle LPS-023 ATAGATCATTTTAAAGTTTCAGTGATTTGAATCTAATTCCACTGCATTTCCTCGCAAA
    CTGGCAGTCAAATAGTATTCCCTCTTTCAGTGACAGGCTGGCAGGTGTTCATTCT
    TATACAAACATGATTATCATAATTCCATTAATTCATGGCGTTTTCTTTGCCAAAAAAA
    AAAAA
    SEQ ID NO:16 Late LPS-024 TTTTTTTTTTTTAGGGAGAAAGGTAACTTCAGCCAGCTTTCAAAGGCAACACCTACA
    AAAGGGGTGACTGAGAACTCAGACACAGACGACAAGTGATCATTCGGGCCAGATT
    TTTGTTGAGAGAGTTGTAGTGTGTAATTGATTCATTTCATACATTTGATATGCAAGC
    CTGTACAATAGCCTGTGACTGTTAAGGGCATTCTTTTGTCTCCCTGTTGCTATTTGG
    GTTTCCGGTGTGTTCATTTTCACTTATTTTTGTGTTTTAGCTGGAAGAATTTGAGAG
    GGTAGAATTGTGTCATCGCTATGGCTTGTGCATGACTCATGAGCCAGCAGTTGAAA
    CTTTTATTTATTAAGTTATAATACTATGTCTTGTCAATTCTCAATAAAAGATATTTTAT
    GCTGTTGGGCAGCATCTAAAATGTTTTGTATGTTAGCATAAAATCCCATTTTCTATA
    AGTTTTTGCCAAAAAAAAAA
    SEQ ID NO:17 All LPS-025 AGCAGGTTCAGTCAGACGTGTAAACGACGCCATGATGTATACGAACTCATATAGGG
    CGATTGGCCTTTAGATGCATGTTGACGGCCCGCAGTGTGATATTCGCAGATCGCT
    TTTTTTTTTTTTAGGCATGGTGCGCGATGAGCTGATAGCGATGATGAAGACCAAGA
    CCACCAAAGGAAGATTCTTCAGAGCAAAAGCTACGGAGACAGAACCAGAGGACTC
    AAAGCCGGAATCCATTGGTGAGGTACCTGCAAATGTGTGATGGACTAACTAAGAAG
    GCTCCTTGAGAGGACCCATTAAGCACAGTGTTTTAGTCCCAAATTCTGTGCAAT
    TCCGTTGAAAATCATTTTTACGATTTTAGGTATGATGTGTGCAATTTAAAGTTGGAA
    TTATTGTGGGCAAAGGCTATAAGTGATTGTCTAATCCATTTAATTTATTATCTTTTGA
    CTAAGAGCATATCTAGGCTGGAAGAAATTAGGGCACATATGTTTTGTGAATTT
    GAACATTCTGGGTTTTGCAATGCAAAACACCACAAATATTTTATAATGTAGAGGTG
    TACTTTTTCTGGCCAAAAAAAAAAAA
    SEQ ID NO:18 Middle LPS-026 GGTACTCCACCAATAATACTGTCTGTTCTGCTCCCTGCTGATCCACTAAGCAGA
    TTATTTCTGTCCACCCCACTTTAGAGTCTCAGTTTGTAAAGCACTCCCTAGGAGCTA
    AACTCATTTCCAATGGATTAAAGCACTCCATAGGAGCTAAACTCATTTCCAAGGGAT
    TTTTGTCCATTTCTCTGTGCTAAAAAAAAAAAA
    SEQ ID NO:19 Early LPS-027 ATGTATACATATATGTGGTACTCCACACACTCAATAACAGCATCACAATCAAAACA
    AGAAGGCGGCCAGAAAGCTTTAAAATGCTAAGCCTACAGGTAATATTCACAACTGC
    ATTAAGCACCCCGCTCCTAGTTCTGAAGAAGCCAGAAAGCTTTAAAATGCTAAGC
    CTACAGGTAATATTCACAACTGCATTAAGCACCCCGCTTCCTAGTAGGCTAGTACTA
    GGACTAGGACCGCATTACCAGTTCCCTTATCTTCTACTCATCCTCTACAGGAAAAC
    TATGACTAAAACTGCATTACCAGTTCCCTTATCTTCTCAACTCGTCCTCTACAAAAAA
    AAAAAA
    SEQ ID NO:20 Early LPS-028 GGTAATTTCCACCCACCACGGGCTTTTTCAATTAACCCATTTCTACCACTCCACAT
    AGGGTTCTAAGTTTTGTGACTCACCCCCAATTTCGCTGATATTTTGCATTGCAGCT
    GTTTATCTACAGGAAATGGCTAATCAGTACTTTCAGAATTGGTTGCTTCTGTACAG
    GAAATGGATAATCAATCAGTACTTCTATACTAAGTTGCTTACGCGGGGATCAGAG
    CCTTACTTCAGAAAATTGAATACATTTTCTTCTTTGTGTATGTATCAGGCATGGAAT
    ATATGTAGCATGCCATGGAATGCGTATTTACTAGATTATCTTTTAATTTAATACATAT
    GTTGCTTACTAATTTGTCCACAAAAAAAAAAAA
    SEQ ID NO:21 Early LPS-029 GGTACTCCACACACTCAAACAACAGCATCACAATCAAAACAAGAAGGCGGCCAGAA
    AGCTTTAAAATGCTAAGCCTACAGGTAATATTCACAACTGCATTAAGCACCCCGCTT
    CCTAGTTCTGAAGAAGGCCAGAAAGCTTAAAATGCTAAGCCTACAGGTAATATTCA
    CAACTGCATTAAGCACCCCGCTTCCTAGTAGGCTAGTACTAGGACTAGGACCGCAT
    TACCAGTTCCCTTATCTTCTACTCATCCTCTACAGGAAAAACTAGGACTAAAACTGC
    ATTACCAGTTCCCTTATCTCTCAACTCGTCCTCTACAAAAAAAAAAAA
    SEQ ID NO:22 Middle LPS-030 GGTACTCCACTATTAGATTGATGCAAGACCAACTGATCATGGCTAGGGTGTATCA
    AGCATTTCCCAGGCTAGGAATAATCTTGATTTATACCATGAATGATGCTTCGTATT
    AAAGAATGTCAACGTACATGGGTGAGACTAATGCCGATCTGATCTACCTCAAG
    GTAATAATTTTTGCATTAGCTGCTTCTAATCAAGAGTAGTAAGTGCTTCCATTTGC
    AAAAAAAAAAAA
    SEQ ID NO:23 Middle LPS-031 GGTACTCCACAAGGCATATATGGGCAATTGATTTTGCCTAGCCCAAATCCTATCA
    AGCTTGCGTATTTCTAAAAGATGCACTATTTTTTGTCCGAGTGTAGGTTTTGAATTC
    ATTGTAACATTCAGCAATATTAATTCAGGGGTAGCATTTCTGGCAAAAAAAAAAAA
    SEQ ID NO:24 Middle LPS-032 TTTTTTTTTTTTAGGGTAGAAAACCATGCTTCACTAACAAGGTATTAAAATTACAATAT
    AATTCTGGGTGTAAACGACCTGATAGATGATCTGCAAGTGCCAGGAGGCAATATCT
    AGCAGAATACGTACAAATAAATTGCCAAAAAAAAAAA
    SEQ ID NO:25 Late LPS-036 GGTACTCCACCAATGATCACCCATGTCCATTTGGTTAATTCAATGTCAAGATTTAGT
    AGTTCCGTATTCCCTTGGGTAAGCTGTAATGGTCCATTTGGGAACAGTCCATGTTT
    GGGACACAAGTTCAATAGAGATGTCATCCATAAATATCCATAAATATGGGTATGAATCTCTTCCTC
    CCTCTCCGCCCAATAATAAAAAAAAAA
    SEQ ID NO:26 Late LPS-037 TTTTTTTTTTTTAGTAGCAATAGCAATCCATTTTAGGGATCTGCAGATCAGTGACTAA
    GTGACCCCTACCCCCAAAGGATTAATTGTACTTTGGCTTAACCACAAAACCTGATC
    AATGTGAAGTTTTTACCCATATTAATTCCCAAAAGTAACTACAAATTCCAG
    AGTACATTTTTACCCAAAAAAAAAAA
    SEQ ID NO:27 Middle LPS-038 GGTACTCCACTATACAATATCAAGGCATATCTGCCGGTGTGAATCATTCGGATC
    TCAAGCACTCTCCGTGCCGCAACTTCTGGCCAGGCTTTCCCTCAATGTGTGTTTGA
    CCACTGGGATATGATGGGATCTGATCCATGGAACCTGGTCCCAAGCTGGGCAG
    CTTGTGACTGATATCCGTAAGAGGAAGGGTCTTAAGGAGAGTATGACTCCCTGTC
    AGAGTTCGAAGACAAGCTGTAGAGCTTTGCTATGTTTGCATGTCGGATGCTGTCAA
    GATTGAGGAACCTCCGAGTATTAACACAGTTTTGTGTGCTAGGACTAAATT
    TATGCTATTCACGTATTTTTGTGATCTGTATTTATGTTATCACGTATTTTTGATTG
    GAAAATACTTTTTACAAGTCATCCATTAATCTTTTAAATGTTACATAATTCTCTCTGT
    C
    SEQ ID NO:28 Late LPS-040 AAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATC
    GGCTTGGTACTCCACTATACAACATCAAGGCATATCTG
    SEQ ID NO:29 M,L LPS-041 CTTTTCTTCGTGCTTTCGTGGAGTACC
    SEQ ID NO:30 Middle LPS-042 GGTACTCCACAAAGTGAGATGAGTGATATGAGGTCAAACACGTAAATGACAATAGC
    TATTATTTCCCCACTTGTTTGTGGCTGTGTATATTATACTTCATTGTCAGGACTTTTG
    TATGGTTGAAGTTGCAAGGTTTTGGCAAAAAAAAAAAAAAAA
    SEQ ID NO:31 Middle LPS-043 GGTACTCCACCTCCAGCTGCTTATCCAAGTACTACGGATAGTTCATACTCCTATTAT
    GCTTCTGCCAAGTGAACCAGAAGGCTTCTGTTCTACACTAGCAAACTGATAGCTC
    GAGCATCTCATTTACTAAGGATGATAATTCAAAATTGTAACATTGCAAACATCAGC
    AAACATCAGCATCAACTCTGTTACTATACAAGCAATGGATGCGTCGCTGATGCTG
    CGGGAGAGTAAATTTTTAGTTTACTGCGGTTGGTAATTGAGTAGGTTGACTTACATT
    TCTGTTGTAAAGCCGTTGTCGGGCATTGTTTATCTGGCCGAGTTAGCGCCAGGAAG
    CTAAATGTACCAAATATTTATTATTTTTATTAAGAATATAAAATTTAGTCGTCTTCT
    GCTGCCCAAAAAAAAAAAAAAAA
    SEQ ID NO:32 Late LPS-044 ATGGCCATGGACTTATGACTTTCAAAACCCTAAAACCTATCTACAACTTTCCACGCT
    GAGATTTTCCGAGGAAGGCATTCTAAGCCATTCCCACCGTACTTTAATAAAATAAAA
    ACAAGAAGATAGTAAAGCTAAGCTACAACCTTCCGCCAAAAAAAAAAAA
    SEQ ID NO:33 Late LPS-045 GACCGCTGTAGGAACACTAGCAGATTCCGGAACATAGGTACTTTGAACATCTTTC
    ACTCCTCACCATATGAATAGTGAGTCGATGGCGGCCTTAACAGTCGAGCATGCTTT
    GATTTCGTCTCTCTCTCTAGTGACCGGAAATCAATCTCATTATATATGTCATTATGCAT
    TCATTCCCACTTCCTAACTTTCATTATTGTTCAAAACTCGCCTTCCTGAAAATGCTA
    TAATAGTAGGGGAATATTGAAAAACTTCCGCCAAGCTAAAAAGGCACTTAAAGCAC
    CTGGATTTGAACGAGGATTTCCCACCCCGATGAGGGGGGGTGTCTTTCCATTGAG
    ACGATGCCTTACTCGGCAGACCCTGTGGGGGTCTTTATAGGTGACTTAATACTTAA
    GTATAGGACTAAGAGAGAGGAAGCGACCGCCTCTCTGATCAAGCCTTTACGTGC
    GACGTGCCCAGGTAAAGGCTGATCTCACCAAATAATTCAGAGAAAGAAGATGACTC
    CACAGTAGCGAAACTCCTACATTGTCTTACATATCGTAACAAGCGGTC
    SEQ ID NO:34 Middle LPS-046 GACCGCTTGTGCGTGGTGTCCAAACTAGGACGCCTTAGTTTTCCTAAGAAGGAAAC
    CCAGGCGTTGACTTGAGGCAGACTTGTGCTTCTGGGTACTCTCATCACTGCGTGA
    CCTTGAGAAAGGGACTTTACCTCCAGGATCCTCAAACTTCTTCTCTGTAAAATGAGC
    ATTGTAATAATTATATCCCAGGCTTATGTTGGGAATATTCAATAAATGCTCCCTTCAT
    TCTTTAAAAAATAAGTAAAGACAGCCTGAATGGGAGCCACGTTCTCATTCTTCTTTC
    TCTTTAAAAAATAAGTAAAGACAGCCTGAATGGGAGCCACGTTCTCATCTTCTTTC
    TCTATGCAAAATGTATTGTGTAATGTTTGTGTACTAGTAGTTCAAGAGCAAATAAGT
    AGTTGGTTAATGGCTAACATATTTCTTAAATTTGTAACTGTTAAGATAAACATTGAAC
    AAGGAAAAAGATTCGTAACTGAAATGTAAAGTCATTTGACCCTGGATAGTCAATGAC
    AATCTTATTCACAGTGTAATAAGTAATTCATAACGAGATGATTATTATGAAATTATCA
    ATAGCCTGCTATATCACTTTATGTTTATGATCCACAAGCGGTC
    SEQ ID NO:35 All LPS-047 GACCGCTTGTGGAAGAAAAGAAAGAATCTCTTTCGGATTCAATAGGCGGTATGGGA
    GAGTCTGCTACTGCCTCTTGGATTCCAGGAATCCTAGAGCTGGGAGTATGAGTTGG
    AGATGATGAAGGTGTCTCTTACCTATTTCTTGAAGTGGATGGAGTTGTGAAAATCG
    ACTTCTAGCTTCAGCTAAAAACCTTCCCCTAGAATCTCTTGCTCTATGCATATCATTT
    TTATTTTTTCTTTCAAGATAGGGTAATAATTCTCTTTCTGATCTTCCAGGTCACTCTA
    GGTGCAAGAAGAGAGCATAGTCAAGGAACTATTAAACCAATAACTTTCTCTTTTCTG
    ATCCTCCAGTTCACTCTAGGTACAAGCGGTC
    SEQ ID NO:36 All LPS-050 GACCGCTTGTGCAAAGTAGATACCGTCCTGTTCCGGTGAATTGAAGTACATTTTCA
    AAATGCGCTACTATGACATTTTATAGGATGTCTGAGTGTAAAATAATGGTACTGGTT
    GTTGCAAAGAATCTGATGTTTGGATGTATGGAACTATAAATAGATGTTATTTTCTGA
    TCCAGAAGGCTTTCCTTACCAACTGATTTCATCTCAGAAACTAAAAGCTCTTGAAC
    TTGTGTAGATGGGGCTTGGTCATTGTAGTTTAAATGCATTATGTAGTGGCAAAAAAA
    AAAAGTTATAGCCTACGTTCAAATGGATTTGCTCGACAATCAAATGAATTACAATT
    GAATATTCATGTATACCCAAATTTTAAATGTAGAATGACATCATCAATGTAGACAAAC
    ACCACTGTGCTTGTCCTTGATATCCTCTTTCACCATATAATTGGTGGCTACTCAAA
    GTCACTATCTGATGCAACTACAAGCGGTC
    SEQ ID NO:37 Late LPS-051 GACCGCTTGTTCAATGCAGAATCTCGAAGAGATGTCTTGGACAAATACTGAACTGG
    CACGATTGGTGTAGTGCGGTTCAAAAGGCGCTCCAGATTCGTCTGGAACGAATCTT
    CATACGCTGAACAATTAGACATCTTGTACGCAAGAGAATTACGATCGGCCATATAAA
    AACCCCAAAGAGAAGAAAGTGTTTCGAAATTCTCCCAGAAAACAGTCTTATGCCAC
    CGATTTGTCTTTTCAACATGCATTTGCAATGAAGTCTTTGGATTCTTACTGTGAGTG
    CTGATCAGCAACGGATTTTCGATCTGTATAGCTCTGCCGATTCCTGGTTAAAGCAG
    CTAAGAGTTAGGCATCCAGATTTGAGTTTTTTGCATCTCACAATGTTTGAATACAT
    TCAAATCCATTGTTGGAGTAACCTAACAACAACTGTACTCTTCTTCCTATTTCTGAA
    GCCCTCTGCCAGTTTAAGGCAGAGAACTGAGTATCTACAAGCGGTC
    SEQ ID NO:38 Late LPS-052 GACCGCTTGTATAATAAAGTGGTACCGCGTCCTGCAAACAGGGTTCTCTTGCCATC
    CTGCTACAACCCTGCAGTGGTCGCAGTAGAGAGAATCGGAGCAACGAACGTTTTC
    CCGAATATATGGAGCGGGAGGAAGAGTTTTCTTGCTGATGATCCAATCGGAGTCGA
    ACTGCCACCGCTGGATGAAGGGCGGCGAGGAAATCTTGGGGGGCAGAGGCCCGT
    CGGCGTAGGAAATAAGAAACGATTTGATATGGAACGAAAGGGCCCGTCCAGGGTT
    CGATCCCCGGCAGGGCAGCCAGCCCCGAACTAAACAAAACAATAAGAACAAACAG
    CAAAGTAAAAGAAAGCACCAGAAGAAACAGCAGCAGACGAAGAGTAAGGAGCTGC
    CCACAAGCGGTC
    SEQ ID NO:39 All LPS-053 GACCGCTTGTAATCCACAGCATTTTCAATAACTTCCTGAGGTGACATCCACCTCCAC
    TCAGAAAACTCGGCTGCATCTGTCCCATCACCAGCTAGATTGATCTCACTCTCGTC
    TCCTCTAAATTTTAGGAGGAACCATTTCTGTGCTTGACCTTTCCATTCGCCTCCCCA
    CAAGCGGTC
    SEQ ID NO:40 Middle LPS-054 GACCGCTTGTATATAATGTGAAGACACAATAAAATTTTGTCCAACAAAGCAACCAAA
    CGACCAAAAATTTAGCTGTGACATCAAAAAGCTCAACCCCTACAATGAATGTAACCT
    TAATCTAGAAAATTGATCCATGATCTCCACTGAATTTCTCGTCATCCTGAAGAAT
    GAGAAACTTAAATGTACCCGATTCCCTCAACCAAGCCCCCACAAGCGGTC
    SEQ ID NO:41 Early LPS-055 GACCGCTTGTAATCCACAGCATTTTCAATAACTTCCTGAGGTGACATCCACCTCCAC
    TCAGAAAACTCGGCTGCATCTGTCCCATCACCAGCTAGATTGATCTCACTCTCGTC
    TCCTCTAAATTTTAGGAGGAACCTGTGATTGGTAGGGGCTTGTCATAAATGATCAAG
    ACGACCCGCATCGTGATGCCAAGCTTAGTCTTTCTACTTACTGTCTATGTAATGGTC
    ACGGGCCCTTCTTATGTTTATGTCTCTTTGAAATGGACGATTTTTTTGTTTTAGGTAT
    ACGGGCCCTTCTTATGTTTATGTCTCTTTGAAATGGACGATTTTTTTGTTAGGTAT
    TCAGTTTCTGAAGCTGTTTTGGTAGTAAACTGGGCTCAATCATTTCTGTTGCTGAA
    CTTTCCATTCGCCTCCCCCACAAGCGTCAGCCGAATTCTGCAGATATCCATCACCT
    GGGGGGGCCGCTCGAACATGCATCTAGAAGGCCAATCCCCTATATGAATTCTATTA
    AATCCCTGGCCTCGTTTTA
    SEQ ID NO:42 Early LPS-056 GGTGCGATCCAGAACTATCATCTCTCACTGCTCGTGAACAAAATGCTGGTCAT
    AGCCATCACTAAGGCTAAGGTACTATCCAGCCAAACTGATCTCAAATAATAATTTCA
    TAAGCTTAAATAAATAGTCCAGCCAGTAGATGGAGCCAAAAAGCCATAGAAGCTC
    AAATACTTGTGGTATCAATCTCTCCTCTGTTAAGGGAGGTATCAGATCAGAAGCACT
    AATCAAATGCATACATAAATGCAGTAGACTGCAATAAAACAAAATCTGCAGATAGCA
    ACAGAGCGCTTAACGAACGGAAAAGAGTTTAACTTGATCTATCACAGGATCGCACC
    SEQ ID NO:43 All LPS-057 GGTGCGATCCACAATAGTTCGTACGAGCGACGTCTATCTGGTTAATCAGAACACAT
    ATCTAATTTGGAAATTTGTGGGCATAAAGCTCCACAGTGTAGGTGGGCTAATCCCA
    TGAAACATTACTCTTCAAACATCATACAACTGAGGTGGAAATGCAAAAGATTATT
    ACTGGATGCTGATCTGGGACTAAGGTGGTGGCCATTGGTAATGTGTGTTTCAGAA
    ATATATCTTCATGATGATCAGTAGTTGCATCTGGTTGGAAGAATGATAAATTCTGGT
    AATTTGTCTTGGGATCGCACC
    SEQ ID NO:44 Late LPS-058 GGTGCGATCCAACTAGAAGAATATAAAGAAAAATTACGGACTACCAGAAAACATCA
    CATCACAGTGTATGCATTCTCAATAATCAGAACTGTACTGGCTAATATCGCTGTGC
    CTGTCGTTTCATTTTCCTGTCATCCGCATAGGGCCCCTCATTTTCCCTATCTTGCAG
    AAATCCAAGAAATGCAAGAAAACCAAAAAGGAAGAAACCCCCAGAGGAAGAGTCCG
    AAGAGGATATGGGTGTCAGTCTTTTTGACTAGATTGGAGGATCGCACC
    SEQ ID NO:45 Early LPS-059 GGTGCGATCCCAGAACATTTCAGACAGATTAAAACAAGATCTAGTCAATCCTACAA
    GGGAAACTTTTGTCAAGATCCGGATCCAGATTTTCCTCAAGTAAAACTAATCTCATT
    AAATCCAAGCCAATCTCTAGCAAAATTCAAACACTTTTTATTAAATCCAAGCCATATA
    TCTGGCAAATTCACCGAAATATGTACAATCGCAGCGCATTGCTTGGCTTGCGACAG
    AAACCATATTCGCACGTCTTCATAAGGCTTTGGATCGCACC
    SEQ ID NO:46 All LPS-060 GGTGCGATCCAACAACACAGCTTCACACTTACTCCATCCTCTGGAACTCTCATCAG
    ATTGTGTTCTTCGTAGACCAAGTTCCTGTGAGAGTCCACAGGCACACTGAGGCTAC
    AAGCGATGTGTTCCCTAAAGAACAGGGGATGTACATGTTTTCCAGCATTTGGAATG
    CAGACGACTGGGCAACCAGGGGTGGGCTTGGGAAGACAAACTGGACTGCCGCTC
    CATTCAGCGGATCGCACC
    SEQ ID NO:47 All LPS-061 GGTGCGATCCCAACACCAAGTGAGAATGAAGCAATATAAATCAGCAGACTCACTAA
    AGCCAAAACAGTGAAAAATGTTTCATATTGGGAATCTGCTCCAGAATGAGCCTTCAA
    GTAAAATGACAAACTAACGAGGAAGAGACATACGGCCATGCCCCCAGATGAGACC
    ATGAGGAGGAGACGTCGTCCGGCTTTATCCATGAGCCATACAGCAACTGCAGTCAT
    GATGACCTGGATCGCACC
    SEQ ID NO:48 Late LPS-062 GGTGCGATCCAGGAAATCATCAAAGGGGAGCACATCCAATGTGCAAAATAAGATCA
    TCATGCAGCAAGATCTCTGAAATATAAGCTCTGTAAGACCAATCTGAAGTGCTGATG
    ATCAATATGAACTGAAACATCATGCCACAATGGGCTGGTACTTGTGCAAAATTCTCT
    GGCATGTGATGAGAATCACATGGTTACCTCTTTGGATCGCACC
    SEQ ID NO:49 Early LPS-063 GGTGCGATCCAAAGAGCCTTCTTGCAGACAATCCGTGAAAACATGGCTATACAATA
    AATTCCCAGTTTGGAATTCTAAATAAAACTGTTCAATATTTGAAGGCCTCTGATATCA
    CAGAGACTGATATTAGAATGGAAGCATGTAGCAACCCTAGAAGCTTTCGCATAAAG
    ATACCAGATTAATTCATAAGAAGGATCTCTCGTTCACCAGTCACATATCACAGTCGG
    ATCGCACC
    SEQ ID NO:50 Late LPS-064 GGTGCGATCCGTTAGATGAGCTGCCAAGTATGGAATTATTGACATTTTTGGACGGG
    TTATGGGCAGAGGGATGTGCCAAGCTGAAGAAGATACCGGGGTTGGAGCAAGCCA
    CAAAACTTCGAGAGTTAGATGTTAGTGGGTGCCCTCAGTTAGATGAGCTGCCAAGT
    ATGGAATTATTGACATCTTTGGACGGCTTGTGGGCAAAGGGATCGCACC
    SEQ ID NO:51 Middle LPS-065 GGTGCGATCCACATAGTTTGAATGCAAGGAAATTGCACATACTTCGTGGGGAATTT
    CGATGGCAAATCAGTCCAGGTAAATGACTTCTCAACATAGGTCCAAAACTCTTTCAT
    AGACCAGATCTTGACCGTGTTGTCCATGCCACAGCTGCAATACGATATACATCTG
    AAGGATGAAAATCTACACTGAGAACTTCATTGCGATGTCCCCCAGCTCCAGCAAAT
    ATCAAAATGCATATTCCAGTTTGAACATTCCAGAGTCGTACAGATTCATCTTTGCTA
    GCAGATAAAATAAGGGAAGGTTTCAGTTGCTTGGGTCCTTATTTCATTCACAGAACT
    CCATGGCCAACGAAACTCTTATGGACTTTTCATTTGCACATCCATTCTCGAATTATA
    CATTGTGACCGCAGCCACTAATAATGGGGAACATCACTCGCCTGCCGACTTATGTG
    TTAAAGAATC
    SEQ ID NO:52 Late LPS-066 GGTGCGATCCCCTCCATTTACCATGGTATACTGTTCCAAAGGTTCCAGAGCCTAGC
    TCTTTCAATTCTTCAAGGTCAGCATTCTTTATATCTGGAAACTTCGCTAGCTGTGT
    CTATAATCACGAAACCCAGACGGGGAACTAATAGGCGATGAAGTTCTCTTATCCA
    TAACCGTTGCAAAGATCTTACACGGAGTTTTCTCTTCTTCTGCGTGGCTTTtCTTTC
    CCGTATTCTCGGATCGCACC
    SEQ ID NO:53 Late LPS-067 GGTGCGATCCATACATGCGAGGGCGCATGAGAGACTACCACAAATCCTACATACCT
    CCATTCACCCCTGGATCGGTTATACAAGGATTTGGGGTGGCTAAAGTGATACTCTC
    AAATCACCCAGACTTCAGAGAGGGTGACTTGTATCTGGTACTATAGGATGGGAAG
    AGTACAGCATAATACCAAAAGGGAGTAACTTAAGAAAGATCAAATATACGGACGTAC
    CACTTTCATATTTTGTGGGTGTTTTAAGAATGCCCGGGTTTACTGCTTATGCTGGAT
    TCTTTGAAGTTTGCTCTCCTAAAAAGGGGGAGCATGTTTTTGTCTCTGCCGCTTCA
    GGAGCTGTTGGCCAGCTTGTTGGGCACTTTGCAAAGTTGATGGGTTGCTATGTTTGT
    TAGGGAGCGCGGGTAACAAACAGAAGGCTGATCTGCTGAAACATAAAATGGGCTTT
    GATGATGATCTCCACCATAACGAGGAGCATGACTTCGATGTGGCTTTAAAAAGGCA
    TTTTCCAGATGGGATTGCACC
    SEQ ID NO:54 Late LPS-069 GGTGCGATCGAACTGAATGAATGAOGTTGCCAAGCTATGTTTGGGAATTAAAACTT
    GAATGCCGTTATTCTCTCCTTTTTCCAAAAGGGCCTTTTCTGCCAGAAAACCTTAAA
    TTTCTGACTGGTTTCCAAGTCCAATTTTTCCAAATATGGATTGGTTTACCATTGAAGG
    CACCACCATGCTCTGAAAGTTATGGACTGCACTTGCCCCAGTGCTATATTTAGTCC
    AGATAGCGCTTGTGTCTCTAAATGCATCTCCCTGCTCGGATATCACC
    SEQ ID NO:55 Late LPS-070 GGTGCGATCCGAACAGAGGGAGCAGATTTTGCCCTGCAAGTATTCACAACATTAG
    AGAAGCCCTGCCAGAGATATGGGAGGAAGAAGATGCAGAGAACACCAAAAATGTT
    GTGGGATCAAGAGGAGCGGATGCAACTATAGAAACTGTTGTCACGGCATAAGCCA
    TCGCCTCATTGAATGAGGGAATGGAGGACTAGACAAATCCCTTTGGATCGCACC
    SEQ ID NO:56 Middle LPS-071 GGTGCGATCCGATTGGGCAGCTGCAGCCTTGGGAAGCTTTAGAATCAAATTGCAC
    TCATCCTCCAGGAGGTATTGAGAAGTCAATTTCTCAAGGTCTACAGTGACAGAAGG
    AACCATCTTGACAATCTTATCAGGTTTCCTGCTCTGGTTAAACACTTCAACTTTGAC
    AGGACGAGAGTATGTGACTAATTCATCTTCTTCATCAGACTCTACATCTCCTGTTT
    CAAGAAACAAAGATACTGATCATCACTAGGGCAAGAATTGATGATTTTGATATCTCT
    GGAGAAGCCAGTGTTTACATTGGTTTGCTTCATGGCCACCAGTCTATGGCATAAAG
    CTTTCCCGAAAGGGTACTTGGCAGATTTAACAGAGCCCAACGTTATATTTAAGGCC
    CATCTCTTTGCTCTCAAAATTTTTCTTGCATCCTCTGGAGAATATAAAACCCCTTGG
    TGTCTCTTTCCACAAACACCTTCTCATTGATC
    SEQ ID NO:57 Late LPS-072 GGTGCGATCCAACTGAGAGGGTGTTGGTGGAAAGATGACACCAAGTGGGTTCT
    ATATTCTCCAGAGGATGCAAGAAAAATTTTGAGAGAAAGAAGATGGGCCCTTAAAT
    TAACGTGGGGTTCTGTTAAATCTGCCAAGTACCCTTCAGGAAAGTTTATGCCATAG
    ACTTGGTGGCCATGAAGCAAACCAATGTAAACACTGGTTCTCCAGAGATATCAAAA
    TCATCAATTCTTGCCCTAGTGATGATCAGGAAGATGTAGAGTCTGATGAAGAAGAT
    GAATTAGTCACATTCTCTCGTCCTGTCAAAGTTGAAGTGCTTAACCAGAGCAGGAA
    ACCTGATAAGATTGTCAAGATGGTtCCTTCTGTCACTGTAGACCTTGAGAAATGAC
    TTCTCAATACCTCCTGGAGGATGAGTGCAATTTGATTCTAAAGCTTCCCAAGGCTG
    CAGCTGCCCAATCGGATCGCACC
    SEQ ID NO:58 Late LPS-073 GGTGCGATCCATGTAGTGCCAACTTACGAGATCACTAACTTTAAAACTATCATGCAA
    TTGGCCAATAGAAGCGACACTTGCTGTGCCAAAGTATCGATAGGCTACTCCCGATG
    GCTCAATCATATATAGTTGGGGCCCATCTCTATCATAACCTCCAAGGATAACTCCAG
    ATCCAAAAGGCCTTAACCACCAATATAGTGTGCACAAATGCACATAACTGGCAACA
    CGTTCACAAAGTTCCTTAAT
    SEQ ID NO:59 All LPS-074 GGTGCGATCCCATGGGATAGTTGCAAGACACACAAATTTGTTGTGAAAGAAGAGAG
    ACACGCACAGACAACCATATGATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
    TTAGCAAAATTCAAACACTTTTTATTAAATCCAAGCCATATATCTGGCAAATTCACCG
    AAATATGTACAATCGCAGCGCATTGCTTGGCTTGCGACAGAAACCATATTCGCACG
    TCTTCATAAGGCTTTGGATCGCACC
    SEQ ID NO:60 Early LPS-075 GGTGCGATCCCACTGTAGTGTCCTTGTTGAGCATAGTTCAAGCTGTTCTGATTCC
    ACCAGTTAGTGGCCCAACACTGCGAGGTGCTGCCATTTCCATTCCATTCACAGACG
    TCAGTGTTGAAATTCATATAGGAAGCCACAAAGGGTGAGGAAGACCAATCTATTTT
    ACTCGCCCCCCTTGAGTTGCCCACTGGTCTCCGCTCCATATGCTAGAGAATACTCT
    CATTGCCTGCTCATTCGGATAGGGAACGCCTATGTTTTCATTGTTTGCAAATACTCT
    GATTGGCAAACCATCAACGAAAATCGCAATTTGCTGGGGGTTCCAGAGAATAGAGT
    AATTGTGGAAATCTGCTGTAGGATCGCACC
    SEQ ID NO:61 Early LPS-076 GGTGCGATCCCACACTCCTAACCCTATTATATGTCTCCCGTCCATGGAGTCATAGA
    AGGAGTACGATAATATGCCCTTCAGCCAAGCGAAGTATGACTTTAGTATGGCCAGG
    CAGCAGTATGAAAGCACATCTTGTTTCTTCCAGGTCGGCATGTATAGTCTCCGGAG
    GCTAACAATGTCACCCAAAGCTAATTGCGCAAACGGAACTCCTCTGCTGATCTCCC
    GGGAACTTAGGCGGAACCACCCTGAATCCACTATTCTCACCGCGCATTTCATCCCT
    TTGGTGAACGCCGCTGCCTCTGGTAGATACAGAGCTGGCTTGTCTCCACTGGAAC
    CCCCTTTCCGGATCGCACC
    SEQ ID NO:62 All LPS-077 GGTGCGATCCAAACTGTGGTTATCGGTGGAGAGATTAAGCAATTTATTGGAGTAGC
    AAGTACGCTGAATTAAGGGGGTCCATCTTCAAGCAAAGGTTCCTTTGGATGACTAT
    GTGTTCTGGAAGTGTTTATGGATCAATCATCTCATAAATTTTGGTAATATATAACAGA
    AGATTATGGCATCCAGTTAGGATGGTAGTTTCATTGAGGTATAGTAAAAACTACACT
    AGTCTTGTGTTGCCACCCACTTTTCAGAGAAGTCAGGAGGTCTCTTTGTGAATCATT
    GATAACTTTATGAGTGGGTACCTAAATGAAATATTTGCATCTTGAGTATATACTCAAT
    TGATCTTACTTGTGGATCGCAC
    SEQ ID NO:63 Middle LPS-078 CTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTTACG
    GCTGCGAGAAGACGACAGAACACCTATCATAACTTGAATTCTGATGCAAATCGGAA
    TTTGCCAAAAACTTGGACGGAAATATAATAGGCAATATCATCCCCGCAAGTAACAAA
    AAAATTGCATGAAAGCTCAAATCCTATGTGCTTTACACCTTGACTGCATACTTTCTC
    ATTGGAAAATACATCTCTTTCTTTTTCTGTCTCTCAGTCTTCAATGACGGCTGATGC
    TGGTAAGGCGTCGCCTGATAGCACGAGTCTTCTTGGGACGCAAATCAAGAGGCAG
    GTACTTCTTTTTTTTGTATGCTTCTCTTAATGCGGATCGCACC
    SEQ ID NO:64 Late LPS-079 GGTGCGATCCAAGATTGTACGGCACAGGCAAATGCTGTTCTTTTTCTTAATCACGA
    TGTGCTTGAAGAATATGAGCGCCGATGTGAACAGATCCACAACCTGGAGTTAAAAT
    TGGAGGAAGACAGAGCAGTGCTGAATAGGAGCTTGGCAGAAATAAATAGTCTTTAAG
    GAATCCTGGCTTCCCACATTGAGGAGTTTGGTTACCAGAATTAATGAAACTTTCAGC
    CACAACTTTCAAGGGATGGCTGTTGCTGGAGAAGTTACACTAGATGAACATGGCAT
    GGATTTTGACAAGTTATGGTATTCTAATAAAAGTCAAGTCAGGCAAACTGGACAGT
    TGCAGGTATTGAATTGCTCATCATCAGTCTGGAGGGATCGCACC
    SEQ ID NO:65 All LPS-080 GGTGCGATCCGAGGGAAGCGATGTAGTCTTGCCCCAAGCGACGACCATGATCCCT
    TATTCTTGGGCAATATGTGCAAGACGTGGACAAATGAAGCGGTTAAAGGGAAGCTT
    ATGGACTATGGAATAGAGGGTCTTGAAGAGCTAACTCTAGTGGGTGATACTCAAAA
    TGAAGGAATAAGCCGTGGTTTTGCATTTATAGCATTTCTACGCACATGGATGCGAT
    GAATGCATACAAACGCCTTCAGAGGCCAGATGTTATTTTTGGTGCTGATCGAACTG
    CGAATGTGGCATTTGCAGAGCCACTGCGTGAGCCTGACGAAGAGATCATGGCCCA
    GGTTAAGTCAGTGTTGTTGATGGGATCGCACC
    SEQ ID NO:66 Late LPS-081 GGTGCGATCCAGTCCTGAAAATGTACTTTACCATTTGTATAATGATGTAAAAATCTT
    GGCCATAGTCTGGTCAAACCAGACTGTATTGTTGCTAAAGTATGGAAATTCTGGC
    CATATTTTTGTCTAACCAGACTGTATTGTTGCCAAAGTTATGGGAATTCCGGCTATA
    TTTTTGTCTTCGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATCATAGGG
    TTGTCTGTGCGTGTCTCTCTTCTTACACAACAAATTTGTGTGTTTTGCAACTATCCC
    ATGGGATCGCACC
    SEQ ID NO:67 Early LPS-083 GGTGCGATCCGCTGGAAGGTGGGCAGCTGGACATCTGGGAATTATAAGTCGAATG
    TCAATTGCTGGGCCATCTGGGGGATGAGCAATAGCATCGGAGGCCAAGTTCTTCT
    GCAGCCGGGCACCAAATGCCATGTGGAGGTCTGAATCTTAGTTTGGAGGTCGAAG
    TTTCAATCCCCTTGTGTTTACTCTGTTTCTGGTTTTATTTGAATAATTTGAGCAATTT
    AATGTGGGTCCTTAGTGCTTCTGTGGATCAGATTCTAGGGAACGCCATCCTGATAA
    GTAAAGATCCGAGTTTTAATGGAGATTCAATCTATCAGAATTCCATGGTGGTTTAA
    ATTCCCTTGTACTGTTGATCTACGTCGCTTTGTATATCAGTGTGTGTTAAGATTTTCT
    CAGAATCCACAGCTTTGTTATGGATCGCACC
    SEQ ID NO:68 Middle LPS-084 GGTGCGATCCAAGCACTTACGACTCCCAACAAGGACGGGAAACTCTAAAATCGG
    AAATATCATATACTGAGGCATCAACTTTGTTGATAAAACTTTAAACAAGAACAATATT
    TGCAGCATATTAGCCCACATGCCATAATGACAAACAAATATGAGAACACTGCCTACA
    GGTTTGCCAAAAGCATGGCCCTCACTTTTGCCCTGAGGTCATCAGGAGCTTCTGAG
    GCTCGAGAAGGAGAAAAAGATTGTGTCACTTCAGGAGCTGAGGCCTCCACATCTTT
    SEQ ID NO:69 Early LPS-086 GGTGCGATCCAAGGTACGAGCGAACAAGTTTCTTCAGCAAGCCACCTGGAACTTTC
    CATGAGTCCAAAACAAGTTGAAGAAGGCTTCTTTGGCTACTTTTAAGATGCTGAAGT
    GATTGTGCTCGCCTCTTGCACAGTTCAACCGCAATAACATTGGGTTTTACAAAACC
    GATTACCTGTTTAACCTGCTGTGCACTCTTTTTCGAAACATGACAAGTTCCAACAAG
    ATAAACTTCGGCCCCATTCTCGCCATTCCGCAAATAAACCACGCTCTCATCTTCTGT
    TATCGAACTCGAGTGCATGCCACGACGCTCAATTGCAGGATTCCAACCCCGGACTT
    GCGAATGGTGCAAAGCGATGCCCGTTTCGTCTCAGCGATACTGCTAAAGATCGGCA
    GACCCGAACCAGTTTGATGCTTCCATTGCCTTAAACATCCAGAGTTTTCCTTCGACC
    TTAAACCCTAACAAGATTACTGATTTCTGGTCCGGATGTTCACTGTCTGTTATACTT
    CTCACAAATCTGTCACACTCCTGATAATCTTCGGTATTGAACTTCATTGAATTGAATT
    TTCCTTCTCATTGGAATTCAATTGTACCTTGTAAATGTCTGGATCCTACACTATACCA
    ATATTTACAGGTCTGAGTATTTTGCCTGTAGTATAATTATCTTTCCTTCGGTCTCGT
    GTTTCCGTATTATTCGTGTAGGATCGCACC
    SEQ ID NO:70 Late LPS-087 GGTGCGATCCCGGGGGGAGGTTGATGTTCTGAGAGAATCAATGAAGGGATTTCAG
    CTGAGCTTGCCTTTTTGAAGACGGAATGCGAACAACCAGTCATTTGCAATAGCGAG
    AATTCTCTTAAGCCACTGCCTGCTGGGGAGGCGAGTTCTGATTCCGGTGATTGCAT
    CACTCAACGGCAGCAGCAGCGGCAGAACCTTTAGTTTCCCATGACAGGTCTCTCTG
    TACAAGTATCTTCCTGTATGATCTAATCCGGGTTGTTCGATTATCGTGATGTCTC
    CTGTATTGACATATTAGCAGAATATTACCATGATACGATGTTAAGTGGCATGGTTTA
    TGCCCTGCATGTTATGTTATGGAGGAGGTGAGGCATGTGGCGCTCATGGGAGGGC
    CCACATGGTCCATGGACGTCTTATTAAACGCATAGTCGTGAATGAAAATAGTTCAAT
    ACATTCAAAATTCCAACACAATTTCATTACAATGGAAGTGACTTCGACTTGAATGTT
    CATTGAAGCATTTGCATGCACAAACAAAGTATACTAGATTAGAAGAAAATTGCAAAA
    AAGGACATTGTGCCCTTCTTAGTGAATATATAAAGATGTTCTTCATGCTGGATCGCA
    CC
    SEQ ID NO:71 Middle LPS-088 GGTGCGATCCCAATAGCCAATATTGCCTCCAAGATAGCCTAGACTGCCTTTGCAT
    AGTTCTAGAAGCCAGTCACCCAACCTCCCAAAAGAAATTGCGCAATCTTTCCCATC
    AGTTTCCCGGGTATGTGTTCTGTCATTCCCCGAATTTTCTTTGGTTTCACTAATAG
    ATTTCTTTCCATGCACATTGCTTGTCTCCAGATCTTTTAGGTGTTCATCCATCTCTTA
    GTAGTACTAGATCGATGGCTTCCAAGAGAACAGGATCATATGACACTGTTGGAAAT
    GTAGCTGGAGCAGCAGTTGAGCAAGTGTCCTCTAGTCTATCTATCTATGAAAGATA
    CACATTGTTTCTAGACATGGATATCAAATTGAAATTGCCAGAAGTCCATGAAACATT
    TGCCGCCTTTTGAAGAAAGGCTCCAAACTGTCAGGGTTCGTGAACATCACATGTT
    CTCGCTGTCTGATCCCCCC
    SEQ ID NO:72 Middle LPS-089 GGTGCGATCCTCAGGGTAATGGCCTGGCTGAATCAAGTAACAAGAATCTTATAACC
    ATTATCTAAGAAGATAGTAGGAGATAACAAGCGGTCTTGGGACAACAAAATCAAGT
    GCGCTTTGTGGGCAGATAGGATAACTAAAAAGAAAGCCACTGGTAAAAGTCCCTTT
    GAACTTGTCTATGGCATGGATTTGACATTACATGCCCATCTTAAATTACTAGCTTAC
    CAACTCCTTCAACATTTTTCTAGTGATAAAGGTGTTGTCCAAAACATGGTTGATCAA
    ATTGTGCAGTTGGATGAAATCCGCAGGAAAGATTTTGATAGTGCAAAAATCAGTCT
    CCATTAAGAAAATCTTTGACAAATCTTCTCGGTCTAGATATTTACAGGTTGGAGATA
    TGGTTTTACTATGGATTCCACC
    SEQ ID NO:73 Late LPS-090 GGTGCGATCCTGCAGGCTTAGATAGTTTCGGCGCTCCTCTGAAAGAAGCACGAGT
    AGGTGTCTCCACATTAGGTGGCCTGATCCCTTGCCTGCACTTGCAGCTTGTCTTA
    CAACATCTCCTATGCTTTGATCCAGGCTTTTCACTGACATAACTTCAGGGGCTTCCT
    TCTCCCAGGGCCGTGCTGCCATCCAGCGTTCTAGCCAGCTCCATCCCCAATTTGG
    CTTGTTTGGGTCAATTTCCATCAGCATAGGATGAGCTGCTCCTCGTGTGCTTTTCAA
    TGACTGATGAGAATATGCGTTATGCCAATGCCCTTTCTCGCTTCATGGCTGCTTCTT
    GCTTGCTTTGCAAACTAGCCTCAATTTCCTCTTTGGATTGCAACTGTCATCCAATCC
    TTTGCTTCCATACTGGATCCAC
    SEQ ID NO:74 Late LPS-091 GGTGCGATCCCAAATGAACATTCAACATTCGATCATGTCAAGCGCTAAATGCCTTG
    GCAGCTTAAAAGCTAGACTCCGCAAGTGACCCTTCTGACTTAGTACACATATTAAG
    CTCATCAAGGGTCCAATTCCATGAAAAGAAATTTTAAAACGGTTACATATTCACAAG
    AACAGCACGAGATTTCCCAGATAGTCAACCACCAACTTGCCCTATCAGCCCAAATA
    TTACTCATTCCATGTTAAAAATAGCAAATTTCCAGATAGAATGTCGAAAGAGATCTT
    CATGCACCATATATGGACTCTTAAAACCAGCCAAAATCTATACTGCCATGCTTGGAT
    CGCACC
    SEQ ID NO:75 Late LPS-092 GGTGCGATCCTGGAGAGAGAAGCAAAAAGCCTACCATCTAAATCTACATCTAAAT
    CAGATATCTTTACTGTGAAAGGAATTGAATGCTGCTTCAGATATCCTACAAGAATA
    AGAAGAAAAGAATGATCAACTCCAAATCAGGCAGATGGCTCAGAATTTCCCGCAGC
    TTCATTTTCGACGGCCTCCACAACACCAACCTCGGCAGGACGTATTACTCTGCCAT
    GAAGTGTATAGCCAGGCTTCAAAACCACAGCCACACTGCCAGGCTGCTTACTAGCA
    TCTTGAACTTGAGATACTGCCATGTTGCATATGAGGATCAAACTCTTCATTTATTGG
    ATCGCACC
    SEQ ID NO:76 Late LPS-093 GGTGCGATCCCCAGAGGTTATTTGGGTTCAAAGTATTCTACACCAGTTGACATGT
    GGTCATTTGCTTGCATAATTTGAACTGGCTACAGGTGATATGTTATTTGATCCTC
    AGAGTGCAGAAGGTTATGACCGCGATGAGGACCACCTTGCCCTGATGATGGAGCT
    TCTTGGAAAAATACCTCGTAAGATCGCCTTAGGTGGGAGCTATTCACGGGAACTTT
    TTGACAGGCATGGGGATTTAAAGCACATTAGACGGCTTCGGTATTGGCCCTTGGAT
    CGCACC
    SEQ ID NO:77 Late LPS-094 GGTGCGATCCTAAACTGTATGTCTCCACAATTGTCTTCAATATAGAAGCAGCTACG
    CCCCTCCTAAGTCATCATAAGTTAAAAACTTCATCTTTCCAATACAATTAAACTATCT
    AGCTTATCAGTTTGGAATAGAGATACAAAATTACAGATAGATTAGCGAAACTGTGCC
    ACAAAACCTCTTCAAAATTAGAAGCATGATTGTCTACAACTCCACTTCAAAAAGGAG
    CTGAACCAGTCCTTCGAAGGGTGTGCTTTGGTTGTGGTGGAGGTACAGAAGGCAG
    CTAATTTCTCCAAGAACTGCTGTTTTTTTAGCCTCTCATTCTCCTCTTTAAGCTGCATC
    ACTTCATTCTCTAGCTCATTTGTGTATGCCTGCTTTCTTGCCCTGGATCGCACC
    SEQ ID NO:78 Middle LPS-095 GGTGCGATCCGAGTGATGGCACAAAGAAAAGCAATGATAGAAAACAAAGAACAGGT
    AGCTCAGAAGGTTCAGCAACTAGAGAGTCAACTTCGAGTTAAGGAGGGCGGGAG
    CAATTGGCAGATTCTTCCAAATTTGTCAAGATCTCTTGGCATGAGATGACCTTATAG
    GATGTTAAGGAGCAAGAGGATTCTAGGAATAATGCCAAGGATAATAAGACTAAAAG
    GATGCTTCAAGACCAGGTGGCAAGGAAGGCTTCTAATTCAAAGGGAGTTAGCAAC
    GGCAACAGATGCAATTCTAGGATCGCACC
    SEQ ID NO:79 Middle LPS-096 GGTGCGATCCTAGAATTGCATCTGTTGCCGTTGCTACTCCCTTTGAATTAGAAGCC
    TTCCTTGCCACCTGGTCTTGAAGCATCCTTTTAGTCTTATTATCCTTGGCATTATTC
    CTAGAATCCTCTTGCTCCTTAACATCCTATAAGGTCATCTCATGCCAAGAGATCTTG
    ACAAATTTGGAAGAATCTGCCAATTGCTCCCGCCCTCCTTAACTCGAAGTTGACTCT
    CTAAGTTGCTGAACCTTCTGAGCTACCTGTTCTTTGTTTTCTATCATTGCTTTTCTTT
    GTGCCATCACTCGGATCGCACC
    SEQ ID NO:80 Middle LPZ-001 ATCTAGATCATCGATCTTGTCCAAATTTTAACTAGTGAATAGTTTAAAAAAAAGCAA
    CTAGCAGAAGAGAACCTAACCACTGACAAATTGCAAATACTCTAGAACACTATTCAT
    CATTTTTTGCGATTCACGCTGGACCCACAAGAACCCCTTGAGCTGAACTTTCTTTTC
    GTTCTCCCTCCTTTTGGATCGCACCATCTAGACCATCGATCTTGTCCAAATTTTAAC
    TAGTGAATAGTTTTAAAAAAAAGCAACTAGCAGAAGAGAACTAACCACTGACAAATT
    GCAAATACTCTAGAACACTATTCATCATTTTTTGCGATTCACGCTGGACCACAAGAA
    CTCTTGAGCTGAATTTCTTTTCGTCTCCTCCTTTTGGATTGGACATCNAATCCTGCA
    GCCGGGGATTCATATTCTTAACGGCGCNCGCGNGGACTCCATNCCCCATATGATC
    TTTTCATCCTGGCGCNTTTAACTCTGAAGGGAAACCGGNTTNCCCTTATCCCTGGA
    NATCCCTTCC
    SEQ ID NO:81 Middle LPZ-002 GTGGAGTGTAAAGGTCAACGTGCCATCCGGGTACAAACTATTGTAGAAAAAATGGC
    AAAGTTAGGTCTGAAAATATCCATTTGGCCTGCTCTAGTTGTACAGTACATGATTTT
    GCACTCGCACAACAATGGACTATAATTATTTTCCTGGCAAAAAAAAAAAA
    SEQ ID NO:82 Late LPZ-003 GGTGCGATCCAGGACATGAGGCCGAGTTTGCCATTGTGATATGATTGAGGAAGTC
    CAGTCCTAAAATAGGTTTATCTTGATGTTTGACAAGAGATATAGAGGGGCATGATG
    ATTCATTGATCTGTTTGCAGATCTGTAACTGCAACCATTCTAATGACATAATAGCGC
    TATTGTTGGGTTCGTGTGATGACATAATAAATTGATTTAATTTAATAACATCTGTTA
    ATGCAATGGCTGTAGCTGCATCATCACCGTATCCATCGAATGTTCCATTTTTCCAAA
    TGTTTGTTTCCAAAACCAGAACACCAAAATGTCCCCTGCGTTTGTNTTGAAAAATAT
    TGGGCCCNTACTATACTATAATNTTTNGGCATACTATACTATAATGTTTCTCCCATTC
    CCCCCAAATGANTCCTATACAATCCTGGCCGNCTTTACACTCCTGACNGGAAACCC
    GGCTTNCCACTAATCCCTGGNCNANCCCTTC
    SEQ ID NO:83 Late LPZ-004 GGTGCGATCCGACTGTGATATGTGACTGGTGAACGAGAGATCCTTCTTATGAATTA
    ATCTGGTATCTTTATGCGAAAGCTTTTAGGGTTGCTACATGCTCTCCTCTTTTGTAT
    GAATTTCCATTCTAATATCAGTCTCTGTGAT
    SEQ ID NO:84 M,L LPZ-005 GGGGAGTGTCAAGGGATAAGTGGTAAGCCAGGTTTCCAGTCAGAAGTGTAAAGGC
    GGCCAGTGATGTAATAGATTCATATAGGGGAATGGAGTCACCGGGGTGCGCCGTT
    TTAGAATAGTGGATCCCCGGCTGCAGGATTTGATGGTGCGATCCTGCCCCTGATAA
    TTTGGTTGCAATGGAAAATGCAGTATTAGGTGCGAGATGTAAAGCCCGCCCGGAG
    CGGTGCATGAAGTACTGCAATATTTGTTGTAGTAAATGTGCTGGTTGTGTTCCCAG
    CGGTCACTATGGCAACAAGGACGAGTGCCCCTGCTACAGAGATATGAAGTCCGCA
    GCCGGCAAGCCCAAGTGTCCCTGATCTAGCACTCAGTCCAGTCGCTCACTTCTT
    TTATTCTTTTTTTTTATAAAAGTGACGAGGCCGTTTTTCTTGTACTTGGTGGCCATAT
    GTAGAGCGGTGGCTACTTCTCCTGTGTTAGGAAATGTTGCAGTACTAATAATAAGA
    ACTTCTTTGGCAAAAAAAAAAAA
    SEQ ID NO:85 M,L LPZ-006 GGGTTTCCTTAAGAGTTAAAGGCGCATGATGTATAGAATCATATAGGGGATGGATT
    CCCCCCGGGGGGCCTTTCAGAATAGGGATTCCCGGCTGCAGGATTGATAGTGCGA
    TCCAAGACACAGTGGAGTACCACAATGGGGATCTGGCCAGTGCTTTGTGGCTATTC
    ACTGCAGCTGTATTAAAACAGGAAGCCGCAAATGGCCAGAAGGCCATTGAACTTGC
    TGAGAGCAGACTATCTAAGGATGGCTGGCCTGAATATTATGATGGGAAGCTTGGAC
    GATATATGGAAAGCAGTCTCGAAAGTGGCAAACCTGGTCAGTTGCTGGATATCTT
    GTAGCCAAGATGATGCTTGAAGATCCATCCCATTTAGGTATGATAGCATTGGAAGA
    GGACAAAAAGATGAAGCCGTCCCTCACTCGATCAGCTTCTTGGATAATGTAAAATG
    GGGAAATCCTAAACTTTCAGGCCACTCTTGAATGTTTTGTCACTTCTGTATGACAAA
    TGAGGCAATTCATAGTACATGTTGTGCAAAAAAAAAAAA
    SEQ ID NO:86 M,L LPZ-007 GGTGCGATCCCAGAGAATATTAGTTCATGTGTTGCTCTCATTTTCTTCAATATGCAG
    GGCAACCATTTGAATGAAATTATTCCTTTCGAATTTCAAAAACTTAATAGGCTAACTT
    ATCTATCTGGAGCCGATTTTCATTGACGAGTAACCTGTAAGCTGGCCAGCAAAAGC
    CAACAGATGTTCAGCTCGTTGGAACCAGTTGAAGATGTAATAGAGATGGTGAATA
    ATCGCGGACGGCTCGGCCAATGGAATATTTGTTGCATCATCATCAAGGGGGTATGA
    ATTCCAAAGAACTTGTTGATTGAAATTCCCAAGCAAAATTCTGTGAAATGAAAAATTT
    ATTGAGACCATTGGGCAAAAAAAAAAAAA
    SEQ ID NO:87 Late LPZ-008 GGTGCGATCCAAAGAACACAAGATGGAGTTACCACAATGGAGGATCTTGGCCAGT
    GCTTTTGTGGCTATTCACTGCAGCCTGTATTAAAACAGGAAGGCCGCAAATGGCCA
    GAAGGGCCATTGAACTTGCTGAGAGCAGACTATCTAAGGATGGCTGGCCTGAATAT
    TATGATGGGAAGCTTGGACGATATATTGGAAAGCAGTCTCGAAAGTGGCAAACCTG
    GTCAGTTGCTGGAT
    SEQ ID NO:88 Late LPZ-009 GGTGCGATCTGTGTGGCTCTGAAACATCCCGGCTCCCCTCTGCACTATAATAATCC
    CAAAATTAAGTGAACCCAACAGAATTTGCTCATATCTCTACAGTTATTGCAGACTGA
    GCAAAACCCTCAAACTCATGTGACCTCTCAATAGGAGCCCACGCCCAAGATTTGTC
    CAGCATGTAACACACCTGATCGCCGCCACTGCAAGCACAACCGCTCACAAATATCT
    TGTCACACCACACTGTTGCGCAAGTTAACAATATTCATGTCTCCAGGAAAGAAATGC
    CACACTTCCCAACATTCTCTTTACTATTATAGAACTTCCTTGTTGCTATGGAAAAAAT
    ACATTCCCAACGCAGAACCCCAACGGGGGTTCCCAATANCCCATTTCCCCCCTNTC
    CAANCCNNTNTGAATGCNCCCCATNCCCTATTGNATNNTTTAAATCCNGGCGCNTT
    ANCTGGAAGGNAACCCGNTTCCCN
    SEQ ID NO:89 M,L LPZ-010 GTTTCCCAGTCAGGACGTGTAAAACGACGGCCAGGGATTGTAATACGATTCACTA
    TAGGCGAATTGGAGGTCGATCCGTATAGGTAGTTGGATGATGAACGGGCAAAGAA
    GGCAAAGGAGTACAGTGATGGATCCTGTAATTCCTGTTTCAGAAAACAGAAAATCT
    GCAATATAAGGATGGCTAAGCTTTTCAGCTATGAAAATATATGGTGCAGTGGCACT
    CATATCAGTTGCAGAGTTGTCAATATAACTTTTGTGAATAGGAAAGTTGTCCTCTTT
    TAGAGTGCAGAAATCCTGCAATATAAGGATGGCTAAGTTTTTCAGCTATATGAAAAT
    ATATGGTGCAGTGGCAAAAAAAAAAAA
    SEQ ID NO:90 All LPZ-011 GGTGCGATCCTACAGAGAGCAGCTTGACGAGGGCCAAAAGGTTAAGGATGAAGAA
    TGACCTCAGCTAGTAAGGTTTACAGAAGCAGCAGAGGCATCTTAACTGTTTTTATGT
    TTTGGCAAAAGTTGTTGCGTCGGTTGTTTAATCCAGGATTTCAGATGTATTTTGTAG
    A
    SEQ ID NO:91 Late LPZ-012 ATTGTAATACGACTCACTATAGGGCGAATTGGAGGGTCCGATCCTGCGAGACCGA
    GGGTTCATTTTCCTTTAGACAACGACGTTCAGTGGCGACCAGAGTTTCCCAATCAC
    TTCAGCGATTCTATTCCTTCGTTGTAATAAAGCTTAAGGAATCCATGCAAATTCCT
    GGAAGGTTTGAATATTTATATTTATTGGCAAAAAAAAAAAA
    SEQ ID NO:92 Late LPZ-013 AGGTGACCGTCAAAATGATTGCAGAGGACTTAGAGAGGGAAAACCGTTCCGATCT
    GGTGAAGCAATTGGATGAAGCAGCTCTGGAATTGATTCCCGTTCTGATGATATCG
    TACGGCTAAGCTCAGCTCTTCAGGCAATTGGCAGAGAATACGATTCTTCAAATGAG
    ATGACAGATTTTAAGAAACTTATAGATGAACATATTTCCAAGCTTGAAGCGGATTCC
    CCTACGGTCACCT
    SEQ ID NO:93 Late LPZ-015 AGGTGACCGTAAAATACTATGAGAAATGCTTTCATCAGGCACCGCTGGTAGGTTTT
    CTTCAAGCTTTTCATTAGGCAAAAGAGGCTCCGTGAGTTGATCGTAATCTCTCCT
    TGAATGGCCATATTGACCAGACACTCTGATTAGAAACTGGAATACAACTGCACATAT
    AGTCATTCTTATATGATCATCCTTCTGCACTTCAGCATCCTGCGGCAACTCTCAT
    CCCGCCATACTGCAGAAAAATTATTTGACTCTTGATCATGTTGTAGATGAATCTTCA
    TGAATCTTCTCATCTTGCATTCTTGTCTTTATATCTTTAGGAAATTGCATCTGGTAAA
    AGTATAAATGCATCTTCACTGGTTGCTTCAGTTTTGCATGCTCCTGTCTTCTTGTT
    TACATGTGATCTACCAAATCATCTAATGTATTCTCTCAATGTCTTGTGGACATCTCC
    TTCATTCCGAGATTACCAATCATCTACCCGAATAAATGTGCCCCGTCAGCAATGCC
    GTTTTGGTCC
    SEQ ID NO:94 Late LPZ-016 AGGTGACCGTAGTAGGCGTCCAGAGGCTGACAAAATCCCAGGCCTGTGCAAATCT
    GGAAGCCGCATGCAGGGCCGTGGCACCTACACTTGCGGCCTTAACAAAGTGGCC
    CGCGGCACCCACTTCTACCAGTGTGTTTATATTCTTGTGCAGCCAACACCAGAGGT
    TATGCAGGCGAATGTGCTGGCCAAGCGTTGTTTCGGCTGTCCGCAAACCCTCTC
    GAGTCTTACATGCCGCATATGAGTCTTGTGTATGGCGATTTGCCTGACGACGAGAA
    AGAGAAGGCCAAGGTTAAGGCGCAGCTAAATTCGATGAACTTATCCGCAACACGG
    ATTCCAAGTCTCCAGCTTGTGCTTGTACTCGACAGATCTGAAAATAATCCTCACTCA
    TGCATAAGTGCAAAATGTGATCTTAACCTGCTCTGAAAATTACATAA
    SEQ ID NO:95 Late LPZ-017 AGGTGACCGTCCACGAGAATTTGGCTTCAAAACCCTAGGAGAGGGATATGAACTTG
    CCAAGGCACAACTGACGCATGAACAAGACGTAAAATGACTCATTAGACACTGACAT
    GATAATGAAAAACCTATGAATGATGATAGACTCAGCTACTTGATGACATCGCCCGC
    CATTTGGACATCTTTATAAGGAGTTTAAGCAAACCCTAGACCTACTGCCTAGTGACC
    AACTTTTGCTTGACGACTCACTGAAATGACAATATTTGACCTTGACACTTCAAAATC
    ACTTTGTAGGAACTCATTTGATCACTGGAGGACGGCTGGAAAGACTGACACTAACA
    GGACTTTATATATGCACCTCGTCTATCCGAACTT
    SEQ ID NO:96 Late LPZ-018 AGGTGACCGTAAGCACAAGTCGTCAAAATTATCTCTATTCCGGCAGTAAAAACCTAT
    AGCTAATGATGGATCAATAGCACTAAGTGGCAGCTGGCGTACATCACTGCAATGAT
    AAGAACCAGTATCAACCCCCATATTATCAGGAGATATCTCCACCACCTGCTGCACT
    ACATGTGGATCTAAGTACAGAGCCTGATCATCCTGAACACCAACAATATACGTTG
    GCTCCAGGCTTTCCACCAGCAATACCAAGACTTTGGGGAAATGTGAACGTTTCACG
    AAGTGATGGTACATACCTGGGTTGATCTCTCTACACCAAGAACAAGCGGCACCA
    AAATCAGGATAGGCACTTGGTCTTCCCCTTCTCCATTGGACCACTCTGAACACAGC
    CTCGCAGCATCATCAATGCAGATAACTGGAGTCCCTCCACGGTCACCT
    SEQ ID NO:97 Middle LPZ-019 AGGTGACCGTGAATATGGTGGGTATTTGCAGGGCAAGATTCAGGATGCTGCTCCC
    GGAGCTTAAGTAAGGTCTTGGACCCTAATAAATTCAGGGTATATGCATTATGTATAT
    GCTCTCATTTAGCTGCTCATCTGATTTCCATTGGGTGAATCAGTTGTTTTGCAGTAC
    GTGGGGGTCTGTTATTTTGTGAGTTATGGTGGAGTCATTTTGTTGTTGTTGTT
    TTTCTTATCTAGGGTTTAGGGTTTTGCCCTGTAATCGGTCTTCCCCTCTCTCCTGCG
    CTTGAATTTGACCTGAAACCTCTTGAAGTAGGCCCTGGTTTTCTGGGCTTTGACGA
    AAACCATGGTTGTGGATCTCCTCTCTCCTGCTACGGTCACCT
    SEQ ID NO:98 Late LPZ-020 AGGTGACCGTCCTACTTCACCGCAGTGACTTCCATCTGGTTTTAGGAAACTATCCC
    TAAATCCTTCACTAGTTGACGAATTGATTGACTCAAATCAACTGTCGGTCAAACCCA
    CTCTCTCTGAAAGTGAATTCTATGAGTCTATACCCAACCCAAATCAATAGGTTGAGG
    TAACAGTTGACCCGATTTCACCTTCAACAAATCATACCTTTCCCGAAGAGAGTGAAC
    ATGATTCAACACAAGTTCTTTTTGGTTCACCAGATTCAAATGAGCTTGGGGGTAATC
    CTCCTGTTCCATCAAGACAAGAAGAAAATCCTCCCACTCTCGTAACTCAAGGGTTAA
    TCCTCCCATTTCTACGGTCACCT
    SEQ ID NO:99 Late LPZ-022 AGGTGACCGTCNCGGGATAGNTGGAGCCNAACAAAGTACNGAANAAANTGAANCG
    CNCTGGGAAGCGNGCNGAAANNTGGNCANACNTGCCCTNCNACTCGGTTACCCAG
    CCNTTCTCTACCNANAATTATNACNNNANAGCNCCATGCTGGGTTTGTNANAAAAN
    AACNGCTNTTGATAAAATTACATAGANTNNNGAACACGTTAAGAGGAATATGGTCC
    ANATNCATTNTNAATNANNANTTAAAAACTNNNTATGTNCTAGNGTCNCCT
    SEQ ID NO:100 Late LPZ-023 AGGTGACCGTACAGCACAGGTATACAAATCATAGAAATGGGCTTCTGTCCAACTGT
    CAGCAGAAGCGATATGAAACCCAGAAGCATCAACTCTGCTTTCAATTTTTCAAGCG
    CTTCATATAGAGCCTTTTTATTTCTCTGGAGAGCCAATTGCTAGCATAATGAATAC
    CATGTTCAAGAAGTAAAGAGATGACCACAAATGCCAAACAAACAACTGCTACTGCC
    CAAGTTAGGAGTTTGCTCTAGAGAACGGTCATTGCCACGGTCACCT
    SEQ ID NO:101 M,L LPZ-024 AGGTGACCGTGGATATGGGAGCAGAGCCGTCCGCAGTGGATGCTGCAATTCAACT
    TGAAGTGGCAGAAGCTGTGAAGACTCTCCAAATGGACAAGGCACGAAGACAAAAC
    CAAGACAAGGATGAGGGCAAGAGTGGCAACGCTGATTCAGATGACTTGAATGAAAT
    GGAAGTCAAAGCTAAAGCAGCCGAACAACTGCTTGCTGTGCATGGGGCAGCATTA
    CTACAGAATGCTCTGAAAGAAAATTGTCGAGTCATGAAATGCGGGTTGGTTCAAA
    TACAAGGGAGGAAGGTGAAGTTAGAAAGAACAGAAAGGGCATCAACGCAGACCCC
    TCACTGATATCGGCAACACTACGGTCACCTAAGCCAATTCTGCAAATTTCCATCACT
    GGCGGGGCCCGCTCCAACTTCCTCTAAAAGGCCAATTCCCCTATATGATTCTTATT
    ACAATCCCTGGCCCTCCTTTTCCACTTCT
    SEQ ID NO:102 M,L LPZ-025 AGGTGACCGTAGCAGGAGAGAGGAGATCCACAACCATGGTTTTCGTCAAAGCCCA
    GAAAACCAGGGCCTACTTCAAGAGGTTTCAGGTCAAATTCAAGCGCAGGAGAGAG
    GGGAAGACCGATTACAGGGCAAGGATCCGCCTGATTAACCAAGATAAGAACAAGT
    CAACACACCCTTGCCAAAAAAAAAAAAAAAA
    SEQ ID NO:103 Middle LPZ-026 AGGTGACCGTATGAGCAAGGAGGGAACAGTATGACAGGCAGTCAAAGCCCACGAG
    GGGTGCCCCACTGCCTGCAGCAGCGCACTTACTTGGACTAACAAACTTGTATCGTG
    ATTAAAACGATGAACATCGTATTGTGGAGTGGAGCCACTCGTGACCTGATTCTGTC
    CTAAGTACTTGGTCCTGGAATACAATATTGCACGGTCACCT
    SEQ ID NO:104 All LPZ-028 AGGTGACCGTCAAAGTACAATGGAGTCATATATCCACTTGAATTGAAACCTCTAATT
    TAAAAGTTCTCAAAAAATATTTTATTTACAAAACAGGGAAAATAAAAAATGACTCTAT
    CAACTATACAATCCTAACATCCATCTCCCGACAGACCTCCAGTATATGTACAAGGC
    GCTGAAAGAAGGCTGATTATTTTCTATTCCAGCTCGCATAACGTGGTCTTCTGAG
    GCTTTGCCTATTCCTTTCTTTAAAATCTTTCGCACGAAAGATTGGCATTGACCTTCG
    GCTAAATCTCAGACTCCAGGGAACCTTGGACTCCCTTTAAAACCTAGAGCTACTTTT
    TACGAACCCCTGCTTCTCTTGAACACTTAGGGAACTTATACTTACAAAACTTCGGGA
    ACTCCACCCCCTAGCTTTGCAGGACTCCAGCAGATTCCCCAAACTGCCAGAAGGC
    TATTTCCATGCACTGTTAGGGGTGAATTCCTACTATCAAAACCCCCAAAACATCATA
    SEQ ID NO:105 Late LPZ-029 AGGTGACCGTATGGGAACAAGTATGGGAACAAGAACGTATTACATAAAAGATGGA
    GATGCAACACAGCATAAATTGATGCTAAGTTGTTACAATGATGCATACAGCTTAAC
    CAAGCTTGGAATGACATCATTAAGTGCGGTCACAGCCTCTGCATAGTATTTCTCT
    GCCTTGGGTGTATCCTTGCTCCTTGCAGCGTAGTCCAGGTTGTCAAGGGTTGTCAA
    AAAGCTTGGTGGTGAAGGTTTTGAGGGGCTTCTTCTGGTCCTTGGGCTTTGAGGA
    GATAACGGTGTTTGAAGTCCTTAGCGAAAGTAAGAAACCTTTGGAACCGAAGTCCG
    TTCTTGACGTTACCGCACGCCTTCCTTATCTATCACTTTTTCACCTCCAGAAATTGC
    TTCCCGAATCCCTTGCTCTCCCACCCCCTGTCCCCC
    SEQ ID NO:106 Late LPZ-030 AGGTGACCGTAGTGTTGCCGATATCAGTGAGGGGTCTGCGTTGATGCCCTTTCTG
    TTCTTCTACTTCACCCTCCTCTCTTGTATTTGAACCAACCCGCATTTCATGACTCGA
    CAAATTTTCTTTCAGAGCATTCTGTAGTAATGCTGCCCCATGCACAGCAAGCAGTTG
    TTCGGCTGCTTTAGCTTTGACTTCCATTTCATTCAAGTCATCTGAATCAGCGTTGCC
    ACTCTTGCCCTCATCCTTGTCTTGGTTTTGTCTTCCGTGCCTTGTCCATTTGGAGAG
    TCTTCACAGCTTCTGCCACTTCAATTGAATTGCAGCATCCACTTGCGGAACGGTCT
    GCTCCCCATATCACGGCACCTT
    SEQ ID NO:107 Late LPZ-031 AGGTGACCGTAGTGTTGCCGATATCAGTGAGGGGTCTGCGTTGATGCCCTTTCTG
    TTCTTCTACTTCACCCTCCTCTCTTGTATTTGAACCAACCCGCATTTCATGACTCGA
    CAAATTTTCTTTCAGAGCATTCTGTAGTAATGCTGCCCCATGCACAGCAAGCAGTTG
    TTCGGCTGCTTTAGCTTTGACTTCCATTTCATTCTAAGTCATCTGAATCAGTGTTGCC
    ACTCTTGCCCTCATCCTTGTCTTGGTTTTGTCTTCGTGCCTTGTCCATTTGGAGAGT
    CTTCACAGCTTCTGCCACTTCAATTTGAATTGCAGCATCCACTGCGGACGGCTCTG
    CTCCCATATCCACGGTCACCT
    SEQ ID NO:108 Late LPZ-032 AGGTGACCGTCGTGAAATAGCGAGAACGGCGTGGAACATCGCAACGGCGGGGAG
    GCTGGCGGACGTTGCACGTTTCTGGAAGGTATGCGGCTCTCTCCTCCGCCTCAGT
    TTCCATGAAGAGGTCCTCCCTGGTGAATCATACGATTGCGATTGATCGAGTACTT
    GCTGTATGGCTCGGCATCGGCATTGTGGAGACATTCTTTCCTATTCCTCGCAGCAT
    CTCTCCGATGGTTGCTCTCTCCGGAGCTCCATGTTATCCCCGGCACTGAGACAGTC
    GCTGCCGAATCGCAAGAGCTTCTTTGTTTTTTGCAGGCTTCTCCAAACATAATGCCT
    CCGGGCCCCTCAACCGAATTCTGCCAAATCCACCCC
    SEQ ID NO:109 E,L LPZ-033 AGGTGACCGTGGACGACAGTGAGTGCAGTCATCATGCTCTCCAGTGGACTTTAAG
    CAATCTGCATCTTTATGGAAGTGATGTATCTCTTGTGGTTTTTCATGCTCAACCATT
    GGCAGTCTTCAACAGTGCTGCAACAATGGGCATAACGTCTCCCGAATTAATTGAAA
    CTATTGTGAATCAACAGATAGGTTTCTGGTCACATCTAGCAATACAAACACAAATAA
    CTGTGGAACAGAGCCACAAAACTATGCTTCAGAGCATCTAATTACACATATCTCTC
    TAAAACCCTTGCATAAAAAATAAACTGAATCTCGACCTTAGCACTATTGCCACCATC
    ATCTCAAGCAAACATTCTCTAGAATACCATCTTCACAATGCACTAAAGTTACATAAG
    CACTGAACTTAAAACATTTCTGTGACGAATGAAGGACCAATTCATCATACTCAGCCT
    TTGCATCCAATCTGTTGAATGTGCTGAAAAATGCCCAATAAACCTCCATCCAACACT
    GTCTTCCTCTCTGAGGTGCACACTGATTTCTGCTGCTGAACCAGTCGGGATTCCCT
    GCTCAACGTCCC
    SEQ ID NO:110 Middle LPZ-034 AGGTGCCCGTGGAACTACTGTTAAATCTGGAATCCCTTGTCTAGCTGTTAAAAACTC
    GACAAGTGCATGTTGGTATAGTAGGGTAACAGAAGGGTTCTTACCCAGATTTAC
    CCCTTTGGCGGAGATATTTAAAAAAAAAGAATTGTCATTATGGTAAATAGGTGTGAC
    AGGTTATCAATAGAATAACTGACGAGAGTAAACTGATAATTATTAAGGTTAAAGTGT
    TCGTAAAGGAGACTTGGACTCTAGGTTGGATGCCTACACTTAGAGCCGTCCCGCA
    CTTGGACGGTCACCT
    SEQ ID NO:111 Middle LPZ-035 AGGTGACCGTCCAGTGCGGGAACGGCTCTAAGTGTAGGCATCCACCTAGAGTCCA
    AGTCTCCTTTACGAACACTTTAACCTTAATAATTATCAGTTTACTCTCGTCAGTTATT
    CTATTGATAACCTGTCACACCTATTTACCATAATGACAATTCTTTTTTTTTAAATATCT
    CCGCCAAAGGGGTAAATCTGGGTAAGAACCCTTCTGTTAACCCTACTAATACCAAC
    ATGCACTTGTCGAGTTTTTACAGCTAGACAAGGGATTCCAGATTTAACAGTAGTTCC
    ACGGTCACCT
    SEQ ID NO:112 Late LPZ-037 AGGTGACCGTATGGGAACAAGAACGTTATTACATAAAAGATGGAGATGCAACACAG
    CATAAATTGATGCTAAGTTTGTTACAATGATGCATACAGCTTAACCAAGCTTGGAAA
    TGACATCATTAAGTGCGGTCACAGCCTCTGCATAGTATTTCTCTGCCTTGGGTGTA
    TCCTTGCTCCTTGCAGCGTAGTCCAAGTTGTCAAGGGTGTCAAAAAACTTGGTGGT
    GAAGGTTTTGAAGGGCTTCTTCTGGTCCTTGGGCTTTGAAGAAATAACGGTGTTGA
    AGTCCTTACCAAAGGTTAATAAACCTTTGGAGCCGAAGTCGTTCTGGACGTACGGC
    CACCCCTTCCTTATCTATCAGCTTTTTCACCTCCAAGAATTTGCTTCCCCGAATTCC
    TTGCTCTCCCAGCCGCCTGGTCCCCCGAAAAGGGCTGAATATAAAACCGTCCTCA
    ACGGCATTCCATTCCTCCCTCGTCTGAAACACTTCCCCGCTGCCCCCGAGGTGAA
    GGGCCATCAACTTGATGAACGGCTTTTGCAAGGCTCTGACCCCGGCCCCGTCACT
    AACCAATTCTGCAATC
    SEQ ID NO:113 Middle LPZ-038 AGGTGACCGTGGGGAACAACTACATGACAAATCATTTCTTTGTGGTGGATGTACTG
    GACACCAAATAAGTGTTGAGAGTCCACTGGCTCTGTACGCGTGGCAGAATCACAAC
    GGACTTGAGAAAGTTGAAGATGGAATTTGTATCGCTAGATGGCCAGACCATGTTGC
    TTCAAGGGATGCACTCGTAACCCCCACAGTCTGTCTCTACCCACTAGATGGAGGCT
    GACATGAGACATGGAGACATTAATTGGGTTGTGGAGTTAAAGATCTCTCACGTTCG
    GGGAAAATCCAAGCCATCATACTTATATATCCGTCCCGTGCATGTAACCTCCTCCA
    CTCTGTCCCTTAGGCCCGTTGTTGCCT
    SEQ ID NO:114 E,L LPZ-039 AGGTGACCGTATGAGCAAGGAAANNACCGCACTGGCTCCCAGCAGCATGAACANC
    CAGGTCCCAACCATANACCNCNTGGAGAANGTGATCAAGATATTAGCGACAGTGTN
    ATTGTACNTCTCNCCAAACACATTATACACGATAAGAGAGCNTAAACTACTCTATTC
    CTTTGACGNAGTGACTACNTGAGTANAAGCGATCATTATCTTGCNAACTTTGCATG
    AAAACAACAAACCCACNTCCAGTTTCTCTATANTCTGGCCCCACNATGAATAANANT
    CCTGCCATAATAATGANTCTTTGTCCCCANAGANAAATTNNATAAGACAGGAGCCC
    ACTGTTGCTTGCATGACTACCANTCACTTTAAGGCGTTGCGAATCCCGGTCCTAAC
    CATCTCCATACCATNGGCANNCTTTACTTTCCAACTGCCCAAGACTGTGAACAGGG
    CGGTTCNNACCCTATAANTTTTAGCCTCTNNTCGAANCNCTTNTTTTCGTTCCCCGG
    AAANCCGNTTCCCACCCTTTGGAACCTTTTTTTTTTGCCGGGCCCCAGGCNAATTC
    TNCAATTCCCCNCTGGGGGG
    SEQ ID NO:115 Late LPZ-040 AGGTGACCGTGGCGGAGGTTAGGGAAGTTTGACTTCTCATTTTCTCACGCACTCCT
    CTCCCTCGTAACCTCGGTCGAGTCGATGGCGGCTTTTTAGTCGAGTGTGCTAACG
    CACCCTCCGGGCCTCAAAATTTCCAGCTACTCGTATTTGATCAATGCTGAAATCGC
    GTAATCACGTAGATAATAAAGCGTAATGAATTCTATAATGAAGCATGTTTCTCTATA
    GTTCATGTTGCCGAGAAGGAATAATGAAAATGAAGCCTTATATATTATCTGGGGCTC
    AAGGAGATGTTATCTTTTCTCTTCCTTGGTTAGAGACCGTCACCTTCACTTTGAATT
    GGATAAAGCTTCATTTGTTTAAGACCTCCCACCCGTAAATACATACGGTAGCCTTCT
    TATGTTAGAAACATACGTCACCTACGCAGAATTGTTAGAATGAAATGA
    SEQ ID NO:116 Late LPZ-041 AGGTGACCGTGGAACAAGATGATTAGTTCTCATGCGGGCCAGGATGATTAGTTCTC
    CTATGGCAACTGTTGGACAGGATGATTCGTTCTCCTGTGGACAGGATGATTAGTTC
    TCCTATCGAGGCATCCTACCCAAGCAGTTTGGGACTCATGGGAAGTACCTCTCATC
    TGATCAATGAGTAGGAAATGGGGTTAGGGACCATTAAGTAGTATTATCGATGGATG
    CATTGTTGTATCTATTGTACTCCCTATGCTAGAATGAACTCCATTGATCTGGGATCA
    ATGAATACTGTTTCTGGGAATCATTGAAAATTTGTATGAACACACTCTGAACACTGA
    ATTTCCGGTTCATTGGAAGAGATGGTTTTAAACACTCTCCTCATCTCATTTCTTCCC
    CTTCCTTATTCCAACCAAATTTGGGCCACCCTGCCAGGAAATTCATTTGATGGTTGG
    AAAATACCACGGGCCCTAACCAATTCTGCAA
    SEQ ID NO:117 Late LPZ-042 AGGTGACCGTNCATCTCTACCATNATNCCTCCCTCCCGNCTGTATCANCNGGCNTN
    NANGTCNTTNNCTANNNNAAGNTAATCCTATCCCNTTANAGTGACGGTCTCTAN
    NCCTAGAAGAGAANCCATAACATCTCCTTGAGCNACACATGGGATATACCGCCANC
    TTATNTAATACTTTCNCNGCACGGTAACNGACCANAANCATTCTTCACTATAGAATT
    CATGTCGCTTCATTATCTACCTCATTNCNCCANATCCCCCTTNATCTCATNNATTTA
    CTAGAAANTTCTGAAGNTCCNNAAGGGTTCGTTTTGCACCNCCCCAANTAAAAAAN
    CCCTNCCGNTTACNTCGAACGAAGGTTTCAAANGAACAGNAATTCCTTTACAAAAA
    TCAANAATTTTAACTTCCCNAATCCGGCCCCCCNGTNCCCGAAACCCNATTTCTAC
    GATTGCATCACCCCGGGGGNCCNCTCAANCCNNCTCTAAAGGNCCATNCCCNT
    NNNTGATCCTCTNCCATCCAANGGCNCCTTTCCACTTTTATTGGAAAACCCCCNTT
    CCCCNTTTTACCCTTNNAAGGCCCCTCCC
    SEQ ID NO:118 Late LPZ-043 AGGTGACCGTGGAACTACTGTTAAATCTGGAATCCCTTGTCTAGCTGTAAAAACTC
    GACAAGTGCATGTTGGTATTAGTAGGGTTAACAGAAGGGTTCTTACCCAGATTTAC
    CCCTTTGGCGGAGATATTTAAAAAAAAAGAATTGTCATTATGGTAAATAGGTGTGAC
    AGGTTATCAATAGAATAACTGACGAGAGTAAACTGATAATTATTAAGGTTAAAGTGT
    TCGTAAAGGANACTTGGACTCTAGGTTGGATGCCTACACTTAGAGCCCGTTCCCGC
    ACTGGACGGTCACCT
    SEQ ID NO:119 Late LPZ-045 AGGTGACCGTGGGGGATGGGGCCGTGGGGAAGACTGTATGCTCATCTCCTACAC
    AAGCAACACGTTTCCAACGGATTACGTGCCGACTGTTTTTGACAATTTTAGTGCAAA
    TGTGGTGTTGATGGCAATACAGTAAACCTTGGCTTGTGGGACACTGCAGGGCAA
    GAAGATTACAACAGACTGAGGCCATTGAGTTATAGAGGTGCAGATGCTTTTCTGCT
    TGCCTTTTCTCTGATCAGCAAGGCTAGTTATGAAAATATATCAAAGAAGTGGATTCC
    AGAACTAGACATTATGCACCAAATGTGCCAATCATTCTTGTGGGAACTAAATTAGA
    TTTGCGTGATGACAAGCAGTTCTTTGCTGATCATCCTGGAGCAGCCCCTATAACAA
    CAGCTCAAGGTGAAGAGTGAAGAAGCAGATTGGAGCAGCAGCATATATTGAGTG
    CAGTTCCAAAACCCAGCAGAATGTCAAGGCTGTTTTTGATGCTGCTGCAATTAAAGTGG
    TTCTTCAGCCACCAAAGCAGAAAAAGCGGAGAAAAAAGCAGAAAAATTGTTCTATTC
    TCTAAGAAAAATGTGGATGTTCTGAACGCNCTTCACTGACAATAANGNTGACGTNG
    GAATATCTTCCTCC
    SEQ ID NO:120 Late LPZ-047 AGGTGACCGTAAGCACAAGTCGTCAAAATATCTCTATTCCGGCAGTAAAAACCTAT
    AGCTAATGATGGATCAATACCACTAAGTGGCAGCTGGCGTACATCTCTGCAATGAT
    AAGAACCAGTATCAGTCCCCATATAATCAGGAGATATCTCCAGCACCTGCTGCACT
    ACATGTGGATCTTAGTACAGAGCCTGATCATCCTGAACACCAACAATATACGTTGAA
    GCTCCGGGCTTTCCACCAGCAATACCAAGACTTTGGGGAAATGTGAACGTTTCACG
    AAGTGATGGTACATACCTTGGGTTGATCTTCTCTACACCAAGAACAAGCGGCACCA
    AAATCAGGATAGGCACTTGGTCTTCCCCTTCTCCATTGGACCACTCTGAACACAAG
    CCTCGCAGCATCATCAATGCAGATAACTGGGCGCCCTCCACGGTCACTT
    SEQ ID NO:121 Late LPZ-049 AGGTGACCGTGCCATAGCGCATGGCGTGTAACTGGATGAGACCGCATGGCTCAAA
    TCTGCTAGGAATCAACATGAAATCAGCTCCAGCTGTTATCATATGAGCAAGTGGCA
    CGTTAAACTTTGCTACTCCCCTGACGTTGTCTGGATATTTCTCTTCAAGCTCTTCAA
    GCTGCTTCTCCAAGTACTTTTTACCGGTGCCTAGGATAATTAACTGCACGTTTCAT
    CTGCAATTAGAGGGACAGCTTCAGCAAGAATATCTGGACCTTTCTGCTCTTCAAGT
    CTTCCAATAAATCCTATAACAGGAATATCTGGATCCACGGTCACCT
    SEQ ID NO:122 Early LPZ-051 ATGTGACCGTCAAAAGGGCATATAAATCGGGGAGCTCAATGGCAAGAATGTACGAT
    TTCTGGCCTCAAGTCGCCCTGAATTTGGTCAACAACATCTTGATAGAGCGAGAGGA
    CGCTCCCAATTAAGATCTGGAAACTGTCGAGAGTGATTGAGGTCATTTTTAATCTAA
    ACTGAATTGTGGGGACAATTTTTCAATTCAGATCCTTCTAGCAAAGCAAAGCAAACC
    TTAACAGTATTGTATCCATGAGAATGGATTCTGCACAGGTCAGGCTCCACGGTCAC
    CT
    SEQ ID NO:123 All LPZ-053 AGGTGACCGTGGAGAAGAGAACGCTTTGCCGACTCTCTGGGATGCCCTTCCCTCC
    ATAGCCGTCGTGGGAGGACAGAGCTCCGGGAAATCCTCTGTGCTGGAGAGCATCG
    TTGGAAGGGATTTTTTACCGCGTGGATCAGGTATTGTTACTAGACGGCCGCTTGTC
    CTTCAACTTCACAAGACTGATGAAGGCAGCAGGGATTACGCCGAATTCCTTCACCA
    ACCCAGAAAGAAATACACCGACTTTGCACTGGTAAGGAAGGAAATTGCGGATGAGA
    CTGATCGAATTACAGGGCGTTCCAAGCAAGTCTCAAGTGTCCCAATCACCTTAGT
    ATTTATTCACCCAATGTTTGTAAATTTGACTCTAATTGATCTCCCTGGGTTGACAAAA
    GTGGCTATTGACGGTCACCT
    SEQ ID NO:124 Middle LPZ-054 AGGTGACCGTGCAATATTGTATCCAGGACCAAGTACTAGGACAGAATCAGGTTA
    CGAGTGGCTCCACTCCACAATACGATGTTCATCGTTTGATCACAATACAGGTTTGT
    TAGTCCAAGTAGGTGCGCTGCTGCAGACAGTGGGGCAGCCCTCGTGGGCTTGGA
    CTGCCTGTCATACTGTTCTCTCCTTGCTTCAGGCTCTACTGCTGTTGCTGCTGCTG
    ATACGGTCACCT
    SEQ ID NO:125 Middle LPZ-055 AGGTGACCGTACATACAAGGTCTTATCACCAGCAGCAAGAATAATCAGTTGGCCAT
    CTTCTGCAGGCTTCTTGCTGCCTGAGACAGGAGCCTCAAGAAATTTTCCCCCCTTT
    TCAATGATTGCCTCATTGATCTTTGTTGAAGTGATAGTATCAACTGTTGACATGTCA
    ATGTATCCTTTTCCTGTACACATTTGCTCTAGGACACCATCCGAGAGGGCAGCAGG
    AGGATCAGACAGGATGGCTATGGTATAGTTGCACTTCTTTACAACTTCGGCAGGAG
    TGCTTCCTATGGAAGCACCTTGCTGAACAAGTTCTTCACACCTAGACATTGTCCTAT
    TCCACACGGTCACCT
    SEQ ID NO:126 Late LPZ-056 GGTGACCGTACATACAAGGTCTTATCACCAGCAGCAAGAATAATCAGTTGGCCATC
    TTCTGCAGGCTTCTGGCTGCCTGAGACAGGAGCCTCATGAAATCTTCCCCCCTTTT
    CAATGATTGCCTCATTGATCTTTGTTGAAATGATAATATCAACTGTTGACATGTCAAT
    GTATCCTTTGTCCTGTACACATTTGCTCTAGGACACCATCCGAGAGGGCAGCAGGA
    GGATCAGACAGGATGGCTATGGTATAGTCGCACTTCTTTACAACTTCGGCAGGAGT
    GCTTCCTATGGAAGCACCTTGCTGAACAAAGTTCTTCACACCTAGACATTTGTCCTA
    TTCCGCACGGTCACCT
    SEQ ID NO:127 Late LPZ-057 AGGTGACCGTGGAGGGGCTCCAGTTATCTGCATTGATGATGCTGCGAGGCTGTGT
    TCAGAGTGGTCCAATGGAGAAGGGGAAGACCAAGTGCCTATCCTGATTTTGGTGC
    CGCTTGTTCTTGGTGTAGAGAAGATCAACCCAAGGTATGTACCATCACTCGTGAA
    ACGTTCACATTTCCCCAAAGTCTTGGTATTGCTGGTGGAAAGCCTGGAGCTTCAAC
    GTATATTGTTGGTGTTCAGGATGATCAGGCTCTGTACTTAGATCCACATGTAGTGC
    AGCAGGTGGTGGAGATATCTCCTGATAATATGGGGGTTGATACTGGTTCTTATCAT
    TGCAGTGATGTTCGCCACTGCCACTTAATGCTATTGATCCATCATTAGCTATAGGTT
    TTTACTGCCCGGAATAGAAATAATTTTGACAACTTGTGCTTACGGCACCT
    SEQ ID NO:128 Late LPZ-058 AGGTGACCGTGGAGGGGCTCCAGTTATCTGCATTGATGATGCTGCGAGGCTGTGT
    TCAGAGTGGTCCAATGGAGAAGGGGAAGACCAAGTGCCTATCCTGATTTTGGTGC
    CGCTGTTCTTGGTGTAGAGAAGATCAACCCAAGGTATGTACCATCACTTCGTGAA
    ACGTTCACATTTCCCCAAAGTCTTGGTATTGCTGGTGGAAAGCCTGGAGCTTCAAC
    GTATATTGTTGGTGTTCAGGATGATCAGGCTCTGTACTTAGATCCACATGTAGTGC
    AGCAGGTTGGTGGAGATATCTCCTGATAATATGGGGGTTGATACTGGTTCTTATCAT
    TGCAGTGATGTACCCACTGCCACTTAGTGCTATTGATCCATCATTTAGCTATAGGTTT
    TGCAGTGATGTACCCACTGCCACTTAGTGCTATTGATCCATCATTAGCTATAGGTTT
    TACTGCCGGAATAGAAAAATTTTGACAACTTGTGCTTACGGTCCCT
    SEQ ID NO:129 Late LPZ-059 AGGTGACCGTGCTAGGACACACAATTTCTCAGCAAGGATTACAGGTGGATCCTAAC
    AATATTGCTATAATTCAAAAGGTTCCACCTCCTTAAAAGGTAAGAGATGTTTGGAGT
    TTTCTAGGCTTGGCAGGATATTATAGAAGATTCATCAAAGATTTCATTAAGCTAGCC
    TCGCCATTGTCTAGCCTCTTAGGGAAAGATGTTGAGTTTCAATGGACTGATGACTG
    CCAAGGGGCTCTGGATGAGTTGAGAGATAAGCTGGTATCCGCCCCGATCTTGAGA
    GGTCTAAACTGGGCCCTACCTTTCCACATCCACATTGATGCCTCGAACAAAGCCAT
    AGGGGCAGCCTTAGGACAAGTTGAAGAGAAAATACCATATGCCATATACTTGTCA
    GCAAAAATCTGTCTAAGGCAGAACTGAACTATACGGTCACT
    SEQ ID NO:130 Late LPZ-060 AGGTGACCGTCATATTCCCCTCTATAGCAGCACTAACAATCCATTTTCTGAGTGCAT
    CAGAAAATCAACACACGGTAAATGTCTTGAGACTAACGAGAAATTAATAATCACGTT
    GTACAAAGAACAGTATGTCCCGTCACGTCACGAGTGCCCTGAGAGATCATCCAACT
    TTCTCTGAACCCTCGTGTTACACGCACGCAAAATCAAGGATCAGTTGTAGTTATTGC
    TGGCGTGACAGACGTGACACCTACTGTTCCGCTACAAACGATATAATTGAATCCAT
    GATCGGATTATGTATTATGATCTTAGCGCAGTGGTTATGAAATTATGATGAATTTGC
    TTATGATTTTCTCAGCGTTTGTGGAAGAATCTCGCTATTGAAAACTTCCCCGTATAT
    TTCCAAACTTATTATCATCCCACGGTCCCT
    SEQ ID NO:131 Late LPZ-061 AGGTGACCGTACAGCATTTATTGATGTTCTATTTTGTTGTTTGCAAGTTTTTCCGA
    CGCTGTGAGGCACGGAAAACGAGATAAGTTGTAAAAGTTTGCTCGCTGATTTGAGG
    CACGGAAAACGAGATAAGTTGTAAAATTTTGCTCGCTGATTTTTTGCTGAATATTTC
    TCTCACTATAAAAAGCATTTTCCAGAAATAAGAAGGAGCTTTCGAACTGGTTTTCCC
    CAAGAGTTGTAGGGGGTTTTTCCACGGTCACCT
    SEQ ID NO:132 Late LPZ-062 AGGTGACCGTATTTATGGTCGCAGGCACAAATTCTGCTACTGTAGAAGGGTTCTTA
    CCAACTTTAGGTAGAAGGCGAGGAGGGCTTTATTAGTACAGTTCTGTGTAATCTTA
    ATGATATTTTTTGCACTATTATTTTATGGTAAAAGGATTGATTTGTCTTTTGCAAAGG
    CCTTAGGATTGTTTATTTACCTTTGGGCTAAGGGAGGAGGTAAATTTTTCACATTGG
    GAAAAAAAATGCCTCGGTCGTTGTCACGGTCACCT
    SEQ ID NO:133 Late LPZ-063 AGGTGACCGTGCCAGTATGACAGATGGAACCATGCAGCTAGCCACCAAATTGTAAA
    CATCAAATTTTGTCTCAATATAAGTTGCAAATTCTTAATTAATTATGATCACCATTTC
    AACGGTCACCT
    SEQ ID NO:134 Middle LPZ-065 AGGTGACCGTGAATAGAAGCGAACACATCCTTGTTGCTGAATCTAACGACCAATCG
    GTATTTGGGTGTGTTGTACTTGTTCTTATCTTGGTTAATCAGGCGGATCCTTGCCCT
    GTAATCGGTCTTCCCCTCTCTCCTGCGCTGAATTTGACCTGAAACCTCTTGAAGTA
    GGCCCTGGTTTTCTGGGCTTTGACGAAAACCATGGTTGTGGATCTCCTCTCTCCTG
    CTACGGTCACCT
    SEQ ID NO:135 Middle LPZ-066 AGGTGACCGTGGTAGAGGAGGCAGGCACTCATCTAACAGTCGAAAGCCCTTTACA
    AAGGGGAATGGTACCAGCATAGAGAAGAAACACAGACGGTTTGAAGAGGATGATG
    GATCTGCCATAGATGAACGATCAAATAAGGTTCAAAAGCTGGAAAATGATGGTGAA
    TTCCATGCATCCCACTTGGCTCTGTCCCTCAAGTTGAATATACCTGGACGAGAGGT
    ATTGCATTTCCCAACGGTCACCT
    SEQ ID NO:136 Middle LPZ-067 AGGTGACCGTACTGATAATAGAAGAGGCAGGGAAAGAGAAATCAATGATAATAGAA
    GAGGCAGGGAAAGGGAGATCAATGGCATCATGCTACTTCTGTAGCTGTTACCT
    TAGTGATGTAATCTTCCATGGCAGACTCGGGGGTTTTATCTTTAAGTTGAATTTCCA
    TGCATCCCCTTGGGCTCTGTCCTCCAGTTGAATATCCTGGAACAAGAGGTTTTGCT
    TTCCACGGTCCCCT
    SEQ ID NO:137 Late LPZ-069 AGGTGACCGTGAGAAGGCAACTTTATCCCCTGCTAAACCAAGTCCAGAAATGAGGA
    AAATATGTGAAAACTGAATTGCTATATATGATGCCTAGTCTTGGCCTCTCAATTACA
    AGTTCAACGTCTTCAAATGATTGAAATATGGACCTTCTTAACCGTTCTGGAAATCTA
    TCAATCTTCAAAATTTTGAAACTTTGCCTCGATCTTGGAGTGATCAGACTTGATTTCT
    AATCCTAGAAATACCCTATCACTGGCTACCTGGTCTGTACGGTCACCT
    SEQ ID NO:138 Late LPZ-070 GGTGACCGTGGGATAGGCAGAAGCAAGAAACACAGAAGTTCTCCGGGAATGTAA
    GCGCTGACAGTGGGGGAGAAAGTAGTGAACAAGGACATGGTCGGTATGAAATACA
    TGGCAGGCGATGGATTTCAAGGGATTAAGCATCTCAATGGATATTTACTATTGGAC
    TGTAGTAACTTTCGCCATCGCTTTTTGAACACATCTGTGGCTTAACTGTCATCTGTA
    ATGGTAAGCGAACCAGGTTTTGTTCTGAACCACTTGTATGTACGGTCACCT
    SEQ ID NO:139 Late LPZ-071 AGGTGACCGTGGTGGAGCGATTAGTGATTGTGATAAAGGGAGCATCAATATCTATG
    TAGACGCCGTATAAAGGTGGAAAAGGTATGTTTTGCAGGTATTTCTTTGTAAATGGT
    TTATAATGGGTTAAGCTCGGATATATGAGGTTTATATATAAGTCCTGTTAGTGTCAG
    TCTTACCAGCCTTCCTCCAGTGATCAAATGTGCTCTAACAAAGTGATTTTGAAGTGT
    CAAGGTCAAATTATGTCATTTCAGTGAGTCTTCAAACAAAATTTGGTCACTAGGCAT
    TAGGTCTAAGGGTTTGCTTGAACTCCCTCTAGAGTTGTCCAAATGGGCGGGCTATG
    TCATCATTTAAGCTGAATCTATCATCCAATCAATAAGGTTTTTCATTATCATGTCAGT
    GTCTAAATGAGTCATTTTACCGTCTTGTTCACGGCTTCACTTGTGCCTTTGGCAAAT
    TCAATTCCCTCCTCCAAGGGTTTGAACCAATTCTCTTGGACGGCCCCTAAACCAA
    ATCTGCAAAATCCAC
    SEQ ID NO:140 Late LPZ-072 AGGTGACCGTGGTGGAGCGATTAGTGATTGTGATAAAGGGAGCATCAATATCTATG
    TAGACGCCGTATAAAGGTGGAAAAGGTATGTTTTGCAGGTATTTCTTTGTAAATGGT
    TTATAATGGGTTAAGCTCGGATATATGAGGTTTATATATAAGTCCTGTTAGTGTCAG
    TCTTTCCAGCCTTCCTCCAGTGATCAAATGTGCTCTTACAAAGTGATTTTGAAGTGT
    CAAGGTCAAATTTTGTCATTTCAGTGAGTCTTCAAGCAAAATTTGGTCACTAGGCAT
    TAGGTCTAAGGTTTGCTTTAACTCCTTCTAAAAGTTGTCCAAATGGCGGGCTATGTC
    ATCATTTACGTCTTGTTCAGCTCAGTGTGCCTGGCAATTCATTCCTCTCTAAGGTT
    TGAACCATTCTCTTGACGGCACTAAGCCAATCCACACTGGGGCCGTCTATTGAATC
    AACCCGGACACTGGGTTACAGGCAAC
    SEQ ID NO:141 Late LPZ-073 AGGTGACCGTCCAAGAAGAAATTGGCTTCAAAACCCTAGGAGAGGGAAATGAACTT
    GCCAAGGCACAACTGAAGCATGAACAAGACGTAAAATGACTCATTAGACACTGACA
    TGATAATGAAAAACCTATGAATGATGATAGACTCAGCTAAATGATGACATAGCCCGC
    CATTTGGACAAATTTTAGAAGGAGTTAAAGCAAACCTTAGACTTAATGCTTAGTGAC
    CAAATTTTGTTTGAAGACTCACTGAAATGACAAAATTTGACCTTGACACTTCAAAATC
    ACTTTGTAAGAGCACATTTGATCACTGGAGGAAGGCTGGAAAGACTGACACTAACA
    GGACTTATATATAAACCTCATATATCCGAGCTTAACCCATTATAAACCATTTACAAAG
    AAATACCTGCAAAACATACCTTTTCCACCTTTATACGGCGTCTACATAGATATTGAT
    GCTCCCTTTATCACAATCACTAATCGCTCCACCACGGTCACCT
    SEQ ID NO:142 Middle LPZ-074 AGGTGACCGTGATAGACCCAAGAAAAATAGATCCAACCCTCAGAGGGACAAAGA
    CTTATAAAGACTAGAAGAGTGAATCAACCTATTCTATTTAGAATATATATTTTTGGGG
    TGCTTGCTTATCGTTTTGGGGGTTAATGTATGTCGTACTACGGTCTTATGCCCTAAT
    TTGCCCATTGAAATCAACTAAATTGACAGTAACCGACTAAAAGTTGGTCCACACTAA
    GATATCGATGACCAACGATCATAAAGGTGTCCATGATCCTAATAGTATATGTGTCAA
    TTAATGTAACTTTGGTGCTACAACATAAAACCATTCGTGGGGATCCTCCTTTTTATG
    CGGTCACCT
    SEQ ID NO:143 Middle LPZ-075 AGGTGACCGTGGGACCGACCTTGACTACAGGCCAAAATTTTGACTGTTGACCAGC
    GTTCACTTCTGTATTTTTGGTTGGTATGAGCAACATTGACTTGCTGGAAATTGACCA
    GGTTTGACTGGTATTTGGACTTGGATTTTGGCACAGATTTCTAGACAATTTGTATTT
    GTAAACCTTACAGAAGAATAATTTATCGAAGAAGAAAAATGCTAGGTTTCCCCTCAA
    GTTTGGGTTTCCCAAGGGAAAAATTGTTGTCCCAATGGTTGAATTTTCCAAAGGTCT
    CCTAACCCGACAATACCTCCTAAGAATTCCTTAATTTAACCTTTCTTGTTTTCACGGT
    CACCT
    SEQ ID NO:144 Middle LPZ-076 AGGTGACCGTGAAGGAGCAGCAACAATTTGATTTTGTTTGGGTAGATCGGGGATTT
    TCTCGTGGAACATACCTGATTGAGTATAAACTAAGTCAAGGTACTGTGCTTGAGAAA
    TTACTTGCTCCTCAGTAACTACTCTGGCCTTAGCTACATCCTCAGTGATCTTGGGTA
    GTAAAGATTTTACAAACCATTCAGCTAAGATCTGATCCGGGATATAAACTTTCACTA
    AACGTCGTCGACGTCTCCATTCATGGATATGATCTGAAATGTAAGTGGACGTTGAC
    TGCTTTAACGAAGTTAATAATTCTGTGCCATTTTCATATCTGACGGTCACCT
    SEQ ID NO:145 Late LPZ-077 AGGTGACCGTACCTAATGGGAAGACACTTCAAGGTAAAAACAAATCATGATAGTCT
    TAAATACCTTTTAGAACAAAGATTATATTCAGAACAACTTGCTGGAAGTGTACCAAG
    TATGACTGGTATTGAGACTTAGATCTTCGCACAGATTTCAAGACAATTTGTTGTTGT
    AAGACTCACTCACGAAAAGTGATGTGGATATGAAGAACTTCCCTGTCGCCTCTTGG
    TTAGGAGTCTCCCACTCATAGGAATTGTGTAACTTATAACTTGGTCCACTAAAGAAG
    TTAGGTACAGTGTGTTCCTTTACCAGGTTCCCTGTTGTAACTTACAAATCTACGGCT
    ACCT
    SEQ ID NO:146 Late LPZ-078 AGGTGACCGTCACTGGAGGTTTGAGATGCTTGATCGGTACTGAAATGAGACATGAT
    CAGAATAGGACCTTGTTGAGGCCGTGTCTCACCCCCCATCCACAATCTTTTGTAAT
    TTTGAGTTTCGTTTAGAACATACTTGTAGGATAAAACTTACCTTACTCATGGATCAT
    GGCTGTATATGTTTATCGACCAGAGACAGATATGCCGAATGAAAGCGAGTCTAGTA
    TTCTAATGCAATATATTGGTAGTATGGGACATAGTACTGAACACTTGTATAGTACGG
    TCACCT
    SEQ ID NO:147 Late LPZ-079 AGGTGACCGTGGTCTCAGTTATGCCATATGTCCGCCCCTCCATATGATGCTCCGCC
    TCTATGGGGGTCTTTGCGATGTTGATATCTAGTAGTACTTCTTGTCCTATTGCAGCA
    ACCTGTACTGGTGTTGGTGTTGGTATGGGTCTCCTACGCGATGGAGATATGAGAC
    ACCCATAGGTCGAACAGGTCTAATATCTGGAATCCAACGCTATTGTTGTAGAAG
    ACGTTGCTCCCGTCCTTTAGCTTTGGCTGGTCACTATCCTTACGCTCCACGTACGG
    TCACCT
    SEQ ID NO:148 Middle LPZ-080 AGGTGACCGTTGGGAAATGCAATACCTCTCGTCCAGGTATATTCAACTTGAGGGAC
    AGAGCCAAGTGGGATGCATGGAATTCACTTAAAGATAAAACCCCCGAGTCTGCCAT
    GGAAGATTACATCACTAAGGTTAAACAGCTACAAGAAGTAGCATGATGCCATTGAT
    CTCCCTTTCCCTGCCTCTTCTATTATCAGTACGGTCACCT
    SEQ ID NO:149 Late LPZ-081 AGGTGACCGTCAAGGCAAAGTGTCATGCCACTCATTGGAATTAGTTAATATAGCTA
    ATTTGAGATATTACAGTCAACTGTGGGTATATGTATGTGAGATCAAGGTGCAGTTTA
    GATATTATCAGTGGTGCAGTTTAGATATTATCAGTGTTTGTGAATCTGCATACTGCT
    TTTGGTTGGTTCTAACTACGGTCACCT
    SEQ ID NO:150 Middle LPZ-082 AGGTGACCGTAGACATATATCATGGAAAACCCAAGTAACATACAAACACAAAACACA
    TGGAACTTCATAAAACCTCCACTCGTCATAAGCTTTATTGCTATGTTATTGTGGTG
    TTGCATCGTACTTAGTGGAGGTTATTGTTATGTTATGTGTTCTATTTTCCTCCCGAA
    CGCCCTTCGGAATTGAGCTAACCGTGGTTAACAACATGTGGGCTTTTTTTCTCGAC
    AGTATATATATAATAAATCTTTATTTTTTTAAAAACTAATGCTATTGCATTTATATACT
    GGAAAAAATGATTTTTCTGTATTATCGAAAATAATAATTTAGTTTCTTGATAATCACT
    TGGAATTAAGAAATTACAAACCCTAACAACATCAAGAAATTTTAAAACACATAAGCTA
    GAAATTTTAAAACACATAAGCGTGACAACAAGAAGATCAAATCTAATACTTGCTTGG
    GCCGGAGATTATGGATTCATGAAGCGATTTGACAGCGTCCATTGATCTTCCTCTCC
    CACGGTCACCT
    SEQ ID NO:151 Late LPZ-083 GGGGGTAGGGGTGTTTATACTGAGCATACTTCGAAAGTGGTTCACCACCACCATG
    TGACTAATTGTTCCTGACTTTGGTAGACCTATAATAAATTCCATAGAAACCTCCGTC
    CATATTGATGCCGGAATGGGCAACGGTTGTAATGTGCCTGGTACTTTGACGGTCAC
    CT
    SEQ ID NO:152 Middle LPZ-084 AGGTGACCGTTGGGAAATGCAATACCTCTCGTCCAGGTATATTCAACTTGAGGGAC
    AGAGCCAAGTGGGATGCATGGAATTCACTTAAAGATAAAACCCCCGAGTCTGCCAT
    GGAAGATTACATCACTAAGGTTAAACAGCTACAAGAAGTAGCATGATGCCTAGACA
    AATAGCTTTGCTCAACACATCCTGATAGTGTACACTAAATCGCACAACTTTACTACT
    ACAAAGAAAGATCGTTGACACCTTGACAAATAGCTTGCTCAACACATCCCAACAAT
    TTGGATTGCGAATACCGACTCCAATTTGTACTTGATCCATATGTCGTTGCGATGTAC
    TAGTTCCTCTATACATATGTTTCTGCAAGAATCGGAGTTGGACCTCTTCTTCCCTGT
    TATCAGCACGGTCACT
    SEQ ID NO:153 Early LPZ-085 AGGTGACCGTGGATAAGAGAACGCTTTGCCGACTCTCTGGGATGCCCTTCCCTCC
    ATAGCCGTCGTGGGAGGACAGAGCTCCGGGAAATCCTCTGTGCTGGAGAGCATCG
    TTGGAAGGGATTTTTTACCGCGTGGATCAGGTATTGTTACTAGACGGCCGCTTGTC
    CTTCAACTTCACAAGACTGATGAAGGCAGCAGGGATTACGCCGAATCCTTCACCA
    ACCCAGAAAGACATACACCGACTTTGCACTGGTAAGGAACGAAATTGCGGATGAGA
    CTGATCGAATTACATGGCGTGCCAAGCANAGTCTCAAGTGTCCCAATTCACCTAA
    TATTTATTCACCCAATGTTGTTAATTTGACTCTAATTGATCTCCTGGGTTGACAAAAT
    TGCTATTGACGGTCACT
    SEQ ID NO:154 Middle LPZ-086 AGGTGACCGTTGGGAAATGCAATACCTCTCGTCCAGGTATATTCAACTGAGGGAC
    AGAGCCAAGTGGGATGCATGGAATTCACTTAAAGATAAAACCCCCGAGTCTGCCAT
    GGAAGATTACATCACTAAGGTTAAACAGCTACAAGAAGTAGCATGATGCCATTGAT
    CTCCCTTTCCCTGCCTCTTCTATTATCATTGATCTCTCTTTCCCTGCCTCTTCTATTA
    TCAGTACGGTCACCT
    SEQ ID NO:155 All LPZ-089 AGGTGACCGTACATACAAGTGCTCAGTACAATGTCATATACTACCAATACATTTGAT
    TAGAATACGAGACTCGCTTTCATTCGGCATATCTGTCTCTGGATGATAAACATATAA
    AGCCTTGATCCATGAGTAAGGTAAGTTTGAAGCTACAAGTATTTTCTAAACGAAGTT
    CAAAATTACATAAGATTGTGGCTGGGGCGTGAGAAACGGCCTCAACAATGTCCTGT
    TCTGATCATGTATCATTTCAGTACCGATCATGCCTATCATACCCGCCTGGTGACGG
    TCACCT
    SEQ ID NO:156 Middle LPZ-090 AGGTGACCGTACTGATAATAGAAGAGGCAGGGAAAGGGAGATCAATGGCATCATG
    CTACTTCTTGTAGCTGTTTAACCTTAGTGATGTAATCTTCCATGGCAGACTCGGGG
    GTTTTATCTTTAAGTGAATTGCCATGCATCCCACTTGGCTCTGTCCCTCAAGTTGAA
    TATACCTGGACGAGAGGTATTGCATTTCCCAACGGTCACCT
    SEQ ID NO:157 Late LPZ-091 AGGTGACCGTATAGTGTCAAGCTTTTCTGGATTGGATAATGGACGGCGGCTTGCG
    CATACATCTACACATTCTGTAACAAGTACACTCTACTGCAACAGCAGACCCAATTTC
    ACCTCTTCAGTCAGCCAGAGATCTCGATGGATTTGGGTTGAGGAGGTTGGGGTTC
    GCCTGCTTCGGCACGGTCACCT
    SEQ ID NO:158 Early LPZ-092 AGGTGACCGTGCTAAGTAATTATCATCTGTACCTGTGCTTGCTGCAGGAAGTAAAC
    CAACCCGACTAGTCTTTTTAATAATACAGGGAGCCTTGCCACCAATTTCCTCTTGAA
    GCACCCATATTGGACGGGTTTGTGTCATCCTCTGTATTATCCTTTTTCATCCCAAGC
    AGGCTGTCTGTTTTTGTAGTAGAAGGATCACAACACAGATCAGGCCCTCCATAGTA
    CAAAGAAGAACCGAGGAAAGTATCATTAACGTTCTGACTCCTGCCATGAAGGCTTC
    CACTATGACCTTGACCCTTTTGTGAATTACTGCCATTTAGACCTTGACTGGCTCTTG
    CAACCAAATGCCCCAGAATGGAACTTCTTTGTGCTCCAGTTCCATTGTGGTTAGTT
    GAATCCCTACCACGGTCACT
    SEQ ID NO:159 Late LPZ-093 AGGTGACCGTGCAATATTGTATTCCAGGACCAAGTACTTAGGACAGAATCAGGTCA
    CGAGTGGCTCCACTCCACAATACGATGTTCATCGTTTTAATCACAATACAAGTTTGT
    TAGTCCAAGTAAGTGCGCTGCTGCAGACAGTGGGGCACCCCCCGTGGGCTTTGAC
    TGCCTGTCATACTGTTCCCTCCTTGCTCCTGCTCTTGCTCTCGCTGGGCTGTGGTG
    AGTTACTAACCTGGTTCGACCCACAAGGGCTTCTCACTAGGGCGTTAGGCTGCATG
    GATCTGCCAGATATTGTGGTTGCAAGGGACAGAGGCATGAGACACAGGCCTTTGC
    TTTGCAGAAACTGCATTGCTGACCCCATGTTCATCCATCAGTTTTGCTACCTCTC
    CTTCTGTTATGGACGGTCACCT
    SEQ ID NO:160 Late LPZ-094 AGGTGACCGTATCCGCAGCAGCAACAGCAGTAGAGCCTGAAGCAGGGGACCTAAT
    TACAGTCAAAAGTCCAGGGCTACCAATGCCTGCTAACAGCGCACTTACTGGACTA
    ACAAACTTGTATTGTGATTAAGACGATGAACATCGTATGTGGAGTGGAAGCCACT
    CGTGACCTGATTCTGTCATAAGTACTTGGTCCTGGAATACAATATTGCACGGTCAC
    CT
    SEQ ID NO:161 Late LPZ-095 AGGTGACCGTATCCGCAGCAGCAACAGCAGTAGAGCCTGAAGCAGGGGACCTAAT
    TACAGTCAAAAGTCCAGGGCTACCAATGCCTGCTAACAGCGCACTTACTTGGAACT
    AACAAAATTTTTATTGTTAATTAAAAACGAATAACATCGTTTTTGTGGGAGTGGAACC
    ACTCGTGAACTGAATCCTGTCCTAAGTTCTGGGTCCTGGGAATAACATATTGCACG
    GGTCACCTT
    SEQ ID NO:162 Middle LPZ-096 AGGTGACCGTTACAGCTAGGGAAGACTTTAAAAGTTTGTAAAACTAAGCATAGCTC
    TAAACACTGAAGTTAAAAGACATGATTGGAATGTGCAAGTGGTTCAGTATCCAAATA
    TTGAAGGTTGCAGAATATGGAGCTACTGTGCAAACGAGTAACTTTATCTATATTTTC
    ACAAGATCATACAATGGGAAACGTTGAGATAACAACTGCATCGGTGAACCAGAATA
    GTTATAAAAGTTCTTGCAAGTAAAGGGATGAATAATTGCATGGTTGGAATTAAGAAT
    GACCATGTAGAGCTGCTATACAGATTCTCCAAGGTTTTATATTTGAGGAGTGCGCG
    CTATTGATGTTGTGCAAAAATTTCAGAAATTAAGTTCTGCGGCATTTATCAAGGTG
    TTTGAGCCATTTAAATAGCAAGTTTTTGTTCTCCAAGTACTTTCAGGAAAGCAGAT
    AGCTCTAGTTATAATGCTCCAGTGACAAACACATCTAGTTGGGGCAGTGAATGACG
    CTTTTGTCATTCTCTTTTGGTTTCAGGCACGGTCACCT
    SEQ ID NO:163 Early LPZ-099 AGGTGACCGTGGACAAACTCTAGAACAGGCATAGCTTTCATGTTCAGTTGTTTTTAA
    AGAGCAGTCCTCGCAGCAGATCGTGCAGCTTCCTGCTTCACTTCCGTTGATTTTCC
    TGATCTGAAATACCCGTAAACTTGCTGAAGAACCCAAATACTTAATAGCGTCTCTAA
    ACAAAA
    SEQ ID NO:164 Late LPZ-100 AGGTGACCGTGCCTGAAACCAAAAGAGAATGACAAAAGCGTCATTCACTGCCCCAA
    CTAATGTGTTTGTCACTGGAGCATTATAACTAGAGCTATCTACAAGCCAAAACAGTG
    TTTGGGAGAGATTCCATAACGTCATTGCCTCTGCTACACATCATTCATTGGTTCCAA
    TAATGAAGCCACGTGCTAAGGACATTGAGAGAATCTTATAAAACAAGAAATATAGTA
    AATTGGGAAATGCATTTTATCGTCTAACCTGCTTTCCTGAAAGTACTTGGAGAAACA
    AAAACTTGCTATTAAATGGCTCAAACAACCTTGATAAATGCCGCAGAACTTAATTTC
    TGAAATTTTTGCAAACATCAATAGCGCGCACTCTTCAAATATAAAACCTTGGAGAAG
    TCTGTATAGCAGCTCACATGGTCATTCTTAATTCACACCATGCAATTATTCATCCC
    TACTTGCAAGAACTTTATAACTATTCTGGTCACCGATGCAGTTGTTATCTCAACGT
    TTCCCATTGTATGATCTTTGAAAATATAGATAAAGTTACTCGTTTGCACAGTAGCTC
    CATATTCTGCAACCTTCAATTTGGATACTGAACCACTTGCACATTCCAATCATGTC
    TTTTAACTTCAGTGTTTAAGAGTATGCTTAGTTTTACAAACTTTTAAAGTCTTCCCTA
    GCTGTAACGGTCAC
    SEQ ID NO:165 Middle LPZ-101 AGGTGACCGTAAAATACCATGAGAAATGCTTTCATCAGGCACCGCTGGTAGGTTT
    CTTAAGCTTTTCATTAGGCAAAAGAGGCTCCGTGAGTTGATCGTTAATTCTCTCCTT
    GAATGCCATATTGACCAGACACTCTGATTAGAAACTGGAATACAACTGCACATATAG
    TCATTCTATATGATTCATCCTTCTGCACTTCAGCATCCTGCGGCAACTCTTCATCCC
    GCCATACTGAGAAAAATATTTGACTCTTGATCATGTGTAGATGAATCTTCATGAAT
    CTTCTCATCTTCATTCTTGTCTTTATATCTTTAGGAAGTGCATCTGGTAAAAGTATAA
    ATGCATCTTCACGGGTGCTTCAGTTTTTGCATGCTCCCGGTCTTCTTGTTTAGCAT
    GTGGATCTAGGAAATCACTAAATGTAGTTCTCTCAATTGGTCTGGTGGAAATTCTCC
    TCAATTCGAGAATTACGAATCATCATACCTGAGTAATATATGTTGCCCTGTACATGC
    ATATGCTGGTTTTTGGCTCCACCATCTCCAAAGGGCTCAAAAACTATGCGACCCC
    TGGTTGCCGTAGTGGAAGGTTATACATTGCGTTCCCAGTAGCCACGGTCAC
    SEQ ID NO:166 Middle LPZ-102 AGGTGACCGTGGAGGGGCTCCACTTATATGCATAGATGATGCTGCGAGGCTGTGT
    TCATCTGGTCCAATGGAGAAGGGGAAGACCAAGTGCCTATCCTGATTTTGGTGCC
    GCTTGTTCTGGTGTACAGAATATCAACCCAGGGTATGTACCATCACTCGTGAGAC
    GTTCACATTTCCCCACTTCTTGGTGGAGCTGGTGGAAAGCCTGGAACTTCATCAAT
    CTATCGTTGGTGTGAGGATGATCAGGCTCTGTACTTATATCCACATGTAGTGCAGC
    AGGTGGTGGAGATGTCTCTGATAAGTTGGGGGTTGATACTGGTTCGTATCATTTGC
    AGTGATGTCCCCCGCTGCCCTTAATTGCTATTGATCCATCATTAACTATAGGTTTT
    TACTCGCCCGGAATAAGACAATCTTTTGACACTTGTTGCTTGGGTCAC
    SEQ ID NO:167 Early LPZ-103 AGGTGACCGTGGCGCCTGACCTGTGCAGAATCCATTCTCATGGATACAATACTGTT
    AAGTTTGCTTTGCTTTGCTTGAAGGATCTGAATTGAAAAATTGTCCCCACAATTCTG
    TTTCGTTAAAAATGACCTCAATCACTCTCGACAGTTTCCAGATCTTGATTGGGAGCG
    TCCTCTCCTCTCTCAAGATGTTGTTGACCAAATTCAGGGCGACTTGTGGCCAGAAA
    TCGTACATTCTGCCATCTACCTGTTATTGAGCTCCCCGATTTATATGCGCTTTTGAC
    GGTCAC
    SEQ ID NO:168 Middle LPZ-106 AGGTGACCGTCAATACCATTAAACTGGGGATTCGTCTCAACAAGTCAACATGCTAA
    CCTCACAGCTCCAATCAAACAACGTCCGTCGAAGGGCGCTCACACTCATCCAAATT
    ACTTCCCTCTGCAAGACTCACAAAATCAGATTCTTCATGAATTGCTCAAACGAGGCT
    GTTATGGATGATGCAGCTGATTACTCAAGTGACAGCACTCTGAATCCCCGTCCCAT
    ATATAGCGACGCGGCGTTTCAGCCGTGACTGGTCGCAACAGCCTCAGTGGGACACAA
    AAGGCCAGAAGCCCCCCAAGGTTCTCACGGTCAG
    SEQ ID NO:169 E,L LPZ-107 AGGTGACCGTGTCGATGTTGTTAGATGTGATTAGGGTTTTATTTCTTGATACAGATG
    CACTGTTTCTCTGTTTATTCTTTTATTTCTTCAATGTATGTTGTCAAATTATACTTAGT
    CA1GATCTCCTTTTATCGTTCGTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAG
    TTTAACAATTAAAAGGGGAAATTAGGCCATATCAGCTTGTCGTATGGACCCACATG
    ACTGTAGGTCAC
    SEQ ID NO:170 Middle LPZ-108 AGGTGACCGTATGCAGAGTCAAGGTTTAGTTCCTTCAGAGCCTGCCCGAGTAGCA
    CTGAGGCAGCTCAAGCCATTTCACGTAGGAAGCCCACAACAAAATAGAAATCAGAG
    TGAGTCTTTGATCGAGTAACCCATAAGTTCTTAGCTCCCGTTCCATCTTAACATAAG
    CATTTTTCTTCGTCTTCTCGCAGCCGT
    SEQ ID NO:171 Late LPZ-109 ATTGCAGAGGACTTAGAGAGGGAAAACCGTTCCGATCTGGTGAAGCAATTGGATG
    AGCGCTCTGGAATTGATCCCGTTTCTGATGATATCGTACGGCTAAGCTCAGCTCT
    TCAGGCATTGGCAGACAATACGATTCTTCAAATGAGATGACAGATTTTAAGAAACTT
    ATAGGATGACATATTTCCTAGCTTGAAGCGGATCCCCCTACGGTCAC
    SEQ ID NO:172 All LPZ-110 AGGTGACCGTCCGATAAAGGATGAGAATATAGGTAGATCAACCCAAAAACACTCTC
    AGAAAACGATTAAAGCCTAACCCCAAGATCGTTGAGTAAATTTAACCCGGTAACCTC
    CACATAAAATATACTTAGCAACAATAAACTCAACAACTAAACTATCCCTTTAAAATTA
    AATTATCCTATTTATTTAAAAAAACAAATCCTTTATATACTAAGGTCCCCTGCACAT
    CTATTACTAAGGTAAAGGAAGGGAATTATATGCTATCATTGTAAACTTTGACTTCCG
    TATTTATGATCAGACCATGAGTTTGATAATTAATTTTACGCTCTTTACTCCCCATTCA
    AGGCACGTGCCTGGTGATATATGAACGCCAAATTATT
    SEQ ID NO:173 Late LPZ-111 AGGTGACCGTAGAATACAATCTATGTATCTTAAATGCTAACAAAGAGAATTTGTTGTC
    TAGCTTGTAAATATACAAAAGAAACTCTCACAAGGAGTGAGAAGCACTAAGGCCCT
    TGGAAAGAATACGTTTCTATTCAGCGGAGTGTATTTTGAGCTACGGCTTGGCACAA
    CTCATCCTATAAAACAAGACTCTGTGAGAGGGCAGAGACCTTGATCCTGGGCGTG
    GCAAGCCGGGTGCCTATTGCGGTAAAATCGAGAAGGGGGACCCTGGAAAAGAGAG
    GCTGAAATTTGTTTCATTCTGCAACTGAAACCTAACCGGAGGCCGAATCTGATCATT
    TCTAAGACCTTTGGGGTCCTGGGCATCCCATTAAAAGAACGCTGCTAACTCTCCCC
    TCCACAAAGGGCCAATGCGCTCAGGTCGGGCTTCTCATCTTCACATTTCTTGCCGA
    AATCTATCTGAATTTGTTGTATTGAATAACACTGCCTCCTACACGGTCAC
    SEQ ID NO:174 Late LPZ-112 AGGTGACCGTGGGCGCCGTGGCTCAAAAGGCCCTCGCAGACGCCCGCTCCATCA
    AGCTCATGGGCCCCCTCCACCCTCGGGGGGCAAGCCGGGAACGTTGCTGTCAGA
    CGAGGCGAGGACCTGGAACTGCCGTTGAAGGAACGGTTCTATATTCAGCCCCTCT
    CGGCGGACCAGGCGCTGCGAGAGCCAAGGAATCCGCGGAAGCAAATCCTGGAGG
    TGAAAAAGCTGATAGATAAAAGGCGTGGCCGTACGTCCAGAACGACCTCCGCTCC
    AAGGCTTCTTACCTTCGCTACGACTCAACACCGTTATCTCCTCAAAGCCCAAGGAA
    CAGAAAAAACCCCTCAAAACCTCACCCCAAAGCTTTTTTGACACCCTTGACAAACCT
    GGACTACGCTGCAAGGAGCCAAGGATACCCCAAGGGCAGAAAAAATACTTTGCAG
    AAGCTGGTGAACCGCCCTTAATGATGTTCATTCCAAGCTTGGTTAAGCTGTATTGC
    ACTCATTGTTAACCACACTTAACGCCAATCCAATCTATGCTGTGTTGCATCTCCACT
    TCTTAGTTAATAACGTCTGTGTCCCAAACTCTGTGCCACACACGGTCAC
    SEQ ID NO:175 All LPZ-114 AGGTGACCGTACAATACAAATAGGTAGTTTATCACATTGTAGCTTATAGAATGTACA
    ATTGAAATCAAATAAATTCAACCAAACTCAAATAATATGATCATGTGCTCCTCACCTT
    CTCAGCAAACTCGTAGAGCAGAAAAAAGGATTATGTTAAATCACAGTTCACACATTA
    GGGTAAATCCCACTAAATGACCTCTCTTCATTATCCAAGTATCTGACACCAACATAT
    TTCAAACAAATAGTGCAAAAAGGAATGGTGAAGTAAAATAGTCAAAACTAAAAAATA
    AGCTTAAAATTTCTCACATGTTTGAATATGTGCACCACAAATTTTGTTAGTGTCATCA
    AAATGCATGTAATCAACTTGCCGTGTATATAATTTCACACAATATCCGTAAAATTTTG
    CAATTCCTTATGAGCATTTCATGTCTAGAGATTGCAATGACTTGGCTACAAACATGT
    TTCTCTACACAAGATCACAATATTTAGTCAGGACACGAATTGCAATGGGGATTCTCA
    CAAGCATCACAAGTCATCTCCCATGTACTAATAAAAAATTGTTTAAAT
    SEQ ID NO:176 Middle LPZ-115 AGGTGACCGTATAGTGCATATTCAGATTGCAATTACAGACGTATTAGAACCAGATTT
    TCGCTTCGATACAGCTCATCGAGAGCAACAGAGATCCAGATCAAAAACCAGACACA
    GTTTAAGAACATCGAAATACCAAGCCCAGGGACAGTTACCAGCATATAGCTCTACC
    ACCAACAGATTATTACAGAACCAAAACATAAGACCACTTGCAGACAAAAATAAACCC
    TAACGCAGAACGTGGCAACTATCTCCTCCAGCTACCACCATCGGAACCACCACCAC
    CATAGCGAGAACCCCACCACCACCATAGCCGCCACCGCCACCACCATAACCACCA
    CCACCACCACCACTGTACCGCCACTACCGCCATAACCACGGTCAC
    SEQ ID NO:177 E,L LPZ-116 AGGTGACCGTCCTTGGAGATACCAGCTTCAAAACCTCCAGTGGTGGAGTCGATGA
    CAATACTGCACAGTCAGCCTGAGATGTTCCAGTAATCATGTTCTTGATAAAATCACG
    ATGGCCGGGGCATCAATCACAGTGCAGTAGTATTTAGTTGTCTCAAACTTCCAGAG
    TGCAATATCATTGTGATACCACGGTCAC
    SEQ ID NO:178 E,M LPZ-117 AGGTGACCGTATAGTAGGAACTTTAGGTGCTTTTGGTGGCACTCTCCAATTTTCATG
    TCCTTACATACCCCACTACGGAGAAGGGTAGCCCAAGATTTGAACCCAAGACTTCC
    GGTTCGTGAGACTTCATTTCCACGGTCAC
    SEQ ID NO:179 All LPZ-118 AGGTGACCGTAAGATCAAGAGCACAGAAAGCAGCCATAGCCCCGCCCATTGAATG
    CCCATAACAATAATCTGTAACCCATCTCTCTGTTTCTGAGCTTTCTGAACTGCTTCT
    ACAACAGTGGTCGTAAGGTTGTGTTGTGATAAGCAGAGTAAAATCCATAATGTACC
    ATTGCACCAGCATATTAGGATAGTTGAGATCAAGTGTCTTACAGAATAAATCCTCCA
    CCCAATTCTGTAGCTCCTTTCTTGAGTACCCCTGAATGCAATTACAATTGCATTGAT
    ATCTTCTGCCACACCACAAAAGCCTGAAGGCAGTGTTGTACATCAACTATAAGCTC
    ACCACCTGAAAACCCCAGTCAAACCATTGCACCTAGAACAAGTCCAAGACATTAGA
    GCACTCAAATCATCCATATAGACCGCAGAAGCATATTGCACAAGTATCTCAGCAAGT
    GTTCGATTATAGACATGGCCA1GGTCAC
    SEQ ID NO:180 Middle LPZ-119 AGGTGACCGTGGGAGGGGAGATTTTTGATTTATATTTCCAATATAAAAGAAAATCTA
    NGTTGTAAGGACATGGCAAGAGCTCTTATTTCCGGGGTTTTAGCCGTGGCCCGGA
    GCGGATGAAAGCAAATGTAAGTCACTCCGTGCTTCTCGGCATTTGGACGCTTCTA
    CTCTACCGCACTACAGACGGGATTGAACCTCGCATCTCTGAGTGTTTGGTCGTTTA
    CATGGCGGACTTGTTCCGCACCTCTGCGGACGTCAAATGCCGCGACGATAATCCC
    TTTGAGAACAGCGATACGGCAGAAAGATCGCCGTTGACGAAGCGAGAAAACTATTG
    AGACTTGCAGATGTGGAGCTGAAGAAGAGCTTGAGTCGACGGTCAC
    SEQ ID NO:181 Middle LPZ-120 AGGTGACCGTCCGTTCGGGGTGTATTTGTCGAACACGTAGGATGGTGCTACGTTGA
    AACCACCGTTACCTTCTTCGATATGTTATAGTTCGAGTTCATACGGAGGGAATACC
    GTTTGTAGTGTTATTCAGCACAACCCCGTCCTGATTAAACACCCCCGCAACCAAGG
    ACGTATTCGACGTTCGGTATTGTTTGACACACTCAAGTTATAACCCTGAATAGGCG
    CTACCCGAAGTAAGCATTGTACCAGTCGTTATTTTTGCCTTCGTATGCGAAGGATT
    TTGAAATATATCCGGACAGGCTGCAACCGATCTTCATAAAACTCTTTCTTAAACTGA
    GCAAACTGAACAGCATTAGCATTTTGACCCGACCTTCATCGGCACCTGCTGCACA
    CCCGCATACGTATTAAAGCTATGTTCGTCTGGCCAGGTTTGCCTTTTTTGGTTGTAA
    TCAGGACAACGCCGTTAGCCGCCCGCGATCCGTAGAGCGACGTAGAAAGCCGCAT
    CTTTCAGCACGGTCAC
    SEQ ID NO:182 Late LPZ-122 AGGTGACCGTGAAATATGTGGGAGATGATATGTGGTTTCCTGAATATTCACCTCTT
    GTGTAGAAAAGTGAGATCCTTAAGATGTTTTTGCTAATAAGACTCTTAGGAATGTTGG
    ACCCCTTTCAGAATGCCATTTGAATAGATTCAAGGTGGTAGCTGTTGCCTGGGGCT
    GTTTTAGGGTTTTAGGCCATGCTCTGTAATTCATTGAGTCAAAATTGGATTAACTG
    GTGTCTTTTACCTCATAATAGCTACTGCAGTATTTGTCGATATAGCTTCCCTATTTAT
    TGACTCTCCTTAGGTACGGTCAC
    SEQ ID NO:183 Late LPZ-124 AGGTGACCGTCCGTCGGGGTGTATTGTCGAACACGTAGGATGGTGCTACGTTGA
    AACCACCGTTACCTTCTTCGATATGTTATAGTTCGAGTTCATACGGAGGGAATACC
    GTTTGTAGTGTTATTCAGCACAACCCCGTCCTGATTAAACACCCCCGCAACCAAGG
    ACGTATTCGACGTTCGGTATTGTTTGACACACTCAAGTATAACTCTGAATAGGCGC
    TACCCGAAGTAAGCATTGTACCAAGTCGTTATTTTTGCCTTCGTACTGCGAAGGATT
    TTGAAATATATCCGCACAGGCTGCAACTGATCTTCGTAAAACTCTTTCTTAAACTGA
    GCAAACTGAACAGCATCAGCATTTTGACCCGACCTTTCATCGGCACCTGCTGCACA
    CCCGCATACGTATTAAAGCAATGTTCGTCTGGCCAGGTTTGCCTTTTTTGGTTGTAA
    CAGGACAACGCCGTTAGCCGCCGCGATCCGTAGAGCGACGTAGAAGCCGCATCTT
    TCAGCACGGTCAC
    SEQ ID NO:184 Middle LPZ-126 AGGTGACCGTCGTCAGAAAAAACGTGATTTCCGCAAACTTTGGATCACTCGTATCA
    ATGGGCAGCTCGTTTGAACGGACTTTCATACTCACAATTGATGCATGGTTTGAAG
    GGCTGAATCGAAGTGAACCGTAAAATGTTGGCTGACTTGGCTGTTAACGATGCAGC
    AGCTTTCAAACTCTTGCAGACGCAGCTAAAGCTAAGCTTGGGTAAATAATTAAAAAA
    AGAACCGAGGTTTCCTTGGTTCTTTTTTATAACTTTTAATGAAAAGTATGAAGAGAG
    AAACAGCCTGTCTTCTACTTATAGTATAAGATAAAAGCTTGTTACTGATAAGACAGC
    TTTCATGGTAAAGCAGTTAAAAATAGGGATTTGCGATATAATAGAAAAAACAGACGT
    TTATGTAAATAAAAAACAGTAGAATGGAGAAATATGTCAGAGAATCGTTTGGCTTG
    GGATCAGTATTTTGCGGCCAGGCTCTCTTAATCGCTAATCGCTCAACCTGTAAGCG
    AGCCAAAGGTGGCTCCGTATTGTCAAGGATAATAAGGGTTATTTCAACTGGGTACA
    ATGGCTCAGTTTCAGGGACTGGAGACTGTATTGACCAAGGAGTGCCTGGTCATTGA
    CGGTCAC
    SEQ ID NO:185 Late LPZ-127 AGGTGACCGTGGCGGAGGTTAGGGAAGTTTGACTTCTCATTTTCTCACGCACTCCT
    CTCCTCGTAACCTCGGTCGAGTCGATGGCGGCTTTTTAGTCGAGTGTGCTAACGC
    CCCTCCGGCCTCAAAATTTCCAGCTACTCGTATTTGATCAATGCTGAAATCGCGTAA
    TTACGTAGTAATAAAGCGTAATGAATTCTATAATGAAGCATGTTTCTCTATAGTTCAT
    GTGCCGAGAGGAATAATGAAAATGAGGCCTTATATATTATCTGGGGCTCAAGGAGA
    TGTTATCTTTTCCTCCTTGGTTAGAGACCGTCAACCTTCACTTGATTGGATAAAGC
    TTCATTTTGTTAAAACCTCCAAGCCAGTAGATACATACGGTAGGCACGTATTATGGT
    AGAGACATACGGTCAC
    SEQ ID NO:186 Late LPZ-128 AGGTGACCGTCCTGTTGCCTTAACCGCGAATCCAAATCGACTTGGGCTGCTTCCTTT
    CGTGCAGATATTTCTGGTTTGGACTCTAGTTCTTGCTCCTGGAAATCATGCTTGAGT
    GCTGGGTAGCTGCCTCCAAGTTTGGTTGACAGGCCCATTCCTTACAGCTTCTCTCT
    TCCGCTTATGACAGAGTAATGACAGGAATTCAACCTGACGGATCCGTCTAGCTCTC
    ACAAGGTTGGGACCCTGTCTTCGAGAGGGTTATTTCTTGAGACTGTTGACTATATT
    TGGATGAGCCCTCAGCTCTGTGTACTATTGTTCATGTACTGGATACTTTGTAAATGA
    TTTTATTCTGGTTTTACCCCGGGGGGGGCATTTTGACTCCTGGGTTTAATACGGTC
    AC
    SEQ ID NO:187 Late LPZ-131 AGGTGACCGTGGAACATGATGATTAGTTCTTCTGTGGGCCAGGATGATTAGTCTC
    TGTGTGACTGTGGGCCAGGATGATTAGTTCTCCTGTGACGACTGTTGGATAGGATG
    ATTCGTCTCCTGTGGACAGGATGATTAGTTCTCCTGTCGAGGCACCCTACCCATGC
    AATTTGGGATCATGGGAAGTACCTCTCATCTGATCAATGAGTAGGGAAATGGGGT
    AGGGACCATTAGAGTACTATCGATGGACACATCGTTGTATCTACCGTCCTATGCTA
    GGACGACCTCCATTGTTTGGGATTAGTGAGAGTGGTATGACACTCTGAGACTGACT
    TTGGGTCAGTGGAGGATGTATGATACATCCTCGATCATTTCTTCTTCTTCATAGTTC
    GAGCAGAGCAGAGCACAACAGGCCAAGTAGTGCAGGGTAGTGCATTTGATGGCTG
    GGATAGTAGCGACGGTCAC
    SEQ ID NO:188 Middle LPZ-133 AGGTGACCGTAAATAAGATGACCCACATGGAGTTTGGCCCTAGTTTCCAATTTTTAA
    CAC1CGCTCTCAACTAGGGAGAACTCCATTCGCTGATCCATTTGTCCGACTATACTA
    TCTCTGCATCAGTGCCCTACACTACTCTGCACTGCTCTGCTCTACTAAACCATGAA
    GAAGAAGAATGACCGAGAATGTCTCATGCCATTCTCTATTGACCTGAAGTTAGTCC
    TATATGAAGAGA1TGTGTCATATCACTCTTATTGACCCAAAGTCAGTTTTATTGATCC
    CAGATCAATATCACAGAGAGTGTCTCAAACCACTCATACTGATCCCAGATCAGTTTC
    ATTGATCCCATATCAAGGAGATCATCCTAGAATAGGGAGTACAGTAGATACAATGAT
    GCATCCATCAATAGTACTTCTATGGTCCCTAACCCCATTTCCCTGCTCATTGATCAG
    ATGAGAGGTACTTCCGATGAGCCCACACTGCATGGGTAGGATGCCTCGACATGAG
    AAATAATCATCCTATCCACAGGAGACGAATCCTCCTGTCCCACGGTCAC
    SEQ ID NO:189 Middle LPZ-136 CTAGGGAAGACTTTAAAAGTTTGTAAAACTAAGCATAGCTCTAAACACTGAAGTTA
    AAGACATGATTGGAATGTGCAAGTGGTTCAGTATCCAAATATTGAAGGTTGCAGAA
    TATGGGCTACTGTGCAAACGAGTAACTTTATCTATATTTTCACAAGATCATACAATG
    GGAAACGTGAGATAACAACTGCATCGGTGAACCAGAATAGTTATAAAAGTTCTTGC
    AAGTAAAGGGTGAATAATTGCATGGTGTGAATTAAGAATGACCATGTAGAGCTGCT
    ATACAGACTTCTCAAGGTTTTATATTTGAGGAGTGCGCGCTATTGATGTTGTGCAAA
    AATTTCAGAAATTAATTCTGCGGCATTTATCAAGGTTGTTTGAGCCATTTAAATAGC
    AGTTTTTGTTTCTCCAGTACTTTCAGGAAAGCAGGTTAGACGATAAAATGCATCTTC
    CCAATTTACTATATTTCTGTTTTAAAAGATTCTCTCAATGTCCTTAGCACGTGGCTTT
    CATTATTGGGACCAATGAAGATGTGTAGCAGAGGCATTACGTTATGGAATCTCTCA
    CCAAGAACACTGTTTTGGGCTTTAGATAGCTCCTAGTTATAAATGCTCCAGTGACAA
    ACACATCCTAAGTTTGGGGCAATTAATGACGCCTTTTGGTCATTCTCCTTTGGGTTT
    CAGGCACGGTCAC
    SEQ ID NO:190 Late LPZ-137 TCCCTTTAGTGAGGGTTAATAGATCTATAGTGTCACCTAAATCGCGGCCGCTCTAG
    AACAGTGGATCCGCAAGCAGGATAGACGGCATATGCATTGGATGCTGAGAATTCG
    ATATCAACTTATCGATACCGTCGACCTCGAGGG
    SEQ ID NO:191 Middle LPZ-138 GGTGCGATCCTAAACATGCAAGCTTTGAGTTTGTAACTTTGTAGAAGTGGACATTTC
    TAAGTTGGATGTACAAATCTACTGTTGGTTGTATTGTCATCCCATAAACAACTGTTT
    GATGAGATGTTTTTTTAAAAACCACATCATAATATTTTTAGGCCTTGTAAAAAAAAAA
    AAAAAAAAAAAAA
    SEQ ID NO:192 Late LPZ-140 ATTCCAAACTTTTCTTCAAGATGTACACCAACATCATTGTCCCCAACTAGTAGAC
    TGACTTTTCACCAGGTCCAAAGAGAGGGGTGGTGGAAGCAGATTTCAGGCTTTCG
    AATAAGTATCAATGATATAAGCATCATCCCCTTGCCAATTGTTCTGGATCGCAC
    SEQ ID NO:193 All LPZ-141 GGTGCGATCCCATCAGGGGTTGTGTTTCTAAGAATCACTTCCATGTTTCAAATTCAG
    CACTTGATCTTGTACATACCCAATTTGTTGCCTGCTACTAGCTAGTATTGTCTTTCA
    GTTTGAACCATTTTTTTGAGTAAATCGTGTTTAGTCTTTGGCAAAAAAAAAAA
    SEQ ID NO:194 Middle LPZ-143 GGTGCGATCCGCATTAGAGAAGCATACAGGAAAAAGAAGTACCTGCCTCTTGATTT
    GCGCCCAAGAAGACTCGTGCTATCAGGCGACGCCTTACCAAGCATCAGGCATCAT
    TGAAGACGAGAGACAGTTAAAAGAAAGAGATGTATTTTCCAATGAGAAAGTATGCAG
    TCAAGGTGTAAGCCACAGGATTTGAGCTTTCATGCAATTTTTTTGTTACTTGCGGGA
    TGATATTGCCTATATATTTCCGTCCACGTTTTTGGCAAATTCCGATTTGCATCAGAA
    TTCAAGTTATGATAGTGTTCTTCGCTTTTGAGCAGTTGATATTGTTTATCTTTTAT
    TCTCTGAATTGCAACATATTCTAATGCAATGAGTGGATTATATATTGTGGTATTTC
    CATGTTGAACTCATATAAATGAGCGTAATTGAGTGGTAGCGCTAGGATATTTACAC
    TTGGCAAAAAAAAAAA
    SEQ ID NO:195 Middle LPZ-144 GGTGCGATCCGTATAGGTAGTTTGGATGATGAACGGGCAAAGAAGGCAAAGGAGT
    ACAGGATGGATCCTGTAATTCCTGTTTCAGAAAACAGAAAATCTGCAATATAAGGAT
    GGCTAACTTTTCAGCTATGAAAATATATGGTGCAGTGGCACTCATATCAGTTGCAG
    GTTGTCAAATAACTTTTGTGAATAGGAAAGTTGTCCTCTTTTAGAGTGCAGAAATCC
    TGCAATATAAGATGGCTAAGTTTTTCAGCTATATGAAAATATATGGTGCAGCAAAAA
    AAAAATA
    SEQ ID NO:196 Late LPZ-145 GGTGCGATCCCATATACAATTACATATATTTTCAACAATTCTTTTGTTGTTATGAAAA
    TCTATTGAAATAAATTGAAATAGTTTGCATCATTTATTTATCGGAATTCGTATTTATAT
    ATTAAATTTCTGATGTCTCAAATCCTTCGTTACTGTAACGATATCATTAATATAATGT
    GTCTGCAAGTTTATTGGGCAAAACAAAATTTATTTTTCGGTCACATCATAAGTTTATT
    TTTGGTCACATCATATGCACCATCACATTAAGCATAAGCATATACAGTAGCGTAAAA
    ATACAATTATTGTTGTTGACTAGGATCGCAC
    SEQ ID NO:197 Late LPZ-146 GGTGCGATCCTAGTCAACAACAATAATATGTATTTTTACGCTACTGTATATGCTTAT
    GCTAATGTGATGGTGCATATGATGTGACCAAAAAATAAACTTATGATGTGACCGAAA
    AATAATTTTGTTTTGTCCAATTAGACTTGCTGTATATGTCTGGAGTCCTACCCTTG
    AATTGACTTGTTTCCC
    SEQ ID NO:198 Late LPZ-147 GGTGCGATCCCATATACAATTACTTATATTTTCAACAATTCTTTTGTTGTTATGAAAA
    TCTATTGAAATAAATTGAAATAGTTTGCATCATTTATTTATCGGAATTCGTATTTATAT
    ATTAAATTTCTGATGTCTCAAATCCTTC
    SEQ ID NO:199 Late LPZ-148 CCACTGCACCATATATTTTCATATAGCTGAAAAACTTAGCCATCCTTATATTGCAGAT
    TTCTGTTTTCTGAAACAGGAATTACAGGATCCATCACTGTACTCCTTTGCCTTCTTT
    GCCGTTCATCATCCAAACTACCTATACGGATCGCAC
    SEQ ID NO:200 All LPZ-149 AGAGCCTTCTTGCAGACAATCCGTGAAAACATGGCTATACAATAAAAATTCCCAGTT
    TGAATTCTAAAGAAAACTGTTCAATATTTGAAGGCCTCTGATATCACAGAGACTGAT
    ATTAAATGGAAATTCATACAAATGAGGAGAGCATGTAGCAACACTAGAAGCTTTGG
    CATAAAGCACCAGATAAATTCATAAGAACTAAATCCATAAGAAGGATCTCTCGTTCA
    CCAGTCACAATCACACTCGGATCGCAC
    SEQ ID NO:201 Middle LPZ-150 GGTGCGATCCCTGGCCCTGATAACTTTGGTTGCAATGGAAAATGCAGTACTAGGTG
    CGAAATGCTAAAGCCCGCCCGGAGCGGTGCATGAAGTACTGCAATATTTGTTGTAG
    TAAATGGCTGGTTGTGTTCCCAGTGGTCACTATGGCAACAAGGACGAGTGCCCCT
    GCTACAGAGAATGAAGTCCGCAGCCGGCAAGCCCAAGTGTCCCTGATCTTAGCAC
    TTCAGTCCAGTCGCCACTTCTTTTATTCTCTTTTTTTATAAAAGTGACGAGGCCG
    TTCTTGTGCTTGGTGCCATATGTAGAGCGGTGGCTACTTCTCCTGTGTTAGGAAAT
    GTTGCAGTACTAATAATAGAACTTCTT
    SEQ ID NO:202 Middle LPZ-151 GGTGCGATCCAATAAAGATATACTTTGCAACAATAATCAAAATATCATATGCAAAG
    TTTAAGATCAAAATAGAATGCAACAAAAAAATGGTTGTAACATAGGAACCAACAATG
    TTGCATTCAAGTAAGACTCTTTGCAAAAAAAAAAAATAAAAAAAAAAA
    SEQ ID NO:203 Middle LPZ-152 GGTGCGATCCACAAGTAAGATAATTGAGTATATATTCAAGATGCAAATATTTCATTA
    GGACCACTCATAAAGTTATCAATGATTCACAAAGAGACCTCCTGACCTCTCTCAAAA
    GTGGTGGCAACACAAGACTAGTGTAGTTTTTACTATACCTCAATGAAACTACCATCC
    TAACTGATGCCATAATCTTCTGTTATATATTACCAAAATTTATGAGATGATTGATCCA
    TAAACACTCCAGAACACATAGTCATCCAAAGGAACCTTTGCTTGAATATGGACCCC
    CTTAATTCAGGTACTTGCTACTCCAATAAATTGCTTAATCTCTCCACCGATAACCAC
    AGTTTGGATCGCC
    SEQ ID NO:204 Early LPZ-153 GGTGCGATCCAGGACATGAGGCCGAGTTTGCCATTGTGATATGATTGAGGAAGTC
    CAGTCTCAAAATTAGGTTTATCTTGATGTTTGACAAGAAATATAGAAGGGCATGATG
    AATCAAGAACCTTTTCCAAATCTGTTACTGCAACCAATCCAATGACATAATAACGCC
    AATGGTGGTTCCTGTGATGACATAATAAATTGGATTAAATTAATAACATCCCTAATG
    CCATGTGGTTAGCTGCATCATCACCGTATCCATCGAGTGTTCAATTTTTGGGATGT
    TGTATCAAAAAAA
    SEQ ID NO:205 Early LPZ-154 AAATATTTTTCAATACAACGCCATGTGACATTTTTGTGCTTCTTGTTTTTGATACATA
    CTTCCAAAAACTGAACACTCGATGGATACGGTGATGATGCAGCTACAGCCATTGCA
    TTACGATGTTACTAAATTAAATCAATTTATTATGTCATCACACGAACCCAAACAATAG
    CGCTATATGTCATTAGAATGGTTGCAGTTACAGATCTGGAAACAGATCAATGAATCA
    TCATGCCCTCTATATCTCTTGTCAAACATCAAGATAAACCTAATTTTGAGGACTGGA
    CTTCCTCAACATATCACAATGGCAAACTCGGCCTCATGTCCTGGATCGCAC
    SEQ ID NO:206 Middle LPZ-155 GGTGCGATCCGTATAGGTAGTTTGGATGATGAACGGGCAAAGAAGGCAAAGGAGT
    ACAGGATGGATCCTGTAATTCCTGTTTCAGAAAACAGAAAATCTGCAATATAAGGAT
    GGCTAACTTTTCAGCTATGAAAATATATGGTGCAGTGGCACTCATATCAGTTGCAG
    GTTGTGAAATAACTTTTGTGAATAGGAAAGTTTTCCTGTTTTAGAATGCAGAAATCC
    TGCAATATAAGATGGCTAAGTTTTCAGCTATATGAAAATATATGGTGCAGCAGAGT
    TGTCAATATAAACTTGTGAATAGGGAAGTTTTGGCAAAAAAAAAAAAAAGAAAAAAA
    AAAA
    SEQ ID NO:207 Late LPZ-157 GGTGCGATCCTCGTTGTGAAGACGTAGTGATGGAAAGGTCATGTTTGTAGGAGAC
    ATAATTATAGGAGTTTCTTTATTATAATAACCAAGAAGTCCGATCCTGGGGGCGTTG
    AGTATATAGTCAGTCTTTGGTAATTTGGTGTGGTGCTGTTTGACCTGCCTTTCCTTT
    GGAGCAATGATCCTTGAGGATGGAAGAGGTTATGTTGAGGCTCAAGAGATGATTGT
    TTGAGTTGTGGAAAGCAAAAGGTTTCCAGATGTAGTCAGATAGTAACTTCTATGCTT
    TTAATAAAATTTAGTCTGTGGGGCATGCCCCTTTTTGCTGGCAAAAAAAAAAA&GAA
    AAAAAAAAAA
    SEQ ID NO:208 Late LPZ-158 GGTGCGATCCGTATAGGTAGTTTGGATGATGAACGGGCAAAGAAGGCAAAGGAGT
    ACAGTGATGGATCCTGTAATTCCTGTTTCAGAAAACAGAAAATCTGCAATATAAGGA
    TGGCTAAGCTTTTCAGCTATGAAATATATGGTGCAGTGGCACTCATATCAGTTGCA
    GAGTTGTGAATATAACTTTTGTGAATAGGAAAGTTTTCCTGTTTTAGAATGCAGAAA
    TCCTGCAATATAAGGATGGCTAAGTTTTTCAGCTATATGAAAATATATGGTGCAGCA
    GAGTTGGAAAAAAAAAAAAAAAAAAAAAAAAAAA
    SEQ ID NO:209 Middle LPZ-162 GGTGCGATCCCAGGAGAATATTAGTTTCATGTGTTGCTATCATTTTCTTCAATATGC
    AGGGCAACCATTTGAATGAAACTATTCCTTTCGAATTTCAAAAACTTAATAGGCTAA
    CTTATCTATCTGGAGCCGATTTTCATTGACGAGTAACCTGTAAGCTGGCCAGCAAA
    AGCCAACAGATGTTCAGCTTGTTGGAACCAGTTGAAGATTGTAATAGAGATGGTGA
    ATAATCGCGGACGGCTCGGCCAATGGAATATTTGTTGCATCATCATCAAGGGGGTA
    TGAATTCCAAAGAACTTGTTGATTGAAATTCCCAAGCAAAATTCTGTGAAATGAAAA
    ATTTATTGAGACCATTGGGCAAAAAAAAAAAAAAATAAAAAAAAAAAAAA
    SEQ ID NO:210 All LPZ-165 GGTGCGATCCGACTGTGATATGTGACTGGTGAACGAGAGATCCTTCTTATGAATTA
    ATCTGGTATCTTTATGCGAAAGCTTCTAGGGTTGCTACATGCTTCCATTCTAATATC
    AGTCTCTGTGATATCAGAGGCCTTCAAATATTGAACAGTTTTCTTTAGAATTCCAAA
    CTGGGAATTTTTATTGTATAGCCATGTTTTCACGGATTGTCTGCAAGAAGGCTCTTT
    GGCAAAAAAAAAAAA
    SEQ ID NO:211 M,L LPZ-166 TTTTTTTATTTTTTTTTTTTCCAACGAGATCACTGTCATTGTTCAATAACTATATGCCA
    AAGAGCCTTCTTGCAGACAATCCGTGAAAACATGGCTATACAATAAAAATTCCCAGT
    TTGGAATTCTAAAGAAAACTGTTCAATATTTGAAGGCCTCTGATATCCCAGAGACTG
    ATATTAGAATGGAAATTCATACAAATGAGGAGAGCATGTAGCAACACTAGAAGCTTT
    GGCATAAAGACACCAGATAAATTCATAAGAACTAAATCCATAAGAAGGATCTCTCGT
    TCACCAGTCACATATCATACTCGGATCGCACC
    SEQ ID NO:212 Middle LPZ-167 GGTGCGATCCGACTGTGATATGTGGCTGGTGAACGAGAGATCCTTCTTATGAATA
    ATCTGGTATCTTTATGCGAAAGCTTTTAGGGTTGCTACATGCTCTCCTCTTTTGTAT
    GAATTTCCATTCTAATATCAGTCTCTGTGATATCAGAGGCCTTCAAATATTGAACAG
    TTTTATTTAGAATCCAAACTGGGAATTTTATTGTATAGCAATGTTTTCACGGATTGTC
    TGCAAGAAGGCTCTTTGGAAAAAAAAAAAATAAAAAAAAAAAA
    SEQ ID NO:213 Middle LPZ-169 TCCCAAAGGCAATTATACATGGATCGCACC
    SEQ ID NO:214 All LPZ-170 GGTGCGATCCCCACTGCAGAAAGATGAGCCAGTACCCTGAAATTTTGCTGTTGTCC
    ATGCCTGGGTCACGGAGGAAAGAACGGCACGGTGCAATATGATTTTGCTACATACA
    AGTTCCAAGAGTGGATGCAGACAGTGCTGGCCATGGCTGATTATTTGCAGGTGACT
    AATGCTCTTTTGGTTATCCTTACCATCATCATCTTCCTGCCATTCTTTTGTACCTCGG
    TATGGAGACGAACACCCACTTTTCAAAGTTTGCAGAGGAAGCATGTATTCATAACA
    GGAGGATCAAGCGGCATTGGCCTTGAGATTGCCAAAGAGGCTCTTCACAGGGTT
    CTTACGTGACACTGGCGTCAAGAAATCTTTCTAAACTTCGTAGGGCTGTTTGAAGAA
    ATCATCCAAGAAGTGGAGTGCGACGGAGACAAGATTAATATCAAGGTAATATACCC
    TGCAAAATGTTGTCTGGAATACAATCCAAAACCAATTTAGCAATTAACCCATTGGCA
    AAAAAAAAAAA
    SEQ ID NO:215 All LPZ-171 GGTGCGATCCAAGTGCGGTATTCTTCCTTTGGCAGTTCTCTGAACTGTTGAGAGAA
    TTTGAGTAGGATAACGACATAATTACTATGCTCACAAGCCCAGACAACACGAATAG
    ACTCCCTTCCGTGCGTCGCCTTCCAGAGGACGCAGCAGCTAAAATCTCGGCCTGA
    CTCACCACATATATATTTAATAGCTTGTATATGCCATATGAACTGTTAGCATGATCTC
    CCTCTAACTGCGAATTGTGTTGCTGTAAACTAATCCCAAAGGATGTTTACTCTGTTG
    CTTTTCCAACTGCTGATGGATTTCGCTCATACAATGACCCGAGAGCACCATAAACC
    ACCCAGCGTTGTGGCCTATGACCCATAGCTTTTTGTTCGCACAGCAATTGAAGACC
    GGCTACAGGAGATGACTAATGCACTTCCGAGAAGGTTTCACCGCGAATGACAGGG
    AAGGACAAGGCAGAGCAGCAGGCCAAGACAGCTTTAGTCGCAGAAGTTCAAGCAG
    ATCTAGATTCATAGTAAATGGAAGTTCTACACTAGTTACAAATTTAAAAACGTACCTG
    CATGGACTACACGGTTTATTTACGAGTGCCACTTGTCTCATTGTTTTCCATCAGATG
    TCTGCTGGATTGTGGTAGTGTGTTCTACCGTATCGGTGCGGGTTTTGTATATTGTG
    CGTCGACAGAGTGACAGGTGGTGATTTTACTGGCAATTAAAAAAAAAAAACAAAAAAAAA
    A
    SEQ ID NO:216 Late LPZ-172 GGTGCGATCCTAGTACAGGCGTTTGGAACAGAGTGGAGAATATGTGGAGTATTGG
    GGGATGCCCCCGGTCGTGTGTTGCTGCGTTTGGGAATTTGTATTTCTTCCATAGGC
    AACAAGTGATGTGTTATAATAGTAAAGAGAATGTTTGGGAAGTGGTGGCATCTCTTC
    CTGGAGACATGAATATTGTTACTTTGCGCAACAGTGTGGTGTGACAAGATATTTGT
    GAGCGGTTGTGCTTGCAGTGGCGGCGATCAGGTGTGTTACATGCTGGACAAATCT
    TGGGCGTGGGCTCCTATTGAGAGGTCACATGAGTTTGAGGGTTTTGCTCAGTCTG
    CAATAACTGTAGAGATATGAGCAAATTCTGTTGGGTTCACTTAATTTTGGGATTATT
    ATAGTGCAGAGGGGAGCCGGGAAGTTTCAGTGTACAGTGATGGGCACCACATGTT
    GCCAGCATTGGGGGTGCCCTGTGAATATGATTTCTATAAGTCCGGATTTTAAATATC
    TAGGCCATCTATCTCATCCAGCCTCTGATTGTGTCTGTACTAAATATATCCTGTATA
    TTCGTGATCCCTGGTTTTGAAGTGAGCAAGTTTTAGTGGAAGAGGATTTTTATTAAA
    TATATATAAAGTTTCTGTATTCAGGGTTTTGGCAAAAAAAAAAAAAA
    SEQ ID NO:217 Middle LPZ-173 GGTGCAATCCGCCATAAGAGAGGCATACAGGAAAAAGAAGTACCTGCCTCTTGATT
    TGCGTCCCAAGAAGACTCGTGCTATCAGGTGACGCCTTACCAAGCATCAGGCATC
    TTGAAGACTGAGAGACAGAAAAAGAAAGAGATGTATTTCCAATGAGAAAGTATGC
    AGTCAAGGTGTAAAGCCATAGGATTGAGCTTTCATGCAATTTTTTTGTTACTTGCG
    GGATGATATTGCCTATTATATTTCCGTCCACGTTTTTGGCAAATTCCGATTTGCATC
    AGAATTCAAGTTATGATAGGTGTTCTTCGCTTTGAGCAGTTGATATTGTTTATCTT
    TATTTCTCTTGAATTGCGAACATATTCTAATGCAATGAGTGGATTATTATATTGTGGC
    AAAAAAAAAAAAAAAAAAAAAAAA
    SEQ ID NO:218 Middle LPZ-174 GCGGACGCCTCAGGATAGCGTTAGGGTTGCCTTAGGATAGCGTTAGCTCTGCCTT
    CTAAGGTTGCCGTCTTATCCTCCAGCGTCTAGGGCTTCCACTCCTAGGATTTCTCT
    TCCACTAAAACCCAAGACAAGTGGAGAGAAATCAAGATAGAAGTGTGTGTGAAATG
    ACTCTTAAGTCATCTCCTTTTAGACTAAAACATTGAGCACATGTGGGGTTTATTTGG
    TTGCTGGCCGTCGTT
    SEQ ID NO:219 Middle LPZ-175 GGTGCGATCCTGAAACAACATATTCCCGATGGCTCTTCCGAAGGAACCATTGCTCT
    ACTGTGTGGCCCTCCCCCCATGATCCAAGATGCCTGCCTACCTAACCTGGCCAAAA
    TGAATTATGACATTCAGAATTCGTGTTTTCAGTTCTAATTACACCCTTCTGGTTAATC
    AAATTGGGACATCCCCTCCCACATCCTGTTATTAATTAAGCCATAGTCTAGTGTATA
    AAATCTGTTGATGTGTACAGCATCAAGTTAATTTCCTCCTTTTCTGTCAAAAAAAAAA
    AAAAATAAAAAAAAAAAA
    SEQ ID NO:220 Late LPZ-177 GGTGCGATCCGATCCTAAGCGGGTGCATATATATAATGACAAGCTGTAGTAACTAA
    CTCTTGTCATGAGGCCATTGCTAACATAGCCTGTCCAATGCACATAGCAGTCAAAA
    AAAGCAAATAGCCGCCATGTTCCCATACACGAAGTAAGTACCCTCCCTATTGAGTC
    ACCTTACCCGCCGAGAGAGATCCCAATTCCATGTATTCGGTTAAGTAAGCCCTGCC
    AGCTATGTCCCACCCATGAAAGAAAGTACTGATCCGAGTGGATCGCACC
    SEQ ID NO:221 Late LPZ-179 GGTGCGATCCAAACTGTGGTATCGGTGGAGAGATTAAGCAATTTATTGGAGTAGC
    AAGTACGCTGAATTAAGGGGGTCCATATTCAAGCAAAGGTTCCTTTGGATGACTAT
    GTGTTCTGGAAGTGTTTATGGATCAATCATCTCATAAATTTTGGTAATATATAACAGA
    AGATTATGGCATCCAGTTAGGATGGTAGTTTCATTGAGGTATAGTAAAAACTACACT
    AAGTCTTGTGTTGCCACCCACTTTTGAGAGAGGTCAGGAGGTCTCTTTGTGAATCA
    TTGATAACTTTATGAGTGGTACCTAATGAAATATTTGCATCTTGAATATATACTCAAT
    TGATCTTACTTGTGGATCGCACC
    SEQ ID NO:222 Late LPZ-181 CAATCTGTCTGCAATTGATATTATTGCATCCAGTAAACCAGATACACATTCACCACA
    ACATTAGAGACTCTAGAAGTTCCTTTGGCGACAGGCAAAACTCATGATTACAGATAA
    TTGGAGTTTCCTCTAACCAGAGTCAAACGATCTAAAGGGATTTGTCTAGTCCTCCAT
    TCCCTCATTCAATGAGGCGATGGCTTATGCCGTGACAACAGTTTCTATAGTTGCAT
    CCGCTCCTGTTGATCCCACAACATTTTTGGTGTTCTCTGCATCTTCTTCCTCCCATA
    TCTCTGGCAGGGCTTGTCTAATGTTGTGAATACTTGCAAGGGCAAAATCTGCTCCC
    TCTGTTCGGATCGCACC
    SEQ ID NO:223 Late LPZ-182 GGTGCGATCCTCTCAGTTACGAGCTCAATTTCGACCAGGGGTCTCGGCAAATTGA
    GGATCATGAGAAGCAGGGTATGCCCTTGAATGCCCTGAAGCCAGGGGAGTCTCAG
    GGCAATCACGAATGAAACCTGACAAACCCTAAGAAAACCCCTAGAGCGTGCCCTGC
    AGAAAGGGAATTCTTTTTGAGGCCGGCGGTCTTTCTGTCGTCTTCTCGCAGCCGTA
    SEQ ID NO:224 Late LPZ-186 GGTGCGATCCAGCAAGAGAACGAAAAAGGTATGAGAATCTATGAAATATTTGTACA
    TCACTGTATTCATATGAGGGCCTTTTTTTACAATGCGGTAGGGTTGTTTGGAGAAT
    AGAACCTGATTAAAATGTAGATGGATTCAAGCTTTTAGTGAAATGAGGCTCGGAAC
    GCAAGTATGCTGTCCACTTTGAGACTCATTCTTCTATAGTATCTGAAGCCAAAGCC
    SEQ ID NO:225 Middle LPZ-189 GGTGCGATCCCATGGGATAGTTGCAAAACACACAAATTTGTTGTGAAAGAAGAGAG
    ACACGCACAGACAACCATATGATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
    TTTTTTTTCACAACTCTGCTGCACCATATATTTTCATATAGCTGAAAAACTTAGCCAT
    CCTTATATTGCAGGATTTCCGCATTCTAAAACAGGAAAACTTTCCTATTCACAAAAG
    TTATATTCACAACTCTGCAACTGATATGAGTGCCACTGCACCATATATTTTCATAGC
    TGAAAAGCTTAGCCAGCCTTATATGCAGATTTTCTGTTTTCTGAAACAGGAATTAC
    AGGATCCATCACTGTACTCCTTTGCCTTCCTTGCCCGTTCATCATCCAAACTACTAT
    ACGGATCGCACCA
    SEQ ID NO:226 Late LPZ-194 GGTGCGATCCTGCGAGAGCCGAGGGTTCATTTTCCTTTCGACAACGACGTTCAGT
    GGCGACCAGAGTTTCCCAATCACTTCAGCGATTCTATTCCTTCGTTGTAATAAAGCT
    TAAGGAATCCATGCTTTATTCCTTGGAAGGTTTGAATATTTATATTTGTTGGCATTAA
    TGCTATATACATCTATACTAATTTTGGGTTGTTCTAAACTTGTTTTGAATAACTTAAA
    SEQ ID NO:227 All LPZ-195 GGTGCGATCCATGGCAAAGAGCTCGTTCAAGCACGATCATCCTCCAGAGAGAAGA
    CAAGCTGAAGCTTCTCGGATTCGAGAAAAGTATCCGGACAGGATTCCGGTTATTGT
    GGAGAAGGCTGAGAGAAGTGAGATACCTGATATTGATAAAAAGAAATATTTAGTCC
    CAGCAGATTTGACTGTTGGGCAATTTGTTTATGTTGTCCGAAAAAAAAAAAA
    SEQ ID NO:228 Middle LPZ-196 GGTGCGATCCCCTGTATTCTTGAAAGGGTTATAACGGAAGATAGCATTTTGCTCAG
    ATTGTAGACAGTCTGCATGATTTGTCAATACTACTATTTCGCATTATTTGTTAATACT
    ACTAATCCTTGTACTCATCTAGACTATTTAATTATTAAATTCTACAGTTTCTTTCTCCT
    AGATGGCAAACAATATGAATAAAATGCCAATAGTTTTGGAACTACTCCATTAAGAGC
    TTTAGATGATTATCATTCATCATTTGCCTGTTTTGAATCGTAAATGAATGTGTCACGG
    TCTTCTTTTCTGTTAGTCTCTATGCTTTCATCAGAAGAGTCTAAGCCAGTTACTGGA
    AGCTATTTGTCATCTCTTTAAACATTGTTTCCGTGCCAAAAAAAAAAAAAAAAAAAAA
    AA
    SEQ ID NO:229 Late LPZ-197 GGCAGAACTTCCAAAGTCTAGTATTTGATTAACTAATATGATGAAGACACTCAGTCT
    ATAACATGACGCCAGAAATCAGACCATATGCATGATAACTAGCACGATTAAAATACA
    ATTCGCAACCTTTAATACACTAAAAACGTTTACTGTATAGTCCACTCAGAACATTTC
    GATAGTATTGTCAGATCGACTTATTTAGCTCATATTCAGCAATCTGAACTGTACGAT
    GCGGCTCATTCAAGGGCATTTGGGTTTGCCCTTGGCATTCTTCATATCCCGATAGC
    AAGGACACGCGTTCTTGTTGCCATATGTCCCTGGGGGATCGCACC
    SEQ ID NO:230 Early LPZ-198 GGTGCGATCCACATTGGCCAGGCCGGTATTCAGGTCGGCAATGCCTGTTGGGAGC
    TTTACTGTCTCGAGCACGACATTCAGCCTGATGGACAAATGCCAAGTGACAAGACC
    GTTGGCGGTGGAGATGATGCATTCAACACATTTTTCAGTGAGACAGGTGCCGGTAA
    GCATGTTCCTCGT181GCCGTGTTTCTGGATCTGGAGCCAACTGTCATTGATGAAGT
    TCGAACCGGCACATATCGGCAGCTTTTTCACCCAGAGCAGCTGATCAGTGGCAAA
    GAAGATGCCGCGAACAACTTTGCTCGTGGCCATTATACCATTGGTAAGGAAATTGT
    GGATCTGTGCTTGGATCGCAGC
    SEQ ID NO:231 Late LPZ-199 GGTGCGATCCCAGCATTGGATGCATTTCTAGCACAAAGCCATCTTGACTAAAATAG
    CACTGCGGGCAACTGCAGTCCATAACTTTCAGAGCATTGTTGCTGCCTCAATTGTA
    TACCAATCCATATTCTAAAAATTAGACCTGGAAACCAGTCAGAAATTTAATGTTTTCT
    TGCAGAAAATGCCCTTTTAGAAAAAGGAGAGAATAACTGCATTCAAGTTCTAACTCC
    CAGACATAGCCTGGCAACGTCATTCATCAGTTCGGATCGCACC
    SEQ ID NO:232 E,L LPZ-201 GGTGCGATCCAGAAAACAGCACAAGCAATCTGTAAGACCAATATTATTATCATCTCT
    CACTGCTCGTGAACAAAATGCTGGTTCATAGCCATCACGAAGGCTAAGGCTACTAT
    CCAGCCAAACTGATCTOCAACAATAATTTCATAAGCTTAAATAAATAGTCCATCCAG
    TGGATGGAGCCAGAAAGCCATAGAAACTTCAAATACTTGTGGTATCAATCTCTCCTC
    TGTTAAGGGAGGTATCAGATCAGAAGCACTAATCAAATGCATACATAAATGCAGTA
    GACTGCAATAAAACAAAATCTGCAGATAGCAACTGAGCGCTTAACGAACGGAAAAG
    AGTTTAACTTGATCTATCACAGGATCGCACC
    SEQ ID NO:233 Late LPZ-202 GAAAATGGGAGCCTCAAATATTCAAAGCCTCATCTCAAGAGTCTCAGATTCGGATT
    CATTTCATTTGGTTCGTAATAAAATAATGCATCAAATAGTTATTATCCACAAAAATGG
    GAGAATTATTACAATCTGTCTTCTCAACATAAAGTCATAGCATAGCATAGAACCACA
    CCACAGTCGTCATCATTTGTTTTGTTCACCACCGAAGGGGCTCTTTACAGCGTCCA
    TGAAGCCCTGTGTAGCACCCTTCGCCTTGTCCCCCGCCTGTTGGAAGAAAGAGCC
    AGTTTGTTCTTTCCCCTCTTGGGCTTTTCCCGTGATGGATCGCACC
    SEQ ID NO:234 Late LPZ-203 GGTGCGATCCTATTATAGAACCATGACTCTTGTCGATGGGGCATAAACTTCTCATTC
    TTAGGCGTGCCTACTGTGACTCTTGCCGATGTGGCATAAACTGCTTATTCTTAGTT
    GTGCCTTCTGTGCAGAACTTGTTGAGTCGGTGGATTACACTGAC
    SEQ ID NO:235 Late LPZ-204 GGTGCGATCCATTAACTAGATTAACGATAACATTCCTCTGCATCCAATCCAATGCTC
    ATCTAAATCTACTTCTACTTAGATCTCTGCCTCATCTTTCTCCACCTCCTCATCCATT
    CTGAAATATTAATTTCTGCATAGATTTTGTTAGGGTCTAGTAATCATTTTCATGAATT
    TAAATCTGTTCTAGTCTCTTATTATTATGCTGCTTATGCTAGCATCAGAACCTGTGTA
    TAATTCATTCATGTATATATTGGATTACACAAATTATACGGATGCCAGAAAAAAAAAA
    AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
    SEQ ID NO:236 Late LPZ-205 CTTGAAGCTGATATGTTTGAACCCGAAATTTTGTTACCCAACTCCAGTGTACATTGT
    GTCACTGTCAAAGAGAACATGAGAGCTGCATGCAAGCTTTTGCATGATAGATAGAT
    TACTGATCACCGAACATTTCTTACTCTACTTTCCTCTCCTATCCCCAGTGATTTTTG
    GGCATTTTCTATACCCTTCGGATCGCACC
    SEQ ID NO:237 Late LPZ-206 CTCATGAACAGCAATATGATGCATTCCTCTTATACACATTTCATATATGTCACCCTT
    GCCGTCATGGCTACTCTAAGAAGAGCAAAACAGACCCATTGAATCTTTACACGCGC
    TTGTTTATATGAATACAAATAATTTAGGCGTTTCTTTACACGCCCTTGTTTACATTAA
    TACAAGTGATTTAGGCGTTGTTACCAGAATAGTGCCACGGATCGCACC
    SEQ ID NO:238 All LPZ-207 GGTGCGATCCCAAGATAGAAAAGGGAACTATGGTCTCGAGGAGTGTCAGGTGCTA
    CAGATCACAATATACATAAGGGTCTGATAGTAGTACTCGGCCCAATGTTTGAGGGC
    TCTAACTAAGGAGGATCAACCGTACCCTTAGCCGTAAAACCCGACTACCCTATCGT
    ACGGGCGAGTAATCTCTCTGAGTGTTGTTCTCGGTGTATCGTAGCAGCAACACGG
    CTGACGGTTTATCTATGGTGAGGTTTCAAAGGAGCTAGGGGGCTTCCAATATACCC
    AGAGGGTACTTGGAAGACAGTTTATACGCGGTTCTGTCTAATGCGCTACTACTCGA
    AGGGGTACCCACAGGGGTACAAGAGAGTGCAACAAGCATGACCACCCCTTGTAT
    TTCTTGCATGTATGCCTCCCCAAATCCGCAGGTTTATGCGCTCATTGACAGATTCC
    GTGGTTTAAAGATGCCGGAACATGTCTCTAGCCAAAAAAAAAAAAAAAAAAAAAAAA
    AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
    SEQ ID NO:239 E,L LPZ-208 GGTGCGATCCTCCTAACCTGCAATGTCCTTCCTGCAATTATCAACAGA
    AATTAGGTTTATTTTTCTTTTTGTCTTTTCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTT
    TTTTTTTTTAAGTAAACGACCATTTCAAACGCCATTCAAATGCTATGAATTAATGTT
    GAATTAATGTTAGCATTAAGTCTTTTAACATTTTATGTAAGGCATATATATCGTTCCA
    ACTACTCTTACAATACACCTGCGGTGTACTCCTGCCACCGCATGTACCACCGTTAC
    ATGTACGCCTGCCAGCACATCTAACAGGTGCCAACTCCTTTGAACTCATCGTCGCC
    ATTTTTGTATGCATATTTGAACTCATCGTCGCCATTTTGGTATCTTCACATATGGCC
    AGTCCAGGATCGCACC
    SEQ ID NO:240 Late LPZ-210 GGTGCGATCCAAGGAGTGGGCGTGCAATGCGTCGAAGATAGCCACCACTGCAGG
    GGCGTGGCATGCTGCCGTGCTTCCCACAGGGAGATCAACACCTGCACCTCCGCCT
    CCTTCCGCGGTTACCACGAG
    SEQ ID NO:241 Middle LPZ-211 GGTGCGATCCAGCCACAGAAAGATTGGTTTACTCGATAATTGAACGGTAGACTTTG
    TGCAGGTTTAGATTGTGTACATGCTGATCAGTATTGTCTACACCATTTTCAATCTTG
    TTTAGTTCTATGGTAATTTATGTAACAAATTCAGCGATGTTGGGGAAATTGGTCACA
    TCAGCTTTGTGCCTATATATTCAAGTAAATCAGGGGATCCATTAATACTGCTTTT
    AATAATTGGGGCAAAGTTGTGGGATGACTGCTTCAGCGGAATACGTGCTTTTCATA
    GTGCTGTATGACATTTTGTTGAATATGAATTTCTTTGTGATACAGTTGCGCGAAAA
    AAAAAAAA
    SEQ ID NO:242 Middle LPZ-212 GGTGCGATCCATGCCAAGAGGGTGACCATCATGCCCAAGGACATCAGCTCGCTC
    GCCGCATCCGTGGAGAGAGGGCATAAACAGTCAGTCAGATCCAATGGTGTGTTTT
    CACACCACCATATGTTTCTTTTACTAAATTTGTTAGGTCCCTTCGGTGGGTCTTTTC
    TTTCCCCCGATTTTAGTATTTTGTTGTTCTTCTGAGTTTCATCATTGCAAGTACAAGA
    TGCAGAATTGATGGTTATTGGGACTTGGAGACTGGTTATTGCTATGTAGAGTATTTA
    TATTAGACAGGTTTCACTTGAAGATATAAAATTG
    SEQ ID NO:243 Late LPZ-213 GGTGCGATCCTCATGTGTATAACCGAAGTTTGCGGGATTCAGATGGTCAGTATCT
    TAAATGTCCAACTTTCGGTACGAATGGGGTGCGTTCTGAAACGTGCCACGAAAGAG
    GTGTTCAGGATCTGTCTGAGGCATCTTTCCGGTATTTTCCACTTCCATGGTATGAG
    AAACTTTCGTCTTGTTGCAG
    SEQ ID NO:244 Late LPZ-214 AGGAGACACAACTTTACGAAAAAGTTCAATCTGGAGTCTTCTAAGTTTTTCAGACTC
    TCTAAATATGAAAAGCGCCGAGTTTCTCCTATACTGGACTCGTTAAAATTTTACAGT
    AAAGGACCTGTTCTATTACAAACAGGAACGGACCGCTCCTCCTTAGGGATCGCACC
    SEQ ID NO:245 Late LPZ-215 GGTGCGATCCAGCAAGAGAACGAAAAAGATATGAAGAATCTATGAAATATTTGTAC
    ATCACTGTATTCATATGAGGGCCTTTTTTTACAATGCGGTAGGGTTGTTTGGAGAAT
    TAGAACCTGATTAAAATGTAGATGGATTCAAGCTTTTAGTGAAATGAGGCT
    SEQ ID NO:246 Late LPZ-216 CTCAACATAAAGTCATAGCATAGCACCACACCACAGTCGTCATCATTTGTTTTGTTC
    ACCACCGAAGGGGCTCTTTACAGCGTCCTTGAAGCCCTGTATAGCACCCTTCGCCT
    TGTCCCCCGCCTGTTGGAAGAAAGAGCCAGTTTGTTCTTTCCCCTCTTGGGCTTTT
    CCCGTGATGGATCGCACC
    SEQ ID NO:247 Middle LPZ-217 GGTGCGATCCCATGGGATAGTTGCAAAACACACAAATTTGTTGTGAAAGAAGAGAG
    ACACGCACAGACAACCATATGATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
    TTTTTCGGGACCAAATATTTTTCAATACAACGCCATGTGACATTTTTGTGCTTCTTGT
    TTTTGATACATACATTCCAAAAACTGAACACTCGATGGATACGGTGATGATGCAGCT
    ACAGCCATTGCATTACAGATGTTATTAAATTAAATCAATTTATTATGTCATCACACCA
    ACCCAAACAATAGCGCTATTATGTCATAGAATGGTTGCAGTTACAAGATCTGCAAA
    CAGATCAATGAATCATCATGCCCCTCTATATCTCTTGTCAAACATCAAGATAAACCT
    AATTTTAGGACTGGACTTCCTCAATCATATCACAATGGCAAACTCAGCCTCATGTCC
    SEQ ID NO:248 Late LPZ-219 GGTGCGATCCTGGACTGGCCATATGTGAAGATAACAAAAATGGCGACGATGAGTTC
    AAATATGCATAGAATAAGCGTTCTGTAATTGGAACGGCCATAGGAGTTGGCACCTG
    TTAGATGTGCTGGCAGGCGTACATGTAAACGGTGGTACATGCGGTGGCAGGAGTAC
    ACCGCAGGTGTATTGTAAGAGTAGTTGGAACGATATATATGCCTTAACATAAAATGT
    TTAAGACTTAATGCTAACATTAATCAACATTAATTCATAG
    SEQ ID NO:249 E,L LPZ-220 GGTGCGATCCCATGGGATAGTTGCAAAACACACAAATTTGTTGTGAAAGAAGAGAG
    ACACGCACAGACAACCATATGATCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
    TTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTTTTTTTTTTTTGTGAAGTGACAAAATCTA
    AACCAAAGATTAAAAGGCTTTGGCTTCAGATACTATAGAAGAATGAGTCTCAAAGTG
    GACAGCATACTTGCGTTCCGAGCCTCATTTCACTAAAAGCTTGAATCCATCTACATT
    TTAATCAGGTTCTAATTCTCCAAACAACCCTACCGCATTGTAAAAAAAGGCCCTCAT
    ATGAATACAGTGATGTACAAATATTTCATAGATTCTCATATCTTTTTCGTTCTCTTGC
    TGGATCGCACC
    SEQ ID NO:250 Late LPZ-221 GGTGCGATCCCAACCAGGTGTCCATGCAATATATGGTGAGCATCAAGTTTGAGGTG
    GTTGATTGAAAGTTACAAATTGGTGACATCTGAAGTCTCATTCAGTTATGTTTTTGT
    TATAAAAACCATAACCAATTTTGTATATAAGATCCATAATCAATTTTGGCCAA
    SEQ ID NO:251 Late LPZ-222 GTTTTCAAGAAGAGCCTGACGGTTTCCTCGGCGGGATGACGGAAACAGGAAGCGG
    CCGGCCGGTTCCGGACCCTCCGCAGGCGGAGCATAGCATTTTGCCGGAACCACC
    GCATGTCGTGCACCCAACATCCGCGTCTGACCAGCGGAGGCACATGCACCCAACC
    CTCCCGGTTCCATGCACCTCGGGCAGCGCGGCCACCCGCCGGCCATCGGCTTAT
    CCATCATGGATCGCACC
    SEQ ID NO:252 Late LPZ-223 TGGGCGAATCATATGGCTTGCATTTTCATTGTAACATGTATACGTTAAGGATTATCA
    TAATGCCTCCAAAACCTTGTATCTTCGTCCTTGCCACAATACATCCAGGATAACTAA
    TGGAAGCTTGACATGTCTTCACCAGTAATAATATATCAACTATAATACATGCCATTC
    TTTATCAGTTTTGAACAAAATAATCGATTTGCATTCTTGACAAAGAACCTCGCGCAT
    AAAAACAAATAAATTCTCATAATGCCTCCCAAACCTTGTAGTCTGGGCCCTCAGTCG
    CCACAATCCATTTAAGAGGAATTTGGGGGTTGATAGTGCCCAGGTCCAATCTTCAT
    GAAAATTCGTTCATCAATCTTTGCTGCATACACATCTCTCTCTGCTTTCACTATCTG
    GGATCGCACC
    SEQ ID NO:253 Late LPZ-224 CCACTATAATGAACATTGATATTACAAATATAATATACATAATATTACAATTCAAATC
    ATTGACAATGAGCAGGCACTACTTGCAGTGCTTTGGAATTCAGACTTCTGATTTGC
    ATTAATTCTTGTAGACGCTTTTCTGGGAGGGCAGGTTTTCCGCTTCAGAGAAAACC
    ACGTACAAAACGATATTAAATAAAAATAGACACATACAAAAAATACTTCATTTTTTGC
    TCTTTCCATTTGGTTTCTTCCTCTATCTCCATTTTGGAGGGCTTAAATGACTCAAAT
    TTAAAAGTCAACAACAGAGTGCAGCACATTCTATTAGCTTTGCTGTAAATATCTGAT
    TGGATCGCACC
    SEQ ID NO:254 Middle LPZ-225 GGTGCGATCCGCATTAAGAGAAGCATACAAGAAAAAGAAGTACCTGCCTCTTGATT
    TGCGTCCCAAGAAGACTCGTGCTATCAGGCGACGCCTTACCAAGCATCAGGCATC
    ATTGAAGACTGAGAGACAGAAAAAGAAAGAGATGTATTTTCCAATGAGAAAGTATG
    CAGCCAAGGTGTAAAGCACAGGATTTGAGCTTTCATGCAATTTTTTTGTTACTCGCG
    GGATGATATTGCCTATTATATTTCCGTCCAAGTTTTTGGCTAAATTCCTATTTGCATCA
    GAATTCAAGTTATGATAGGTGTTCTTTCGTTTTTGAGCAGTTGATATTGTTTATCTTT
    TATTTCTATTATTAATCTTCTAAGTTGGATCGCAC
    SEQ ID NO:255 Late LPZ-226 AAACAGACAAATATAGAAATATGCATACATAAGTCCCTGCAGAATTGTTTTCCGCAA
    TGAATTCTGGTTTATGGCAACATTACCTACTTAGTACTAACCCTAAGATTATTTTCAG
    CTCTGATAAGTGGCATACGTGTATCAATCTTGCATGAGTCTATCCCTGTTTTAATCT
    TTTGTTGGGATCGCACC
    SEQ ID NO:256 Late LPZ-227 GTGGAAGCTTCATTGTAAAACACTACTGGTTTTGAGAGAACAAAATATATACGCTAG
    CCGAGTGGATTATAACAAAATATAGGCTTTATTTCTATTGGATCGCACC
    SEQ ID NO:257 Late LPZ-228 GGTGCGATCCCATACATTAACATAGCCATCACAGCCCCCAGTGGCAAAAGTACCAT
    AGCTGCAAAACATTATAAAACTAACATTCCTACAAGGAAATAAAATACAACTAAAAA
    AGCAAGCAATAGGCATTAGGGGAGGGAGAAGCTAAAACTATTAAGCAACTTACATG
    GGATGAAAGGCAATTGCGTTTACTGGATAAACAGTATCTCTGCCAGCCTCTGACTT
    GCGATGACATTTAAAGGCATATTTTTTAAGCTTGACCAGCTTCAGATACATCATAAT
    ACTCCATAGCCATGCGAGCTTCCACAGAACTAAGGGGCAAAACCTGTCCATTGG
    ATCGCATCA
    SEQ ID NO:258 Late LPZ-231 GGTGCGATCCAACTGAGAAGGGTGTTGGTGGAAAGATGACACCAAGTGGGTTCT
    CTATTCTCCAGAGGATGCAAGAAAAATTCTGAGAGCAAAGAAGAATGGGGACTCAA
    ATATTACGTTGGGTTCTGTTAAATCTGCCAAGTACCCTTCAGGAAAGCTTTATGCCA
    TAGACCTGGTGGCCATGAAGCAAACCAATGTAAACACTGGCTTCTCCAGAGATATC
    AAAATCATCAATTCTTGCCCTACTGATGATCAGGAAGATGTAGAGTCTGATGAAGAA
    GATGAATTATTCACATTCTCTCGTCCTGTCAAAGTTGAAGTGATTAACCAGAGCAGG
    AAACCTGATAAGATTGTCAAGATGGTTCCTTCTGTCACTGTAGACCTTGAGAAATTG
    ACTTCTCAATACCTCCTGGAGGATGAGTGCAATTTGGTTCTAAAGCTTCCCAGGGC
    TGCAGCTGCCCAATCGGATCGCACC
    SEQ ID NO:259 Middle LPZ-233 GGTGCGATCCAGCTAATCAAACTTAATGGAGAGCCCTCCCAGGAAGAGTAAATGG
    TAGTCACTTGAAGCCCTACACGGGTGGGCTGGCGGTCTGACTAACTGACCAAAAC
    ATAGTCTTCGCGACCCAACAAGCCAGACAGAGGTGTGGGACTATAAGCACAAGTAC
    TAGAAGCTAGCATCAAAGTAGAGAATTAAGTTAGATACAGATGATTCAGAAGCA
    ATGGAGCAGATCCAGACCACGGTAGCATGGTGAGTTACGAACCTTCACGCCACAC
    CAACGCAATTGGTTAAGACTTCGCACTAGGATCGCACC
    SEQ ID NO:260 Late LPZ-234 GGTGCATCCATAGTTCCTTTGCTAAGCGACTACTCTATCTCTTTTGACATTCTCC
    AAATATTGGGTCTTTCAGTTCCTTCAAATGCTAGAATCATATCAACATGGGATTTAG
    TGAGGCCGCAATACTAACCAGGGCATTAAAATAATACATTTCATTGATCCTATTCCC
    AAAACATTTCCCGCTATCGTACGTTGACTCAGCATATTTAGAGCAATTCTCTTACA
    AACCTTAAGAAGGTTGTTCATGATAGTCTTTCCGTCTGCAATATTGGATCGCACC
    SEQ ID NO:261 Late LPZ-235 GGTGCGATCCCACCCAAGAGTTAAATTCACTTCTCCGCCTTTCTGAGGAAGAGCAC
    TCTTTGGATGATATGAAAAGTGGTCCACTCTTAACCGTATTCGGAACCCTGTTC
    CGCGGACGGTCGTATGGCGTAACCGGCGCAGACATTTTATCTCCTCACACAATATC
    AACATTCAAGTCCCCGCTGTTCCCCGTTGCCTTTCTCTGCTCCCGACCGTTAAACA
    AGAACGACCACAAGAATGAACAACACCGCAACCGAAACCTGACCCTCCACGTTGTC
    TTCGGTTCGGATCGCACC
    SEQ ID NO:262 Late LPZ-237 GCGGACGCCTGGCAAAAACAGAGGGTATGCTCAAGCCTTACAGAAATTGAAAAATA
    AGAGAACGTATGACCATCAATCTCAATCTCAAGAAAAGAAGTTGCAATACGACTCCA
    ACACTTTTGAAAGTTGGAGGTTTGCTCTTTCTAGCGTTGCAGACATGGTTGGTTTTG
    AGCTGGAAGCGTGTAACGGGCACTTTACAGTTGCGGGAATTGGAGATTGAGGACC
    CCCTCTCAAACGTCGATAGGGAGGCTAAGCATCTATAGAGGATTGTGATTGGTCCT
    TTTCCGCTACATGGAAAGTTTGTCAAACTCAGAAAATTACCAGAAGAATTCTGTCGT
    CTTCTCGCAGCCGT
    SEQ ID NO:263 Late LPZ-239 GACGTTGTAAAACGACGGCCAGTGTAAAGAGCAGCCCCGATGCGCCGAAGCTCGC
    GAGGGAAAAGCTGCAGAAGATGGGACCGATGACCAAGAATGAGATCATCATGAGC
    GGCACGCTACTGGTCACGGTGGGTCTTTGGATATTTGGGGGTAATGCTGAACGTGG
    ATGCTGTTACTGCAGCGATCCTTGGTTTGTCTGTCCTACTCTGCACAGGCGTCCGC
    SEQ ID NO:264 Late LPZ-240 TACGGCTGCGAGAAGACGACAGAAGCAGAACCTGCCAATATAGGATCAATTGAATG
    TTGTGGGATTGCTGCATGCCCACCTTTCCCAGTTATTACTGCCTTGAAGAACCCAC
    AGCCAGCGAGTAAGGGCCCGGGTTTCGAACCAATCACAGATGTAGGATAATCGCT
    TGAAACATGCATAGCGAATATGCCTTCCACATTTTCCAGTGCTCCCTCCTCTATCAT
    TCTTTTTGATCCTGCACCTGATTCCTCTGCAGGCTGGAAGAGTAATATGACAGTTCC
    CTGTAACAAATGCTGACGTTGTTGCAAAATCTTTGCACCACCAAGAAGCATGGTAA
    CATGTGCATCATGTCCACAGGCGTCCGC
    SEQ ID NO:265 Middle LPZ-241 TACGGCTGCGAGAAGACGACAGAAAAGAGGCAAACCGAGCTCGACACCTCCACTC
    AGAGCATTTGCAAAAATCCACAACAAATCTGGAGCCAAGGTCTTTCCCTCATTGAAA
    ACATTTATCGGACACATCAATGTCTGTAGTCTTTCCCATGGTCCATCCAGAGTAATC
    ACGGGAAGAACAATGCACTTCAGTCAGAATTTTTGATGACAGCTATCAGCTCCTG
    ATCCTTTGAACCAGGTATATAATAATCTTGACCTGACTCCTGTTTCAACAGTGTAGA
    GGTTCTGTCAACCTCAAGCAATGAATCGGCAGAACTTCCATTTGCTGTTTTGTCAAT
    ACAGGCATTGTTTTTACCAAGACTGTGACGCATCTTCTGTCCTTGTCTATACAGTGC
    AGTTTGTTCAAGCATAGACTTATGTGCTAGAACATGTCTTCCTTTTAAATTGTAAGA
    GAAATGTAGGGGTTGACTGCTTTTACTGAGGCGTCCGC
    SEQ ID NO:266 Middle LPZ-242 ACGGCTGCAGAAGACGACAGAACCCTGGCTGACTACAACATTCAAAAGGAGTCTAC
    CCTGCATCTGGTGCTCCGTCTAAGAGGAGGCATGCAGATTTTTGTTAAAACCCTTA
    CAGGCAAAACAATTACTCTGGAAGTGGAAAGCTCGGACACTATTGACAATGTAAAA
    GCTAAGATCCAGGACAAGGAGGGAATCCCACCTGACCAGCAGAGGTTGATCTTTG
    CCGGAAAGCAGCTAGAAGATGGTCGTACTCTGGCCGATTACAACATTCAGAAGGA
    GTCGACCCTTCACCTGGTGCTCCGTCTCCGTGGTGGCTTTTAGGTTGGCTGTTGT
    GTGTCAATGTAGTCTGGTGATGTTCAGTGGTTTTCCTGCTTAATCCTTTTTATGTAT
    GCATGTGTTTGTTGTGTTTGTGTTTTGTCTCTATGTTTTTTCTACTTGGTTTGTCGGT
    CGGTTGAAGCCCGGCTGGTGTCCTGGTAGGCGTCCGC
    SEQ ID NO:267 Middle LPZ-243 GCGGAGGCCTGGACAAACACAGAAGGCGAAGTAAAAGCCAGTCTTACTTTTCATGT
    AAATACTATCAAACTGCATGGCCGTTCCGCTGGTTGGCAATACCACACCTGCGCCG
    GTAGTGCCAATGAACACTGCACCGGCAGCTCTTTCAGAAGTTGCAGAGGACTTACC
    ATTTTAATTTTCACGGCATCCCGTCAAACGGCGGGATGCTTTTAATTTTTTAATCAA
    AAAAAATATTAATTATGGCACACAATATTGTTTTCAACGAACAGACAGGCAAACACA
    GTTTCTTTAGTGTAAAAGAAAAAGCATGGCATGGTTTGGGGCAAATTGTACAGGAC
    TATCCCAACAGTAAAGAAGCATTGCAATTTGCAGGGCTTGATTTTGAAGTTTGCAAA
    AGGCCCAATATTCACAGGCTTGATAATGGTAATGAGATTATTTCTACCAGTTCATTC
    TATACTTACCGTCCTGATACCAACGCCATATTAGGCGTCCGC
    SEQ ID NO:268 Late LPZ-244 GCGGACGCCTGAACATAGGAGCATTCTTAAGCATATCAGGTATAACCATAAACCTG
    ACTTTGCTGCCCCGAATAAAGACATGCTCCAATTGGGATACTTTTCCATCCTTGGC
    GTGTNTGTGATGCCCTCGAGCTGGCAATTCCAGTTATCTTCGCATTCGATCATGCT
    ACCCCTGTACAGCTCGCCACTTTTGAGTTCAACTGTCACAACATGCCCGGCTGCTT
    CATGGAGCAACTTCACAGGAATCCCCAAACTTCTGCTCATTTTTTTGTCACTGCTCA
    AAAACCCTAAACCCCAGATAAAACCCTCGGTTCTGTGCCTTTTATCCCCGGGTGGC
    TTATTGTTGCAGTAGTTGGCAACGGCTAGACTTACTCACATTTTGATTTCAATCTTT
    CTAAGTTTGCCCTTTTGGGTTTTCCTCACAGTAGATCCTATTTTATGTATTTTCTCGT
    CTTCTCGGCAGCCGTA
    SEQ ID NO:269 Late LPZ-246 GCGGACGCCTGCAGGAATCGGCCGATTTGCAGTTCGAGGCATAAGCGCATCGAG
    GTCGCGTTCGATGTAGCAATTAAGCGCGCATGAACCGCCGCTAAGCAAGCCAGTC
    CCAATCAAAGCACATGCAAAGCGGATGCAATCAAATCTTCCGTTGTAAGCAAGCAC
    AAATCCAACTGCACATGAGATCACCACCATGAATGCAATTCGAGTGCGAGCTAAAT
    CCCAAAACGCTGCGAGTGTCCCCTGAAGGCGATTCGTATGTAATATTTGACCGCTG
    CTCAACACAAGCAGTACTCCAAACACCAGTGCTTCCGCCGTCAATTCTGTCGTCTT
    CTCGCAGCCGTA
    SEQ ID NO:270 Late LPZ-247 CTGCGAGAAGACGACAGAACACAGACACAAAATTTGGAAACTACAGAAAAGACCAT
    GTCATGAAATCTTCATAATTGGGCTTCAGATGCAGAGGGGGTCGGTTTTGGATTAA
    GCAATGGCTGAAGTGCTTTGACAACAATACTCATGTTAGGACGAAAATCTGCTTCAT
    ACTGCACACACAATGCCGCAACAGCAGCCATCTTTGCAACAGCCTTTGGAGGATAT
    TCACTCTTCAACTTGGGATCAACACACTGCTTTACTTTGTCTTCACTCAATCTTGGA
    GTTGCCCAAGTAACAAGGCTTTGTTGTCCCCTAGGCATTGTATGGTCCACAGGCGT
    CCGC
    SEQ ID NO:271 Late LPZ-248 TACGGCTGCGAGAAGACGACAGAAAGAGACAGGCTTGGACTTCGTGGCCTTCTTC
    CACCACGCATTATTTCTTTTCAGCAGCAATGTGATCGTTTCATGGTTTCTTTTAGAT
    CCCTGGAGCATAACACTCGAGATGGTTCAGCTGACTTAACAGCTCTGGCAAAATGG
    CGTATTCTTAACAGATTGCATGACAGAAATGAAACACTATACTACAAGGTTCTTATA
    GATCACATTGAAGAGTTTGCTCCAATAATCTACACTCCAACTGTAGGATTGGTTTGT
    CAGAATTATGGTGGGCTGTCAGGCGTCCGC
    SEQ ID NO:272 Early LPZ-249 GCGGACGCCTCAATAGTATGGAAGGGCAGCTGCACTACTCAGCATGAGTGGAG
    GCCTAAAAGTTTTGTTAATCTTTCTGGTGAGGTGGACACCAAAGCCCTTCACAACA
    GTGCAAAGGTGGGGCTATCTCTGGTTTTGAAGCCTTGAAGGATATGCACTATTTGG
    TACAGATTTAAGCGAAGGTCTGTGCCAAATTTTTATTGGAATTTTTGAGTTTTTCCTT
    TCAGAATAATTATTTCAATGCCTGTGTTTTCTGTCGTCTCTCGCAGCCGTA
    SEQ ID NO:273 Late LPZ-250 GCGGACGCCTTTTGCCCAATTAACATCCCTGCATCTGCGCATTAAAAATTGATTGC
    AGACCTGAGGTTTAAGTGGAAGCTTCTTCCACCATCTCTCCCCTGTTTAAGGAAGA
    CCCGAAACCCTAGCCACTGTCTCCTCTGTGACTTAAAATTCCAGTTCACCAACCTTA
    ACTCTGCGTCCGTTAAAATTCTGGGCAAACTGCACTGCCAATTGGTCATCATATCCT
    CTGAATTTGGCAAAGAAAACATAGGTCATTCTGTCGTCTTCTCGCAGCCGTA
    SEQ ID NO:274 ND LPZ-251 GCGGACGCCTCGTCAATCCATGGTTGTAAACATGCCTTCAAAACTGTTTCCTTATGT
    CGCACAATGTCTACATGTTCCTTGAGCGATTTTTCCTGCTGCATTGCGAGCCTCTG
    TGTAAGTCCCACTATCTGCGCTGTCCCTTTTACTTCATAATACTTCTGTCGTCTTCT
    CGCAGCCGTA
    SEQ ID NO:275 Late LPZ-255 TACGGCTGCGAGAAGACGACAGAAAAAACTGTATACGAGTAGGCAGCGAGTCCTG
    GCAGTATGGGAGATTGAACTCCAATTACATTTAGTTACAAGTAGCATCAACAGTGAC
    TGAGCCAAGAGCTCTACACAGAAAAATAAAATAAAAACTGTATATATTTACAGGAGA
    AACCCCTATGGCCTCAGGGCCTGAATAAATCAATCGCAGCGGTGGTCGATGTGGC
    CTTTTCAGGGCTGCAAATCTTGCAAGGGGAAGCCATCATCCTTGTTCCGTATCCTT
    TTTGAGGGATAGCGAGCCACGCAGCCAAGATTTGAAGCGATTGAATACTTTGGGGT
    GTCGAGAACGCACCAGAACAATGCCACTCGAGAAATACTACTGTGATTACTGTGAC
    AAACAATTCCAGGATACTCCCTCCGCTAGAAAGCGACATCTACAAGGCGTCCGC
    SEQ ID NO:276 Late LPZ-256 GCGGACGCCTGTACCGTATTGGAATTCTAAACCCTTCCTTGGTATAGGGTTTTCGC
    CACCCTTGCGTTCATTTGGTTTTGTATTACGTCCGATTCCTCCGTCTGCGAGCTCTC
    TGCAACTTGGCAATTTCATTGTGATTTTATCCTATGATGCTTCGTATTTGTTTGAAGC
    TCGTCCTCCTAGTTCTCTGTGATACCAGTTGGTAGTCTGCAAGTTTCGATGTGGGT
    TCTTTTAGCTGGTCTGGGGTTTTGTTGCTCTGAGTATGTTGAGCTGCATGCTCGTG
    GCGGTCTTCACGGCTCCATTTGTTCGGAATCTGTTGTGGAAGTGTCTCGGTCATCT
    GTGGAACTGTGGAAACCTGGTAAGATTTGTTTATCTGCTTGTGTCTAAACTGTTCTT
    GAGTTTTCTGTCGTCTTCTCGCAGCCGTA
    SEQ ID NO:277 Late LPZ-257 GCGGACGCCTGCTGTTGAAGAAGGATGAAGTCATTGTCTGCGGCCCTGTTCAGCA
    TGATTTCGGCATTCTTAATCTGGTCAACCAGTCAGAAGGTGGCGCTGAAGGTGACG
    AAGAGGCAACCTGGGTAGCTGCACTGGAAACTCAAGCTGCAAGGGGCACCGACCC
    TCAGACTTCGCGCGATAACTTCTCCCTCTGGGTAAGTCGATGCCAAGGTCCTTGT
    TCTGGGTTCTTCTCTCTGTTTCGCATGTTGTTCTTCTCTCTGTTTCATTTGTTTTTCT
    TCTGTCGTCTCTCGC
    SEQ ID NO:278 Late LPZ-258 GCGGACGCCTGCACATACAAAGAACGACAAAAACAAAAGCATAAAATCCAATAGAT
    GCAACTATATATCAAGTCAGAAATGATATAACTCATCATTATTACAAAGAACAATAAG
    AGTGGAACCATAATAATAGTCGTCTATTATTGATAAATAAAGAAGAATACAACCATA
    GTTCTGTCGTCTTCTCGCAGCCGTA
    SEQ ID NO:279 Late LPZ-260 GCGGACGCCTGTATAACATGCACCAAGAGACCCAATCAAAGCACATGCAATCTGTA
    TATATAGCAGAATAACAGCCAGGGATTGCACTCTATCGTAATCGCGAAACCACGCA
    CTAATATGTGCCCATGCTGATGATGCACACAGCATGTTCTGTCGTCTTCTCGCAGC
    CGTA
    SEQ ID NO:280 Late LPZ-261 GCGGACGCCTGAACTGTATAGAGTTGAAACTTGAGGGAAGGCTTGCTGCCACCAA
    AGCCTCCCTCCTCTTTCCTTGGCGGTTCGTCACCTCCTTTCGCGTCAGAGCCCCAA
    TTCCCCTCCTGCGCACACCAGCAAACTGCATCGAATGTTTTTTCCACCATTCTGTAA
    ATTCCCTCGGAGTTACCTTGGGGCAGAAGCCGCATTGAAGAGCATTGAATGCTATT
    CATTATCCCACCGTAAACTACCATTGCAACCTGCCTGTGTATCGACCCGCTGTCCT
    CTACGCGTGGCTGGCACATGGCGTCGTTAATTGCATGTTGACACCCGTATCCGGG
    TGTGCTTGTGTGCTCGTCTGCATATCATGTTTTAGGATCTCATAGAAGGTGGACCA
    TTCTGTCGTCTTCT
    SEQ ID NO:281 Late LPZ-264 GCGGACGCCTCTTACAATGTCTCTAAAGATTGGAAAGATTGTCTTGTCTGCAACC
    ATAACTTCCGCGTGCTTTCTTATTAATGCAACCCACTGTGATCCTTTCCGCCATTTA
    TCCTTTCGAATGGTTGGAGCCATTTTTGGGTTGTACCGACTAGCTTTTGGGTCTAC
    AAAGCTGTCTACAAAACTCTTTGGAGATGACATTACATAATCATATGTATAGCTGAA
    GTTGTACAAAGGTACACAACTATCTGAAACCAAAATGAATCTCTCGTTAGCTGGATC
    CTCGAGTGCTTTCCTAAGTAGAATACGCTCCGCTTCTATCATACTGGCTTCTCCCC
    AAGTACCTGTATGCTATCACTAAGCTGCCAGCCGTAACAAAATGTACATTCTGTCGT
    CTTCTCGCAGCCGTA
    SEQ ID NO:282 E,M LPZ-265 GCGGACGCCTTGCTAGGAGAGCTCTACGCCATTATTTGAACGATTGAGCCGAAGTT
    TCACCGTTTAAGGCATTTGTGTCCCAGAGGTTATTGGAGATTAGCAGCTTGGATTT
    GGCTGCTTCGCTCAGCGCCGTGATTCAGCTTTTGATTGATTCTCTCCAGTTTCAT
    CCTGTAACGACAATGGCAATGAAGACCTACACATTGCAGTGGCAGCTGCGTACGC
    TGTAGTCCTGATGTTCGCTCTCTTTGGCATCGCAAAGGCTGCTGATGCACCGTCTC
    CCAGCCCCGTTACTGGCGCGGGTTCCATGGACTTCGTTCCTTCTGTCGTCTTCTCG
    CAGCCGTA
    SEQ ID NO:283 Middle LPZ-266 GCGGACGCCTTATCAGCTGGGGGCATTCATAGGTATGGAAATTCAGATCAACTTCA
    GTGGACAGTATGTGGATTTAGGCGACCTGTGACAGTTCACGATATCTATTCATTTCT
    ATCCAGAGACAGATTCCCATACTCACCTCCGTCCTTCCCATATATTTTCTGGAAGGC
    ATCATGTCCTCCCAAATTTACTCATTTTGCCTGGCCGTCGTTTTACAA
    SEQ ID NO:284 Late LPZ-268 GCGGACGCCTGTTGCCACAGAAGAATGAATAATGCTTCAAATTTGAGACCTCTTC
    GGAGGAAAATCCTTGTTCTTACTGCCTAACCACTCATGATGATCTGCGTCACGCTG
    ATTATGAGCTGCAATTTAAATTATTTCAGATGAAACATTCCCATATTGAGCTTGCAG
    CAAGTTGCAGACCCTTCAATTTCAGTTCTGTCGTCTTCTCGCAGCCGTA
    SEQ ID NO:285 Middle LPZ-269 GACGTTGTAAAACGACGGCCAGGATTAAGGTTCATGAGCTCCGCAACAAGAGC
    TCAG
    SEQ ID NO:286 Late LPZ-270 GCGGACGCCTCTAGGAGCCGGCGGAATTCCTGTGAGCTCGAATTTGCCGAGCAG
    GTTATTGTCCTTCGTCCGCGCTCGCTCACCTTCATATACTTGAATTAGAACCCCAG
    GCTGATTATCTGAGTAAGTTGAGAAAATCTGCTCCTTCTTGGTTGGAATGGTGGTG
    TTCCTCGGTATTAATACTGTCATTACACCTCCCGCTGTCTCCAACCCCAGACTTAAT
    GGCGTGACATCTAGCAACAGCAGGTCCTGCACCTCTCGTTGCCTTCGCCGCTGA
    GAATGGCAGCCTGCACAGCTGCACCATATGCCACGGCTTCGTCTGGGTAATGCT
    CTTACAAAGCTCTTTGCCATTGAAGAAATCTTGGAGCAATTGTTGTACTTTGGGGAT
    ACGAGTCGAACCCCCGACCAAGACGACATCATCTATTTGGCTCTTGTCCATCTTAG
    CATCTTCGCATACATTTCTCCACAGGCTCCATACTTCTCCTGAAAAGATCCATGTTG
    AGTTCCTCGAAGCGAGCTCGCGTAATTTGTGGCGTAAAAATCAATTCCTTCATATAG
    AGAATCAATCTCAATCGTTGTCTGTGTAGTAGAAGACAGCGTTCTTTTGCCCTCTC
    ACATGCTGTTCTCAGCCTGCGAAGAGCTCTGGCATTCCCGCTGATGTCTTTTCTGT
    GCTTTCTTTTGAATTCCTGCACAAAGTGATTCACCATTCTGTCGTCTTCTCGCAGCC
    GTA
    SE Q ID NO:287 Late LPZ-271 TAGCCATCGCCATTCTATAATCTTAGGATCCTTGCTGAACGATAAGCCCATAAAAT
    TGATGCACTGCCTCGCTATCCCTGGCCGTCGTTTTACAACGTC
    SEQ ID NO:288 Middle LPZ-272 GACGTTGTAAAACGACGGCCAGGAAATTACAGCTACCTCTAACTGGTTTGACGGCG
    TTGCATCTATGAGCCGCAAGGGTTCGAATCCTCTGCGGGCCAGATCTGCGATGG
    AACCCTGGGCGAGTGCAATGATGATGAAGAAGAGTTTGCGATGGATTCTGAAGCG
    CACGGGAGGCTTCTGAGGAGGATCCGTTACTATATCAGCTACGGAGCATTGGCTG
    CTAATCGCGTTCCTTGCCGACCTCGGTCTGGGAGGTCTTATTACACTCGGAATTGT
    TACGGCGCAACAGGCCCCGTCAGACCTTACCACAGAAGCTGCACTGCTATCACTC
    GTTGCAGGCGTCCGC
    SEQ I D NO:289 Middle LPZ-273 GCGGACGCCTGGGAAGCAATGGATGGGTGGCTAGACGCCATCCGTCTGTGTATA
    CTATTTTTGCACGCGGAAAGAGTGATGTCCTGGCCGTCGTTTTACAACGTC
    SEQ ID NO:290 Late LPZ-274 GACGTTGTAAAACGACGGCCAGATTCAAAAGAAAAAATCCTCACTTCTTGGCTCCG
    TTTGCGCTCCCGCCGAAGCTCCTCTGCAACCCCTCTGCAGCGTACACTGCATCCC
    GCTCGCGGTGCTGGCTCACCTCGCAGGTCCGCTGACGGTAAATGGTTTCCAATAA
    AGCTATTTGTCCTCTACCCAAAATCCATCTAGCATTCGTTGTGGATTGACATTCTGC
    CATTTCTCTGCTTTTCTGGTTGATATGCAAAGATTGAAAGCCCAATTGCAAGCAGTG
    GTCGTGGATTCACTATAAGGCGTCCGC
    SEQ ID NO:291 Late LPZ-275 GACGTTGTAAACGACGGCCAGGAATAAAACAAAGCATCACTGCAAAATTTCAAAC
    GTGGTAATAACGGCTAGCCAGCTCGACGTGAAGGCAGTGGGGGCCTTGAGGTTGC
    CTTTTGGCGTTCAAAATTGGCTAGACTACCATAACATAAATATTGATTTCTCAGTGA
    CATCACTGGTTTGGAGTCATCCACAGCCTGTGCACCAGTACGGCAATTGCCTTTTA
    CATGAAGCCATCCTTTCACTTTTACTTTTGAGATTCTCAGAACTGAGGGGCTAGGC
    GTCCGC
    SEQ ID NO:292 Middle LPZ-276 GACGTTGTAAAACGACGGCCAGCACCTTCCTAGTCCCCTGTCCATTCTCCTGAAA
    TAGGAGCAGTTTGACCCAGTCCAGTTTTCAGAATTGAGAATATGAAACAAAGAACC
    AAGCATATGAGAGAACATACAAAGACTTTGTATAAACTACTTTTCACAGGATCTCAA
    CAGCCCTCTGCTGAGATCCATTTGATACAAGGCCCCTTGCATCTCCACCCTCTCCC
    TTATCACCTCCACTAGAAAGATGATGGAAAGCAGACACATGGAAATGTTGCTGCAG
    GCGTCCGC
    SEQ ID NO:293 Middle LPZ-277 GACGTTGTAAAACGACGGCCAGTTAGGTTGTATATTGATTGATGACTCTTTGACTCC
    ATTTATGAAAACATCTTTGTTCTCGAGATTTAATCAGTATTAAGCTTTCAGAGTGAAG
    TTCAGTTTGATCTGCATAAACCTGATCCACCATATCTACATCACATCTAAAATTACTA
    AAATGTGAGGAGATGGAATTTGTTTCTTGAGAATCCCTATTCCTCATCGACACTGTT
    TACTGGATCAGATCCAATCAAACTCTTGAGAAGTAATCTCTGGAAAGAAATTAAAAA
    GTCTTTACCTGAATTATCTCGATATCAGAAGCAGAAATTATGATACATAGACTTCTTA
    ATAATGAAGAGTCATTTTGCCAACGTTGTCTTTGCCACCCCACCAATCCCCATGATC
    CCAAAGATCTGAGGTTTCCATCTCTATGTGGCTGTGATAACACTGGATTTTTCAAAA
    ATCTTCTACTTTCGCATCCAAACCTTTTTGGGATATTT
    SEQ ID NO:294 Late LPZ-278 GACGTTGTAAAACGACGGCCAGGGGGATGGGAGATACAGAAAGATTCCGGATAAA
    AGGGAGCAATGAACGGCTGGTTAAAGCGTAGTCCACCACACTAGCCCCACCTCCA
    TGAGGCCTACACGTGAAGAAGCAGGATCTGGGAAGCGCGAGAGGCCGTCAAGA
    TTATCAGCTCATGTGATTCGCCCAACTGCAAAAGATGTCTACCGTAGGCTGTGATG
    GGGCCCAAGGCGTCCGC
    SEQ ID NO:295 Late LPZ-279 GCGGACGCCTATCAGATGGGTGAGTTGACCGACATTTATCGTCCGATAAATGTTTG
    AGGCTGATGTCATGGCAATCCACGTGTCTGCACCATATTTCATCGGAGCCCCTCGT
    CGGAATATTCCATCGCCGGAGAGCTGGCGCGATAGGTTTCAGGCGGCCGGTTTCT
    GGTTTGCAGCTGTGGCTTCCCGCGCGCCTTAACTGTTGGCCCGCGCGCACAGGG
    GAAATTACAAATTTCAACATATCCAATACCATCATATAACCCAACAACACTAGCAACA
    GATCCTGTTCTGTGCCATCGTCCAACTCTGA
    SEQ ID NO:296 Late LPZ-280 GCGGACGCCTTAATTCGACTACAAAGATACTGAAGCCAATGATGACAGGTTGTGCC
    ACTTTCCCAGCTGATAAAGACAGCTCTGAAATTGATAGAGCCAGAACTCCAGCTGC
    AATGCTCCCCAGAGCCTGGTTGAAGCGCTTGCTAAAGGTGGCACTTTATAGACCGA
    CCCAAAACCTCCCTGGCCGTCGTTTTACAACGTC
    SEQ ID NO:297 Early LPZ-281 GCGGACGCCTACTGGAAACCCGGTCCACCGAAGGCTGAAATTGTCCTGCTTTGTA
    TACCGAATGGCAGGAAGGTTGTCGAGCATCAGGTTCACCTGGTAAAGATTATCGAT
    CCTATGCTTCAATACCTTCAGCTGCTCTGCCCCAAGGACAGTAGTATTGCACAGGT
    AAATTCAGATTCATTGACATTCATCCGGAAGCGATATGGTGAGTTCTCGATCCTGT
    CCCCCATGAGGAGCTCCCCAAGATTTTCTGCCATGTCCTTCACACCATCCAAGGGC
    TTGCAGAAGGGCAGGCTGTAATAGCTGTAGGGAAGCTCTGTCTCGACTGAGGTAA
    GGGAATTGACGTTCACCCATAAATCTGACCCCTGGGAGAATATGATGTGAGGAATA
    CAGTGCCCAGTAAATATAACTCCGCATTATACGTTTGTGTGTGCCTTCCCCAATATT
    GCCCCAACATAATCAAAACCCACAATCCCAAATCCTGGACCGTCGTTTTTACAACTG
    TC
    SEQ ID NO:298 Early LPZ-282 GCGGACGCCTTGTCAGGACCAAATGTGTAAGAAACACCTCTGTCATTCGAGCCCC
    TCCTTGAATTGCATTGCAGGGGTCTGACCAAAGAAGATCACATTAACAACCCTGTAT
    CTGGCACATCTGTAGGTCGAGGTATATTCTTTATTTGTTCCAAATTGGTCAGTTCAG
    GCGAAAGACCACCATGCATGCATAGGATCTTTTCATCTATAAGTGCAGCAACAGGC
    AGGCAGTTGAAACAGTCTGTAAAAAGTTTCCATAGTCTTACATTGAATCTGCGCTTG
    CACTCATCATAGAAACCATATATGCGATTTATTGAGGCACATTCATGATTTCCCCTC
    AGAAGGAAAAAGTTCTCTGGGTATTTAATTTTGTAAGCAAGGAGGAGGCATATTGT
    CTCTAGGCTTTGTTTGCCCCGGTCCACATAATCTCCCAAGAAATAAGTAATTTGATT
    CTGGTGGGAAGCCACCATATTCAAAAAGCCTTAGACAGATCAGAATACCGGCCTGT
    CGTTTTACAACGTC
    SEQ ID NO:299 Early LPZ-283 GACGTTGTAAAACGACGGCCAGGAGACGGGAATACCTATTTTTGGGAGGATTATTG
    GGCTCGGGAATCAGCATATTGATGTGGCTGCAACTCGCATCCTCGATCTTTGGTGG
    TTCTTCGGCGATTTACACATTTGAGATCTACTTCGGTCTGCTAGTTTTCCTTGGGTA
    TATTATATTTGACACACAGATGATCATCGAGAAAGCGGACCATGGAGACTATGATTA
    TTTAAAACATTCACTGGACCTCTTTATTGACTTCGTTGCTGTATTTGTTCGCCTGAT
    GGTCATAATGGCAAAGAATGCAGACAGTAAATCCAGGGAAGGGAAAAAGAAGAGA
    AGGGCTTGAACTATGTGAGATACAAAAATATCGAGAATAGAAGGGCTTGAACTAGG
    GCTTGAAAGCGTCCGC
    SEQ ID NO:300 Middle LPZ-284 GCGGACGCCTATCAGACAAGGGTTGTTGACCGAACTTTATCCTCTGAAAAGTGCTT
    GAAGCTGATGTCATGGCAATCCACGTGTCTGCACCATATTTCATCGGAGCCCCTCA
    CACGGAAACAACCTTAAGCCAAAAGGTGGTGCGATGACTTACCGGCCGTTTATGGT
    TTGCTTCGGTGGTTTTCTGTTGGGTGGTTTCCCGCGCGCGTTAACTGCTGGCCGT
    CGTTTTACAACGTC
    SEQ ID NO:301 Late LPZ-286 GACGTTGTAAAACGACGGCCAAGAGGGGGAAACTCCCAAAACACTTTTCCATTTTT
    CTTCTTTTATTAAACTTCAAAGTATTTTCCAACAGAGTTACAAGGGGCCAACCATGT
    CCAAATCCATGCATTTACCAAGTACAAAGAATGGTAGTCCTTGGCTTGACCTATCG
    ACTAGCCAAAAGTGCCAAGTCCACAACTAGGGTGTGCCCAACCTAAGGTGACACC
    TTGCCTAGAAAAAACCCCAAACTTGGCACCACAAATAACACAGAAACACAACTCTTG
    ACCTCTGCCAGAAACCAGGCTCTCTTGGGAAAGCCACACCTCTCTCTGTGATATGT
    CTTATCTCCAATTTCCCTTTTTGTGATGCACTCCCTTGCTTGTGGTTCTGCGATATC
    ACACAAACTTACATTTCTGCGATTTTTGTTTCTTGCTTCTCCAAATCATGCGATCTTA
    TTTTTAACCCTTGAGACCCTTCACACTTTCCATCCATGACGTCACTTCATCGTTTTA
    GCCAATTCGTCATTTGGGCATGTTGGGCGTTGGGTCTACCCGTATTCCGGTCGTAC
    AGGCCAAATTGACCATTTTGGTCCAGGTGGGTGCACCCATTCCTGGAGGGCGTTC
    GGC
    SEQ ID NO:302 Late LPZ-287 GCGGACGCCTCCACAGAGCTCACACATACAATATACTATGATGCCTCCAGAACTAT
    GGCACTCTGTATGCCGCTTCAATATGGATTAGCCCACACTGCGCCATCCAATTAGG
    CGAATCAACCTTATAGCACCATCCACAACCTCCAGCGCTCTCTTTTTCACGCTAGAT
    TGGCCAACTACAGGCTTTACAACACTACTCATATACAACTCAACTCGGCTCCTCTGC
    TCACCACTAAATCACACAGGCTCCAATCGCTAGACAGAGCCACTACACAGGCACTA
    ATAGCCACTACACAGGCACTAATCTTGGCGTCCTCCACCAGGTTCCAACAACAACC
    CCAAATTGCATATGCACTCCACAGTGAGCACCAACTAGGTCCACACAATAGGCCAC
    ACCAACAACACTCCAAGGACCCTAGATCCTGCCTCACCCAGACACCACTAGGCCTT
    CCTCACAGCTCACCTAAGTGAGCCAACAACTGGCTGGGCACACAGCTCCCAACTAT
    ATGAGCACACAGCCCAACTACAGCTCCAGCACACGCACAGCTACACGCACAATGC
    CTTCTCAAGTTCACAGCCACACCATAACGCAGCACAGTTCTTACAAACATATCTCTC
    CAGGCGTCCGC
    SEQ ID NO:303 Middle LPZ-288 GACGTTGTAAAACGACGGCCAGGATAATGGACACGAGAAACCTTTGGATGTGCCT
    CTAAAGTGCGGGCAATCCTTAAAGCTGTTGAATTTGTGCTGTACACGAAGGTGC
    AGGGTCTTTATGCCACGAAGAATCAAGTACGCTGCATTTGGACTTAATACACCTCC
    CAAGACATTGTGCAAAGCACGTACTGTGCCAATAACCTTGTTTGAACCACTCAAACT
    GCCTGCAAGAACATCATTATGACCTGCAATATATTTAGTTACCGAATGCAATACAAT
    ATCTGCGCCGAGTGCTAACGCTTTCTGGTTAACAGGCGTCCGC
    SEQ ID NO:304 Middle LPZ-289 GACGTTGTAAAACGACGGCCAGTCATTATTGACAATAATCCTTTCAGCTTTTTACTG
    CAACCTTTAAACGGTATACCTTGCGTTTCTTTCACTGGAGCACACTCAGATGATAAT
    CAGCTTTTACAGGTGCTCTTACCTCTGTTGAAGCATCTTGCCACTCAGGAGGACGT
    GCGCCCTGTGTTGTATGAAAGATTTTACATGCCCGCATGGTTTGAAAAGCGTGGCA
    TTCCAGCATCTGAGTGGCCCTTGTGACTTGGTTTTGATTTTGGATACTCTTTGTCAT
    TTTGGGTCAAGGTAAAGGTGTACGTATCCAAGTGATGCAAGCGTCCGC
    SEQ ID NO:305 Middle LPZ-290 GCGGACGCCTGATAGCACGAGTCTTCTGGGACGCAAATCAAGAGGCAGGTACTT
    CTTTTTCTTGTATGCTTCTCTTAATGCGGATCGCTGGCTCTGAGAAATCACAGTCAG
    AACCTGAGCTATTGATAGCCTCACGACCTTGATTTTAGAGAGTTTGTTGGGCGCTC
    CTCCAGTGACCTTGCAACTCTGAGCAAGGCAAGCTCAGCCTGAGCTCCTTGACC
    TGGCTTAACAGCTCGGATTTGCCCTTGTGGCGGACTCAAGGACCTTTAACCTGGG
    CGTTCGT
    SEQ ID NO:306 Late LPZ-293 GCGGACGCCTGGTGTCGCTGGGCCAGTTCAAGTATTTTAGCAACAGTGTTCACACT
    TATTCCCTGTGATATTCTTGACTCACACAACCACCTAACTGACGCAGACCATATCG
    ATCTGCTGCTGTAAGCAAATGTTCGATCATTGTCTCAGGTGTCAAAAAGCAAGGGG
    ATGGATCAGAAAGCTCTTCTAAATCTGCATGCTCCTCTAAATCTGGAAGGGTATCTT
    TGTAAATAAAGTGTAACATAGCCTTAAACACCTCTGGCCGTCGTT
    SEQ ID NO:307 Late LPZ-294 GACGTTGTAAAACGACGGCCAGAGGTGTTTAAGGCTATGTTACACTTTATTTACAAA
    GATACCCTTCCAGATTTAAAGGAGCATGCAAATTTAAGAAAAACTTTCCTGATTCAA
    CCCCCTGCCTTTTGGCACCCTGAAGATGGTTCAACAATTTGCTAACGGAACCAATT
    CAAAAGGGCCGCCTCCATTTAAGGTGTTGTGTTAGTCCAGAATATCACAAGGAATA
    AGTGTTAACACCGGTGCCAAAATACCTGAACTGGACCAACGACACCAAGCGTTCGC
    C
    SEQ ID NO:308 Middle LPZ-295 GCGGACGCCTTGTAATCCAGGGCCTTGAATATTGTAAGAGAAGATCGAGAAATAAT
    AGTTTTCTTATTATCAGGAATCACAGCTTGAAGAAGGCAGACCATGGACTCCCACT
    GGCTTCGTGATATTGAGTCCCCAACAAACATTAGTCGTTTTCCCCTCAATCTCCACA
    GCAAGTCTCTGGCATTGAATCTGCGAAAGGAACACCCGAGTGGCTTCCACCTCCAT
    TTCTCGTAATCAGAATCTGGCCGTCGTTTAACAA
    SEQ ID NO:309 Late LPZ-297 GACGTTGTAAAACGACGGCCAGCAGAAGACCAGTGCAGTATGCTGCAGCATAGTT
    TGTAAGCCCTACTTCGAGTCCATAACGAGGCAACTCCCTAGAATAAGCAGCCGACA
    TAACAACATCTCCCGCAAGAGTTGCATAAATGATCTGTGCCACCACATCCTTGTTG
    TGAATCTAACGACCAATCGGTATTTGGGTGTGTTGTACTTGTTCTTATCTTGGTTAA
    TCAGGCGTCCGC
    SEQ ID NO:310 Late LPZ-299 GACGTTGTAAAACGACGGCCAGCATCCATTGCAGAAATTTTGGGGGCTATATTTAG
    CAACAGATATCACAGCTGTAAGTCAAAGTTGGACCCTTCTTCTTCGACATCTTTTC
    CAGCTGTGCAATAAACTGAACACTGTCCTTTTGGATAAGCTCCTCAACATATTTAG
    AAAGTTCAACATCCAAGACATTGCGGTACTCCTCAACATATATGGATGCAAGTTCAT
    CATCTGCAGCTGGTCTCACCGCTGTACAAACTGTTTAACATGGTTGACAGTTGCA
    AGTTGAGCAGTCCGTGGATCCAAATAATGAGTTCCGTCAAGCTCACTGAACTCAGT
    CACAATCACCTGGCCACTTTGATTGGGCATCTCGAGGGATATCATGTGAGACTTGT
    TGTGGATGGGGAAAGCGTCCGC
    SEQ ID NO:311 Early LPZ-300 GCGGACGCCTGCATAAACATCGCTACCCTGGGGATGATTAATAATAGTACCAGGGT
    TAGGATTTTCTTCATCTTGAGCGATATCATCATACATAAAGACCACAATGTTTTCCTC
    TTTCAAACCGCCTTTCCTCAGAATTTGGTAGGCATGGCAGATATCAGCCTGATGCC
    TGTAGTTCCAATAACCGGAAGAACCAGCCAACAGAATAGCCCACTGAGTACCGATC
    GTATCACTATCATCAACGATATGATCGGTGGGCATTTTCAGTACTGAATCCCAACCC
    CTTCTGGCCGTCGTTTTACAACGTC
    SEQ ID NO:312 Middle LPZ-301 GCGGACGCCTAGACTGGGCATACCAACTACCTTCCTCATGCCAGGCCATGGGCCA
    CCTACCTGGTACTTAGGCATAACACCTTACTTACGAGCATGCCAGGCTCAGTCAGA
    TAGGCATGCATCCCACCCACCTAGCTATGACCCAATCCTTATAAACACTAGATATTC
    TCCCTGGCCGTCGTT
    SEQ ID NO:313 Late LPZ-303 GCGGACGCCTAGACAATCATACTGAAGATCTGTAAGCCATGACAAGACGAATAA
    AACGAAGCACGGCGCAACCAGCGTGAATATTGACGCCTTAATTTCATTCAACTGGG
    TTGCGGATTCTTTATTCCTCAACAAGTGTTCGATAGCTTCACATACGCAAGGCCCCT
    TTTACTCTCACCTTCATGGTTTAATGCTGTAACCGTCGAAGGTTGATGAAAGGACTT
    GGATGATGATGTTGCCAAAAAAAAAAAAA
    SEQ ID NO:314 Middle LPZ-304 GCGGACGCCTGCTCAACACCTGTATAGTCATTTCTTGTTTCCTTTTCTCAATTTTC
    TCTTTCGAATGACCGCATTGAAATTCAGGCTGCCCAACGCGTTTTTGTTTTCACAAT
    TAATTTTTGAATCATACGCGAAGATCATGATGAGAATGGTTGTGGAAAAAAACTGTT
    TGTAAATATTTAG
    SEQ ID NO:315 Middle LPZ-306 ATATCACATTACCATTCAAAAAATAAACATTTTACAAAATACAATTCCATAACAATTTT
    CTTCCCTGTTCCAACCTCCACAAAAGTAAATGATCGTATAAGAAATTAACTACCAAC
    AAAAATCCCAAAGTTAAAGGAAGACATCCCCAAAAAAGATGTAACTTTCAAAACCGG
    ATGACTTCACTCCTGCCATTGCACCTAGTCATTTACTTCTCAGAGGAGTTTGGCCCT
    TTCTTCTTTCCAAAAGTAACCACTGCGGTAAGAAACCGGCGGTTGTATTGCATTCG
    CTTGTAGGCGCGGCCTCTAGGCTTCTTCTTCTGTCTTGTTTGGCCACCTTAGGGTC
    CGC
    SEQ ID NO:316 Middle LPZ-307 GCGGACGCCTTGGTACAATGGACTTGCAAAAATAAAATGAGTTCTCATTGTGGGT
    GAGATGCGGATATTTTATGCATAGGCACTTCATGGAGATGTGGTTATAAACGCCA
    TCTTAATATCTGTACCTATTACTTTCAAAATATGAAGGCAAGATGGAAAGCTACTCAT
    CTGTTGTGAAGTCAGAATGTTGGTAGCGGTTGGGCTCTGAAAGTAAGAAACTTTTT
    GATTGGTTTAATTAAATGAGGGAATTTGCCTGGTTTCCCTCTTCCTTCCGAAAAAAA
    ATTTATTTA
    SEQ ID NO:317 Late LPZ-308 GACGTTGTAAAACGACGGCCAGACAATATTGGAAGGGAGAAAGGCGCCAGCAGGG
    TTGAGGGGAAGAAATGCATAATGACATATATAATGAGATCTATTTGTATACGATATT
    ACGGGTACGATCGATGATTCGAGCTACGATCCCATACGACGCTAAAGCGTAATTAC
    ATATATAATAGATGCATTTCAGAATGACTTATCTATTTCATTACGCGATATTATATAC
    GTAATTACGTATATAATTGCAGAGATCTCACCGACCAACCAAATAGTCTTTCATTTC
    ATCCCAGGCGTCCGC
    SEQ ID NO:318 Late LPZ-309 GCGGACGCCTGTATCACTAGAGGTGAATACTCAGCAAGCAAAACTGAAGGATATTA
    TTGAAAAAGCTGTCAAGGCTAAATTGGGTGTCAATTCCCCATTGATCATGCATGGTT
    CTACACTTTTGTTTGAGTCCGGTGATGACATGAGGAAGATGTTGCTGCACATTAT
    GCACAAAACTTAGAGAAGACGTTAGCAGAATTTCCAGTTCCAATCACAAATGGTGTT
    ATTCTTACAGTAGAGGACTACCAGCAAGAGTTCTTATGCAGTATTAATATTAAGCAC
    AGAGATGACTTTGATGAGGAGTCAGGTGGCATTGTACTGTCTGGAGGCGTCCGC
    SEQ ID NO:319 Late LPZ-310 GCGGACGCCTCCTTGTAGATACGATACATGAGTCTAAGATCAAAATCATACAAGAA
    GAGCTTCATTTCCGGGCCTCACCTTTTCTACAAGCTCCTTTTTGGCTGGTGGAAAGC
    CAAACACTCTGTATCGGAAACACTCCTGCCTAGTTTCAGAATTACACATAAAAATCA
    AGCCGGCAAACCTATCTTTGCCACTGCCATCTCATTGTTTGCGTCCTGGCCGTCG
    TTTTACAACGTC
    SEQ ID NO:320 Late LPZ-311 GCGGACGCCTTACTAAAACGACGGCCAGATGTGTAATGGGGAAAATGTGTCATGAT
    AGTTGGGTACAAATAACGAGCCACCTGCTCTATGTTTTCGAAGTTTTCTGTTGGATT
    TGTCCGGGTGAGAGAGCGTTCGTTCGTTGCGCGAGAGGGGCAAAATGCTGAGCG
    TGGGGAATTGCCATTGCCGCCCCTGGAAGTGCCGCACGAACGCGATCACATTTAA
    ATCACCATTTACTTCATCATCACCATGGTTAAATGCAGTCCCTGCTCCTCAAACAG
    GACTTCAGATCCTTCAAGCTCGAAATCTCCGCCTCTGCTTCCTCGAAGACAAGAC
    TCTGTGAGGAGGAAGCGCAGCAGCTGAGCTTAGCGGATCTGCTGAAGCCCGGTG
    GCCTCGCCCCCGATGGGTTCTCGTACAAGGAGAACTTTACCATACGCTGCTATGAA
    GTCCGAGTTAAACCGCACTGCCACCATTGAGGCGTCCGC
    SEQ ID NO:321 Middle LPZ-312 GACGTTGTAAAACGACGGCCAGCAACCAAATAAACCCCACATGTGCTCAATGTTTT
    AGTATAAAAGGAGATGACTTAAGAGTCATTTCACACACACTTCTATCTTGATTTCTC
    CCACTTGTCTTGGGTTTTAGTGGAAGAGAAATCTAGGAGTGGAAGCCCTAGACGTT
    GGAGGATAAGAAGGCAACCCTAGAAGGCAGAGCTAACGCTATCCTAAGGCAACCC
    TAACGCTATCCTAAGGCGTCCGC
    SEQ ID NO:322 Late LPZ-314 GCGGACGCCTGCTCAGCACCTGTTATAGTCATTTCTTTTTTCCTTTTTCTCATTTTTG
    TCTTTCGAATGACCGCAATGAAATTCAGGCTGCCCAACGCGTTTTGTTTTCACAAT
    TAATTTTTGAATCATACGCGAAGATCATGATGAGAATGGTTGTGGAAAAAAACTGTT
    TGTAAATATTTAGGTGACCAACAATTTTCATGATTGCAATCTAAAGTTGATAATTGAT
    TTATCGGGTCGACATTTGTAATTATTAACACGGAAAATCTGAGGCTTACAATTTTTG
    GATTGTAAATATTTAGGTGACGAACAATTTTCATGATTGCAATCTAAAGTTGACAATT
    GAGTTATCGTGTCGACATTTGTAATTATTAACACACAAAATCTATGAGGCGTCCGC
    SEQ ID NO:323 Late LPZ-315 GCGGACGCCTCATCAATCCATGGTTGTACACGCGCCTTCAAAGCGGCTCCTTATG
    TCGCGCAGCGTCTACTTGTTCCTTGAGCGCTTTTCCCTGCTACATCCGCGCGAGCC
    TCTGTGCAAGGGCCACTGTCTGCGCGGTCCCTTTAACTTCGTCGTACTTCTGCTGC
    AGCTCACGTGTCTCTATTTCTAAGTGCTATATATTTGGGTCCTCCTGCATAGTAGTG
    AACTTCGAACGACTCCTCAAATAGCCAGGTGTAGTCTTTCATTGCACTATTGATCTC
    CACTATTCCTGCTATAATGGCGCTAACATGCTGTTCCTTCACCTTTGGCGGAGTG
    AAGGCTGCGCCTCTGGAGCTCGGTTATTTGAAGCTGAACCTTGGGCATATCTTC
    CTTCACCTCGTGCATCCCCTGCTTCGAGTTTCTGGATGCACGCCTCCACTGGGTCT
    TCTGCTGGGATGGGCAACTCTAAGACCAACTGGTATGCGTCGC
    SEQ ID NO:324 Middle LPZ-318 GCGGACGCCTTCTTCAATCCATCAGGCCTGATTAATGTATTGACCTTCTTTGTCTGA
    ATGTCATACATTTTTTTCACTGCATCCTTGATCTTCTTCTTGTCTTGCTTTCTATCCT
    TTCTCTTGCTTTCTATCCTTTCTCTGGC
    SEQ ID NO:325 Late LPZ-320 GACGTTGTAAAACGACGGCCAGCAAAATTGATATAAAGAATAGACACATCGACTCA
    AATGAAGTGACTCAACAGTTCATAATTCATGTCAGCTTGAATGCATGGACATACAC
    CCATAAATAGGCAGTTGGGGTCACCCAAAAGAACATAGAAACATCTCGCATCTCTC
    TGAAGAAACTCGGATGGGTACAGGTCTGTGACTTCGCATATTTTGAAGGAGCACTC
    TCTTGGATAAGTACAATATAGGTACCATCTCGGACTCGCCTGAAATCTCGCAAAGA
    AGTCTCATTCTCCTCCTTGTTACAGGCGTCCGC
    SEQ ID NO:326 Late LPZ-321 GACGTTGTAAAACGACGGCCAGAAGCATCAATAAACAAAATGACAGATTAACAAGT
    TCTCTCTTAATCTTAAGAGAATACATCAACATCCAAGTAAAGTCATAACACATTTACA
    AAATGGTGCCACGGTATCCATTCTCTGTAACAAGGTTTTTCTGAAAATAGTTTTCCT
    CTTATCTATGTAACTCTTCATAGGGATGCCTGTGTCAACGTGCCATATTCCCAAATT
    TGGCCACAATCAAACCTTCCTCATTAGAAGAAACAATCTCTGGTCTAGCTCAAAATT
    GGCAAAATTTCCAGCATCTCCCTTTAACATCATTAGAAGGCGTCCGC
    SEQ ID NO:327 Early LPS-097 GGGAGATGCTAATTTGAAGCCCTTCTCTGAAGGTGGACAATTCCAGCAGCAGTGGT
    CTAAAGCCCCAATATGGCTATAGAAATTCTTCTGGGGGTTGCACCTATGGAAGAGG
    GTCGGAGAGGACGAAGCTGTGGATCGCTCTTACCATCTGTGCGGAAGGTGGTAGC
    AGAATTCATTGGAACGTTCTTCCTCATATTTGTAGGATGCGGATCTGTCGTTGTTGA
    TAAGATAAGCAACGGTTCCATAACTCATCTTGGTGTGTCGCTTGTATGGGGAATGG
    CGGCCATGATTGTAATTTATTCCATAGGCCATATTTCTGGAGCTCATTTGAATCCTG
    CAGTGACGTTGGCCCTTGCGGCTGTGAAGAGATTTCCATGGGTTCAGGTTCCAGG
    CTACATAGTAGCTCAAGTATTTGGATCGATATCTGCTGGGTTTCTCCTACGTTTCAT
    GTTTGGAGAAGTGGCATTCATGGGAGCCACAGTCCTTCAGGCTCAGAAATGCAGT
    CTTTCGCTTTGGAAATTATTACTACGTCATTGTTGGTGTTTGTGGTTTCTGCAGTCG
    CCACTGATACAAAAGCGGTGGGTGAATTGGGAGGTTCAGCAATTGGAGCGACCAT
    CGCAATGAATGTAGGCATATCCGGACCAATCTCAGGAGCTTCAATGAATCCAGCAA
    GGACAATAGGATCCGCAGTGGCTGGCAACAAATATACAAGCATTTGGGTTTACATG
    GTTGGGCCTGTAATCGGTGCGCTAATGGGTGCAATGAGTTATAACATGATTAGAGA
    GACAAAAATGTCCGAAAGGGAGATTATGAAGAGTGGGTCATTTGTTAAGGACATGG
    GCTCCAGCGAATCAACAGCATAACAACTTAGAGATTTNTTGCATTCCCGAGACGGT
    ATCCAGTGATAGTGGAGAGTAGTCATAATAAGATTTGTGAAAATGTTTGTGTAGATT
    AATGTGTAAAATTCAATCCATCAACCATGAAGCGAACTGCATTCCGTTTTTAAATGT
    TTATTGGATTTGAATTAATAAACAGCTTATACGTGAAAATCCCTACTTTATGTACGGA
    SEQ ID NO:328 Early LPS-098 ACTATAGGGCACGCGTGGTCGACGGCCCGAGCTGGTATCCGATGAAGCTAGATTC
    AATGGTTCAAGTCCTATGAAAGCTAGATTGGAGAATTGCAAAGAAATCTAATCTCCG
    TTAGTTGTCCCAACCACTGACTCGCACCCAATCAGAGTATATTAAAGTTAAAGATTA
    TATAAAGGTAAATTGAACATTTATAAAATCTTAAATGTATTTTTAGAGTTAAACATTAT
    ATAGAATATTTAATGTAGTATAGATATAATAAAATATTAAAAATTAATTTCTCTTTACT
    ATCAAGTGAATAAAAATAAAAAATAAATGTAAGACAATATAATAAAAGACTTGTTTTT
    AGTGCATTTTTTGGACTCTTCGTTATTGTGTGGTATTGTGTTATTTAAACTGATCTTT
    TTACTGTATATATGGATGGGTTACCCATCAAACTTGTGATTTCAATAAATTCCTCCC
    GGATTTTAGAGAAATTAGACCATAAAAACTCACGAAAAAAATTTTAGACCATAAAAAC
    TCACGAAAAAAACTTCCCCAAAATCACGCTAAAAACAACTAGATAAAAAAATACCCA
    TCTTTGATGATGTGGATAGTGACAGCCTATTCCAAACTATCACCTAAATTGTAAGTT
    ACATGCATAACACGATGACCTCATCTATACGTTGTGCCAAATAAAGGTATGACCGTT
    CAAACTAAAGAATCAACGAGCTCCAACGCATCTTTTGCTGTGGGGGGATTTCTCACG
    GCTTAACNTTCATGGANCCGATTACCTTNCTANCCAACCAAGGGTTTTAACCTGG
    CAAATNCCAAACCAATTACCAGCTTNACAAATCAACCGAGCCGCCCNACCGGGATC
    ATTTTGGTCAAGTCTCGAAAACNGGCATTGGGTATATGGNATATGGAATTGGAATT
    GGATCAATGGTAACCTTGGGANAAGCTTAANTTGGAAANCCCTTTTTTTTGANGGG
    GGCCAANTTCCCGNNCCCCCGG
    SEQ ID NO:329 Early LPS-099 ATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCTATGGTCGACCTGCAGG
    CGGCCGCGAATTCACTAGTGATTAGATGGTAAGAGCGATCCACAGCTTCGTCCTCT
    CCGACCCTCTTCCATAGGTGCAACCCCCAGAAGAATTTCTATAGCCATATTGAGGC
    TTTAGACCACTGGTGCTGGAATTGTCCACCTTCAGAGAAGGGCTTCAAATTAGCAT
    CTCCAAGTACATTGATCTATTCTATTCATATACATATAACAATGCTGCTCGAGACT
    GACAAAATGATCCGTTGGCGCTCGTTGATTGTTAGCTGTAATTGTTTGGATTGTTCA
    GTTAAAGCCTTGTTGGTAGGAGGTAATCGGTCATGAATGTTAGCCGTGAGAATCCT
    CACAGCAAAAGATGCGTTGGAGCTCGTTGATTCTTTAGTTTGAACGGTCATACCTTT
    ATTTGGCACAACGTATAGATGAGGTCATCGTGTTATGCATGTAACTTACAATTTAGG
    TGATAGTTTGGAATAGGCTGTCACTATCCACATCATCAAAGATGGGTATTTTTTATC
    TAGTTGTTTTTAGCGTGATTTTGGGGAAGTTTTTTTCGTGAGTTTTTATGGTCTAAAA
    TTTTTTTCGTGAGTTTTTATGGTCTAATTTCTCTAAAATCCGGGAGGAATTTATTGAA
    ATCACAAGTTTGATGGGTAACCCATCCATATATACAGTAAAAAGATCAGTTTACCAG
    CCCGGGCCGTCGACCACGCGTGCCCTATAGTAATCGAATCCCGCGGCCGCCATG
    GCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTAC
    AATTCACTGGCCGCGTTTACACGTCGTGACTGGGAAACCCTGCGTTACCACTTAAT
    CGCTTGAGCACATCCCCTTTTCCAGTGNGTAAAACGAAAAGGCCCCNCCATCGCCT
    TTCAAAAATTGGCAACTGAANGGGAAGGACCCCCT
    SEQ ID NO:330 Early LPS-100 ATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAG
    GCGGCCGCGAATTCACTAGTGATTAGATGGTAAGAGCGATCCACAGCTTCGTCCC
    CTCCGACCCTCTTCCATAGGTATAAAACCCAGAATTTGGTGAGCAGGAAGAATTTC
    CATAGCCATATTGAGGCTTTACACCACTGCTGCTCGAATTGTCCACCTTCAGAGAA
    GGGCTTCAAATTAGCATCTCCAAGTTACATGGATCTATTCTATTCATATATTTATAAC
    AATGCTGCTTCGAGACTGACAAAATTATTTGTTGGCGCTTGTTCATCGTTAGCTGTA
    ATGGTTTGGATTGTTCAGTGTAGGACCAGCCCGGGCCGTCGACCACGCGTGCCCT
    ATAGTAATCGAATTCCCGCGGCCGCCATGGCGGCCGGGAGCATGCGACGTCGGG
    CCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGT
    CGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCC
    TTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG
    TTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGG
    CGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGC
    CCGCTCCTTTCGCTTTCTTCCTTCCTTTCTGGCCACGTTCGCCGGCTTTCCCCGTC
    AAGCTCTAAATCGGGGGCTTCCTTTAGGGTTCCGATTTAATGCTTTACGGCACCCT
    CGACCCCAAAAAAACTTGATTAGGGGTGATGGGTCACGTAGTGGGCCATCGCCCT
    TGATAGACGGTTTTTCGCCCTTTGACGNTGGAAGTCCACGTTTNTTTAATAGNGGG
    ACTCTTGGTTCAAAATGGGACAACACTTCAAACCTTTTTTGGGGNTATTTTTTTGA
    TTATNAAGGGATTTTTGCCGNNTTTNGGGCCTTTTGG
    SEQ ID NO:331 Early LPS-101 ACTATAGGGCACGCGTGGTCGACGGCCCCGGCTGGTTTCAATAAATTCCTCCCGG
    ATTTTAGAGAAATTAGACCATAAAAACTCACGAAAAAAATTTTAGACCATAAAAACTC
    ACGAAAAAAACTTCCCCAAAATCACGCTAAAAACAACTAGATAAAAAAATACCCATC
    TTTGATGATGTGGATAGTGACAGCCTATTCCAAACTATCACCTAAATTGTAAGTTAC
    ATGCATAACACGATGACCTCATCTATACGTTGTGCCAAATAAAGGTATGACCGTTCA
    AACTAAAGAATCAACGAGCTCCAACGCATCTTTTGCTGTGAGGATTCTCACGGCTA
    ACATTCATGACCGATTACCTCCTACCAACAAGGCTTTAACTGAACAATCCAAACAAT
    TACAGCTAACAATCAACGAGCGCCAACGGATCATTTTGTCAGTCTCGAAGCAGCAT
    TGTATATGTATATGAATAGAATAGATCAATGTAACTTGGAGATGCTAATTGAAGC
    CCTTCTCTGAAGGTGGACAATTCCAGCACCAGTGGTCTAAAGCCTCAATATGGCTA
    TAGAAATTCTTCTGGGGGTTGCACCTATGGAAGAGGGTCGGAGAGGACGAAGCTG
    TGGATGCTCTTACCATCT
    SEQ ID NO:332 Early LPS-102 ATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCATATGGTCGACCTGCAG
    GCGGCCGCGAATTCACTAGTGATTAGATGGTAAGAGCGATCCACAGCTTCGTCCT
    TCCGACCCTCTTCCATAGGTGCAACCCCCAGAAGAATTTCTATAGCCATATTGAGG
    CTTTAGACCACTGGTGCTGGAATGTCCACCTTCAGAGAAGGGCTTCAAATTAGCA
    TCTCCAAGTTACATTGATCTATTCTATTCATATACATATAACAATGCTGCTTCGAGAC
    TGACAAAATGATCCGTTGGCGCTCGTTGATTGTTAGCTGTAATTGTTTGGATTGTTC
    AGTTAAGGCCTTGTTGGTAGGAGGTAATCGGTCATGAATGTTAGCCGTGAGAATCC
    TCACAGCAAAAGATGCGTCGGAGCTCGTTGATTCTTTAGTTTGAACGGTCATACCT
    TTATTTGGCACAACGTATAGATGAGGTCATCGTGTTATGCATGTAACTTACAATTTA
    GGTGATAGTTTGGAATAGGCTGTCACTATCCACATCATCAAAGATGGGTATTTTTTT
    ATCTAGTTGTTTTTAGCGTGATTTTGGGGAAGTTTTTTTCGTGAGTTTTTATGGTCTA
    AAATTTTTTCGTGAGTTTTTATGGTCTAATTTCTCTAAAATCCGGGAGGAATTTATT
    GAAATCACAAGTTTGATGGGTAACCCATCCATATATACAGTAAAAAGATCAGTTTAA
    ATAACACAATACCACACAATAACGAAGAGTCCAAAAAATGCACTATTTACAAGTCTT
    TTATTATATTGGCTTACATTTATTTTTTACTTTTATTCACTTGGATAGTAAAAGAGAAA
    TTAATTTTTAATATTTTATTATATCTATACTACATTAAATATTCTATATAATGTTAACTC
    TAAAAAACATTTAAGATTTATATATGGTCAATTACCCTTATATAATCTTTAACTTTAAA
    TCCCTGATGGGGGCCAATAANGGTNGGGAAACTAACGGAAN
    SEQ ID NO:333 Early LPS-103 ACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGTTTCAATAAATTCCTCCCGG
    ATTTTAGAGAAATTAGACCATAAAAACTCACGAAAAAAATTTTAGACCATAAAAACTC
    ACGAAAAAACTTCCCCAAAATCACGCTAAAAACAACTAGATAAAAAAATACCCATC
    TTTGATGATGTGGATAGTGACAGCCTATTCCAAACTATCACCTAAATTGTAAGTTAC
    ATGCATAACACGATGACCTCATCTATACGTTGTGCCAAATAAAGGTATGACCGTTCA
    AACTAAAGAATCAACGAGCTCCAACGCATCTTTTGCTGTGAGGATTCTCACGGCTA
    ACATCATGACCGATTACCTCCTACCAACAAGGCTTTAACTGAACAATCCAAACAAT
    TACAGCTAACAATCAACGGGCGCCAACGGATCATTTTGTCAGCCTCGAAGCAGCAT
    TGTTATATGTATATGAATAGAATAGATCAATGTAACTTGGAGATGCTAATTTGAAGC
    CCTTCTCTGAAGGTGGACAATTCCAGCACCAGTGGTCTAAAGCCTCAATATGGCTA
    TAGAAATTCTTCTGGGGGTGCACCTATGGAAGAGGGTCGGAGAGGACGAAGCTG
    TGGATCGCTCTTACCATCT
    SEQ ID NO:334 Early LPS-104 ATACTCAAGCTATGCATCCAACGCGTTGGGAGCTCTCCCTATGGTCGACCTGCAGG
    CGGCCGCGAATTCACTAGTGATTAGATGGTAAGAGCGATCCACAGCTTCGTCCTCT
    CCGACCCTCTTCCATAGGTGCAACCCCCAGAAGAATTTCTATAGCCATATTGAGGC
    TTTAGACCACTGGTGCTGGAATTGTCCACCTTCAGAGAAGGGCTTCAAATTAGCAT
    CTCCAAGTTACATTGATCTATTCTATTCATATACATATAACAATGCTGCTTCGAGACT
    GACAAAATGATCCGTTGGCGCTCGTTGATTGTTAGCTGTAATTGTTTGGATTGTTCA
    GTTAAGGCCTTGTTGGTAGGAGGTAATCGGTCATGAACTGTTAGCCGTGAGAATCCT
    CACAGCAAAAGATGCGTTGGAGCTCGTTGACTCTTTAGTTTGAACGGTCATACCTT
    TATTTGGCACAACGTATAGATGAGGTCATCGTGTTATGCATGTAACTTACAGTTTAG
    GTGATAGTTTGGAATAGGCTGTCACTATCCACATCATCAAAGATGGGTATTTTTTTA
    TCTAGTTGTTTTTAGCGTGATTTTGGGGAAGTTTTTTTCGTGAGTTTTTATGGTCTAA
    AATTTTTTTCGTGAGTTTTTATGGTCTAATTCTCTAAAATCCGAGAGGAATTTATTG
    AAACCAGCCCGGGCCGTCGACCACGCGTGCCCTATAGTAATCGAATTCCCGCGGC
    CGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGT
    CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGCG
    TACCCACTTAATCGCCTTGGAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGA
    AGAGGCCCGGACCCGATCGGCCCTTTCCAACAAATTGCGCAACCCTGAATNGGGA
    AATGGGCCCCCCCCTNTTACCGGNGCAATTAAACCCCGGGGGGGNGNGGGGGTT
    CCCCCCCCCGTGGACCT
  • TABLE II
    Clone SE1-SE2 SE3 SE4 SE5 SE6 SE7 SE8 SE9
    LPS001 0 249.4 1400.9 827.6 1683.8 2019.4 189.2 4303.9
    LPS003 701.2 555.9 2815.2 2445.1 3249.9 3094.7 227.1 3111.6
    LPS004 466.1 335.5 2652 2701 2644 2329.6 218.5 2332.4
    LPS006 753.1 332.7 3287.3 2964.5 2832.2 2688.9 182.1 1591.9
    LPS007 685.2 226 2010.2 1911.3 2600.4 1730.1 181.5 2737.7
    LPS008 652.8 274.8 2415 2219.3 2607.1 2294.9 155.7 1292.1
    LPS010 558.3 356.1 2667.6 2881.1 2584.3 1573.4 161.7 1041
    LPS011 3536.1 424.7 4021.5 3793.8 3590 3182 160.5 1471.7
    LPS012 809 408.4 2206.7 2187.1 2282.2 2422.5 462.4 1483.2
    LPS013 1211.1 391.6 2294.7 2652.6 2005.4 2167.8 166.8 1570.5
    LPS014 2191.9 432.5 2651.8 3013.5 3341.2 3586.7 178.8 3527.1
    LPS015 1197.9 306 5651.4 14828.6 20242.8 21558.2 1427.2 34472.3
    LPS019 1830.2 334.5 3329 3954.4 4347.5 4658.2 312.1 4743.1
    LPS020 675.2 327.8 2258.3 2284.7 2542.7 2321.4 171.9 1609.8
    LPS023 451.3 337.5 1401.9 1106.8 1766.2 1842.6 109.6 1365.2
    LPS024 4585.8 444.5 3006.3 3431.1 3548.8 3759 157.3 4062.3
    LPS025 5102.3 397.1 4322.9 4699.6 5067 4973.2 262.4 5240.4
    LPS026 1568.7 285.9 1809.9 1830.4 2829.9 2381.7 164.9 1404.9
    LPS027 5499.9 458.4 4853.9 5218.6 2598.4 1756.6 457.9 2375.3
    LPS028 4812.9 314.9 2368.8 2616.5 3113.3 3292.4 557 4146
    LPS029 4464.6 251.2 2334.4 2058.1 2930.3 3219.3 472 3814.4
    LPS030 1142.2 352.5 2519.8 2460.9 2499.8 2634.5 378.3 2147.8
    LPS031 1067.7 481.6 3510.8 2799.2 3568.2 3257.2 287.9 2209.7
    LPS032 1120.2 332.3 3153.1 3032.4 1769.2 1816.7 146.6 2689.9
    LPS036 1498.2 1072.9 4633.6 5524.2 5465.1 6350.7 918 14058.5
    LPS037 1890.3 320.9 3719.1 3618.9 4138 4518.1 513.4 5087.5
    LPS038 2899.5 310.3 4530 4226.1 4491.6 3969 268.4 4245.3
    LPS040 527.4 238.1 1433.4 1611.2 1984.5 1506.5 143.9 1988.7
    LPS041 506.1 265.5 1958.9 2843.2 2065.3 2016.2 147.4 2781.7
    LPS042 1432.1 1140.3 4379 4973.3 4525.4 4340.8 319.6 3009.6
    LPS043 696.9 776.2 3933.1 4894.3 3512.2 3664.7 340.6 3098.4
    LPS044 57.8 275.1 3365 4261.2 4773.5 4979.9 974.4 10645.5
    LPS045 536.1 211.1 1559.5 1415 1498.5 1584.8 562.1 1912.3
    LPS046 796.3 231.7 1023.9 306.4 1417.8 1328.2 83.8 946.4
    LPS047 5029.9 518.2 3632.5 4262.1 4755.5 4087.9 386.3 4933.8
    LPS050 6333.5 2620.8 5271.4 5242.1 5586.4 5560.1 980.1 11444
    LPS051 1378 224.4 2328.8 2221.8 2260.5 2715.1 123.7 3670.4
    LPS052 1526.4 267.5 2046 1856.2 2186.5 2416.3 99.3 2010.1
    LPS053 4438.3 361.6 4087.6 3959.9 4786.5 3666.8 379.6 4256.7
    LPS054 1992.9 269.9 2734.2 2388.1 3143.8 2337.7 177.6 2803.9
    LPS055 4587.8 334.4 3488.6 3474 4018.3 3101.6 196.2 4309.4
    LPS056 5960.7 1333.7 5338.8 5670.3 5674.4 5533.5 446.4 5593
    LPS057 2219.9 301.9 2397.3 2356.1 2218.1 2085.6 184.4 2657.8
    LPS058 4070.4 299.9 3485.4 3721.3 4113.8 4142.2 239.8 4945.6
    LPS059 8729.3 279.2 3885.7 3636 2720.4 3346.7 165.7 3734
    LPS060 4580.2 323.7 3027.8 4713.4 4929.1 5047.5 161 4704.8
    LPS061 2831.9 366.8 2392 2327.7 2546.5 1991.8 177.9 3036.7
    LPS062 1674.1 353 2711.2 2526.1 1847 1830.3 124.5 3584.2
    LPS063 5514.4 419.8 5238.9 5020.3 5417.4 5041 250.1 4812.6
    LPS064 7417 3166 5229.5 7497.4 7933.1 10261 1088.3 16829.6
    LPS065 5634.9 343.5 5527.8 5099.4 7833.4 5356.6 237.5 4696.7
    LPS066 1015.9 244.5 1702.6 1650.5 2895.1 2437.2 128 2514.1
    LPS067 2796.8 240.4 3931.5 4810.3 5407.8 5418.3 202.5 9403.8
    LPS069 533.4 189.9 1635.8 1816.4 2114.2 1646.8 119.8 3208.8
    LPS070 2516.9 240.6 1909.5 2519.6 2156.7 1777.4 186.4 4362.1
    LPS071 592.8 196.4 1789.2 2189.2 1981.1 1304.5 127.6 3430
    LPS072 444.2 217.6 1422.9 1509 2065.3 2289.9 122.7 2678.8
    LPS073 4362.8 273.1 3094.9 3348.1 3771.8 4075.3 137.7 4259.6
    LPS074 32072.9 6816.3 33531 25258.9 38176.4 32687.7 14607.1 37529.6
    LPS075 7013.9 472.7 4759.7 4933.9 5452.2 5408.7 409.4 5397.1
    LPS076 4236.1 362.6 3131.9 2882 3368.5 3354.6 119.5 3141.9
    LPS077 2958.7 276.6 4380.4 4862.5 4475.1 4958.7 218.9 4426
    LPS078 23685.3 2642.5 35458.6 25869.6 42378.9 33047.1 25402.2 37189.8
    LPS079 4794.3 547.8 4628.6 4821.8 5257.2 5277 829.5 5449.7
    LPS080 30454 10527 33713.7 23785.4 32590.9 32210.7 16224.4 37659.2
    LPS081 30405.9 28677 35358.3 25873 22338.1 31715.3 36436.4 36650.5
    LPS083 5040.1 460.8 3251.7 3487.3 2688.9 2565.9 190.5 2979.7
    LPS084 2031 298.9 2843.7 2718.4 2352.2 2165.5 164.9 3398
    LPS086 3571.7 320.1 2715.8 2648 1989 2528.4 143.9 2969.7
    LPS087 3302.3 337.4 4873.1 5695.8 5407.2 5450.6 670.8 18404.9
    LPS088 826.8 302.1 2389.2 2871.1 3180.8 2635.2 138.6 3141.5
    LPS089 796.4 321.2 1987.7 2640.6 3299.1 2285.1 143.7 3176.6
    LPS090 4031 235.9 3867.3 4064.4 4503.3 4798.4 341.7 4697.7
    LPS091 2423.3 196.5 2836.8 3101.3 4049.1 4172 295.2 4612.2
    LPS092 2914.9 208.5 4005.3 3138.4 3911.6 4036.1 270.4 4842.9
    LPS093 793 195.5 1619.2 1331.6 1909.3 1843 147.1 2772
    LPS094 1374 221 2205.5 2028.5 2240.9 2632.2 163.3 2849.1
    LPS095 728.7 174.1 2022.6 2112.1 2335.8 1264.6 117.5 2957
    LPS096 333.3 168.5 1531.9 1393.4 1893.3 869.1 118.3 1691.1
    LPZ001 2008.6 185.4 2535.9 2937.9 3472 1981.8 118.9 2421.7
    LPZ002 3529.3 384.6 4579.3 4474.6 3236.7 3855.8 313.8 3237.5
    LPZ003 4076.8 275.4 2651.2 2966.7 2829.2 4177.4 378.5 4369.7
    LPZ004 5595 687.4 5468.2 5615.9 5243.6 5699.6 601.6 5889.9
    LPZ005 5680.5 3353 34994.7 26121.9 42555.1 33144.5 16193.7 37798.2
    LPZ006 1199.8 299.4 3013.7 3099.8 3517.3 3397.1 140.6 3370.8
    LPZ007 1159.1 462.2 3292.7 2992.5 3121.4 2936.7 235.5 3238.6
    LPZ008 1874.3 237.7 3110.8 3236.7 2516.5 3182.2 325.3 4330.1
    LPZ009 3331.1 296.3 2348.5 3414 2478.2 3309.5 348 5658.1
    LPZ010 3216.3 1186.8 4977.3 5024.7 4564.4 4992.4 442.6 4454.5
    LPZ011 4613.4 910.9 4510.7 4515.7 3729 4357.3 371.4 4695.9
    LPZ012 1531.5 469.5 2915.3 2611.1 2012.3 3481.4 270.3 3804.3
    LPZ013 3495.1 268.8 2125.9 2584.7 3194.7 3787.4 125.1 4929.6
    LPZ015 2040 257.6 1971.1 2966.7 2191.1 3056.7 227.1 4156.6
    LPZ016 5307 2761.1 8451.7 17219.7 22792.7 15567.3 1073.6 35074.1
    LPZ017 2476.4 354.3 3175.5 4330.8 4496.2 4061 273.2 5328.9
    LPZ018 3929.4 417.5 12420.2 14916.1 18116 17637.5 2541.6 31981
    LPZ019 5404.2 427.3 32190.3 24710.4 42102.7 32342.6 19528 36969.5
    LPZ020 576.9 142.9 1451.4 1505.4 3534.8 2679.8 210.9 3046.2
    LPZ022 1408.2 155.2 2406.7 2845.7 3042.5 3074.8 189.9 3829.2
    LPZ023 562.1 152.8 2096.7 1710 2045.5 2078.9 200.8 2874.3
    LP2024 496.7 158.1 1681.3 1264.7 2102.9 1857.1 132.1 1818.4
    LPZ025 5431.3 464.1 13492.2 9726.2 11911.5 13462.8 1262.5 11780.6
    LPZ026 1663.2 139.7 2464.8 2760.1 3113 2219.4 159.1 3183.5
    LPZ028 5029 190.7 5367.2 5339.8 5483.9 5205.5 482.3 5565.9
    LPZ029 961.3 119.2 1805.4 1989.6 2298.5 1998.4 126 2576.9
    LPZ030 1457.4 177 2444.7 2687.5 1966.4 1857.2 178.5 3312.8
    LPZ031 3092.8 361.7 3564 3925.3 4627.8 5171.4 506.7 5920.5
    LPZ032 1906.5 156.8 5542.3 24342 42917.8 33386.1 30058 37998.6
    LPZ033 12934.5 354.7 5280.1 7301.2 5638.9 9238.7 375.4 15843.5
    LPZ034 1307.4 177.5 1737 2208.4 3213.1 1984.1 150.2 3228.3
    LPZ035 556.5 201.9 880.2 1280.1 1654.5 915.1 74.1 1422.1
    LPZ037 1356.8 269.7 2072 3110.5 2912.8 2488.2 211 4119.3
    LPZ038 4027.9 426.9 5639.9 5872.3 5476.8 5614.6 796.8 5583.3
    LPZ039 5059.1 550.6 3807.9 4393.8 3825.6 3889.8 342.2 5164.2
    LPZ040 1226.1 236.5 1566.4 1889 1679.1 2263.6 140.6 3331.1
    LPZ041 944.2 219.3 1629 543.1 1148.2 1416 90.2 2524.6
    LPZ042 570.6 206.1 1129.5 806.5 1448.8 1423.1 75.1 2013.8
    LPZ043 1190.2 236.7 1878.8 1024.4 2834.6 2767.4 241.7 3236.2
    LPZ045 5315.3 465.7 4933.2 5580.2 5151.1 5205.1 557.3 10754.3
    LPZ047 859.5 285.2 1606.2 2099.3 2059.4 1992.6 68.3 3054.8
    LPZ049 3232.7 108 1278.6 2834.2 3657.8 3944 244.2 5459.6
    LPZ051 3048.1 146.9 2373.2 2067.3 2745 2383.2 179.1 2837.6
    LPZ053 2580.3 135.6 2625.8 2088.7 2468.5 2297.2 156.8 3001.4
    LPZ054 1838.1 159.5 2657.8 2759.7 2658.1 2224.7 170.4 3444.2
    LPZ055 2181.8 151.1 2381.2 2262.7 3228.3 2983.9 139.3 2673.9
    LPZ056 4028.3 219.5 2884.6 3416.6 3779.6 3789.9 208 4518
    LPZ057 1470 121 1676.5 1629.6 1702.7 1703 112.2 2272.1
    LPZ058 1923.3 122.5 2453.5 2169 3127.3 2465.4 160.6 3319.6
    LPZ059 1760.4 113.8 2180.6 1832.4 1997.2 1530.8 174.4 3366.6
    LPZ060 3296.4 139.3 2571.1 2250.2 2721 2976.9 221.3 3896.5
    LPZ061 2495.6 182.8 2663.9 2235 3265.9 4227.1 498.1 4915.1
    LPZ062 1992.7 194.9 3296.7 3975.8 3861.5 5642.6 497.6 5606.2
    LPZ063 2167.1 145.9 2733 1843.9 3066.6 4961 305.6 4773.2
    LPZ065 5641.2 251.7 13690.3 9269.2 8562.8 13254 986.3 9554
    LPZ066 6307.3 652.4 12630.8 6968.4 4918.9 5062.2 400.7 5456.8
    LPZ067 10838 1548.1 16986 11776.8 5633.2 7054 1014 15262.2
    LPZ069 1481.9 209.6 2239.8 1480.9 2496.7 2542.4 250.5 3717.2
    LPZ070 1932.5 263.8 1895.1 2221 1555.9 1570.4 145.5 3471.3
    LPZ071 3672.6 378.6 4185.5 3050.5 4166.8 4246.2 553.7 5333.4
    LPZ072 744.5 210 1210 676.7 1420.2 1393.4 95.8 1997.1
    LPZ073 1997.9 235.9 2275.1 2141.7 2613.2 1989.9 170 3489.4
    LPZ074 1375.9 237.4 1899.1 1787.3 2472.9 1623.7 125.6 2435
    LPZ075 831.4 247.9 1536.4 1773.1 1886.9 920 80.6 1053.5
    LPZ076 345.7 251.8 854.8 564.6 1747.1 526.2 55.9 1058.3
    LPZ077 2466.3 102.2 949.4 820.9 3093.9 3179.6 202.9 3314.8
    LPZ078 3102.1 197.1 3654.2 3261 4204.3 4433.6 400.8 5559
    LPZ079 1584.4 108.3 2389.2 2243.3 2624.8 2677.1 208.3 3675.6
    LPZ080 12206.5 2043.1 25021.4 8579.5 11707.8 8717.6 1172 18663.9
    LPZ081 1368.7 103.6 1902.8 1349.9 2166.1 1597.7 103.5 2709.6
    LPZ082 2601.3 140.3 3264.3 2853.9 2799.6 1742.3 251.1 4288.2
    LPZ083 1311.9 76.7 1622.4 1071.1 1733.9 1878 104 2007.7
    LPZ084 9974.7 801.3 14255.3 8399.1 5763.9 8852.9 542.2 5714.3
    LPZ085 4609.8 158.4 3923.3 3729.7 4082.8 3867.3 219.3 4075.1
    LPZ086 10874.1 987.4 19189.5 8284.6 5646 9109.8 1116.4 14988
    LPZ089 3505.8 211.6 4010 3430.6 3762.1 3770.8 224.3 5341.2
    LPZ090 5780.9 581.8 13217.4 6303.4 4694.8 4779.9 425.2 5408.9
    LPZ091 5316.1 148.4 2263.4 2139.8 2382.2 4067.2 256.8 14732.6
    LPZ092 5448.7 209.4 3631.6 4152.7 2934.1 3403.7 174.9 4943.6
    LPZ093 1169 159.4 2097.9 1187.4 2050.8 2350.7 109.4 2605
    LPZ094 1245.5 139.7 1547.5 1650.5 1875.2 2009.9 80.2 2376.9
    LPZ095 711.2 177.9 900.9 1253.3 1013.8 1395.3 48 1586.1
    LPZ096 2122.2 249.7 2929.3 3271.3 2132.9 2224 232.8 4443.8
    LPZ099 4306.4 211.2 2603.1 2144.4 3479.2 3488.5 138.1 4085
    LPZ100 3373.5 297 3941.3 3149.6 3790.4 3857.5 443.8 5028.1
    LPZ101 3007.7 272.4 3546.9 2291.3 4299 3232.1 306.1 4819.6
    LPZ102 2092.7 324.7 3167.5 2109.3 3524.3 2829.4 279 4297.4
    LPZ103 3602.1 285.7 2923.3 3112.9 2812.9 1318.3 87.9 1739
    LPZ106 1359.7 305.1 2680.3 2391.6 2838.5 2097 173.7 3009.6
    LPZ107 28560.8 4989.5 20821.7 17880.4 39173.1 27035.1 11973.3 36123.4
    LPZ108 4136.8 179.4 4259.8 4978.2 5553.2 4862 837.2 5597.5
    LPZ109 3708.3 202.4 3842 3510.4 4638.4 4453.7 469.5 5107.4
    LPZ110 4557.2 291.4 5020.6 4801 4487.4 4481.1 552.3 5484.2
    LPZ111 1625.6 130.9 2242.1 1982.7 2740.6 2455.4 164.6 3722.3
    LPZ112 2887.4 195.8 3813.2 3759.4 3984.8 4167.1 409.7 5461.8
    LPZ114 5029.5 213.4 5016.7 4678.8 5036.9 5168.1 302.1 4316
    LPZ115 24434.4 2637.1 27958 23684.2 41104.3 30920.9 2153.9 36902.6
    LPZ116 8682.9 235.7 5647.3 5316.6 5805.6 9313.7 466.6 16018.9
    LPZ117 30879 4843.7 36277.1 24358 24673.1 20545.7 4669.9 5652.6
    LPZ118 4023.6 171.1 3743.5 4568.2 3845.4 3783.9 254.3 4782.5
    LPZ119 2580.4 114.1 2507.2 3114.1 2544.6 1963.8 127.6 3195.4
    LPZ120 1998.8 157 1987.2 1503.1 2331.8 1805.1 131.5 3522.3
    LPZ122 2041.4 119.6 2145.6 2430.9 1998.6 2171.8 101.3 2677
    LPZ124 2795.6 185.4 2980.4 2672.5 2495.2 3459.4 173.1 3081.5
    LPZ126 2559.7 181.8 2560.1 2349.8 3500.6 2362.1 224.9 3646.9
    LPZ127 1993.5 169.1 3161 3180.8 3382.5 3321.3 180.6 4058.4
    LPZ128 2866.7 263.2 3556.8 3597.4 3545.7 3813.8 306.7 4071.3
    LPZ131 1993.5 171.7 1983.9 2069.6 2565 2607.2 80.3 2527.8
    LPZ133 2446.7 290.4 3218.6 2847.2 3830.1 2889.5 245 4252.4
    LPZ136 1952.3 281.1 2956.9 1870.6 3167.6 2680.6 215.9 4291.6
    LPZ137 2833.8 281.9 3264.4 2350.2 3874.4 3532.8 420.8 4935.3
    LPZ138 2932.9 1791 5211.5 4502.1 5409.9 4832.8 543.1 4741.3
    LPZ140 2284.7 337.4 3680.2 2810.9 3196.1 3191.2 271 4613.6
    LPZ141 4726.2 368.5 4792.5 4412.5 5368.1 5466.3 722.1 4956.4
    LPZ143 25290.6 2692.2 35967.9 25679.9 43668.3 32612.1 25456.9 36344.4
    LPZ144 2620.9 286.6 3948.7 3394.6 4505.7 4142.8 488.7 4776.7
    LPZ145 3472.5 171 3949 3194.2 3430.5 3539.9 327.9 4487.2
    LPZ146 2612.8 127.3 2482.4 2080 3000.8 2979.1 135.1 3391.3
    LPZ147 2447 106.3 2855.1 2237.7 3134.2 2841.8 261.6 4388.1
    LPZ148 2036.8 77.7 2559 1932.3 4296.1 4699 359.6 3982.3
    LPZ149 5720.7 267.4 5377.3 5408.2 10999.7 5717.7 1078.9 13033.2
    LPZ150 5861.7 772 35541.7 26314.8 44633 33238 13126 37853.6
    LPZ151 5550.3 3499.3 9012.8 8380.4 11968.1 5716.5 715.8 5536.9
    LPZ152 4746.6 352.8 5169.3 5647.7 5384 5394.2 408.5 5382.1
    LPZ153 21881.2 2773.2 14738.2 15979.5 16996.8 15756.8 2388.5 30812.9
    LPZ154 4869.8 265.9 3244.3 3497 3948.6 3703.3 303.8 4119.1
    LPZ155 3904.2 1596.3 5078.5 5482 4631.7 5314.1 553.4 4112.9
    LPZ157 4726.5 1732.8 5427.1 5369.5 5213.3 5705.9 756.4 5462.2
    LPZ158 15297.4 3817.1 17993.9 17405.3 25168.8 22056.6 2337.4 22375.3
    LPZ162 5725.8 4204.8 10380.1 11364 17948.2 14250.8 1934.6 10535.5
    LPZ165 5615.2 666.7 5274.6 5486.6 5560.3 5310.9 637.1 5405.9
    LPZ166 5889.1 2603 9503.5 10943.7 13743.3 14080.4 1772.5 5772.8
    LPZ167 5347.2 1948 5708.9 6769.6 5742.3 5347.9 370 5279.7
    LPZ169 3043.8 267.4 1976.6 2851 3451 2451.2 189.8 3420
    LPZ170 3507.3 301.5 3532.3 3391.4 4481.4 3398.7 130.2 5604.2
    LPZ171 3762.3 780.7 4554.8 4311.7 4936.4 4511.3 398.6 5030.5
    LPZ172 5098.2 947.4 5550 6287.9 5135.4 5323.9 1242.5 8539
    LPZ173 22580.5 3313.9 35486.9 24974.9 42874 31828.8 26531.8 36066.9
    LPZ174 4115.7 221 5241.5 4262.4 5765.8 5554.9 872.5 4815.2
    LPZ175 4388.3 1360.6 5563.7 5504.9 5165.3 5182.9 583.2 4602.3
    LPZ177 1371.6 94.5 2119.4 2218.6 2730.7 2431.7 143.2 2893.2
    LPZ179 3643 195.1 4409.9 4898 5458.3 5319.8 797.9 5677.7
    LPZ181 5573.3 215.9 4799.6 5272.2 5825.3 5554.4 1573.5 13689.7
    LPZ182 4118.9 107.6 3491.5 3182.1 4617.5 4543.6 478.1 5527.8
    LPZ186 5792.2 325.5 4965.1 5182.6 12373.6 11191.4 1804.9 37336.4
    LPZ189 33820.3 5188.5 30941.4 24955 43453.2 33115.2 17929.3 38055.6
    LPZ194 2807 151.1 2915.9 2955.1 3306.8 3120.2 142.9 4101.6
    LPZ195 5345.7 532.7 5597 5628.7 5540.4 5491 545.7 5756.7
    LPZ196 4805.1 3512.9 5183.2 6968.6 5465.4 5052.4 786.4 5694.5
    LPZ197 7268.6 153.8 5398.9 5673.5 13582.6 15111.9 3499.6 34684.8
    LPZ198 7208.3 210.6 5800.8 8043 5439.2 5183.6 409.3 5042.7
    LPZ199 3058.3 186.1 2749.6 2667 3713.6 3704.3 243.4 3917
    LPZ201 7175.3 236.7 4827.6 5029.9 5523.4 5802.2 1981.9 14614.5
    LPZ202 3603.3 1113.9 35531.5 26035.1 44762.8 33837.3 63521.8 38225.4
    LPZ203 4325.4 424.4 5517 5387.3 9934.8 5662.1 2104.8 9370.7
    LPZ204 32355.9 34690 36443.6 26004.3 44546.1 33680.5 55702.6 37890.3
    LPZ205 4904.1 519.6 5162.4 5398.7 5427.6 5325.6 281.4 5770.2
    LPZ206 3504.4 319.8 3124.8 4561.7 4192.2 3899.9 255.7 5489.9
    LPZ207 32035 24978.7 34825 23371.8 42639.9 32686.4 30672.2 37674.8
    LPZ208 25174.6 3118.6 14244.4 13906.3 16694.7 21111.9 2190.1 34542.4
    LPZ210 3885.1 422.3 3895.8 4551.5 4205.7 5108.7 258.6 5514.3
    LPZ211 2569 176.7 3689.2 2943.5 4001.9 3860.9 250.2 3113.1
    LPZ212 5988.8 1244.3 32684.4 11154.1 19853.4 13654 618.3 10736
    LPZ213 3406.9 106.8 3964.1 3876.6 4236.4 4294.4 274.2 4874.6
    LPZ214 1668.3 55.3 2136.3 2394.8 2390 2269.3 105.8 3436.2
    LPZ215 5019.8 139.3 5020.1 5024.8 11013.9 13747.1 1991.7 36930.6
    LPZ216 3336.8 1085.4 35895.6 26245.3 44980.1 33834.4 64482.7 38238.1
    LPZ217 23512.1 26363.3 36065.8 24685.4 43193.4 31422.4 21462.1 35990.6
    LPZ219 4011 256.9 3193.5 3326.3 4509.5 5258.5 455.2 5841.9
    LPZ220 8696.5 2383.3 5064.7 5171.3 4923.7 5340 951.7 17530.6
    LPZ221 1221.4 83.1 1201.8 707.6 1556.5 2083.9 182.1 3948.8
    LPZ222 1885 146.1 2834.7 2253.2 2557.7 3382 196.7 4225.1
    LP2223 1048.5 121.2 2339.6 2642.1 2663.8 3573.5 383.5 4579.2
    LPZ224 3190.6 118.8 3049.8 2833.2 4373.8 5139.9 858.6 5285.7
    LPZ225 25428.2 4079.7 35724.5 25423.6 43300.5 32574.8 38888.3 37219.9
    LPZ226 1044.9 130.4 1776.9 1210.8 2757.7 3388.5 326.6 3520.3
    LPZ227 1078.3 133 1461.7 973.5 7032.1 9452.8 2043.5 4705.3
    LPZ228 3961.6 213.5 3373 4050.7 5575.6 10714.4 2428.6 5928.9
    LPZ231 3475.2 230 4096.3 3841.1 5009.3 5690.3 959.9 5514.3
    LPZ233 2404 218.2 2170.9 1531.4 4362.7 4198.1 673.7 3350.1
    LPZ234 1688.3 312.3 1887.6 1486.5 4228.6 4715.6 724.8 3170.1
    LPZ235 2661.6 199.9 2422.2 1852.6 3078.9 2886.6 98.2 3143.5
    LPZ237 3174.5 324.2 3032.5 2988.2 3931.1 4587.9 314.4 4588.3
    LPZ239 4061.3 309.3 3175.1 2932.1 4131.7 3892.6 122.3 5083.5
    LPZ240 3799 316 3730.6 3314.6 3379.8 3538.8 212.4 4784.5
    LPZ241 2559.2 62.1 2610.4 1794.5 4165.6 3754.4 134.8 2915.1
    LPZ242 29360.5 3262.9 35254.6 25196.9 43028.8 31468.2 7308.4 36768.4
    LPZ243 3405.3 88.8 3015.7 2683.4 3678.7 2990.6 121 4001.4
    LPZ244 4856.9 483.6 4842.2 5235.3 5317.6 5432.1 205.4 5712.9
    LPZ246 1274.8 65.9 2301.7 1922.8 4332.2 4628.8 672.1 4232.4
    LPZ247 3894 69.8 2522.8 3389.9 4451.4 4937.1 939.3 5522.8
    LPZ248 3016.7 268.6 2883.2 3805.2 3791.7 3777.6 487.1 4585.6
    LPZ249 5224.1 138.3 3524.5 4091.2 3022.4 3393.2 149.9 4101
    LPZ250 1060.6 46.5 1400.9 1246.9 1419.5 1411.2 118.8 2908.4
    LPZ251 1336.8 248.5 1354.6 1049.3 657.6 924.3 70.3 2064
    LPZ255 3787.8 171.8 4801.8 5076.3 4608.1 4965 340 5636.6
    LPZ256 536.6 61.6 865.5 971.6 1130.8 1327.7 82.7 936.9
    LPZ257 844.5 112.6 1507.4 1537.8 2337.9 2745.8 341.5 1610.7
    LPZ258 2588.5 142.3 3443.1 2902.2 4576 4976.4 1182.9 3619.6
    LPZ260 837.7 113.7 1677.9 944.3 1217.6 1286.6 170 2928.2
    LPZ261 981.3 132.1 1499.6 743.4 1590.8 1953 67.2 1652.1
    LPZ264 4559.3 231.3 4348.5 2856.3 4869.2 5179.7 412.3 4698.3
    LPZ265 21063 2793.9 26928.5 12365.5 13816.5 12134.4 691.6 17954.8
    LPZ266 1642.4 130 1767.5 1463 1633.8 1410.5 59.4 1444.5
    LPZ268 2451 114.2 2803.3 2495.4 3126.5 3433.7 79 4261.3
    LPZ269 15670.7 3660.5 35782.4 21720 40375.2 31597.3 2024 35213.6
    LPZ270 3541.2 240.8 3803 3132.4 4827.8 5213.6 79.9 5473
    LPZ271 5590.7 677.2 5465.4 5197.1 5703.4 5615.2 309.2 5732.7
    LPZ272 27369.6 3445.9 35824.6 22832.6 40684.8 27398.4 1732.9 37016
    LPZ273 1107.3 46.1 456.7 336.3 1879.3 1654.1 65.4 971.9
    LPZ274 3936.2 114.5 3192.3 3024.1 4983.3 4907 293.9 4933.5
    LPZ275 2567.2 42.9 1760.4 2091.8 3656.5 3800.5 77 2585.4
    LPZ276 560.9 32.9 1075.4 1878.9 1889.9 1766.2 66.8 1294
    LPZ277 423.7 34.6 1199.1 1169.8 1376.8 1383.9 91.9 1123.7
    LPZ278 323 39.7 937 382.9 770.1 935.2 66 1403.4
    LPZ279 965.9 70.7 1907.5 1368.2 1783.7 1603.9 133.2 2438.8
    LPZ280 390.7 19.6 175.4 42.3 464.9 631.5 29.9 2074.9
    LPZ281 84.3 8.2 0 0 0 0 9.7 0
    LPZ282 1849.7 28.4 315.1 34.2 664.3 1097.3 21.5 1229.5
    LPZ283 10678.6 329.2 5134.7 5311.1 4772.3 8591.1 226 9633.6
    LPZ284 996.1 39.8 236 147.2 2349.5 981.1 26 719.7
    LPZ286 563.8 77.1 1031.1 945.9 1347.4 1601 81.2 1303.6
    LPZ287 1045.7 123 2057 1475 1730.9 3003.6 149.9 2493.5
    LPZ288 1201.7 116.2 1797 1448.8 1648.3 670.4 80.6 3700.4
    LPZ289 1922.3 113.3 2515.4 3395.3 3460.7 3369.4 70.8 2183.8
    LP2290 14629.5 3945.8 34659 24047.3 40474.8 27786.2 1348.2 27566.4
    LPZ293 4364.8 385.4 4664.2 3170.9 4321.6 4789.8 74.6 5095.8
    LPZ294 564.7 171.4 1257.5 705.2 1357.7 1610.2 18.6 2027.6
    LPZ295 823.1 97.3 2102.7 1056.2 2899.7 2698.3 39.4 2448.2
    LPZ297 5273.4 169.1 5229 5074.4 5727.8 11512.9 423.2 10966.8
    LPZ299 1564 161.1 1743.9 1752.3 2764.1 2660.5 63.5 2791.9
    LPZ300 3068.3 205.4 2406.8 1881.8 2898.6 2758.4 0.2 2007.2
    LPZ301 1979.7 233.1 3207.1 2109.3 4343.5 3713.8 40.4 2690.6
    LPZ303 509 32.7 281.3 877.7 893.1 751.5 30.1 1373.8
    LPZ304 2531.1 289.3 3809.4 3406.7 3674.8 3517.4 158 2652.7
    LPZ306 22632.7 2861.2 34933.8 25435.6 40453.9 30906.9 1505.9 34032.8
    LPZ307 2604.4 1395.2 4780.6 6945.3 4419.2 4416.9 232.6 4299.8
    LPZ308 1093.9 60.5 2028.1 1751.6 1770.8 1891.9 92.4 3245.8
    LPZ309 286.1 26 480.4 378.4 589.6 731.4 38.4 1062.4
    LPZ310 2284.1 129.5 1622.7 1091.7 1207.1 3089.4 101.2 3624.9
    LPZ311 3309.9 43.6 2782.6 2956.3 2828.8 4446.7 95.8 5593.4
    LPZ312 446.3 52.7 1577.7 1221.4 542.2 518 56.1 1952.6
    LPZ314 378.6 26.9 333.9 355.8 682.2 701.2 61.7 732
    LPZ315 3897.5 115.2 2611.9 3145.8 4296 5240.2 151.3 4499.3
    LPZ318 9709.6 767.1 19964.9 15678.3 20611.8 19600.2 475 18079.9
    LPZ320 1126.7 82.8 1215 1002.7 1502.5 1555.3 67.5 2964.4
    LPZ321 2944.7 85.4 2590.7 2597.6 2550.3 2962.7 72.1 5481.4
    Clone ZE1 ZE2 ZE3 ZE4 ZE5 ZE6 ZE7 ZE8 ZE9.1
    LPS001 369.9 369.9 369.9 369.9 369.9 369.9 369.9 369.9 369.9
    LPS003 600.3 363.9 0 243.7 1565.3 2624.5 1942.7 242 1892.5
    LPS004 522.3 254 0 74.6 907 2638.8 1933.6 274.9 4209.2
    LPS006 444.6 161.2 0 174.6 793.6 2651.4 1991.5 206.5 598.8
    LPS007 528.9 136.3 0 244.9 1623.3 1202.1 2044.7 245.1 213.9
    LPS008 534.5 215 0 281 1231.2 783.4 1760.3 178.5 832.4
    LPS010 469 183.6 1.3 240.1 947.7 591.6 2208.3 161.2 482.6
    LPS011 468.7 93.3 0 142.3 1544 1021.5 2334 254.6 1223.7
    LPS012 511 278 17.7 197.2 2129.6 68.9 1362.7 478.9 960.4
    LPS013 478.2 407.1 192.1 235.6 2470.6 9 1163.9 885.7 1109.4
    LPS014 579.7 369.1 0 272.7 2799.6 1525.7 2222.4 606.9 2638.6
    LPS015 419.7 254 0 2380.7 7188.1 4998.4 16519.6 5245.1 15550.4
    LPS019 1068.4 279.6 0 396.2 3848.5 3074 3866.9 959.1 3664.7
    LPS020 314.2 109.1 0 102.7 2036 234.4 1504.2 319.6 1053.2
    LPS023 364.9 104.2 0 100.7 1151.8 0 1253 175.5 570.5
    LPS024 804.7 213.8 0 346.3 3248.5 2523 2722.6 915 1987.4
    LPS025 1374.7 407.8 0 857.1 4731.2 2584.2 4119 1138.4 2458.4
    LPS026 337.6 86.1 0 100.2 1242 0 1052.9 242.9 988.9
    LPS027 440.5 182.5 0 118.5 1318 691.2 1274.1 226.1 385
    LPS028 369.5 166.2 0 168.7 2587.7 2597.8 4035.5 565.9 1883.1
    LPS029 323.4 141.9 0 165.3 2524.3 2147.2 3031.3 567 2263.9
    LPS030 362.3 226.5 0 169.6 1528.2 422.9 1236.7 239.2 1049.1
    LPS031 591 536.7 4.9 383.6 1768.3 850.4 1013.3 399.8 781.4
    LPS032 443.9 327.3 0 328.1 3200.9 1880.1 1832.8 265.6 1391.6
    LPS036 1093 781.8 24.5 680.8 3911.3 3750.9 3746.7 661.8 3856.4
    LPS037 501.6 180.4 0 200.9 2664 2369.8 1960.5 339.9 2892.9
    LPS038 1180.1 471 155.9 1679.7 4392.3 2103.5 3019.9 800.3 2819
    LPS040 398.8 108.6 0 103.9 1030.7 195.9 1566.1 144.4 682.1
    LPS041 384 153.8 0 149.7 989.2 1257.5 2235.4 143.3 1228.4
    LPS042 1381.9 951.7 44.9 716.1 3682.1 2755.7 4011.7 508.5 2963.4
    LPS043 1211.6 704.8 74 613.6 3494.4 2435.3 3362.1 391.4 1544.7
    LPS044 361.3 100.2 0 142.1 2244.4 3031.8 2653.5 393.4 1620.1
    LPS045 285.7 75.2 0 64.4 856.5 223.5 1616.1 216.8 609.7
    LPS046 325.8 217.2 0 70.2 1758.6 0 1280.9 284.9 1115.5
    LPS047 2041.3 1347.8 768.4 1080.5 4169.9 3927.9 4263.5 1831.6 4804.9
    LPS050 3226.4 3356.4 6064.5 3347.5 9841.4 3046 5362.2 2924.8 5821.2
    LPS051 377.1 96.4 0 156.8 2452.6 2286.5 3035 396 2238.1
    LPS052 330.1 80.1 0 162.6 2418.1 0 2097 352.5 1677.3
    LPS053 402.6 160.1 0 146.3 2249.5 56.9 1986.1 464.3 1349.6
    LPS054 497.4 147.5 0 184 2188.4 379.1 1976.1 308.6 1558.9
    LPS055 1168.2 645.7 0 354.9 3901.1 1476.5 2607.5 774.8 3026.1
    LPS056 1549.5 1243.3 37.9 752.6 4770.9 3403.7 4086.8 1204.2 4958.4
    LPS057 387.5 154.3 0 262.7 2612.2 502.6 2317.5 365.1 1418.8
    LPS058 671.2 198.9 0 434.8 4189.9 2258.6 3366.3 586.3 2190.5
    LPS059 726.2 207.5 0 304.6 2974.8 2054.3 2712.8 395.1 1331.6
    LPS060 534.2 215.8 0 221.7 2896.9 718.3 2693.3 477 2474.9
    LPS061 530.8 369.4 0 204.3 1801.1 1286.4 1533.6 298.7 1327.2
    LPS062 407.4 305.2 0 226.4 1509 0 1413.1 212.5 954.6
    LPS063 619.4 280.8 0 282 3987.4 1805 2589.9 642.1 1650.4
    LPS064 3689.2 4982.4 10201 3080.3 8359.8 3622.3 8304.6 2997 13781.1
    LPS065 466.4 189.7 117.3 817.1 4336.3 2332.6 4393.4 1092 3866.8
    LPS066 269.5 104.6 0 131.4 1006.2 76.7 1834.6 185.9 668.5
    LPS067 426.4 179.7 49.3 341.3 4153 4077.7 5101.3 1195.9 3894.1
    LPS069 367.8 136.7 0 128 1456.6 0 2685.6 308.7 1234.3
    LPS070 438.5 137.3 0.4 111.6 1932.5 25 3005.3 210.4 721.5
    LPS071 283.9 83.2 8.5 109.2 1831.5 0 3634.2 302.8 708.3
    LPS072 301 147.2 5.7 132.9 1600.8 592.3 3051.5 331.5 1173.5
    LPS073 692.1 485 251.5 497.9 4205.3 2827.4 3777.9 740.1 3882
    LPS074 36280.3 66359.2 63362.2 44047.1 47176.9 20938.9 64534.5 33666.4 78457.8
    LPS075 3204.8 1250.6 650.9 1033.7 4976.3 4377.1 4632.6 1617.1 5570.9
    LPS076 434.7 127.9 0 204.7 1731.3 419.1 2737.8 298.8 2175.1
    LPS077 416.6 107.6 0 327.2 3360.7 1950.5 4020.1 609.1 3713.9
    LPS078 5164.5 1194.3 906.3 6556.7 20779.8 7364.3 28847 8680 22339.1
    LPS079 1317 501.9 304.2 893.4 5047.6 3196.6 4887.5 1058.8 4992.1
    LPS080 27721.4 56038.2 70896.6 26826.4 43426.4 20557.3 47819.2 16935.5 68145.5
    LPS081 36397.3 66337.3 48195.9 41685.3 46187.5 20628 66138.1 31620 78253.7
    LPS083 844.6 534.2 123.6 305.3 3724.2 1699.7 2524.6 583.8 3266.4
    LPS084 665 249.4 0 334.5 2570.6 1491.7 2893.2 342.8 2061.7
    LPS086 456.6 155.8 0 165.1 1962.7 754.5 1931 130.2 2176.5
    LPS087 967.5 450.7 17.2 633 4238 3720.3 5373.9 1754.7 19094.1
    LPS088 468.4 276.1 0 151.1 1109.4 0 1779.7 302.8 2497.2
    LPS089 329.2 316.9 0 133.6 988.7 0 1619.6 320.7 1616.9
    LPS090 478.9 272.3 0 218 4486.4 2182.3 2923.1 584.6 2640.7
    LPS091 385.7 177.9 0 290.7 2923.3 2008.6 2453.2 441.5 2246.6
    LPS092 396.3 164 0 345.2 2249.1 1219.1 2906.7 413.9 1589.3
    LPS093 308.3 164.5 24 98.7 262.3 427.8 2140 175.4 661.4
    LPS094 331.6 179 54.2 146.4 773.3 948.2 1729.1 116 1030.7
    LPS095 363.7 157.7 46.3 142.5 967.7 341.4 2639.7 199.3 1055.4
    LPS096 266.9 90.6 0 59.3 676.5 0 2616.2 136.5 215.6
    LPZ001 270.9 49.7 21.1 121.2 1958.2 496.8 4495.7 325.6 557.7
    LPZ002 491.7 231.8 157.9 345.8 1929.4 1183.4 3243.9 305.5 932.6
    LPZ003 632.6 407.2 342.3 343.5 2630.6 2108.3 3212.8 423 4663
    LPZ004 2034.4 2260.7 1487.4 1442.5 5730.7 2135.4 5424.1 1804.4 5786.1
    LPZ005 6301.3 4683.6 2801 10127.5 31972.9 7747.5 51335.5 17767 52067.8
    LPZ006 471.7 131.2 0 179.4 1964.1 1552.2 2977.9 333.5 2954.9
    LPZ007 584.6 383.7 69.3 294.3 1424.1 531 2664.9 265 3021.7
    LPZ008 325.4 87.5 14.4 176.8 1966.6 1588.5 2420.1 199.5 3481.2
    LPZ009 451.3 281.6 143.4 362.3 3149.2 4280.4 3006.5 357.5 4395.9
    LPZ010 1324.5 1442.8 621.8 931.8 4310.4 3238.7 3926.9 617.4 4912.5
    LPZ011 1740.9 2073.5 1436.2 1075.8 4631.4 5232.7 4563.3 1080.6 5456.6
    LPZ012 424.4 217.8 50.1 271.8 2286.7 713.5 1791.4 390.6 3209.9
    LPZ013 395.4 123.8 33.1 181.1 3456.8 2121 2898.4 428.9 2673.3
    LPZ015 490.7 210.7 60.8 130.8 2889.2 330.5 2123.4 230.2 2680.6
    LPZ016 2411.9 710.9 346.5 1201.2 6897.9 4057.6 13340.1 3246.1 16664.2
    LPZ017 635 257.4 36.2 247.2 2797.3 1219.1 3508 558.7 3953.8
    LPZ018 1405.4 474.9 214.9 3188.7 8143.7 4992.8 12908.9 4208.2 14318.9
    LPZ019 3487 975.9 698.4 8916.5 23680.5 15131 31656.3 14191 45343.3
    LPZ020 251.9 195.6 0 40.9 1448.5 860.4 1113 305.5 971.2
    LPZ022 250.1 112.4 0 133 2085.9 1282.5 2538.5 532 613.2
    LPZ023 355.5 122.6 22.9 47.8 224 879.4 1419.1 132.3 605.4
    LPZ024 366.5 108.9 45.7 94.8 225.3 723.1 1276.8 81 319.5
    LPZ025 705.4 278.6 202.1 716.7 5145.5 4563.4 7215.5 1581.9 4195.8
    LPZ026 268 100.6 9 92.1 1164.5 750.4 3973.6 275.5 642.3
    LPZ028 386 174.2 96.8 374.9 4188.4 2733.8 7024.6 1186.8 2375.6
    LPZ029 221.9 86.8 0 47.8 225.3 264.4 2172 162.4 957
    LPZ030 319 166 67.6 146.9 801.6 1385.4 2283.4 145.8 1189.4
    LPZ031 2010.6 881.7 538.1 754.3 4625.8 3395 4051.7 1327.9 5202.1
    LPZ032 36097.5 35972.1 13659.1 19975.4 45544.5 20035.8 63759.8 31117.4 78268
    LPZ033 1813.8 433.6 243.7 1171 7402.4 2278.7 10670.5 2204 5643.8
    LPZ034 332.6 97 34.6 181 1097.2 0 2704.1 262.6 3481.4
    LPZ035 248 60 0 71.4 1188.8 0 1332.5 153.6 3028.4
    LPZ037 375.8 133.8 0 114.6 3024.3 909.1 2350.6 248.2 3546.8
    LPZ038 577.7 237 44.3 370.7 4484.6 3572.5 4266 907.9 4571.4
    LPZ039 965.6 406.7 361.2 537.9 4020 2304.1 4269.9 816 3717.5
    LPZ040 399.9 127.4 0 88.3 1200.5 365 2123.2 244.3 1955.4
    LPZ041 318.4 105.1 0 136.6 856.3 716.6 1528.5 245.6 1538.5
    LPZ042 289.3 77.6 0.9 189.5 441.5 365.4 1007.7 239.2 1212
    LPZ043 417.3 166.7 57.4 158.2 1197.2 1617.9 793.5 569.1 2018.7
    LPZ045 754.8 310.3 152 691.5 4810.4 3305.3 4043.7 1476.3 3925.7
    LPZ047 270.5 155.4 53.1 39.1 2165.6 579.6 980.9 361.7 1036.3
    LPZ049 809.6 381.9 0 461.4 4406.4 2277.4 4764.6 2257.8 5528
    LPZ051 333.1 121.5 0 56.6 1597.8 1677.8 891 270.7 1134.6
    LPZ053 271 119.7 0 16 1662.4 2447.8 1202.4 201.1 827.1
    LPZ054 345.4 131 61.7 79 1181.1 2238.5 1426.5 156.5 627.5
    LPZ055 291 78.1 102.9 63.5 551.3 2343.8 1433.5 193.6 814.2
    LPZ056 364.6 167.9 83.6 130.3 1816.9 2580.4 2589.4 343 1579.8
    LPZ057 250 76 0 11.9 426 457.6 2589.6 113.4 709.8
    LPZ058 231.1 40.8 6.2 44.4 454.7 163.4 3403.4 208.5 1300.4
    LPZ059 239.2 78.6 0 15.7 189.7 267 3272.7 141 1439.2
    LPZ060 235.1 26.7 29.4 35.6 524.8 1238.2 2231.2 182.5 1908.8
    LPZ061 402.1 268.4 141.6 254.6 1694.1 3088 2343.5 557.3 4157.7
    LPZ062 727.7 146.8 0 203.3 2873.3 2418.9 3109.6 926.6 5812
    LPZ063 316.7 108.2 25 190 1837.3 1025.1 2727.2 421.8 4195.9
    LPZ065 512.5 59.9 94.9 583.5 5694.4 3741.6 5366 1221.6 3911
    LPZ066 622.2 66.8 23.8 405.6 6932.1 3089.5 4804.9 642.2 4079.9
    LPZ067 883.7 218.7 0 358.7 4518.2 2342.9 5200.8 1318.4 5539.6
    LPZ069 335.6 100.2 0 46.8 0 0.7 1736.5 255.4 2910.4
    LPZ070 422.6 143.3 49.6 168.8 1231.5 1170.6 2432.2 262.7 2122.5
    LPZ071 356 71.9 0 299.7 2303.6 1909.8 2575.9 447 2827.6
    LPZ072 206.5 32.6 0 71.7 0 0 1362 189.6 907
    LPZ073 374.2 129.6 32.1 125.8 307.4 691.8 1617.6 282.4 1306.9
    LPZ074 434.7 86.5 0 76.6 1671.1 0 1112.5 232.7 663.5
    LPZ075 298.5 173.5 0 53.5 4083.2 0 1143.9 113.7 335.1
    LPZ076 209.9 83.1 7.2 7.4 2185.9 0 490.5 226.6 362.9
    LPZ077 813.4 558.3 0 339.1 3733.4 4279.8 1115.9 671.7 4136.8
    LPZ078 532 349.8 0 265.8 4460.1 3290.7 2776.8 686.3 3283.3
    LPZ079 347.8 183.6 11.6 53.3 2115.4 1191.7 1451.2 160.5 1233.2
    LPZ080 948.1 264.9 178 455.1 4633.9 2869.1 4230.5 1378.5 5566.5
    LPZ081 313.7 96.1 100.2 59.7 1161.6 1470.2 1119.7 98.5 710.9
    LPZ082 260.9 120.3 18.4 68.3 1734.5 1430.6 1581.6 160.1 379.4
    LPZ083 284.9 134.6 12.6 7.8 1067 906.1 1482.7 107.2 790.5
    LPZ084 1493.3 437.1 0 788 4497.3 4608.3 5078.9 2267.8 5087.7
    LPZ085 468.1 139.4 0 62.1 1618.8 1355.9 4314.9 340.5 2421.5
    LPZ086 601.3 91.6 11.9 332.9 3540.3 3354.6 6675.2 781.2 5545
    LPZ089 457.1 124.1 26.2 267.7 2378.1 3364.6 3390.2 448.7 2930.7
    LPZ090 436.6 50.7 0 149.1 3135.3 2087.8 3090.4 483.8 3408.1
    LPZ091 350.2 91 96.2 99.2 1524.3 1976.3 3215 315.9 4532.8
    LPZ092 387.6 33.6 0 114.4 2386.7 2600.9 2845.7 274.6 2036.7
    LPZ093 195.6 25.8 0 27.1 2343.1 321.7 2875.2 252.7 1428.2
    LPZ094 274.8 42.8 0 35.2 886.9 0 2502 152.6 1571
    LPZ095 288.6 91.5 0 14.1 3.5 0 2317.2 144.4 1427.4
    LPZ096 475.4 138.7 13.1 199.2 1690.8 1415.2 3939.2 207.2 1889.7
    LPZ099 428.1 73.8 0 185 2531.8 2596.6 3925.9 399.8 1967.5
    LPZ100 474.3 131.1 24.6 266.6 2710.6 2276.7 3358.6 503.5 2245.1
    LPZ101 492.1 101 81.9 233 2341.4 2076.1 2199.9 423.1 2022.4
    LPZ102 477.3 133.2 0 167 3722.9 1604.4 2432.1 386.5 1199.8
    LPZ103 353.5 90.7 0 108.9 4756.8 214.4 1447.5 203.9 760.7
    LPZ106 534 199.4 58.6 117.6 3052.4 1259.2 1610 277.6 1224.3
    LPZ107 29718.3 56194.4 31132.5 35651.5 44972.5 20589.6 38396.3 32828.7 75965.1
    LPZ108 852.4 433.1 161.4 417.4 4354.8 2259.1 3536.9 1168.5 3161.8
    LPZ109 554.2 248.6 83.8 254.6 3303.4 2521.1 1972.8 608.5 2858.5
    LPZ110 614 203.3 166.1 182.1 3236.7 2792.6 2764.1 455.5 3139.2
    LPZ111 349.5 167.7 77.4 82.9 1407 1386.1 1469 200.9 1397.5
    LPZ112 497.3 279.2 65.3 242.6 3553.5 2504.1 2454.6 328.3 2182.2
    LPZ114 890.2 346.7 0 399.3 4520 3367.9 2902.7 1229.6 3474.1
    LPZ115 24782.8 12016.1 1401.9 12188 32718.2 17087 38203.9 17191.1 25318.5
    LPZ116 1388.1 392.7 0 884.6 8895.4 3131.2 15554.1 2195.7 5401.6
    LPZ117 6228.4 4810.2 389.7 1298.8 4199 2671 5473.5 1488.2 3911
    LPZ118 424.5 267.3 39.9 196.2 2507.4 2210.1 2856.3 370.3 2640.4
    LPZ119 295.1 183.4 0 59.8 2443.3 1153.2 2040 158.7 1132.1
    LPZ120 213.5 88.4 49.2 185.9 1336.6 1390.4 1604.5 186.7 1360.8
    LPZ122 317.6 120.3 0 92.1 1390.2 2045.1 1701.2 112.5 1307.5
    LPZ124 346.6 173.2 38.9 119.4 2097.9 543.9 2679.2 221 1941.7
    LPZ126 436.1 185.4 0 142 1762.2 0 2164 283.2 2343.6
    LPZ127 455.7 208.6 0.1 109.8 1091.7 1925.5 3143.4 345.4 2565.8
    LPZ128 421 602.3 25.1 651.1 4496.3 2712.3 5456.8 1177.7 3875.7
    LPZ131 287.1 401.2 0 58 1931.2 422.4 3510.2 232.2 1275.8
    LPZ133 377.8 399.9 141.9 139.5 2396.5 1989.9 4287.5 388.9 1119.6
    LPZ136 455.2 191.7 103.3 239.9 2289.7 1775 2586.1 322.4 1429.8
    LPZ137 398.1 123.1 0 214.7 2480.2 1118.7 2138.5 411.3 1935.9
    LPZ138 1987.8 1102.2 11.9 1112.7 4785.6 4242.8 3044.7 1298.3 2786.7
    LPZ140 401.4 205.9 115 316.7 2737.2 1950.6 1491.6 414.2 2031.4
    LPZ141 917 621.2 0 726 4938.4 3889.9 3237.4 1109 4165.8
    LPZ143 4702.9 1483.9 774.8 6791.2 20187.4 6913.4 23175.5 10795.1 26322.7
    LPZ144 529.7 392.6 25.7 303.5 2644.9 1902.4 2380.9 486 2422.7
    LPZ145 410.2 206.5 25.4 152.3 1618 2219.1 2012.8 357.8 2494.3
    LPZ146 294.2 152.3 0 125.3 1063.5 1142.1 1129 215.1 1927.1
    LPZ147 366.5 238 0 246.5 2120.4 956.1 1343.4 260.2 1539.3
    LPZ148 390.6 212.9 0 170 2107.1 2195.4 1832.6 1240.9 3839.8
    LPZ149 1872.8 1139.7 218.5 1748 6204.2 1641.3 5144.7 3526.5 5193.6
    LPZ150 1958.1 1284.1 645.6 10615.2 29419.9 2976.7 52052.2 21478.7 41125.9
    LPZ151 3477.3 1936.4 155.4 1423.9 5253.1 1925.1 5434.9 2248.3 5180.5
    LPZ152 963.2 481.9 42.2 658.9 4770.8 3607.7 4661.8 1003 4661.9
    LPZ153 13685.9 27883.7 11205.9 13827.1 24872.9 15412 26204.7 12163.3 41713.9
    LPZ154 621.9 470.5 45.6 381.1 2965 2584.3 2802.2 416.8 2738.4
    LPZ155 2004.5 1513.1 388.4 1358.9 4265.3 4159.3 4386.4 1022.6 4543.9
    LPZ157 2978.4 1332.8 407.8 1400.6 4650.6 3692.3 4760.8 1195.9 5002.5
    LPZ158 12352.4 18933.2 12155.7 9376.4 23120 15280.6 18384 10293.8 50995.4
    LPZ162 3778.2 4069.3 426.4 2285.5 6458.4 3004.6 5794.3 4161.4 13171.9
    LPZ165 1181 805.1 0 756.4 4641.9 2702.4 5464.6 1470 4822.6
    LPZ166 4624.9 5016.4 0 2508.8 9907.8 1939.3 5655.3 2999.9 5667.6
    LPZ167 3339 1655.9 319.8 1101.8 4807 4980 4678.8 1968.2 4248.1
    LPZ169 787.5 556.1 226.4 461.9 2830.2 2225.4 3549.7 684.4 3249
    LPZ170 851.1 501.5 0 589.6 4405.2 4440.6 4652.4 1622.7 4556.9
    LPZ171 1325.6 612.2 0 697.6 3647.5 3148.7 3446.8 1053.8 4043.4
    LPZ172 748.6 490.4 0 802.4 3953.2 2939.1 3550.9 848.3 3809.5
    LPZ173 4460.6 3415.7 1050.1 5769.8 16078.4 6658.8 16861.6 8623.4 21007.1
    LPZ174 501.9 308.6 36.2 358.6 3054.1 1731.2 1954.9 862.9 2773.9
    LPZ175 1476.5 1057.1 181.6 836.9 3731.4 3120.9 3879.4 755.9 3687.2
    LPZ177 302.3 228.3 18.6 107.1 753.6 1430.4 941 165.4 1411.2
    LPZ179 616.8 314.9 8 379.4 4544.7 2954.4 3425.3 1361.5 5310.3
    LPZ181 1103 430.7 0 671.3 4917.1 3821.3 4976.3 2646.1 5785.8
    LPZ182 992.8 435.1 0 468.1 3930.1 4177.6 3287.5 909.9 4820.2
    LPZ186 2455.7 1428.7 760 2414.4 9679 4431.1 5537.9 3859.3 5921.7
    LPZ189 40770.3 68311.4 75133.7 45673 47303.1 21072.6 66542.7 34849.1 78485.4
    LPZ194 612.8 572.1 155.6 376.3 3673.5 2240.2 3365.2 469.5 3608.6
    LPZ195 676.9 346.6 32.5 459.9 4278.1 4622.4 4442 1006.8 4754.3
    LPZ196 2923.1 1787.1 448.6 1537.1 4490.3 3684 4875.6 1426.9 4447.8
    LPZ197 592.3 177.5 343.1 629.4 5825.4 3639.7 4308.7 649.9 2838.2
    LPZ198 801.7 295 8.2 511.1 4384 3156.4 4823.8 1125.6 3947.2
    LPZ199 402.5 165.1 62.7 265.9 2158.3 2363.7 3531.7 423 2360.1
    LPZ201 1338.5 361.2 209.6 1053.7 4997.8 4565.6 5107.6 2350.9 5882.2
    LPZ202 8178.9 2795.3 2359.9 12950.9 39890.9 20247.9 62085.4 31826.4 78541.6
    LPZ203 1983.5 1083.8 1146.8 2044.1 9173.9 2174.8 11135.5 4073.8 19571.3
    LPZ204 38154.8 66241.1 88440.7 45372.1 47166 21054.3 65826.6 33875.6 78581.7
    LPZ205 1079.5 521.7 589.1 758.4 4742.5 3837.7 5220.2 1453.2 4888.6
    LPZ206 947.6 700.1 505.1 697.8 4089.6 2774.1 5242.9 1190.3 4578.7
    LPZ207 39899.6 60813.3 95637.5 45997 47123.6 21073.2 63996.4 35149.4 73080.5
    LPZ208 27268.6 27230.4 39428.3 29579 40216.5 20480.2 42465.8 26526.9 76872.4
    LPZ210 683.8 538 429.6 439.8 2402.5 3285.4 2419.1 636 3965.6
    LPZ211 603.1 530.3 87.2 434.6 3653.3 2234.5 1744.1 359.8 1644.7
    LPZ212 1018.4 580.7 155.2 1734.1 9212.3 2338.9 5061.9 2257.3 4303.3
    LPZ213 465.6 327.1 33.1 296.6 3012.3 2419.4 2198.4 570.9 2013.9
    LPZ214 245.6 267.4 0 160.5 912.5 756.5 880.7 133.9 936.8
    LPZ215 981.1 492.8 52.3 828.3 5390.6 4771.4 4868.1 2893.1 15622.6
    LPZ216 9410.5 3834.8 1843.2 14492.9 43137.1 21097.9 63778.6 34830.7 78538.8
    LPZ217 31119.9 47126.3 30689.1 28515.4 38695.3 19884.5 37913.9 21282.9 69590.2
    LPZ219 1300.2 886.5 475.3 946.3 4898.3 4361.6 3705.4 1784.6 5810
    LPZ220 5695.5 7233.6 9375 4579.9 6540.8 4340.1 5612.6 2993.6 5852.3
    LPZ221 242.6 209.5 0 186.3 2553.4 2132.5 1632.9 258.2 3202.9
    LPZ222 186.9 130.5 0 163.4 2138.4 1386.3 1855.4 223.3 2407.6
    LPZ223 289.9 168.4 0 164 2019 1582.7 2374.4 250.3 2309.2
    LPZ224 308.3 114.3 20.5 333.9 2467.7 2535.2 2722.9 518.9 3054.9
    LPZ225 5540.8 1287.2 919.3 7087.6 18926.6 9277.7 20292.9 8705.5 19974.7
    LPZ226 179.1 111 9 136.8 1049.8 2071.1 3332.8 463.4 2517.8
    LPZ227 243.5 136.2 0 341.4 2502.5 2597.6 4869.5 3226.2 16356.7
    LPZ228 470 249.6 0 349.2 4062.6 3388.6 5688.3 2586.2 15112.1
    LPZ231 480.4 296.3 87.4 326.6 3832.4 2343.1 5557.3 1542.5 5679.2
    LPZ233 468.6 332.9 161.7 350.3 2519.3 1967.1 5201.8 996.1 4833.8
    LPZ234 525.8 368.6 287.8 380.3 1626.6 1285.8 4231.8 1235 5329.5
    LPZ235 310.7 233.3 0 370.2 1746.3 1474.1 4135.8 541.1 3382.9
    LPZ237 683.5 468.8 487.2 506.9 3683.2 2108.7 3966.8 1369 5254.5
    LP2239 546 192.6 339.2 331.5 2584.8 2384.8 3215.7 754.4 4041.1
    LPZ240 353.7 280.8 163.9 274.8 2281.8 1696.4 2345.8 319.2 2180.8
    LP2241 283.3 260.2 0 206.4 2481.1 1787.8 775.1 426 1729.3
    LP2242 7478.4 2664.4 1039.1 7024.1 22393.3 11120.5 20928.9 8581.6 22951.9
    LPZ243 242.4 167.1 0 157.2 2175.8 182.4 806.9 237.7 977.2
    LPZ244 350.3 206.1 0 409.4 4522.7 3997.1 4486.3 1022.8 3387.1
    LPZ246 260.9 200.9 0 251.8 1930.9 1335.1 995.1 1033.7 4712.9
    LPZ247 438.3 274.4 0 341.4 2994 2325.3 2926.7 1508.7 5494.3
    LPZ248 748.4 714.1 291.3 878.4 3814.2 2737.7 3489 1155.1 4689.5
    LPZ249 373.3 375.6 0 613.1 4798.1 2088 3560.8 433.5 2486.3
    LPZ250 159.5 201.7 0 317.6 2037.8 2085.9 1721.8 220.5 2051.7
    LPZ251 141.7 157.9 0 178.9 1377.4 1723.8 1136.8 91 1271.1
    LPZ255 220.8 176.1 0 646.5 4160.8 2725.5 5110.3 1217.2 5217.7
    LPZ256 94.6 101.7 0 149.5 821 812.6 1989.5 65.4 432.2
    LPZ257 147.9 118.4 0 135.1 1206.9 1208.1 2197.2 207.9 745.8
    LPZ258 168.3 124 0 174.7 2264 2172.4 2672.8 532.8 2245.2
    LPZ260 213.5 172.9 0 141.3 1172 2974.6 4118.3 232.4 1057.4
    LPZ261 147.2 78.5 0 126.6 1212.5 2349.9 4604.9 139.4 741.6
    LPZ264 318.3 174.1 0 201.7 3104.4 2387.6 5505.8 582.7 3775.1
    LPZ265 1566.8 449.9 129.7 646 4992.3 3477.7 5635.5 988.7 4307.2
    LPZ266 92.8 287.2 0 132.5 931.4 0 4689 190 726.3
    LPZ268 171.3 217.2 0 206.8 2142 2135.8 5156.3 296.8 1912
    LPZ269 1530.1 571.5 419.3 2333.1 11130.7 4947.4 13881.5 5155.2 5755.6
    LPZ270 162.3 291.4 0 450.4 3822 3736.4 4342.8 978.4 2987
    LPZ271 454.6 266.6 45.9 381.8 3194.7 2859.7 3598.7 1277.5 4054
    LPZ272 2943.2 763.9 613.2 1451.3 7894.9 2900.8 5222.6 3222.4 16317.5
    LPZ273 215.5 178.7 0 112.2 1288.3 908.3 145.5 182.5 544.8
    LPZ274 271.5 189.3 0 322.8 3311.7 1141.5 1301.4 620.8 3182.2
    LPZ275 174.5 152.8 0 99.6 1052.3 0 0 109.3 887.1
    LPZ276 146.8 139 0 129.4 1165.3 123.5 505.4 82.6 461.9
    LPZ277 201.8 137.5 1.8 57.1 761.5 931.9 497.5 106.1 1427.3
    LPZ278 177.9 152 0 76.6 588.2 1424 311.2 107.3 1178.8
    LPZ279 183.3 179.3 0 304.9 2458 1032 524.7 276 1530.9
    LPZ280 142.1 125.5 0 125.6 1116.5 623.5 1147.9 125.4 770.5
    LPZ281 18 109.8 0 58.9 563.4 850.5 564.6 11.5 317.8
    LPZ282 54.3 164.1 48 95.5 1493.6 1874.9 1033.3 54.7 844
    LPZ283 1607.8 392.6 48.4 1220 6358.1 2922.6 5552.8 1970.3 5032.3
    LPZ284 42.5 119.4 0.3 48 804 748.4 1365.8 66.6 0
    LPZ286 34.3 164.5 0 74.8 973.6 1463.4 1205.7 81.2 329.3
    LPZ287 118.8 186.4 0 116.4 1573.7 1568.6 2124 252.9 884.4
    LPZ288 103.4 162.8 0 78.3 1328.1 2890.4 4192.3 196.9 598.1
    LP2289 137.1 87 0 113.6 2096 2101 5147.9 332.3 913.7
    LPZ290 1598 425.5 186 1782.8 10543.9 4598.5 16706.4 3923.8 5648.6
    LPZ293 155.8 225.7 0 129.9 3228.2 2291.5 4891.2 316.9 2001
    LPZ294 65.5 180.6 0 66.6 2237.5 527.7 4397.4 127.5 688.2
    LPZ295 119.9 258.8 0 149.7 1964.6 560.6 5062.1 141.5 897.3
    LPZ297 333.9 277.6 59.4 830 5667.2 3950.7 5680.5 1452.6 3614
    LPZ299 102.6 225 12.2 268.1 734.7 898.2 4025.5 371.2 913
    LPZ300 231 271.7 97.1 42.6 113.2 1713.7 3264.5 4295.2 631.1
    LPZ301 272.7 378.2 155.6 97.2 77.2 2110.4 1733.8 485.6 1392.8
    LPZ303 145.6 184.3 641.8 52.8 1562.5 1072.8 365 55.5 358.3
    LPZ304 422.5 346.5 108.2 350.8 3262.7 2215 1102.8 264.1 1534.7
    LPZ306 2207.6 484.7 609 3182.2 9671.4 3639.8 17157.3 3556.4 15015.7
    LPZ307 1761.1 1119.4 454.7 846.1 4114.9 2673.3 4082.5 554.8 2983.5
    LPZ308 153.5 213.5 85.5 113.3 1369.9 1433.3 133.8 123.2 1146
    LPZ309 132 192 14.3 49.2 1137.6 1626.7 126.9 85.1 805.1
    LPZ310 325.9 353.6 311.3 253.1 4210.4 2703.3 1846.5 513.3 3102.9
    LPZ311 176.9 217.7 72.3 245 3652.2 4352.6 4175.7 734.7 4625.7
    LPZ312 70.4 177.2 139.4 53.2 2094.2 1461.1 945.5 57 274
    LPZ314 247.5 221.2 217.2 66.9 1767 753.8 872.4 93.1 751.2
    LPZ315 167.6 220.4 322.5 172.9 3442.4 1985.7 2505.1 698.5 3667.7
    LPZ318 912.5 297.8 441.9 957.6 7473.1 2682.6 6826.4 2471.8 5148.9
    LPZ320 7.3 212.8 154.8 55.8 1682.1 1548.5 1038.5 111.1 822.5
    LPZ321 199 259.6 157.2 96.8 2588.8 2465.4 3436.8 409.2 3989
    LPS001 369.9 656.8 1322 4095.5 4733.4 7892.2 3248.2 5064.2 6260.9
    LPS003 442.5 392.9 262.1 648.7 2035.7 570.6 4524.4 1332.8 543.9
    LPS004 811.4 552.2 515.2 1694.9 2438.2 425.1 1218.8 714.3 86.8
    LPS006 271 141.2 200.2 0 662.1 1245.1 261.5 1282.6 405.7
    LPS007 212.1 91.8 139 47.7 243.4 0 929.2 568 267.1
    LPS008 197.6 196.8 224.7 0 235.1 2144.5 1034.5 985.2 307.3
    LPS010 152.7 133.4 253.8 241 299.5 1667.7 242.1 1218.8 1170
    LPS011 186 136.1 257.9 816.5 257.4 2372.2 77.7 1377.7 1346.8
    LPS012 317.2 238.4 236.4 817.5 1316.7 1626.1 0 1324.1 564.7
    LPS013 349.5 403.6 412.9 1840 1582.6 2769.8 215.4 1196 1328.8
    LPS014 511.4 1574.1 257.3 4169.4 5035.7 4543.5 1016.4 3744.8 3779.2
    LPS015 2495.9 3076.7 1720.8 7899.3 3640.5 7103 1228.1 5376.7 11989
    LPS019 1028.2 604.5 239.7 3074 2189.3 2161.1 1239.9 2955 2426.7
    LPS020 324.1 222.9 120.5 1214.3 1056.8 676.2 872.7 916.8 1058.1
    LPS023 160.9 116.3 50.3 623.9 1984.1 0 640.7 349.7 694.3
    LPS024 359.3 324.3 198.9 1596 2789.8 1253.1 1212.2 2644.1 3158.3
    LPS025 614.7 616.8 493.6 3452.4 3736.3 3726.5 1796.1 4133.4 5569.5
    LPS026 153.7 494.7 0 3053.4 3077.5 2157.2 806.2 3886.5 2133.1
    LPS027 132.2 267.1 0 1309.2 2323.4 1330.2 1501.6 1931.3 1355.1
    LPS028 214.3 446.5 155.7 2472.5 3336.1 2467.9 2987.9 4507.5 3530.8
    LPS029 202.7 384.3 223 2040.4 2060.5 2600.8 7214.8 4150 3867.7
    LPS030 113.5 132.5 0 515.6 2793.9 110 2158.7 2684.7 1237
    LPS031 168.7 123.5 11 557.6 3086 812.5 6212.6 2534.1 3290.9
    LPS032 145.2 160.9 1.7 650.5 2144.2 1070.8 1474.2 2819 3561.4
    LPS036 582.6 616.4 200.9 2224.2 2656.4 1723.6 3073.1 1866.1 2955.4
    LPS037 502.6 620.4 219 1626.6 3359.2 2705.7 2125.3 2456.9 2004.1
    LPS038 962.6 216.5 375.7 0 1256.5 930.9 1492.9 1578.3 1406.9
    LPS040 228.9 86.1 158.6 0 256.8 0 245.1 758.3 1.7
    LPS041 222.7 149.2 123.3 0 252.4 0 965.1 1065.9 553.4
    LPS042 447 661.1 489.9 1672.8 2520.7 492 1046.8 2834.7 1940
    LPS043 333.7 264.6 407.3 656.8 1046.4 546.5 1412.3 1097.9 948.4
    LPS044 249.8 277.8 652 1327.3 907.5 1110.5 1892.3 1353.4 1674.8
    LPS045 250.7 107.2 177.9 302.6 231.9 944.7 1881.7 0 485.4
    LPS046 232.4 285.6 224.5 1302.1 1872.6 1104.6 2610.7 1128 1744.2
    LPS047 2649.2 6969.7 2792.6 14436.1 10141 5428.8 4057.3 2999.2 13647.7
    LPS050 2428 7502 3442.1 12204.8 7385.4 9850.6 3395 8052.4 14726
    LPS051 478.2 219.7 175.1 1861 2222.1 1876.9 1922.5 800.9 2052.9
    LPS052 328.2 196.2 138.3 984.6 1458.3 1324.4 1585 1112.9 1726
    LPS053 264.2 111 0 1370.2 1524.9 1548 2692.1 2941.5 3096.1
    LPS054 285.4 231.1 8.5 1370.4 2138 1273.8 2930.8 2762.2 2604.2
    LPS055 1192.2 1118.8 180.5 5840.8 4203.1 3690.8 1865.1 3205.9 4824.3
    LPS056 1541.6 2959.9 1354 9519.2 8084 6331.3 1820.6 5902.8 11668.9
    LPS057 214.5 406.9 0 2028.8 2744.4 756.5 2136.1 1844.8 2454.6
    LPS058 277.4 244.5 70.8 1623.9 1826.1 1626.5 2708 2514.4 4077
    LPS059 115.3 135.2 1.5 996.9 1194.7 1022.8 1723.1 1265.3 2390.7
    LPS060 163.3 268.5 0 1866.5 1707.3 1953.7 2184.7 2422.6 2990
    LPS061 222.7 255.6 53.4 1448.5 2146.6 1600.7 1956.2 2511.7 3332.5
    LPS062 136 228.5 99.1 627.1 863.2 467.7 1610.3 2304.9 2842.2
    LPS063 299.7 251.8 226.3 796.8 1427 1771.5 1174.1 930 1433.9
    LPS064 3079.8 4014.9 3039.3 7349.2 10807.8 7372.1 10515.8 4426.8 13038.7
    LPS065 434.1 644.6 313.7 1147 1456.3 3097.5 2632.9 3695.2 1575.1
    LPS066 214.7 171.7 134.2 0 69.3 249.7 726.1 871.9 586.6
    LPS067 706.6 686.2 488.8 3013.5 2498.9 4522.1 4844.3 4782.1 5775
    LPS069 199.6 172.1 123.9 75.4 19.4 269 874.1 854.6 0
    LPS070 143.6 186.9 117.9 289.4 685.5 222.6 528.3 582.6 322.6
    LPS071 180.4 170.2 187.2 157.2 183 882.3 326.7 508.4 310.2
    LPS072 235.9 170.8 169.6 449.9 290.5 777.5 456.3 283.3 479.2
    LPS073 900.4 1318.7 629.3 3416.1 4420.4 3894 4010.1 3367.3 4106.8
    LPS074 27858.2 33812.3 32162.2 44513.2 111430.2 87262.8 47575.8 18233.4 66903.6
    LPS075 2119.6 3296.9 1347.3 9540.3 5518.1 6367.8 10437.2 4054.1 9821.4
    LPS076 347.6 336.3 218.5 2343.7 2326.5 1569 2415.2 1580.7 1990.4
    LPS077 568.1 612.4 550.1 2908.9 1727.9 1660.2 2164.5 1798.6 2588.1
    LPS078 3174.9 3137.6 3222.6 7616.1 6945.1 9024.5 11397.6 7995.6 26362.3
    LPS079 1049 1066.4 302.2 4400.8 4126.8 4404.7 8203.7 4645.6 9377
    LPS080 21208.7 28180.7 9065.4 39068.1 63741.1 37523.8 35948.5 11444.8 57266.6
    LPS081 27381 33419.5 10292.3 43529.2 63629.5 28119 42128.9 15984.6 62043.4
    LPS083 711.4 825 64.8 3306.9 3733.1 1898.2 3688.2 3407.5 3959.9
    LPS084 216.4 211.2 21 1350.5 1724.2 1394.5 1965.6 2089.4 3604.5
    LPS086 185.6 214.1 0 1808.2 1476 2915.6 2342.5 932.2 3339.1
    LPS087 3404.7 5840.3 4144.1 12101.2 12860.9 14601.5 24953 5018.8 19643.5
    LPS088 165.2 224.6 62.5 1497.7 2813.8 1593.8 3740.9 4017.3 3934.9
    LPS089 223.8 213.7 0 1318.9 1574.5 2141.4 2443.5 3799.4 4185.6
    LPS090 398.7 693.2 142.2 2593.7 2695.2 3465 3755.9 3638.7 3587.8
    LPS091 391.2 700.6 270.7 1469.2 2092.2 3047.2 3754.2 3524.1 3149.7
    LPS092 286.1 376.1 254.9 235.2 433.1 1353.8 1747.8 2658.1 3246.6
    LPS093 185.6 273.7 126.3 114.7 296.8 254.7 412.1 1076.9 483.7
    LPS094 232.8 261.8 151.7 274.3 249.9 641.1 891 577 1102.1
    LPS095 199.3 191.7 91.4 25.7 169.9 433.7 813.4 1394.3 807.7
    LPS096 97.9 139.2 63.2 0 162.4 159.2 504.6 752.3 150.6
    LPZ001 150.8 207.8 257.7 202.6 485.5 704.2 555.2 1924.3 528.5
    LPZ002 154.6 167.7 323.6 937.4 717.4 755.6 1068.8 1089.8 1107.7
    LPZ003 1348.1 1781.1 609.3 5019 4261.6 6148.5 5600.4 3419.6 4858.4
    LPZ004 2798.4 5533.1 2921.8 10703.8 8020.5 9958.4 10424.8 4135.9 14742.8
    LPZ005 8403.4 19547.7 8589.6 32699.4 31426.9 21580.2 19437.9 8059.6 14660.3
    LPZ006 360.6 2106.1 1173.8 7812.9 7966 11310.2 10516.8 4661.6 4618.2
    LPZ007 272.5 409.9 454.7 2038.3 1107.6 2043.7 2073.5 2249.9 2751.3
    LPZ008 207.7 258.2 212 1939.1 1482.6 1926.1 2243.5 1036 3324.9
    LPZ009 745.1 496.5 633.9 4276.8 7474.5 9130.4 9814 5721.3 14116.8
    LPZ010 893.7 1464.1 326.6 3759.5 4034 4261 4672.2 4388.6 9625.3
    LPZ011 1829.5 2488.7 350.4 5922.7 4285.4 2984.2 5579.8 4236 9972.7
    LPZ012 227.8 289.9 28.6 1885.9 1660.5 843.6 1913 1434.5 2785.5
    LPZ013 247.7 213.8 84 1553 2015.3 1547.3 2567.6 3196.5 4347.7
    LPZ015 261.7 315.8 55.6 2254.3 2409.8 2190.1 2562.4 1291.2 3237.3
    LPZ016 2750.3 2151.8 3003.8 8316.2 6689.1 9147.4 9444.8 3349.6 8167.8
    LPZ017 582.2 701.3 227.4 3830.1 3650.3 3828.2 4552.3 4574.5 4476.8
    LPZ018 2867.6 6184.2 2746.8 10513.4 9443.1 10880.7 12748.3 5491.2 19422
    LPZ019 7551.3 15875.3 9232.8 20440.4 22870.4 31026.1 33842 12823.6 38075.4
    LPZ020 293.1 896.2 143.1 1661.8 2519.9 2987.1 4132.4 4022 3145.1
    LPZ022 213.3 493.5 173.5 82.5 467.8 1355.1 1041.1 1481.9 1035.5
    LPZ023 191.2 616.7 118.1 78.4 184 955.2 516.3 1254.6 574.4
    LPZ024 142.6 321.7 118 0 81.9 826.8 195.8 763.9 491.7
    LPZ025 661.9 764.4 536.9 1885.6 1791.8 3200.6 3017.8 4028.4 3897.8
    LPZ026 194.7 221 150.4 1102 513.6 1714.4 1291.2 1625.6 980.7
    LPZ028 301.3 424.2 210.9 1467.7 1654.3 2848.4 1937.1 4092.7 3086.2
    LPZ029 132 151 124.8 319.5 644.1 478.7 452.4 564.8 709.3
    LPZ030 305.3 616.6 170.6 2800.7 2572 2485.4 1960.4 1425.4 2381.1
    LPZ031 1945 3098.6 3636.7 12422.3 7673.2 8643.5 11552.7 4295.4 4600
    LPZ032 26761.5 33518.4 33623.2 45482.1 106536.1 114284.5 46968.1 16371.6 42282.1
    LPZ033 2068.3 1779.5 6651.8 7887.8 5249.8 9848.7 7632.1 4500.7 7710
    LPZ034 221.4 363.3 216.3 2503 1949.2 1674 2078.9 1428.3 1774.9
    LPZ035 110.7 156.8 85.2 836.6 512.1 1355.7 1217.4 294 1525.3
    LPZ037 229.5 206.2 186.5 1422.1 1962.7 2742.9 3023 614.1 2895.5
    LPZ038 605.7 722.8 352.5 3551.8 3072.2 3614.2 3266 2494.6 4039.7
    LPZ039 366.4 964.6 177.1 3755.2 2744.9 4599.4 3589.7 2407.8 3925.2
    LPZ040 185.6 278.3 100.8 2131.2 1321 1479.7 1654.6 773.7 2087
    LPZ041 119.9 120.8 5 1199.8 1220.8 1090 1431.2 630 2206.1
    LPZ042 61.9 121.9 2.6 731.4 1897.5 986.4 1366.1 458.4 2625
    LPZ043 357.9 355.1 0 3236.5 2746.8 2960.1 3138.2 911.1 3345.6
    LPZ045 738.6 1003.7 565.3 3866 3168.1 6406.2 4028.7 4526.2 4573.4
    LPZ047 139.7 133.1 0 481.3 857.9 831.8 954.1 1926.3 3129.6
    LPZ049 1396.5 2125.5 1496.9 4514.4 3629.8 5942.4 6898.6 3610.7 9214.2
    LPZ051 264.6 610.7 205.1 826.3 1819.9 2243.8 3000.9 3400.7 2810.3
    LPZ053 174.9 827.9 152 161.2 563.8 1149.7 1277.9 1243 1383.5
    LPZ054 205.7 951 128.4 976.1 1901.4 1626.6 1265.8 1437.6 1328.8
    LPZ055 135.2 389 168.5 420.2 524.3 1650.6 848.4 1200.6 914.2
    LPZ056 190.2 323.3 229.5 439.9 664.2 1613.1 1014.2 1727.3 1126.2
    LPZ057 87 199.9 180.1 2154 863.7 3059.1 2994.9 2696.3 2990
    LPZ058 139.3 227.5 55.3 1695.1 902.5 2426.6 2195.6 1925.4 1598.2
    LPZ059 173.4 289.6 189.4 891.7 759.7 1835.3 1332.9 962.5 1286.6
    LPZ060 301 464.8 114.1 2296.5 2860.4 2786.6 2974.4 1629.6 2301.9
    LPZ061 1212.2 1711.1 794.4 7468.5 5190.1 7957.5 5857.5 2819.8 3922.3
    LPZ062 2078.9 3648.3 2499.3 14932.7 7691.9 9294.1 9213.3 3077.8 4850.3
    LPZ063 641.6 984 2114.2 5547.2 3688.6 6191.3 4844.5 4058.7 4162.4
    LPZ065 520.2 443.3 332.3 2720.4 1816 2848.5 3320.4 4501 3948.4
    LPZ066 663.7 357 356.6 3458.6 2196.3 3567.6 3081.4 1325 2388
    LPZ067 1469.5 2582.6 3152.4 8674.9 8080.1 9367.6 8556.2 4135 7240
    LPZ069 211.4 283.6 0 1921.6 913.1 1567.5 1866.5 1043.3 2269.1
    LPZ070 229.6 334.9 2.8 1659.3 1254.6 1681.6 1883.6 1360.7 2442
    LPZ071 332.3 633 15.7 3126 2729.9 3290.2 2998 2011.7 2744.8
    LPZ072 39 38.9 0 581.2 1401.7 1307.1 1089.7 710.4 1866.3
    LPZ073 131.3 250.9 4.4 1176.4 2903.2 2356.7 1718.3 985.9 2700
    LPZ074 92 116.5 0 355.6 1643 1041.3 1027.4 1042 2553.6
    LPZ075 195.9 0 0 0 474.7 488.1 847.6 1488.4 2755
    LPZ076 232.4 134.5 730.7 0 268.9 0 568.3 1007.4 2624.4
    LPZ077 1143.1 2350.7 187.5 6551.1 5960.9 5520.2 7189.6 3483.6 7931.5
    LPZ078 851.5 1021 873.3 3011.7 4619.7 5273.6 6408.3 3950.1 8831
    LPZ079 281.6 779.7 315.9 1296.6 2065 2090.8 2287 2271.9 1824.5
    LPZ080 1653.5 3124.5 3778.7 8321.1 7987.7 10470.3 8085.7 4454.9 8067.4
    LPZ081 92.6 292.7 161.8 746.8 903 1558.5 1410.8 746.1 1230
    LPZ082 123.2 430.5 240.1 1907.9 1283.9 2707.5 1801.7 948.9 1634
    LPZ083 77.8 183.2 120.2 4010 2437.2 4390.6 3649.7 3983.1 7032.5
    LPZ084 1272.3 956.1 1297.2 5881 4505.3 10642.2 9798.6 3938.5 8446.3
    LPZ085 321 466.2 457.6 4324.8 4008.4 6890.5 4599.7 5672 10082.2
    LPZ086 1529.6 3587.2 3236.8 10729.4 10010.2 10739.2 10634.9 4674.2 11980.7
    LPZ089 614.3 500.4 601.9 4196.2 3890.5 4405.8 4331.9 3066.9 3960.3
    LPZ090 643.8 1177.5 1315 4017.8 4456.2 6394.5 4824.4 3000.1 3538.3
    LPZ091 1006.7 1754.2 4090 11615.8 11728.5 16837.8 14461.9 5005.1 14726.4
    LPZ092 419 528.6 1336.8 4158.4 3568.1 8393.4 8192 4638.4 3939.4
    LPZ093 162.2 453 90.9 1436.5 899.2 2218.6 1798 741.8 1789.8
    LPZ094 123.1 197.1 0 1355.7 779.4 1360.5 1713.8 794.4 1597.8
    LPZ095 94 130.6 3.5 1099.9 737.4 558.3 1473.4 871.4 1809.3
    LPZ096 314.3 327.5 22.1 2574.2 1620.9 2748.1 2533.9 1858.4 2979.9
    LPZ099 231.7 359.8 6.2 1456.9 1335.7 1672.4 2170.8 2160.8 2755.8
    LPZ100 375.6 650.4 136.7 2834 2518.6 3053.2 3159.8 2841.2 3453.7
    LPZ101 217.1 425.7 11.8 2769.3 3312.6 2556.3 5262.5 1501.2 2947.3
    LPZ102 294.3 289.2 55 1803.6 2764.5 2532.5 2613.4 2447.6 3377.6
    LPZ103 224.2 92.8 17 989 1850.6 1643.7 2303.4 3729.5 4029.5
    LPZ106 328.4 158 57.5 912.5 1239.6 1214.5 1669.6 2062.3 4720
    LPZ107 25137.4 28865.6 19438.9 43316.7 75674.1 58296.4 45927 3332.1 64391.6
    LPZ108 1132.8 2084.1 330.6 4299.2 3629.8 4480 6406.5 3235.2 9993.6
    LPZ109 548.8 1356.1 417.7 3237.9 2581.9 3177.5 3977.1 1874.2 4107.7
    LPZ110 379.7 1132.3 220 3941.9 2720.7 4103.6 3563 2245.7 3031
    LPZ111 157.5 383.2 200.3 1188.3 749.8 1675.7 1926.1 1685.1 1655.3
    LPZ112 332.1 522.9 220.7 2950.9 2223.9 2257.5 2856.4 2632.8 2711.8
    LPZ114 590 151.8 217 4659.6 3824.5 6848.2 7228.9 2831.3 9446.9
    LPZ115 6268.6 3368.3 8724.6 25695 19481.5 33011.8 32267.9 2903.4 52760.4
    LPZ116 891.2 766.7 1481 6455.1 3684.7 5597.5 8456.7 1682.9 10139.2
    LPZ117 529.8 826.6 126.5 3706.8 2663 2897.5 3996.1 1807.3 2635.2
    LPZ118 437.8 628.1 122.1 3903.5 3245.2 3529.9 3500.1 3506.1 2867.9
    LPZ119 331.3 755.1 83.1 3761.2 4072.6 4024.9 3449.9 3742.2 2533.9
    LPZ120 196.5 796 181.4 4219.4 3297.7 4322 4539.3 2622.7 3763.6
    LPZ122 154.4 208.4 158 2332.3 1468 3044 2838.8 2112.5 2323.6
    LPZ124 172.9 420.1 136.6 2319.5 1539.3 1722.7 2453.6 3109.1 2297
    LPZ126 446.5 604.2 187.3 3859.4 3120.8 2816.9 3324 2181.4 3237.7
    LPZ127 439.3 499.4 53.5 3781.8 3083.3 3769.8 3848.4 1729.6 2902.2
    LPZ128 1022.3 1063.8 447.9 3904.9 3753.5 5579.6 4534.2 2133.1 5843.6
    LPZ131 249.6 373.8 27.3 2170.1 1551.6 1611.4 3077.3 2946 2409.5
    LPZ133 325.3 328.7 42 2706.9 2358.1 2131.3 3257.2 2598.7 2144.3
    LPZ136 263.6 384.1 0.9 2377.3 3386.4 2044.7 2726.4 2398.2 2570.6
    LPZ137 351.6 402.6 91.9 3435.9 3122.8 2406.9 3054.7 3392.1 3216.6
    LPZ138 1047.7 936 530.3 3920 3091.2 2507.9 4249.5 3109.9 4758.4
    LPZ140 379.9 456.6 105.6 1936.7 3674 2908.4 3678.1 3663.2 4481.7
    LPZ141 715.4 1341.8 536.4 4712.8 3615.6 3746 4815.5 4533 8229.3
    LPZ143 3251.1 4810.8 2848.5 9675.1 8289.6 9486.6 12270.6 4424.9 28251.9
    LPZ144 386.2 1338.3 294.1 3433.8 3015.7 3333.7 4618.9 3699.7 6825.1
    LPZ145 277.5 1064 536.5 3072.3 1155.6 2977.7 3188.2 7388.2 3013.6
    LPZ146 128.5 362.8 139.5 2224.4 1364 2125.6 2428.3 3465.8 2351.1
    LPZ147 266.5 544.2 241.3 3318.1 2241.9 2162.6 2908.8 3252.6 2525.6
    LPZ148 725.8 725.5 290.2 5002.2 3682.1 6542.3 6927.9 2903.1 10410.9
    LPZ149 2492.7 3102.7 1052.7 8980.9 7669.2 7185.3 9921 7375.4 12848.8
    LP2150 5257.3 7586.3 3221.3 15156.2 12776.8 7401.2 14686.5 1570 20635.9
    LPZ151 1384 2049.2 1128.3 5576.7 3853.7 3780.3 7227.3 3206.7 7702.7
    LPZ152 975.8 1300.2 546.4 5096.1 4344.9 4302.5 4960.3 1566.2 4223.4
    LPZ153 13378.6 22235.1 10073.1 33495.1 50802.7 28291.5 29602.5 3224.1 40232.7
    LPZ154 663.3 738 430.3 3847.3 3880.2 6253.9 4707.7 5341.9 3393.8
    LPZ155 1121.7 612.7 748 3902.3 4289 4676 5328 6964.2 5353.5
    LPZ157 1157 957.9 762.2 4218.8 4266.6 3777.2 4583.7 5017.5 4396.6
    LPZ158 13278.5 17582.9 9898.7 32456.5 42805.1 24534.6 31442.6 4152.5 36015.3
    LPZ162 3407.5 5943.1 5126.6 10324 11710.9 7727.6 7572.2 2312.6 9394.1
    LPZ165 1419.4 1550 693 4519.9 3692.9 6102.1 5617.1 3061.3 4706.4
    LPZ166 2642.1 3504.9 1288.6 7134.8 6994.5 5170.5 10453.3 4932.9 15993.7
    LPZ167 980.4 1518.1 564.7 4902.6 4803.7 3343.6 4869.5 4421.2 6582.2
    LPZ169 621.8 792.7 93.4 3562 4266.3 1794.9 3496.3 4238 3143.7
    LPZ170 1009.3 1405.3 695.2 5793.7 4469.2 4952.7 6239.7 3914.1 6782.3
    LPZ171 1064.6 968.6 1050.7 4110.8 3903.7 5467.2 5659 3164.1 6795.3
    LPZ172 1233.3 1113.3 401.8 4207.9 7922 8417.5 10419.2 7983.5 15065.2
    LPZ173 3333.8 5236.8 6072 9552 8880 8653.2 13461.6 2408.7 25719.1
    LPZ174 486.7 1263.6 143.3 3318.2 2027 3632.3 4245.3 6086.4 8781.2
    LPZ175 594.7 1487 520.8 3051.5 3610.8 1846.4 3642.9 4048.5 4329.5
    LPZ177 167.3 481.7 234.5 1955.9 1139.2 907.8 1452.9 4462.5 1762
    LPZ179 1231.4 1583.2 835.8 5029.7 3654 3871.7 3248.8 2741.9 2853.2
    LPZ181 2259.5 4282.9 774.8 8911.6 8788.6 6833.3 5825.5 3398 3922.8
    LPZ182 1194.7 2289.6 673.8 8220.3 5601.2 5869.3 5572 1808.4 7607.9
    LPZ186 6255.6 6866.5 6841.1 24861.8 16742.4 23502.2 17304.6 1750.4 27328.9
    LPZ189 27581.8 34787.4 31889 45673.7 106688 95043.9 46998.5 3548.1 67943.9
    LPZ194 673.9 904.9 528.7 4064.5 3530.2 3160.6 5095.2 4531.1 3492.1
    LPZ195 898.9 889.6 675.7 5606.3 4004.3 5230.5 5721.4 4908.1 4940.9
    LPZ196 1073.7 1935.1 558.2 3941.1 3672.6 3681.6 5977.8 3059.7 5050.3
    LPZ197 488.4 522.5 386.6 2613.4 1684.4 3541.4 3507.4 4980.8 2763.1
    LPZ198 575.6 733.1 299.6 4152.3 2411 2916.8 3872.4 6530.7 2931.8
    LPZ199 390.7 442.5 222.4 2704 2176.2 3159.7 2957.1 5357.6 2983.4
    LPZ201 2255.7 3876.6 347.6 10222.9 6897.7 6294.3 7324.7 3549 3857
    LPZ202 25939.2 34864.8 28937 43395.9 85136 71116.8 38688.5 2676.2 14449.1
    LPZ203 4917.3 4458.1 2603.2 10348.3 6571.1 8325.7 11034 5453.1 6598
    LPZ204 27637.2 31853.9 22475.7 43983.9 89520.6 46504.2 44333.5 7963.8 58054.1
    LPZ205 1184 1084.2 327.7 3901.8 4402 3125 4598.2 4501.5 4714.2
    LPZ206 1309.8 1509.5 367.3 3961.5 3983.3 3079.6 4196.2 3641.4 3247.6
    LPZ207 27569.1 30446.2 27094.6 45211.6 90196.1 58153.8 46488.1 9879.3 64709.2
    LPZ208 22722 28208.9 30019.5 40314 74354.1 37339 33919.6 3989.8 56683.6
    LPZ210 1015.1 1789.8 196.8 5006.3 6159.7 3067.8 4944 4347.5 6846.4
    LPZ211 277 327.3 519.4 2540.7 1788.5 3048.6 1110.8 2577.4 3506.6
    LPZ212 1095.5 865.5 930.6 4051.6 4491.6 2857.6 6348.1 4078.7 16174.9
    LPZ213 376.6 539.3 339.6 2503.1 1540.8 1333.7 3082.2 6024.3 4163.1
    LPZ214 137.5 306.1 190.1 1915.6 866.1 1280.9 1240.8 6372.4 2111.8
    LPZ215 3519.4 3120.9 3300.9 16939.5 15489.1 10948.6 12502 3515.1 16236.4
    LPZ216 26761.3 34226.4 28477.9 42274.8 67630.3 41420.3 36331.4 1433.8 17109.4
    LPZ217 15563.1 21739.4 12259 26824.9 34266.3 9429.4 28156.7 1339.2 41568.4
    LPZ219 2404.9 3704.5 2084 8575.1 8573.2 6237 11757 3255.4 13484.9
    LPZ220 3617.2 6998.6 7957.2 13960 9400.8 3432.3 10805.5 3551.7 12867.4
    LPZ221 482.6 478 1405.6 3296.5 3079.8 3312.5 4143 4429.4 3267.3
    LPZ222 318.3 524.5 406.6 3011.7 2309 3811.8 4199.4 5319.9 3292.3
    LP2223 367.7 633.2 437.7 2752 1970.3 3767.4 2514.2 3672.3 2163.1
    LP2224 337.9 1030.2 317.2 2701.2 1798.7 7393.6 2962.5 7876 3353.7
    LPZ225 3288.1 3590.3 2912 8781.8 7400.7 2317.1 11370.2 3186.6 22244.6
    LP2226 325.6 361.4 128 2467 1263.7 10190.1 1636.3 3808.8 1166.9
    LP2227 2175.5 6375.8 458.8 6316.2 6632.1 9013.9 6614.3 3901.2 2588.2
    LPZ228 2638 3701.7 500.1 5991 4819.8 5747.8 7102.5 3182.5 4185.4
    LPZ231 1631.7 2090.4 260.7 5811.5 4749 2530.8 5033.1 3191.3 3810.5
    LPZ233 1596.6 1223 296.6 4355.6 3818.9 2988.8 3749 3324.7 3855.2
    LPZ234 1734.3 1479.2 219.6 5058.5 4614.4 2034.9 4992.1 1979.9 5152
    LPZ235 626 635.9 185.9 4066.8 3255.5 4035.7 3368.7 2880.8 3643.5
    LPZ237 1677.8 1385.3 847.4 4536 3702.8 2943.6 4886.5 2307.8 5136.8
    LPZ239 673.4 407.8 245.8 2981.9 3199.2 2781.6 4235.6 2342.6 4863.7
    LPZ240 387 247.4 254.8 2075.8 2317.4 2894 2721.7 2054.5 4317.9
    LPZ241 258.3 337.8 110.9 3503.1 3829.6 22593.5 1889.5 1315.9 8842.6
    LPZ242 4315.9 2560.2 22.5 12510.2 12605.3 2345.2 16197.1 1114.8 39684.4
    LPZ243 174.8 274.4 23.1 2193.6 346.2 2395.7 1366.5 2568.2 3103.8
    LPZ244 417.5 269.1 3458.5 3545.1 1831 2834.7 1781.1 7589.2 5662.7
    LPZ246 889.5 918.7 2302.7 3920.3 3228.9 4409.3 3536 1258.8 2645.3
    LPZ247 1203 2088.9 46.7 4956.9 4253.2 3559.7 4570.8 1702.6 3350.6
    LPZ248 973.3 1338.1 86.2 3977.4 4392.8 2033 4094.9 2062.6 4279.9
    LPZ249 361.3 324.3 206.7 1948.6 1764.3 2098.7 2762.1 1643 2862.4
    LPZ250 267.6 487.8 118.7 2690.4 1522 2989 3121 1928.9 1809.7
    LPZ251 245 279.7 168.5 1409.9 555 9932.3 2552.5 3050.3 1371.1
    LPZ255 2021.3 2488.5 334.1 7289.5 7773.7 1269.1 9020.3 4492.4 10134.6
    LPZ256 67.1 72.2 296.7 412.2 229.8 922.7 570.3 5040.6 1263.9
    LPZ257 167.3 146.9 482.6 521.8 102.5 2699.4 599.8 2362.1 1553.4
    LPZ258 247.5 236.5 69.7 1429.8 974.6 971.9 2668.7 2990.8 3445.9
    LPZ260 98.1 188.8 463.1 377.1 337.2 880 808.9 1552 1084.6
    LPZ261 73.7 20.5 386.3 1143.6 50.3 4443.8 903.3 2309 1341.6
    LPZ264 482.7 528.5 1151.3 3659.7 1972.6 9892.4 2831.4 1584.4 3208
    LPZ265 534.6 647 457.6 4473 4089.9 656 5899.6 2972.2 5649.8
    LPZ266 16.9 61.2 1062.3 876.1 1183.4 1624.3 663.3 622.4 1609.7
    LPZ268 143.7 142.9 255 1983.5 810.3 9809.8 1293.5 1757.3 2177
    LPZ269 1747.5 1271.8 1636.4 7364.1 5108.7 6903.3 11401.2 3774.1 14643.8
    LPZ270 373.8 77.9 1901.2 5015 3872.6 3485.9 5621.1 4284.8 5197.8
    LPZ271 705 473.1 315.6 2863.8 2625.4 2120.5 4048.2 1424.9 4291.1
    LPZ272 2809.4 2423.8 300 5056.2 2463 3534.6 3496.9 609.7 3996.8
    LPZ273 219.8 162.4 242.4 90.2 130.2 3251.3 166 1193.5 1836.5
    LPZ274 489.2 367.7 284.6 991 1104.2 395.9 2282.3 747.2 4535.7
    LPZ275 93.5 140 156.8 433.3 217.1 0 837.9 1056.2 2352.3
    LPZ276 53 109.7 106.8 0 0 0 369.1 1303.6 1897.6
    LPZ277 105.9 159.4 68.7 0 0 230.1 236.2 706.1 1337.9
    LPZ278 65.7 48.3 156.4 0 0 1788.2 406.3 1442.3 1564.1
    LPZ279 214.7 212.2 75.3 1356.3 790.8 5213.3 1722.4 496 2426.2
    LPZ280 156.2 247.7 1553.6 3510.5 2515.1 289.7 3182.1 612.7 3123.7
    LPZ281 34.6 92.1 73 0 0 1648.3 565.5 522.8 543.3
    LPZ282 200.9 187.7 218.1 536.6 205.9 7324.8 1145.4 2977.5 957.5
    LPZ283 1833.5 1880 775.8 3303.2 5113.2 527.6 5281.2 324.5 3806.8
    LPZ284 215 0.6 148.8 0 29.6 848 408.4 148.8 1294.3
    LPZ286 219.7 21.9 13.6 234.9 78.6 2703.6 198.4 947.9 1233.2
    LPZ287 112.6 126.2 36.5 1170.3 459.9 306.3 157.5 1173.7 1821.3
    LPZ288 23.6 62.2 37.5 774.1 639.3 792.6 715.8 1422.6 1169
    LPZ289 44.1 13.1 107.4 323.4 95 9975.4 889.5 2240.6 1894.9
    LPZ290 1324.7 1572.4 1838 6941.6 4616.9 2995.3 11538.8 407.1 12699.5
    LP2293 45.2 246.3 145.6 2785.5 1923.1 0 3185.6 0 3550.4
    LPZ294 0 19.8 0 403.3 280.4 89.1 785.9 551.6 1378.4
    LPZ295 40 24.5 0 169.9 26 1324.9 1058 848.8 1406.5
    LPZ297 385.6 127.6 17.4 1238.5 941.5 0 2680.9 2084.3 4065.3
    LPZ299 106.9 36.2 0 0 926.2 0 1060.7 1854.9 1575.9
    LPZ300 73.2 93.2 80.2 0 1143.6 1053.3 1034.5 2304.9 2120.8
    LPZ301 126.2 0 5.8 161.2 1245.7 516.3 1612 761.3 2826.1
    LPZ303 83.1 488.8 98.6 0 73.5 979.9 538.7 510.7 1214.7
    LPZ304 213.7 498.3 137.6 1028.6 0 5405.8 860 2212.1 2201
    LPZ306 1439.4 1735.3 2526.4 4212.7 3140.4 2090.1 8128.5 4874.6 14413.9
    LPZ307 534.1 710.5 515.5 2785.3 734 0 2137.3 1692.8 3540.3
    LPZ308 116 304.4 137.7 151.8 28.2 364.2 621.1 631.4 851.2
    LPZ309 80.1 137.2 92.7 0 0 2648.1 529.4 192.6 735
    LPZ310 430.8 584.9 799.2 1887.2 1887.1 6161.2 2974.3 3575 2426.6
    LPZ311 690.5 995.7 208.4 3725.8 2843.8 0 4329.3 3620.8 4170.1
    LPZ312 109.8 334.2 34 72.5 4.5 1489.3 140.1 431.6 744.8
    LPZ314 26.5 200.1 3.3 181.2 0 1231.5 331.5 440.1 804.6
    LPZ315 305.8 211.3 147.5 811.2 1008.1 3797 2231.8 1438.8 1881.8
    LPZ318 621.3 715 337 3488.2 2480.9 781.9 4326.1 4824.7 6969.2
    LPZ320 214.8 92.2 9.9 1170.9 54.5 4501.5 1122.3 1169.4 1696.6
    LPZ321 880.4 755.2 1899.3 6166.2 5105.8 411.6 6096.5 4853.6 6057.2
  • TABLE III
    LSC Media Multiplication Media Maturation Media
    Components (mg/L) 16 1133 923
    NH4NO3 603.8 603.8 200.0
    KNO3 909.9 909.9 454.95
    KH2PO4 136.1 136.1 136.1
    Ca(NO3)2.4H2O 236.2 236.2 59.05
    MgSO4.7H2O 246.5 246.5 246.5
    Mg(NO3)2.6H2O 256.5 256.5 256.5
    MgCl2.6 H2O 101.7 101.7 101.7
    Kl 4.15 4.15 4.15
    H3BO3 15.5 15.5 7.75
    MnSO4.H2O 10.5 10.5 10.5
    ZnSO4.7 H2O 14.4 14.4 14.4
    NaMoO4.2 H2O 0.125 0.125 0.125
    CuSO4.5 H2O 0.125 0.125 0.125
    CoCl2.6 H2O 0.125 0.125 0.125
    FeSo4.7 H2O 6.95 6.95 41.7
    Na2EDTA 9.33 9.33 55.9
    Sucrose 30,000 30,000
    Maltose 20,000
    myo-Inositol 1,000 1,000 100
    Casamino acids 500 500 500
    L-Glutamine 450 450 450
    Thiamine.HCl 1.0 1.0 1.0
    Pyridoxine.HCl 0.5 0.5 0.5
    Nicotinic acid 0.5 0.5 0.5
    Glycine 2.0 2.0 2.0
    2,4-D 1.1 1.1
    BAP 0.45 0.45
    Kinetin 0.43 0.43
    Polyethylene glycol 130,000
    ABA 5.2 5.2
    Gelrite 2,500* 2,500* 2,500
    pH 5.7 5.7 5.7

    *For solid media only
  • TABLE IV
    Description of clones used in hybridization study shown in FIG. 9.
    ID
    with
    Clone # Homology Description Arabidopsis Score E-value
    PC04B12 Lotan et al.. 1998. Arabidopsis Required for embryo 79% ID, 171 7e−44
    (‘LEC’ in LEAFY COTYLEDON 1 is maturation & Cotyledon 93% + ve
    figure) sufficient to Induce Embryo identity. Ectopic over 96aa
    Development in Vegetative expression induces
    Cells. Cell 93: 1195-1205 embryonic differentiation
    traits in transgenic
    seedlings.
    ST17B05 PICLKE/CDH3, Chromatin The pickle mutants 50% ID, 166 1e−41
    (‘PLK’ in remodelling. Ogas et al. 1999. express embryonic traits 74% + ve
    figure) PICKLE is a CHD3 chromatin- after germination. over 155aa
    remodeling factor that Represses lec
    regulates the transition from expression
    embryonic to vegetative
    development in Arabidopsis.
    PNAS. 96(24): 13839-13844
    PC08C06 FIE, fertilization-independent Fie mutants initiate 61% ID 92 8e−20
    (‘FIE’ in endosperm protein. Ohad, et al endosperm development 75% + ve
    figure.) 1999. Mutations in FIE, a WD w/o fertilization over 67aa
    polycomb group gene, allow
    endosperm development
    without fertilization. Plant Cell
    11 (3), 407-416
  • TABLE V
    488 499 499 500 500
    (Liquid (Liquid (Liquid (Liquid (Liquid)
    Cell Line Suspen- Suspen- Suspen- Suspen- Suspen-
    (Stage of sion sion sion sion sion
    Develop- Culture: Culture: Culture: Culture: Culture: 260 260
    ment) Stage 1-3) Stage 1-3) Stage 1-3) Stage 1-3) Stage 1-3 (Stage 7) (Stage 9)
    Media 1133 16 1133 16 1133 Maturation maturation
    # Embyros
    Figure US20050125161A1-20050609-C00001
    118.5
    Figure US20050125161A1-20050609-C00002
    187.75
    Figure US20050125161A1-20050609-C00003
    Na na
    ‘FIE’ ++++ + +++ +++ +++ +++ +++
    ‘LEC’
    Figure US20050125161A1-20050609-C00004
    ++
    Figure US20050125161A1-20050609-C00005
    ++
    Figure US20050125161A1-20050609-C00006
    + +
    ‘PKL’ ++++ + +++ +++ +++ +++

    Table 5. Table of data from Fig. 9a & b. Numbers (488, 499, 500, 260) refer to different cell lines Liquid Suspension Culture contains early-stage embryos (stage 1-3) Embryo number refers to the number of late-stage (stage 8-9) embryos
    # produced by each cell line when matured according to Pullman and Webb (1994). + = low expression, ++ medium level of mRNA, +++ = high level of mRNA, ++++ = very high level of mRNA. Circles
    # around certain + signs, see text. Na = not applicable. Levels of mRNA are relative and refer to the experiment depicted in Fig. 9a & b.

Claims (61)

1. A relational database comprising the data of Table I.
2. A method of staging embryos comprising:
a) providing at least one embryo;
b) detecting the expression in the embryo of at least one RNA transcript of Table I; and
c) correlating the expression of said transcript to one or more embryonic stages.
3. The method of claim 2 wherein at least two RNA transcripts are detected or determined and correlated to one or more embryonic stages.
4. The method of claim 2 wherein expression of the at least one RNA transcript is analyzed by hybridization with at least one probe of Table I.
5. The method of claim 2 wherein expression of the at least one RNA transcript is analyzed by hybridization with a variant of at least one probe of Table I.
6. The method of claim 5 wherein said variant hybridizes to at least one probe of Table I under conditions of high stringency.
7. The method of claim 5 wherein said variant hybridizes to at least one probe of Table I under conditions of moderate stringency.
8. The method of claim 2 wherein expression of at least one RNA transcript is detected or determined by at least one member of the group consisting of PCR, Northern Analysis, and in situ hybridization.
9. The method of claim 2 wherein expression of said at least two RNA transcripts are detected by a DNA array.
10. A database comprising a multiplicity of nucleotide sequences shown in any one of Table I, including variants thereof, wherein said variants hybridize under conditions of high stringency to either strand of a denatured, double-stranded DNA comprising any of SEQ ID NOS: 1-327.
11. The database of claim 10 wherein said variants hybridize under conditions of moderate stringency.
12. A DNA array comprising a multiplicity of nucleotide sequences shown in Table I, including variants thereof, wherein said variants hybridize under conditions of high stringency to either strand of a denatured, double-stranded DNA comprising any of SEQ ID NOS: 1-327.
13. The DNA array of claim 12 wherein said variants hybridize under conditions of moderate stringency.
14. A method for staging plant embryos comprising:
a) selecting total RNA from a multiplicity of embryos of known developmental age;
b) correlating the embryonic expression pattern to the developmental age to develop a relational database;
c) determining levels of expression from embryos of unknown developmental age by hybridization to a DNA array comprising a multiplicity of the nucleotide sequences shown in any one of SEQ ID NOS: 1-327;
d) correlating the expression pattern from step 3 to the relational database to determine developmental stage for the unknown embryo.
15. The method of claim 14 wherein the embryos of step 1) are zygotic embryos.
16. The method of claim 14 further comprising the step of altering the embryonic growth conditions to approximate the expression pattern of zygotic embryos.
17. An isolated nucleic acid variant of the nucleotide sequence shown in any one of SEQ ID NOS: 1-334, wherein said variant hybridizes under conditions of moderate stringency to either strand of a denatured, double-stranded DNA comprising any of SEQ ID NOS: 1-334.
18. An isolated polypeptide encoded by a nucleic acid molecule of claim 17.
19. An isolated nucleic acid encoding the polypeptide of claim 18.
20. Antibodies that specifically bind to the peptide of claim 18.
21. The antibodies of claim 20, wherein said antibodies are monoclonal.
22. A recombinant vector that directs the expression of a nucleic acid of claim 17.
23. A host cell transformed with the vector of claim 22.
24. The host cell of claim 23, wherein the host is a somatic pine embryo.
25. A method for staging plant embryos comprising:
a) selecting total RNA from at least one embryo of known developmental age;
b) determining the level of expression of a multiplicity of genes which hybridize to one or more of SEQ ID NOS: 1-327;
c) correlating the known developmental ages of the embryos from step 1) with the profile of expression measured in step 2);
d) applying the correlation of step 3) to a sample of embryo RNA from embryos to be staged; and
e) determining the embryo stage.
26. The method of claim 25, wherein the measurement of gene expression is by RT-PCR.
27. The method of claim 25, wherein the measurement of gene expression is by nucleic acid hybridization.
28. The method of claim 25, wherein the measurement of gene expression is by determining the level of protein expression.
29. The method of claim 28, wherein protein expression is measured by antibody binding.
30. A method for selecting advantageous plant clones comprising:
a) selecting one or more samples of embryonic RNA from multiple clones of plants;
b) determining that at least one sampled clone has an advantageous characteristic;
c) comparing the embryonic levels of expression of genes which hybridize to one or more of SEQ ID NOS: 1-327 in samples from the advantageous clone with expression levels in at least one clone that does not show the advantageous characteristic; and
d) selecting additional clones which show an embryonic gene expression pattern more similar to that of the advantageous clone than to the pattern of at least one clone that does not show the advantageous characteristic.
31. Method of claim 30 where the clones to be sampled or compared are from about the same developmental age.
32. Method of claim 31 where the development age is visually detected.
33. The method of claim 30, wherein the measurement of gene expression is by RT-PCR.
34. The method of claim 30, wherein the measurement of gene expression is by nucleic acid hybridization.
35. The method of claim 30, wherein the measurement of gene expression is by determining the level of protein expression.
36. The method of claim 35, wherein protein expression is measured by antibody binding.
37. A method of determining embryo fitness comprising:
a) creating a relational database with RNA expression values for genes listed in Table I for embryos of known developmental stages;
b) isolating total RNA from embryos of unknown stage development;
c) measuring expression levels of genes identified in Table I from the solated total RNA; and
d) correlating the database of step 1) with the pattern of expression determined in steps 2) and 3) to assess proper embryo development.
38. The method of claim 37, wherein the measurement of gene expression is by RT-PCR.
39. The method of claim 37, wherein the measurement of gene expression is by nucleic acid hybridization.
40. The method of claim 37, wherein the measurement of gene expression is by determining the level of protein expression.
41. The method of claim 40, wherein protein expression is measured by antibody binding.
42. A method for selecting advantageous growth conditions for embryo development comprising:
a) determining RNA expression profiles for staged embryos under control culture conditions;
b) altering culture conditions;
c) determining RNA expression profiles for staged embryos under altered culture conditions; and
d) correlating culture change to developmental effect in embryo.
43. The method of claim 42, wherein conditions are selected which produce RNA expression profiles most closely approximating late-stage embryo profiles.
44. The method of claim 42, wherein the culture conditions are altered by operatively linking one or more stage-specific embryo promoter(s) to one or more sense or antisense nucleic acid molecules.
45. The method of claim 42, wherein the culture conditions are altered by operatively linking one more stage-specific embryo promoter(s) selected from SEQ ID NOS: 328-334 to one or more sense or antisense nucleic acid molecules.
46. The method of claim 42, wherein the change in expression profiles is correlated by a relational database.
47. A recombinant nucleic acid molecule encoding a product during embryo development comprising:
a) a first nucleic acid sequence which is the LP2-3 promoter; and
b) a second nucleic acid sequence encoding a product,
wherein the first nucleic acid is operatively linked to the second nucleic acid molecule whereby its expression is directed by the promoter sequence.
48. The recombinant nucleic acid molecule of claim 47 wherein the second nucleic acid sequence encodes for GFP, or a variant of GFP.
49. The recombinant nucleic acid molecule of claim 48 wherein the second nucleic acid sequence is linked to one or more additional nucleic acid molecules.
50. The recombinant nucleic acid molecule of claim 49 wherein the additional molecule encodes a protein product normally expressed, by a developing embryo at a known stage.
51. The recombinant nucleic acid molecule of claim 47 wherein the second nucleic acid sequence encodes an embryo-derived molecule.
52. The recombinant nucleic acid molecule of claim 51 embryo-derived molecule is stage-specific.
53. A plant cell comprising the recombinant nucleic acid molecule of claim 47.
54. A method for producing a protein product during embryo development comprising:
a) operatively linking one more stage-specific embryo promoter(s) to one or more nucleic acid molecules that encode a protein product,
b) delivering construct to developing embryos.
55. The method of claim 54 wherein the operatively linked nucleic acid molecule is a reporter or indicator gene.
56. The method of claim 54 wherein the operatively linked nucleic acid molecule is GFP, or a variant of GFP
57. The method of claim 54 wherein at least one stage-specific promoter is selected from SEQ ID NOS: 328-334.
58. A method for staging embryos comprising:
a) providing one or more stage-specific embryo promoter(s) operatively linked to one or more nucleic acid molecules that encode a protein product to developing embryos,
b) monitoring expression of the protein product as the embryo matures through stage in which promoter functions.
59. The method of claim 58 wherein the operatively linked nucleic acid molecule is a reporter or indicator gene.
60. The method of claim 58 wherein the operatively linked nucleic acid molecule is GFP, or a variant of GFP.
61. The method of claim 58 wherein at least one stage-specific promoter is selected from SEQ ID NOS: 328-334.
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