WO2000042210A2 - Microsatellite dna markers and uses thereof - Google Patents

Abstract

Microsatellite, simple sequence repeat (SSR), markers have much potential for enhancing genome mapping and genotype identification research in forest genetics and tree breeding. SSR markers were developed by isolating and sequencing 1539 loblolly pine SSR clones for 11 SSR motifs. After screening out redundancy among the sequences, 566 oligonucleotide PCR primer pairs flanking the SRRs were synthesized and evaluated for their ability to amplify genomic DNA from loblolly pine. The three SSR motifs that yielded the highest proportion of informative markers from sequenced clones were (AC)n, (AAAT)n, and (AAAC)n. Eighteen polymorphic tri-a nd tetranucleotide SSR loci were genotyped in 20 loblolly pine trees using automated fluorescent marker analysis. The average number of alleles per locus observed was 6.4, and the average polymorphism information content (PIC) was 0.547. Subsets of the 566 primer pairs were evaluated for their ability to amplify DNA from six other pine species, and 54 primer pairs amplified markers thatwere polymorphic among the species. The present invention also concerns the methods of using the identified SSR loci as genetic markers.

Description

Microsatelite DNA Markers and Uses thereof

This application is a continuation-in-part application of U.S. Application Ser. No (not yet assigned) filed January 15, 1999, entitled Microsatelite DNA Markers and Uses thereof (Attorney Docket No. 4481/0E188), which is hereby incorporated herein by reference.

Field of the Invention

The invention relates to identification and isolation of the simple sequence repeat (SSR) loci in the higher eukaryotes, such as the plants, and particularly the pines. The

SSR loci of the invention are particularly useful as genetic markers for genetic mapping, population genetics studies and inheπtance studies in various plant breeding programs

Background of the Invention Loblolly pine (Pinus taeda L.) is an important, expeπmental and commercial forest tree species native to the southeastern United States. Loblolly pine is planted extensively in the southeastern United States and to lesser degrees in other warm temperate regions of the world. In the United States, plantations are managed and utilized for a variety of products including raw mateπals (wood, fiber, and chemicals), ecosystem components (wildlife habitat and water and soil conservation), and recreational activities

Most of planting stock oπginates from production seed orchards established by various loblolly pine improvement programs. To date, such programs have completed one to three cycles of selection using progeny testing for parental selection and seed orchard development, and family and within-family testing and selection for population improvement. Loblolly pine breeding has various limitations, such as, long generation times to flower (>5 years) and harvest (>15 years), low tolerance to inbreeding, large size of individual trees, variable sites for testing and replanting, difficulty of vegetative propagation, low heritability of important traits, and uncertainty of trait values.

Marker-assisted selection (MAS) using DNA-based markers has much potential for improving the efficiency and effectivenes of tree breeding programs (O'Malley and

McKeand 1994 For. Genet. 1:207-218.). Important improvements afforded by MAS include reducing the time-to-selection and improving the accuracy of selection. An important goal of such research is to identify DNA markers or other measures that predict performance of mature trees. With this information, tree breeders could more confidently select trees at an early age, induce them to flower, and breed them to produce the next generation. In addition, selections made at an early age could be vegetatively propagated in mass using rooted cutting or tissue culture based technologies (Bradshaw and Foster 1992 Can. J. For. Res., 22:1044-1049.). Vegetative propagation and deployment has the potential to greatly improve wood and fiber yield and quality by capturing within-family genetic variation and providing better performing varieites for plantation establishment.

Several of the fundamental limitations to MAS applications in loblolly pine (Strauss et al. 1992 Can. J. For. Res., 22:1050-1061.) have been overcome in recent years. Most notably is the application of randomly-primed, PCR-based genetic markers (e.g., RAPD) to parent- or family-specific genome mapping (Tulsieram et al. 1992, Biotechnology, 10:686-690; Nelson et al 1994 Journal of Heredity, 85:433-439; Plomion et al. 1996 Theor. Appl. Genet., 93:1083-1089., Wilcox et al. 1996 Proc. Natl. Acad. Sci. USA, 93:3859-3864.). Although family-specific mapping and MAS approaches have potential, these methods are limited to situations where small breeding (<10 parents) populations are maintained with progeny established in large-family (n>500) tests. In practice, however, most loblolly pine breeding programs do not fit this situation. More typical is large breeding populations, sometimes several populations per program, and always relatively small-family (n<150) progeny tests. In addition most programs now include many pedigrees of at least three-generations, with nearly mature third-generation trees in the field. Utilizing existing extensive pedigree and progeny test information is essential for developing better MAS technology and improving breeding programs.

Currently available marker systems are not optimal for detecting QTL variation across families and across multi-generation pedigrees. Reviews of current marker technologies and their limitations to use in QTL mapping and MAS is provided by Neale and Harry (1994 AgBiotech News Info., 6: 107N-114N.) and O'Malley and Whetten (1997 Molecular markers and forest trees. DNA Markers: Protocols, Application and Overviews ed. G. Caetano-Anolles and P.M. Gresshoff. John Wiley and Sons, New York., 237-257.). Given a genome size of about 2000 cM(K) for loblolly pine, a large number of highly polymoφhic, co-dominant genetic markers will be needed for genome mapping and QTL analyses (Echt and Nelson 1997 Theor. Appl. Genet., 94:1031-1037.).

Accordingly, there is a need in the art for new genetic markers. In an effort to develop such markers for loblolly pine, the pines and the plants in general, the present inventors developed simple sequence repeat (SSR) markers described herein. The markers of the invention are also useful for other eukaryotic organisms.

Summary of the Invention

Simple sequence repeats (SSRs), which are also known as microsatellite DNA repeats, have now been discovered in the pines and have been shown to exhibit length polymorphisms. These repeats represent an abundant pool of potential genetic markers. Accordingly, in one aspect, the present invention relates to the plant SSR motifs, such as for example, di-, tri- and tetra-nucleotide repeated motifs.

In another aspect, the invention relates to the polynucleotides containing one or more such SSR motifs and the primers for the amplification of the fragments containing

SSRs. The primers may be cloned polynucleotide fragments or chemically synthesized oligonucleotides, and contain at least a portion of the non-repeated, non-polymorphic sequence flanking SSRs on either 5' or 3' end.

The present invention is also directed to a kit for the rapid analysis of one or more specific DNA polymorphisms of the type described in this application The kit includes oligodeoxynucleotide primers for the amplification of fragments containing one or more SSR sequences.

In a further aspect, the invention provides for a method of analyzing one or more specific SSR polymorphisms in an individual or a population, which involves amplification of small segment(s) of DNA containing the SSR and non-repeated flanking

DNA by using the polymerase chain reaction, and sizing the resulting amplified DNA, preferably by electrophoresis on polyacrylamide gels. In yet another aspect, the invention provides for a method of determining the sequence information necessary for primer production by isolation and sequencing of DNA fragments containing the SSRs, using hybridization of a synthetic, cloned, amplified or genomic probe, containing sequences substantially homologous to the SSR, to the DNA.

In a further aspect, the present invention is directed to a method for detecting the presence of a specific trait in a subject, such as a plant. The method includes isolating the genomic DNA from the subject individual and analyzing the genomic DNA with a polymoφhic amplified DNA marker containing one or more SSR sequences. In yet another aspect, the SSR markers of the invention are used in commercial plant breeding. Traits of economic importance in plant crops can be identified through linkage analysis using polymoφhic DNA markers.

Detailed Description of the Invention All patents, patent applications and references cited in this specification are hereby incoφorated herein by reference in their entirety. In case of any inconsistency, the present disclosure governs.

Definitions The following terms and phrases are used throughout the specification with the following intended meanings.

The abbreviation "SSR" stands for a "simple sequence repeat" and refers to any short sequence, foi example, a mono-, di-, tri-, or tetra-nucleotide that is repeated at least once in a particular nucleotide sequence. These sequences are also known in the art as "microsatellites." A SSR can be represented by the general formula (N,N₂...N_j)_n, wherein

N represents nucleotides A, T, C or G, i represents the number of the nucleotides in the base repeat, and n represents the number of times the base is repeated in a particular DNA sequence. The base repeat, be., N,N₂...Ni, is also referred to herein as an "SSR motif." For example, (ATC)₄, refers to a tri-nucleotide ATC motif that is repeated four times in a particular sequence. In other words, (ATC)₄ is a shorthand version of

"ATCATCATCATC."

The term "complement of a SSR motif refers to a complementary strand of the represented motif. For example, the complement of (ATG) motif is (TAC).

The term "permutations of a SSR motif" refers to all possible combinations of a motif found within the repeated sequence of that motif. For example, permutations of the (ATG)₅ motif (i.e., ATGATGATGATGATG) are TGA and GAT as both can be found in this repeat.

The term "perfect repeat" refers to a repeated SSR motif without interruption and without adjacent repeat(s) of a different motif. However, the repeats may be "imperfect" when a repeated SSR motif is interrupted by a number of non-repeated nucleotides, such as for example in (AC)₅GCTAGT(AC)₇ Other possible variations of SSRs would be known to those of skill in the art. These repeats, including compound repeats, are defined by Weber, J.L., 1990, Genomics, 7:524-530.

The term "compound repeat" refers to a SSR that contains at least two different repeated motifs that may be separated by a stretch of non-repeated nucleotides. An example of a compound repeat is (ATC)₅(AT)₆. The term "SSR locus" refers to a location on a chromosome of a SSR motif; locus may be occupied by any one of the alleles of the repeated motif. "Allele" is one of several alternative forms of the SSR motif occupying a given locus on the chromosome. For example, the (ATC)_n locus refers to the fragment of the chromosome containing this repeat, while (ATC)₄ and (ATC)₇ repeats represent two different alleles of the (ATC)_n locus. As used herein, the term locus refers to the repeated SSR motif and the flanking 5' and 3' non-repeated sequences. SSR loci of the invention are useful as genetic markers, such as for determination of polymoφhysm.

"Polymoφhism" is a condition in DNA in which the most frequent variant (or allele) has a population frequency which does not exceed 99%. The term "heterozygosity" (H) is used when a fraction of individuals in a population have different alleles at a particular locus (as opposed to two copies of the same allele). Heterozygosity is the probability that an individual in the population is heterozygous at the locus. Heterozygosity is usually expressed as a percentage (%), ranging from 0 to 100%, or on a scale from 0 to 1. The term "informativeness" is a measure of the utility of the polymoφhism. In general, higher informativeness means greater utility. Informativeness is usually defined in terms of , either heterozygosity or "Polymoφhism Information Content" (PIC) (for PIC see Botstein, D., et al., 1980, Am. J. Hum. Genet., 32, 314-331). The PIC represents the probability that the parental origin of an allele can be determined from the marker genotype of the locus in any given offspring. The PIC values range from 0 to 1.0, and are smaller in value than heterozygosities. The formulas for calculating H and PIC are disclosed in the examples. For markers that are highly informative (heterozygosities exceeding about 70%), the difference between heterozygosity and PIC is slight.

"Primers" are short polynucleotides or oligonucleotides required for a polymerase chain reaction that are complementary to a portion of the polynucleotide to be amplified. The phrase "primer adapted for detection of a SSR marker" means that the primer is capable of amplyfying a particular SSR locus to be used as a marker, wherein the primer is complementary to either the 5' or the 3' non-repeated region of the SSR locus and is of a length suitable for use as a primer. For example, the primer is no more than 50 nucleotides long, preferably less than about 30 nucleotides long, and most preferably less than about 24 nucleotides long. The term "polynucleotide" is intended to include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense strands together or individually (although only sense or anti- sense stand may be represented herein). This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.

An "isolated" nucleic acid or polynucleotide as used herein refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring). An isolated nucleic acid or polypeptide may contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated. A polynucleotide amplified using PCR so that it is sufficiently and easily distinguishable (on a gel from example) from the rest of the cellular components is considered "isolated". The polynucleotides of the invention may be "substantially pure," i.e., having the highest degree of purity that can be achieved using purification techniques known in the art.

The term "hybridization" refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing.

Polynucleotides are "hybridizable" to eacli other when at least one strand of one polynucleotide can anneal to a strand of another polynucleotide under defined stringency conditions. Hybridization requires that the two polynucleotides contain substantially complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated. Typically, hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of 0.5X SSC at 65°C) requires that the sequences exhibit some high degree of complementarily over their entire sequence. Conditions of intermediate stringency (such as, for example, an aqueous solution of 2X SSC at 65 °C) and low stringency (such as, for example, an aqueous solution of 2X SSC at 55 °C), require correspondingly less overall complementarily between the hybridizing sequences. (IX SSC is 0.15 M NaCl, 0.015 M Na citrate.) As used herein, the above solutions and temperatures refer to the probe- washing stage of the hybridization procedure. The term "a polynucleotide that hybridizes under stringent (low, intermediate) conditions" is intended to encompass both single and double-stranded polynucleotides although only one strand will hybridize to the complementary strand of another polynucleotide.

The term "% identity" refers to the percentage of the nucleotides of one polynucleotide that are identical to the nucleotides of another sequence of identical length (excluding the length of the SSR) as implemented by the National Center for

Biotechnology Information. The % identity value may be determined using a PowerBlast program (Zhang and Madden 1977 Genome Res. 7:649-56.).

The term "% homology" between the sequences is a function of the number of matching positions shared by two sequences divided by the number of positions compared and then multiplied by 100. This comparison is made when two sequences are aligned (by introducing gaps if needed) to give maximum homology. PowerBlast program, implemented by the National Center for Biotechnology Information, is used to compute optimal, gapped alignments. Alternatively, the % homology comparison may be determined using a Blast 2.0 program implemented by the National Center for Biotechnology Information.

SSR Motifs and SSR Loci of the Invention The present invention relates to SSR motifs and SSR loci useful as genetic markers in various organisms, particularly plants. In a preferred embodiment of the invention, the SSR motifs and loci originate from the pines, such as the pines of the Pinus genus, for example P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, and P. sylvestris. As seen from the list of exemplary species, the pines and SSRs thereof of the present invention can belong to either of the two subgenera of the Pinus genus. P. strobus (white pine) is a species of the Strobus subgenus, and P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, and P. sylvestris are exemplary species of the Pinus subgenus. The SSR motifs of the invention have the general formula (N,, N₂...N,)_n, wherein:

N represents nucleotides A, T, C or G; i represents the number of the last nucleotide in the SSR motif; and n represents the number of times the SSR motif is repeated in the SSR locus. In one embodiment of the invention, the total number of nucleotides in a motif (i) is about six, preferably four, three or two. The total number of repeats (n) may be from 1 to about 60, preferably from 4 to 40, and most preferably from 10 to 30 when i = 2; preferably 4-25, and most preferably 6-22 when i = 3; and preferably 4-15, and most preferably 5-10 when i = 4. Any SSR motif of the above formula is within the scope of the invention, however, the following SSR motif are preferred: AC, AAC, AAG, AAT, ACC, ACG, AGG, ATC, AAAC, AAAT, AGAT and all complements and permutation of said motifs, such as for example ATG, CAT, TTG, TTA, TTC, ATT, and TAT.

Compound repeats are also within the scope of the invention. Examples of such repeats are: (A)_n...(ATG)_n; (ATG)_n...(C)_n; (CAT)_n...(A)_n; (ACC)_n...(ATC)_n; (TTG)_n...(TTA)_n; (C.)_n...(ATT)„; (TAT)_n...(A)„; (ATIj_n...(AAT)_n; T C)_n...(T)_n; and (A)_n(AAAC)_n(A), The SSR loci of the invention are preferably a maximum about 500 nucleotides long. In another preferred embodiment, the SSR locus of the invention is a minimum of

50 nucleotides long.

The invention further provides for isolated polynucleotides comprising at least one SSR motif and having the nucleotide sequences as shown in Table 3 (SEQ ID NOS: 237 to 354). These polynucleotides may be of the same length as the sequences shown in Table 3 or alternatively comprise additional sequences on their 5', 3' or both ends. The latter polynucleotides may be less than about 500bp, less than about lkb, less than about 2kb or less than about 3kb long. In an embodiment of the invention, the polynucleotides comprising the sequences of SEQ ID NOS: 237-354 do not containing any functional gene or coding sequences.

Further within the scope of the invention are polynucleotides that (i) hybridize under the conditions of low, medium or high stringency to the polynucleotides comprising the sequences of SEQ ID NOS: 237-354 and (ii) contain SSR motifs. In certain embodiment of the invention, these hybridizable polynucleotides are less than about lOOObp long, more preferably less than about 500bp long and most preferably less than about 200 bp long. In one embodiment of the invention, the hybridizable polynucleotide is about the same length as the polynucleotide to which it hybridizes. Also within the scope of the invention are polynucleotides that contain SSR motifs and have at least about 75%, preferably at least about 85%, and most preferably at least about 95% identity to the polynucleotides having the sequence of SEQ ID NOS:237 to 354.

Polynucleotides that contain SSR motifs and have at least about 75%, preferably at least about 85%, and most preferably at least about 95% homology to the polynucleotides having the sequence of SEQ ID NOS:237 to 354 are also within the scope of the invention.

In one preferred embodiment of the invention, polynucleotides that align to polynucleotides of SEQ LD NO:237-354 under the following conditions are also within the scope of the invention: alignment done using PowerBlast network client on

PowerMacG3, when the search is set to high stringency (M=l, N= -5, S=80, S2=80) for blastn, without gap alignment. Most preferably, these polynucleotides are not of human origin.

In another preferred embodiment of the invention, polynucleotides that align to polynucleotides of SEQ ID NO:237-354 under the following conditions are also within the scope of the invention: alignment done using either PowerBlast or Blast 2.0 program using the following parameters: match=l, mismatch= -2, gap open=5, gap extension =2, x_dropoff =50, expect =10, and wordsize =9. Most preferably, these polynucleotides are not of human origin. Isolated polynucleotides comprising at least one SSR motif and having the property of being amplifiable from a genomic DNA using PCR and any of the primer pairs disclosed in Tables 2 and 7 are also within the scope of the invention. These polynucleotides may be identified and isolated by amplification of any genomic DNA. Prefereably, genomic DNA used is a plant DNA, more preferably the pine DNA and most preferably the DNA from the Pinus genus. For example, genomic DNA may be from P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, or P. sylvestris. In one embodyment of the invention, these polynucleotides are less than about 500bp long.

However, the length of the amplified DNA fragment is generally limited only by the resolving power of the particular separation system used. The thin denaturing gels, for example, are capable of resolving fragments differing by as little as 1 base up to a total fragment length of about 300 bp. Use of longer gels and longer electrophoresis times can extend the resolving power up to about 600 bp or more. However, the longer the fragment, the lower the proportion of its length is occupied by the SSR sequences, and hence the resolution is more difficult.

Oligonucleotide primer adapted for detection of SSR marker are also within the scope of the invention. A suitable primer comprises at least the sequence of SEQ ID NOS: 1-236 and 367-390.

The present invention also provides probes specific to at least part of the aforesaid SSRs for delecting SSR markers using methods other than polymerase chain reaction, such as for example hybridization with labeled probes. The probes useful in the invention may be any sequence comprising at least the sequence of SEQ ID NOS: 1-236, as well as any other probe that a person of skill in the art can construct based on the information of

SEQ ID NOS: 237-354.

The SSR loci of the invention may be polymoφhic. They may have a PIC of at least 309c (0.3); ρreier÷n>ι_. ... aι least ^" • • . (0.7); and mosl prefernhiy of al least 90% (0.9). The polynucleotides and primers of the invention may be subcloned and introduced into various host cells according to methods well known in the art. The resulting clones and host cell are also within the scope of the invention. A person of skill in the art can make all such constructs and host cells using methods known in the art. However, the following non-limiting examples are provided below. A large number of vectors, including bacterial, fungal and plant vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech, Palo Alto, CA), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), or pRSET or pREP (Invitrogen, San Diego, CA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.

Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl₂ mediated DNA uptake, fungal infection, microinjection, microprojectile transformation, or other established methods. Appropriate host cells include bacteria, archaebacteria, fungi, especially yeast, and plant and animal cells. Of particular interest are E. coli, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora, CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines. Preferred replication systems include M13, ColEl, SV40, baculovirus, lambda, adenovirus, and the like.

The present invention is also directed to a kit for the rapid analysis of one or more specific DNA polymoφhisms of the type described in this application. The kit includes oligodeoxynucleotide primers for the amplification of fragments containing one or more SSR sequences.

Development and Use of Polymorphic DNA Markers

The present invention provides for the methods of identifying and isolating SSR loci and iheir use as genetic markers.

In one embodyment, a method for the identification from genomic DNA of a fragment comprising a SSR locus comprising the steps of: (i) contacting a DNA library with at least one hybridisation probe so as to identify a population of DNA fragments enriched for simple tandem repeats; (ii) isolating and cloning said population; and (iii) screening of the resulting DNA library so as to identify an individual fragment comprising a simple tandem repeat locus. The DNA library may be a genomic DNA library; the genomic DNA library may be any convenient population of DNA fragments such as pine DNA, or subgenomic DNA libraries such as those generated by PCR from flow soiled chromosomes (see Telenius, H., et al., 1992, Genomics 3: 718-725). The genomic DNA library may be obtained by restriction digestion of genomic DNA. The average fragment size within the DNA library may be less than 1.5 kilobases and may be less than about one kilobase. The fragment size may be from about 400 bp to about 1000 bp. The hybridisation probe or set of probes may be immobilised on a solid phase such as a nylon membrane and may be used to identify a particular class of SSRs. Such classes may include dimeric, trimeric, tetrameric, pentameric and hexameric repeats. Particular oligonucleotide probes for use in the present invention may include oligonucleotide probes comprising a repeated region of greater than 200 bp. The probe may comprise repeats having at least 70%, such as 85% or 100%, identity to a given repeat sequence. The hybridisation probe may be a set of probes comprising mixed trimeric or tetrameric repeat DNA or any other combination of various SSR motifs.

The population of DNA fragments enriched for SSR may be amplified prior to cloning and this may be effected by PCR amplification. Universal linker sequences may be ligated to the ends of individual fragments, possibly prior to the enrichment procedure, and linker sequence specific primers may then be used to amplify the enriched population. Linker sequences may then be removed, for example by restriction digestion, prior to cloning.

In another embodiment, a method for the identification from genomic DNA of a fragment comprising a SSR locus comprises the steps of: (i) ligating universal linker sequences to the ends of fragments comprised in a genomic DNA library so as to form a library for PCR amplification; (ii) contacting said PCR library with at least one hybridisation probe so as lo identify a population of library fragments enriched for simple tandem repeats; (iii) separating and amplifying said population by PCR; and (iv) cloning and screening the resulting amplification products so as to isolate an individual fragment comprising a simple tandem repeat locus.

Cloning may be effected using any convenient cloning procedure and vector (for example pBluescriptll (Stratagene, LaJolla, CA)) such as those described by Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press.

Screening may be effected using any convenient hybridisation probe or set of probes comprising SSR sequences. These may be the same as those disclosed above in respect of the enrichment procedure.

A more detailed description of possible ways of detecting SSR loci is provided in the Examples.

Individual clones comprising SSR loci may be analyzed using conventional techniques to determine, for example, specific sequence information. Such techniques may allow the generation of individual "identities" specific for one or more polymoφhic loci. The generation of such individuals "identities" may be used to identify and characterise family relationships and may be used for e.g. trait tracing in a breeding program and in any other technique which uses SSRs and their polymoφhisms. According to a further aspect of the present invention there are also provided methods of genetic characterisation wherein sample DNA is characterised by reference to at least one of the SSR loci, primer sequences and probes of the invention. The method of genetic characterisation may comprise either the use of at least one hybridisation probe or it may comprise the use of polymerase chain reaction (PCR) primers specific to at least one of the SSR loci in order to amplify selectively the SSR locus. The PCR primers may comprise at least one of the primers and probes of the present invention. The method of genetic characterisation may be used in genetic mapping studies such as linkage studies, and may be used in the genetic analysis of inherited traits.

In one embodyment, the present invention is directed to a method for detecting the presence of a specific trait in a subject, such as a plant. The method includes isolating the genomic DNA from the subject individual and analyzing the genomic DNA with a polymoφhic amplified DNA marker containing one or more SSR sequences. The analysis comprises amplification u.>mg the polymerase chain reaction of one or more short DNA fragments containing the SSR followed by measurement of the sizes of the amplified fragments using gel electrophoresis.

Examples of using SSR markers of the invention for detection of polymoφhism in various pines are provided in the Examples. Any other known uses of such markers will be apparent to persons of skill in the art.

Throughout the present application, the standard IUPAC nucleotide representation was used. It should be noted that in these, K = G or T (keto); Y = C or T (pYrimidine); R

= A or G (puRine); M = A or C (aMino); S = G or C (strong 3H bond); B = C, G or T; D = A, G or T; H = A, C or T; and V = A, C or G. The present invention is further described in the following non-limiting examples.

Examples

Materials and methods Genetic stocks

The source of DNA used for clone library construction was needle tissue from a vegetative clone of the P. taeda tree known as 7-56. Allele diversity surveys were based on needle tissue DNA from vegetative clones of 26 trees that were selected from breeding populations established from five geographic origins. The sample origins included South and North Carolina Atlantic Coastal Plain (15 trees) and Piedmont (2 trees), central

Florida (4 trees), southeast Louisiana (4 trees) and central Mississippi (1 tree). For some analyses the trees were classified into two groups— South and North Carolina Atlantic Coastal Plain (ACP) and the others (non-ACP).

SSR cloning, and sequence analysis

The primer extension and uracil N-glycosylase selection procedure of Ostrander et al. (1992), as described by Echt et al. (1996) Genome, 39:1102-1108, was used with minor modifications (use of exonuclease I and lambda exonuclease as described below) for small-insert, SSR-enriched clone library construction. Enriched libraries were individually constructed for the following SSR motifs: AC, AAC, AAG, AAT, ACC,

ACG, AGG, ATC, AAAC, and AAAT. Some commercial preparations of Taq DNA polymerase contained DNA fragments that provided non-specific polymerase priming sues during the primer extension step, thus primer exiension reaction components were treated with exonuclease I and lambda exonuclease to remove extraneous oligonucleotides and increase the proportion of SSR-specific extensions.

Alkaline phosphatase-conjugated oligonucleotide probes specific for each SSR motif were used for chemiluminescent detection and identification of bacterial clones carrying SSR inserts. Probe hybridizations were done on either colony lift, or 96-well arrayed, nylon membranes. Only a single round of SSR clone identification and isolation was used prior to DNA sequence analysis. Di- and trinucleotide primers and probes were all 30 nucleotides in length, while the tetranucleotide primers and probes were 32 nucleotides long. SSR-enriched libraries constructed using a different SSR enrichment strategy were obtained from Genetic Information Services, Inc. (Chatsworth, CA). In brief, genomic DNA was partially digested with a cocktail of blunt-end-generating restriction endonucleases, and size fractionated and purified by agarose gel electrophoresis. The purified fragments ranging from 350 to 650 bp. were ligated with adapter oligonucleotides to provide common PCR priming sites for all fragments, and to provide a H dIII restriction endonuclease site for subsequent cloning into a pUC19 plasmid vector. The adapted fragments were denatured and hybridized to SSR oligonucleotides bound to magnetic microbeads. Non-SSR bearing fragments were washed away from the beads. SSR bearing fragments were released by denaturation, PCR amplified, and used for clone library construction. SSR probe hybridizations and detection to identify the SSR-bearing bacterial clones were done on 96-well arrayed nylon membranes, as described above.

Plasmid DNA for sequencing was prepared according to manufacturers' instructions using either Wizard Miniprep columns (Promega Coφ., Madison, WI) or

QIAPrep Spin Miniprep columns (Qiagen Inc., Valencia, CA). DNA cycle sequencing reactions were analyzed with either ABI 373A or 377 automated DNA analyzers, or with a LICOR 4200-2 automated DNA analyzer.

Duplicated or repeated clone sequences were identified with the contig assembly function of the Sequencher 3.0 program (GeneCodes, Inc., Ann Arbor, MI). Similarity searches in the non-redundant DNA sequence and EST sequence databases at the National Center for Biotechnology Information were done with the Power BLAST network client program (Zhang and Madden 1977 Genome Res. 7:649-56.). Primer pair sequences specific to regions flanking SSR sites were determined by the Primer 0.5 and Primer 3.0 programs (The Whitehead Institute, Cambridge, Massachusetts, USA), and primer oligonucleotides were synthesized by Research Genetics, Inc., Huntsville, AL. SSR locus names refer to the institutions and species of origin (i.e., RIPPT = Rhinelander and International Paper, Pinus taeda), followed by a clone identifier number.

Marker amplification and analysis

PCR amplification and agarose gel electrophoresis were done as described by Echt et al. (1996). PCR amplification success and locus polymoφhism were evaluated on high-resolution agarose gels containing 3% TreviGel-500 (Trevigen, Inc., Gaithersburg, MD). Precise allele sizing and locus genotyping were done by fluorescent marker analysis on an ABI373A Automated DNA Analyzer running GeneScan software (PE Applied Biosystems, Foster City, CA). Polymoφhism potential in P. taeda was evaluated for each marker using one of two methods. In the first, 18 or 20 loblolly pine trees were genotyped for each marker (RIPPT1 through RIPPT89). Methods described by Liu (1998, Statistical Genomics: Linkage, Mapping, and QTL Analysis, CRC Press, Boca Raton, Florida. 611 p.) were used to calculate heterozygosity (H) and the polymoφhism information content (PIC). The H is the probability that an individual in the population is heterozygous at the locus.

The PIC is the probability that the parental origin of an allele can be determined from the marker genotype of the locus in any given offspring (Botstein et al. 1980 Am. J. Hum. Genet., 32: 314-331.). Following Liu (1998, Statistical Genomics: Linkage, Mapping, and QTL Analysis, CRC Press, Boca Raton, Florida. 611 p.):

H = 1 - V pii ι = l and

/ t= l

PIC = 2∑ [pipj(\ - pipj)] i=2 y=2

where / is the number of codominant alleles at the locus, p„ is the frequency of homozygous genotypes, and p is the frequency of ith ory^'th allele.

The second method used a two-step strategy to identify polymorphism. For the primary screen, an individual tree and a pool of eight other individuals from both ACP and non-ACP geographic origins were PCR amplified for each marker locus (RIPPTIOO through RIPPT9325), giving four samples of template DNA. Polymorphism was recorded when, following high resolution agarose gel electrophoresis, a pooled sample displayed more alleles than the individual sample from the same pool, or when size differences were observed between the ACP and non-ACP samples. For the secondary screen, single, polymoφhic SSR loci identified from the primary screen were amplified from eight individuals (four trees each from both the ACP and non-ACP groups), and allelic size differences were scored from high-resolution agarose gels.

Results and Discussion

Enrichment cloning

Relative abundance of certain SSR motifs in the loblolly pine genome was previously shown (Echt and May-Marquardt 1997). However, the results described herein establish that there is no correlation between the abundance of a certain SSR motif and its polymoφhism and the value as a marker.

The proportion of SSR clones in each of the enriched libraries varied from 1% to 15%, depending on the target motif, specific sequence of the oligonucleotide extension primer, and experimental conditions. No correlations were found between the SSR motif and the level of enrichment of a particular library. A total of 644 SSR clones were sequenced, with an average insert size of 400 bp.

Generally higher levels of enrichment for SSR clones were found in libraries constructed by bead capture enrichment method, where the fraction of positive clones varied from 90% for the (AC)„ enriched library to 1.5% for the (AAT) „ enriched library. For the six motifs targeted for this method of enrichment, AC, AAG, AAT, AAAC,

AAAT, and AGAT, the mean fraction of SSR-positive clones in the enriched libraries was 34%. A total of 995 cloned inserts were sequenced, with an average insert size of

465 bp. Similarities between DNA and protein database sequences and the 110 cloned P. taeda sequences that were developed into informative marker loci were evaluated using a PowerBlast network client (Zhang and Madden 1997 Genome Res. 7:649-56.) running blastn and blastx alignment functions. No significant or functional similarities were found.

The best motifs for yielding informative markers were AC, AAAT, and AAAC, although the rate of conversion from sequences SSR clone to polymoφhic marker is still rather low (Table 1).

Table 1 Frequency of sequenced cloned inserts, unique cloned sequences, primers pairs synthesized, single loci that were PCR amplified, and polymorphic SSR loci, by motif.

SSR clones unique primer primer pairs polymoφhi polymoφhic motif sequenced sequences pairs amplifying a c single loci per single locus loci sequenced clone (%)

AC 605 430 315 121 76 12.6

AAC 58 22 20 11 0 0

AAG 55 43 67 28 2 3.6

AAT 187 90 40 26 7 3.7

ACC 7 0 16 4 0 0

ACG 12 10 10 1 0 0

AGG 14 12 2 1 0 0

ATC 298 108 53 34 11 3.7

AAAC 117 67 15 13 6 5.1

AAAT 98 59 21 15 8 8.2

AGAT 68 26 7 1 0 0 total 1539 887 566 255 110 3.35 (mean) a = among 18 P. taeda trees, as evaluated in high-resolution agarose gels

Clones of (AC)„, (AAAT) „, and (AAAC) „ loci had respective conversion frequencies from sequenced clones to single locus polymoφhic markers of 12.6%, 8.2% and 5.1%. (AGAT)_n, which yields many informative markers in mammalian species, produced no markers for loblolly pine.

The trinucleotide repeats that were examined did not, in general, prove to be a very good source of polymoφhic markers despite their relative abundance in the pine genome reported by Echt and May-Marquardt 1997. The three trinucleotide motifs that did produce polymoφhic markers had a sequence-to-marker conversion frequency of about 3.7% (Table 1). The ATC motif, the most abundant trinucleotide SSR in loblolly pine (Echt and May-Marquardt 1997), accounted for relatively few informative markers. It appeared to be associated with a repetitive DNA fraction in the genome, as only 36% of the (ATC)_n clones sequenced were unique sequences. (AAT)„ SSRs produced similarly few polymoφhic loci, even though in soybean they are a good source of informative markers (Akkaya et al. 1995 Crop Science, 35: 1439-1445; Rongwen et al. 1995 Theor. Appl. Genet., 90:43-48.).

SSR locus polymorphism

For both enrichment methods, the total numbers of clones sequenced, PCR primer pairs designed, and polymoφhic marker loci discovered are presented in Table 1. Of the 566 primer pairs evaluated, 164 gave no amplification, 255 amplified a single fragment, 77 amplified two distinct fragments, and 70 amplified more than two distinct fragments. A list of 119 primer pairs used to amplify P. taeda SSR loci is represented in Table 2.

TABLE 2

Locus Forward sequence Reverse sequence Nr loci Expected Null allele

P=polymorph Size (bp) segregating

RIPPTl GCATGCCAAAAGATCTCAA (SEQ ID NO 1 ) AGTGAACTCGGGAGGCTTCT (SEQ ID NO 2) IP 263 N RIPPT6 TTTGGACAAGTGGCTTGTTG (SEQ ID NO 3) ATGTTTGATTGCATGGGGAT (SEQ ID NO 4) IP 295 N RIPPTl 1 GGCTTCTCTCCAAGCTTTTTG (SEQ ID NO 5) GAATGAGCCTCCCAACTCAA (SEQ ID NO 6) IP 171 N RIPPT22 CΓCAGTTTCATAATCTTTGTCGC (SEQ ID NO 7) TTTTAGAAAAGAAGGAAATCTTCA (SEQ ID NO 8) IP 250 N RIPPT24 GACACCGGATACTGAGGTGG (SEQ ID NO 9) CCCGCAACTTCGTAAGAGTC (SEQ ID NO 10) IP 151 N

RIPPT31 CCAACCA ATGTGGTTCATCA (SEQ ID NO 11 ) AGGAAAATAGAAGGGAATAAGACC (SEQ ID NO 12) IP 261 N RIPPT32 TAGCAGGTTACAACCTGGGG (SEQ ID NO 13) AGCCCAATTGATGGGAAATT (SEQ ID NO 14) IP 188 N RIPPT33 TTGGAGAACATGCTTGCAAG (SEQ ID NO 15) TGGAGCATTTTCCACAAAAT (SEQ ID NO 16) IP 181 N RIPPT64 GCAGCGTAATCAGATGGTCA (SEQ ID NO 17) CGGAAGGCGAGTTGAAGATA (SEQ ID NO 18) IP 258 N RIPPT65 CCAACAGCACTTACCCAAAA (SEQ ID NO 19) AGCCTCATGAAAGCCCAGTA (SEQ ID NO 20) IP 142 N

RIPPT66 GTTGATAGAGTTTCATGTGGTGC (SEQ ID NO 21 ) TGGATGAAGAATTTTGTAGTCAA (SEQ ID NO 22) IP 1 14 N RIPPT67 AGCCCTCCAAGACCAAGATT (SEQ ID NO 23) CCATTTGCAAATACCCCAAC (SEQ ID NO 24) IP 227 N RIPPT69 TCAAGAATGGGGGATGATTC (SEQ ID NO 25) TTGCATCCAACAACTGCTTC (SEQ ID NO 26) IP 150 N RIPPT71 CTACTCAAAGTGCTTGGGCA (SEQ ID NO 27) CCCCTTCCCTTTCTATCTGC (SEQ ID NO 28) 1 246 N RIPPT77 ACACCGGATACTGAGGTGGA (SEQ ID NO 29) GGTTGTAGCCTCCCGTAGGT (SEQ ID NO 30) 1 175 Y

RIPPT79 TGATTTGATCCCTCTAGGCG (SEQ ID NO 31) AATCTTGAAAAGAAATTCAATATGAGA (SEQ ID NO 32) 1 153 N RIPPT80 CACACAACCAAAATTAAAACATTCA (SEQ ID NO 33) CACAAACAAGGGGGTCTCAT (SEQ ID NO 34) 2 251 Y RIPPT89 ACGAAACCCCGAGTTGATAA (SEQ ID NO 35) TAAGCCCTTGAACATGGTGG (SEQ ID NO 36) 1 225 N RIPPT101 ATGTTTGATGGGGTCGTCAT (SEQ ID NO 37) CATCATCCCATCAGACAACG (SEQ ID NO 38) 1 100 N RIPPT103 CCCCTTGGTGGAACAACATA (SEQ ID NO 39) TTGGAAAATOGCGGAATTTA (SEQ ID NO 40) 1 210 N

RIPPT104 TGCATTTCATTTTTGCGTGT (SEQ ID NO 41) AGGACATGGAGAGTTTACACATG (SEQ ID NO 42) 1 164 N RIPPTl 06 ATCAGATTGGTGGATCGGAG (SEQ ID NO 43) TGACTGATAAGGGTTTCGCC (SEQ ID NO 44) 2 180 N RIPPTl 17 GCTTCATGATTTCTCGATCG (SEQ ID NO 45) TCTGCGTGGATAAAGGAATTT (SEQ ID NO 46) 2 208 N RIPPTl 23 TCGTGTCGAAACATTGGAAA (SEQ ID NO 47) TATCACCTATAGCCCCGTCG (SEQ ID NO 48) 1 ! 29 N

RIPPTl 26 TCATACCGAGAGAGGTCTTTG (SEQ ID NO 49) GAGCTTAATTTGTGCCTGCC (SEQ ID NO 50) 1 174 N RIPPTl 28 CGACCCTAGTCTCTTGTGCA (SEQ ID NO 51) TTTTGGACCCTAAGCCAGAG (SEQ ID NO 52) 1 175 Y RIPPTl 32 AACCGTGGTGCTCTGATACC (SEQ ID NO 53) TGCAAGTCAAGAGCTAGAGACAA (SEQ ID NO 54) 1 113 N RIPPTl 34 GTTTACATTTTCCTGGGGCA (SEQ ID NO 55) GATTTACAAAAATCCCTGCCA (SEQ ID NO 56) 1 145 N RIPPTl 35 CACGCATGAGCTGAGTCATAA (SEQ ID NO 57) TGTGTTTCCCACTATGCTAAGC (SEQ ID NO 58) 1 218 N

RIPPTl 39 ACCAACCGAGGGAGCTAAAT (SEQ ID NO 59) AAAAACGACATTCACTTCAACA (SEQ ID NO 60) 1 121 N RIPPT158 GTGTGCCACGGATGTATGAG (SEQ ID NO 61) TTGCTGAAAGGGCCAGTAGT (SEQ ID NO 62) 2 211 N RIPPT159 ATATGGCTTACCTCGGGTCC (SEQ ID NO 63) CATAAACCCATTGGGTCCAG (SEQ ID NO 64) 2 131 N RIPPT165 TGGAAGCCACAATTTGTTGA (SEQ ID NO 65) TGCAATAAAACCATGCAACAA (SEQ ID NO 66) 220 N RIPPT166 TTTTGAGAATGTCCGTGCG (SEQ ID NO 67) TGATGCATTGCAAAATCATG (SEQ ID NO 68) 155 Y

RIPPTl 71 TGATCCTAAGCCTTAGAAACCC (SEQ ID NO 69) TTTTGTCACCCATGCATATGA (SEQ ID NO 70) 207 N RIPPT179 TGTAGGAGCACAAGCCATTG (SEQ ID NO 71) AACACAGTTGGACCGTTTGA (SEQ ID NO 72) 170 N RIPPTl 85 TGTTTGCAAATCATGGGGTA (SEQ ID NO 73) CCAGTGTCCATGCCAATTTT (SEQ ID NO 74) 300 N RIPPT193 GATCCCTTGTCCCAGAAACA (SEQ ID NO 75) TGTTGATGTTATGCCTGGGT (SEQ ID NO 76) 163 N RIPPT211 GAGGGGGTCTCATACACCAA (SEQ ID NO 77) TGCATAGAGGATGTATTTCTTGGA (SEQ ID NO 78) 159 N

RIPPT255 TCCTCCTGAGTGGTCCCATA tSEQ ID N0 79) ATGGATATGAGGCCTGTTGG (SEQ ID NO 80) 123 N RIPPT263 TTGGATTGGACCTGAATCAA (SEQ ID NO 81) TTGGCAGTCTTCGAGGTCTT (SEQ ID NO 82) 183 N RIPPT274 TGTTCCTCTCAAGTGACCCC (SEQ ID NO 83) CTTCAGCTTCCCACCAGAAG (SEQ ID NO 84) 264 N RIPPT287 GGAATGTATTCCCGGTTCCT (SEQ ID NO 85) CTCCCGGATATTGAGGAGGT (SEQ ID NO 86) 224 N RIPPT293 CGCTACTATTGGCCGAATCT (SEQ ID NO 87) CTGTGAGGAAATCCCTGGAA (SEQ ID NO 88) 184 N

RIPPT298 CTTTTCCCTTTCCATGACCA (SEQ ID NO 89) GAGTCGAGTAACCAGGTGGC (SEQ ID NO 90) 319 N RIPPT305 TCAATCACCAATTATTTGGCT (SEQ ID NO 91) GGAGTGGATGAAACTATGCCA (SEQ ID NO 92) 230 N RIPPT367 CCAATGCATAATGCAACCAC (SEQ ID NO 93) TAGCCATGGTGCTCAGTCTG (SEQ ID NO 94) 209 N RIPPT369 GGTTGTTGTGCACGAGCTTA (SEQ ID NO 95) TCAGTGAAGTTCAAGGGAGGTT (SEQ ID NO 96) 161 N RIPPT376 AGGAATTGGTGATTCATGTGG (SEQ ID NO 97) ATAAAAGAATCGGCCCTGGT (SEQ ID NO 98) 189 N

RIPPT388 CACAACACTCAAACATGCTCAA (SEQ ID NO 99) AAGAGGATGTGAGGTCCCAA (SEQ ID NO 100) 203 N RIPPT467 CTTGGCGACCTTGTCATACA (SEQ ID NO 101) GGGTCCTTAGGGATCATGGT (SEQ ID NO 102) 178 N RIPPT496 GTAAGAGTGCCTCGGGTCTG (SEQ ID NO 103) GGTGGTAGGTAGATCGGCAA (SEQ ID NO 104) 203 N

RIPPT508 GGCACAGGTTGGACATCTCT (SEQ ID NO: 105) GTGGTGGAAGGGAGATTTCA (SEQ ID NO: 106) 90 N RIPPT538 AAACACTTGGACTGGATGGG (SEQ ID NO: 107) TTTGGAGGATGTTTGTTGCA (SEQ ID NO: 108) 212 N RIPPT540 TGTTGTCATTAGTGGTAGGATCA (SEQ ID NO: 109) AAGCGATGTCACTTGTTGAGAA (SEQ ID NO: 1 10) 200 N RIPPT548 TTTTGTGGTCATTCGTTGGA (SEQ ID NO:l l l) TCACATGGAAGATTATCTCCAAA (SEQ ID NO: 112) 207 N RIPPT556 TCGTGATTACATTGCTGCCT (SEQ ID NO: 113) TCCACAACAATGATCGCTTC (SEQ ID NO:l 14) 183 N

RIPPT560 CATTGGAACTTCACCGAAGG (SEQ ID NO: 115) GTGCTATTGGGTCCAGCAAT (SEQ ID NO: 116) 108 N RIPPT567 GTTGGTGAGGAGACTTGGGA (SEQ ID NO: 117) AAGAACAATTCCA ATATGGATGA (SEQ ID NO: 118) 152 N R1PPT584 GCGAGACAGAAACGGAAAAG (SEQ ID NO: 119) CTCTGCTAGACCGCTCAGCT (SEQ ID NO: 120) 136 N RIPPT609 CAAAATGCAGAGGGGCTTAA (SEQ ID NO:121) CCAGTCCATCGAATCACGTA (SEQ ID NO: 122) 154 N RIPPT619 CAGCTCTCTTAATAGCCTCGG (SEQ ID NO:123) GCACATAGCAACGCTG A AGA (SEQ ID NO: 124) 191 N

RIPPT621 GCAAAGGGAAGCAAAGTCAT (SEQ ID NO: 125) TTCGTCCTCTTTTGAACGAGT (SEQ ID NO: 126) 154 N RIPPT627 GACAAACA ACCCTTGCGTTT (SEQ ID NO: 127) GACCCATCAAGCCAACATG (SEQ ID NO: 128) 168 N RIPPT629 GGTTGTGCTTTCCCAGAGAG (SEQ ID NO: 129) GAATGCAAGGTAGCCAGGAG (SEQ ID NO: 130) 157 N RIPPT630 CGCA AGCTATG ATACA ACGC (SEQ ID NO: 131 ) TGTTGGCTGAGTGTGAAAGC (SEQ ID NO: 132) 157 N RIPPT644 GTTGTGATCCAAGTCCCCTG (SEQ ID NO: 133) TGGTCCATTCGGTCCTATTC (SEQ ID NO: 134) 204 N

RIPPT647 TGGCCATCGAACTTGTGTTA (SEQ ID NO: 135) CACGACCACCAGTCACCTTA (SEQ ID NO: 136) 214 N RIPPT649 TAGTCGAATCGGGCCTGTAC (SEQ ID NO: 137) TTGCTCCTCTGTGTCCTTCA (SEQ ID NO: 138) 218 N RIPPT658 TGCATGCATTACAAATGTCA (SEQ ID NO:139) CGCTTTTAAATCAACCAAACG (SEQ ID NO: 140) 219 N RIPPT675 ACAGATGTCAAGGCCAAAGG (SEQ ID NO: 141) CTGCATTCAAATTACCCGCT (SEQ ID NO: 142) 172 N RIPPT683 TGAAACCAATCCTTCTGCAA (SEQ ID NO:143) CTGATTCCTCTGGCTTCTCG (SEQ ID NO: 144) 187 N

RIPPT688 TTCAGTTATGCATTCACGAGC (SEQ ID NO: 145) GTCCTCCTGGGTTATCCCTC (SEQ ID NO: 146) 141 N RIPPT689 GAAACTTTCCCCTACGAGCC (SEQ ID NO: 147) TTCCCCAAAAGTTCACAGGT (SEQ ID NO: 148) 158 N RIPPT690 ATTCCTAGATGGACCTGGGG (SEQ ID NO: 149) CGACATAAGCCCACCAAATT (SEQ ID NO: 150) 142 N RIPPT692 TGG ATCGTG ATCCTCTGTGA (SEQ ID NO: 151 ) GCTTCCATCACATTGGGATT (SEQ ID NO: 152) 166 Y RIPPT700 TTGCAATTGCGATTAACTGC (SEQ ID NO: 153) ATAATGGCATAGCCGAATCG (SEQ ID NO: 154) 180 N

RIPPT767 TGCATAGAAAGTCGCCCTCT (SEQ ID NO: 155) ATGCATGAGGTAACTTGGGG (SEQ ID NO: 156) 186 N RIPPT789 CATCCCAAGCATCCTCAAGT (SEQ ID NO: 157) TCAAAAATGTGGTTTAATGGAAAA (SEQ ID NO: 158) 170 N RIPPT790 TTGTGAATTGTGTCCATGGG (SEQ ID NO: 159) ATCGGTGAGGCTTAAACACG (SEQ ID NO: 160) 182 N

RIPPT791 ATGGAAGGATCCACAACCAA (SEQ ID NO: 161) GGGCTTGTTGCTGGTCTATG (SEQ ID NO: 162) 2 168 N RIPPT792 GGTTGATGATGTCGATGTTGA (SEQ ID NO: 163) TTCTTGCAAACACAGCATGTT (SEQ ID NO: 164) 2 202 N

RIPPT799 TGATCCTAAGCCTTAGAAACCC (SEQ ID NO:165) TTGTCACCCATGTCATATG ATACA (SEQ ID NO: 166) 1 209 N RIPPT814 A A AA AGAATGAGGCGCACAC (SEQ ID NO: 167) CCCGTTTATGGCATTGATTC (SEQ ID NO: 168) 2 100 N RIPPT815 GAAAACGAACAAGCCATGGT (SEQ ID NO: 169) TGTTTACTTGCATGCATGTGTG (SEQ ID NO: 170) 2 162 N

RIPPT841 GTGCTTCCCTTGCTTCAGAC (SEQ ID NO: 171) GCAAATGCAAACTTTGGGTA (SEQ ID NO: 172) 1 202 N RIPPT846 CATTCATGGTTCCAATGTGG (SEQ ID NO: 173) TGATAAGCGTGGATCTCGTG (SEQ ID NO: 174) 2 109 N R1PPT852 OTTATCCCCCATGTTGTTGC (SEQ ID NO: 175) GGGTAGAAGCACTATGCTTTCATT (SEQ ID NO: 176) 2 213 N RIPPT860 TTGAGCAGACATCATCAACACT (SEQ ID NO: 177) CCAGGTTATGCCTCAAAGAG (SEQ ID NO: 178) 1 217 N RIPPT905 CACGGATCTCTGGAAACCAT (SEQ ID NO:1 9) CGCTGGTTTCCCTCAGAATA (SEQ ID NO: 180) 1 194 N

RIPPT921 GGATTTTGTTTTCCTCATAATCA (SEQ ID NO:181) GGGCATAGCATATGCCACTT (SEQ ID NO: 182) 1 219 Y RIPPT932 GCAAGACCGACTGGATTAGC (SEQ ID NO: 183) GAGGTCATGATATGTGGTGGG (SEQ ID NO: 184) 2 130 N RIPPT941 CTGCGTAGCAAATCACTGGA (SEQ ID NO: 185) TGATCTGATGTGGGATCAACA (SEQ ID NO: 186) 1 151 N RIPPT947 CCATTGCCCGAGCTAGTTTA (SEQ ID NO: 187) TTATATTGGACCCAAGGCCC (SEQ ID NO: 188) 1 214 N RIPPT958 TGGAGTCTCGAACACTGTGG (SEQ ID NO: 189) AATCATCCCAATGGCAACAT (SEQ ID NO: 190) 1 111 Y

RIPPT960 GCATCCATCTTCAGCATCCT (SEQ ID NO: 191 ) TTCATACGACACCTTTGAAATG (SEQ ID NO: 192) 1 188 N RIPPT961 CCATTAG AC A AGTGCGCATG (SEQ ID NO: 193) TGAAAAAGGAATTTCCCCAA (SEQ ID NO: 194) 1 213 N RIPPT968 TCTACGACAAAACCACGTAGTG (SEQ ID NO: 195) CATGTGGCTTTGTGGCATAT (SEQ ID NO: 196) 1 201 N RIPPT984 TGTGACCTGAAAATTCCCCT (SEQ ID NO: 197) GGCTTGCAACCAGTTCCATA (SEQ ID NO: 198) 1 220 N RIPPT990 GACCTAAAGAGGTTCACGCG (SEQ ID NO: 199) TCAAATCTTGGGTTAGTATGCAGA (SEQ ID NO: 200) 1 220 N

RIPPT1013 ATCCCTGTGGGGATGAGTTA (SEQ ID NO:201) TGCCTCTTAAGCATCAAATGTT (SEQ ID NO:202) 1 124 N RIPPTl 023 GAACCCGATGGATTTTCAAA (SEQ ID NO:203) CAAACTGTAAGCTCAGGAGGA (SEQ ID NO: 204) 1 175 N RIPPTl 027 CAGTGTTGATTGTGTGCCAG (SEQ ID NO:205) TCTGCCACAATTTGGAAACA (SEQ ID NO:206) 1 220 N RIPPT1035 AGCATAATGAGCCCTTCTCG (SEQ ID NO:207) AGAATATGTGTCCCTCCCCC (SEQ ID NO:208) 1 174 N RIPPTl 036 TGGTTGTGCGAGATCACAAT (SEQ ID NO:209) TTGAGGGAATTGAAATTGGG (SEQ ID NO:210) 1 211 N

RIPPT1037 TGCTCAATATAGACCACTTGCA (SEQ ID NO:211) AGCCATAATTCAACAAAAGGAA (SEQ ID NO:212) 1 152 N RIPPTl 040 TCAAGGAATTCATTGGAGCC (SEQ ID NO:213) TTTGGCCATATCAAACCCAT (SEQ ID NO:214) 1 192 N RIPPT1066 AAAGGGGGTGTTTGATGGAT (SEQ ID NO:215) GATCGAAATCAGCGAACACA (SEQ ID NO:216) 1 175 Y

RIPPTl 072 TTTCATGACCTTGGAGTGGA (SEQ ID NO:217) ATTGATCCCATTGTTGCTCC (SEQ ID NO:218) 1 209 N RIPPTl 076 TGTGTAAACCCAGGCTAGGC (SEQ ID NO:219) ATGATTTCACAAAGCCCCTC (SEQ ID NO:220) 1 167 N RIPPT1077 AACATTCTAGCATGCCCCAC (SEQ ID NO:221) TTGTGGTGGATGTCTCTCCT (SEQ ID NO:222) 1 220 N RIPPTl 125 GAGCCACACAAACATGCATC (SEQ ID NO:223) TTTCCCAAAAGTTCACGAGG (SEQ ID NO: 224) 2 197 Y RIPPTl 137 CCCATGCAACTGCCTAGAAT (SEQ ID NO:225) AAGCTCGCACGTGGGATA (SEQ ID NO:226) 2 165 N RIPPT9058 CCCGCTCCTATTCAAGATCA (SEQ ID NO:227) AGGCGCCTAGAGGCATAATT (SEQ ID NO:228) 1 206 N RIPPT9104 TTCCTATCGTCAGCGTCCAT (SEQ ID NO:229) GTTCACAGGGGTCATGCTTT (SEQ ID NO:230) 1 155 N RIPPT9138 TGAAACCAATTTTTCCCCTTT (SEQ ID NO:231) CCAAGAAAGACAAGGAGCCA (SEQ ID NO: 232) 1 229 N RIPPT9238 CCCTGAGACATCCAATCCAT (SEQ ID NO:233) ACTTTACATGAGTTGGGCGG (SEQ ID NO:234) 1 119 N RIPPT9315 GGCTTAGGCATAGAGGGACC (SEQ ID NO:235) AACAAGTTGGAAGCCACCAT (SEQ ID NO:236) 1 219 N

Polymoφhism among P. taeda individuals was scored using high resolution agarose gel electrophoresis. Heterozygous marker alleles having a 3 bp size difference could be resolved and 2 bp allele size differences between samples in adjacent lanes could be detected. Since single bp allelic differences were not detectable some 2 bp allelic differences were possibly missed, the number of polymoφhic loci reported may be slightly underestimated. In table 1, the number of polymoφhic single loci reflects the number of primer pairs generating a single major DNA fragment, or a heterozygote fragment pattern. For most primer pairs, there was only one fragment amplified. In a few cases additional amplification of a weakly amplified fragment, or fragments, well outside of the expected size range was observed, but did not compromise inteφretation of the single locus marker phenotype. SSR loci of P. taeda identified using the primer pairs in Table 2 are represented in Table 3.

TABLE 3 SSR loci

RIPPTL LOBSEQ3-27-97ATC441R (SEQ. ID. NO. 237) ATTAATTTTTTTTGAAAAAAAAAGAGTTTTGAGAAAAAGTCTAATATATACTTGGTGGCATGCCAAAAGATCTCAAAAA

TTCCT TCATATATTTGATTAGACAAGAAAATATATTATATTATAATCGTTTAACΓTTTΓATAATTTTAAAAAATATATTA TAATTATTTTAAGTTTATGATGATGATGATGATGATGGTGGTTAACGTCCACTGAGACCAAATAATGATCATCGGACCT AAAAAGACAAATTATTTATTTTGATTTAAGATRTTATTCCTATGCTCAAAAAGCRTGCAGAGAAGCCTCCCGAGTTCACT ATAATTTTGGCATTGTAAAAGGNTAGGAAAGGTCATTGGTGGTTACAAAGGGTGGTGAAATTGAAATCTAATGTTGGTG TTTGCCGGGGCTTCC

RIPPT6 LOBSEQ5-2-97ATC402R (SEQ. ID. NO. 238)

CCGAGACCAAGCAGGCTTGTAACAGGGCTACAGGTGAGTGGCTCCTCAAGACCGGTGGTGTCTTCAGGAACTGAACTTT

CATGATGATCATGATGATGATGATGATGACTGGCTAGCCGTGTTCCAAATAACGAGTCCACACTCGCCCCCCGATGATC GATTCTCGTCGTCCGATGGACGCGACGGACGATACGAGATCTCTGTCTAGGCGGGATCGAACGATCGATGGACGAGCTT

GCACT"ACCAAATGTACCTGCGGTTTCATATCTCACGGTGGCTTCGACACTGGTCGNCNAAACRGACTTTGTTCTTCTGTG TTGTTCTGTTNGGTTTTTTITGGGANGTTGGTCCCGGACAATTTTTCGNCATCTTGTNAAAAATGTGGANCNTNCNCCGA AGGTCCGCGTTGGCTTΓAAAGCCCNCTGGGCGGGNCGNTCCAAACNTTGCATCTAAAGGGCCCNTTCCNCCTTNTAGTT AATTCCT'NTTNCAATCCCCNGGGCCGGCNGTTTTCANCGTCGTNATGGGAAAACCCGNGTTNCCCACTTNATCNCTTGC ANNNATCCCCTTCCCACTGGNGTATACCAAAAGGCCGCCCNTTNCCTCCCNANGTTGGNCNCCTGANTGGAANGGCNN

CCTTTTGGGGCTNANCC RIPPTL 1 LOBSEQ3-8-97ATC229 (SEQ. ID. NO. 239)

GCGCAATTTGTTATTCCTCCTATTTCACAGCTTGATCAAAGTGNTCTAGCTGCACTGCCTGATGCTATACGAGATCAAAT TTTGAAGAAGCAAGCAGGCAGTGCCAACCTGACCTCTGTCGCCAAGCATGAAGAAGNGAAAGAAGNTTTGTCAATGCA AAGTCCATCATCATCATCATCATCTGTGACAATCACACCCAAAAAGCAACGAATAATTGATCCATTTGAACGAATGCGT GCAGCTTCAATTACACCAACGAAAAAAGGAAAATTGAAAAAAGITACAATTAATTCAGCTCCTTCTACACCAAGTGGA

TCACAAAAACGTTGCAAATGTTAGANAATCATGGGAACCTACATGGTCGCCAGTTGATTCCAAAGTTTTATCCGAACTA CCGATAGAAAT

RIPPT22 LOBSEQ5-2-97ATC272 (SEQ. ID. NO. 240) ACAACCACATTAGATCTCAGTTTCATAATCTTTGTCGCAATACTGACCTTCCTAGCCTTTTACGATGTCATAATTATAGT

GAGCTCGGGAGGCTTCTCRCCAAGCTTTTTGAGCATAGGAATAAAATCTTAAATCAAACTAAATAATCTGTTTCTTTAGG TCTGATAGACCAAAGTTTGGTCTCAGTGGATGTTAACCACCACCACCACCACCATCATCATCATCATATCTTTTGAGAA AATGAAGATTTCCTTCTTTTCTAAAAT

RIPPT24 LOBSEQAAT10 (SEQ. ID. NO. 241)

ACGCACTATTAATGAACACAAACAATGTATATGTAGATTACTTGATTTTTCACAATTCTATAAATTTATCTAAATCATTA TATTATTCGCCATACATTATGCAATAACCATGAGCAGTATCATCATAATAGAATATAAGCACATCATCAACACATCAAC ACAAAATTCATATTGACACCGGATACTGAGGTGGAAACCTAATTTGGGAGAAAACCATTGTTGTTGTTGTCTCTTATTAT TATTATTATTATTATΓATTATTATTATTATAAAGAAAAATTCTTCTTACATCTTGCACAATCACAGACTCTTACGAAGTTG CGGGCTCCTACCTACGGGAAGNTACAACCTCTAGAAATTATCCAGCTCCACTGGAANGAAGCTACTACTCCCTAATCAA

GTTTACCAGCTCCNACTGAAAGGAAC

RIPPT31 LOBSEQAAT18 (SEQ. ID. NO. 242) ATTGTTCΓTCTGGATTAATTACACTAGTAATTTΓTCAAATCAAAGTTTCAAACCAACCAATGTGGTTCATCATCAAAATA TAGATGAGGGAGGTTGAACTAAGCCATCGAGATTGATAAGAGGACTGGCAATCTGAACATAGATAATGGGTGGAAAAT

TAGGAGTAGTTGGATTCCTACCCTGACTTCΓTAGGTGGCTTTGTCCCCCATCTAAAATTTAATTTAATTATTATTATTATT ATTATTATTATTATTATTATTATTATTATTATTATTAT^^

TATGATTATTACATATAATTATTTACTTCTAGTTTAACCCTCTTCCTTTTTTNATTTCΠ^ TAT^^R^CCATATCTCTAGTTAATAATTTATTATTAAT ANTTCAAGGATGTTITAATTATANTRC NAAGTT^ CCTAATTTATATTTTATTTCCCAACTCNCAATCCNTTACTAA

RIPPT32 LOBSEQAAT49 (SEQ. ID. NO. 243)

ACTTGTAGTCAGTTCAAGAGTTAACGCAAGGGAAACCTAGCAGGTTACAACCTGGGGTCAATCGGATTGGTAGCTACCT GTTCGAGATATTCTGATTTCACATTAAAAAGTGAAAAGTGAGTGTITTAAGTCRATTATTATTATTATTATTATTTATGA CCTTCTCAATTATGCCTAAAACATCTTGTCTATTTCCAGAATCTGAAATTTCCCATCAATTGGGCTATACTACAGACTCC

TCTGCATACACCTTCCTCACTTTGAACATCGAGAGTTCAACTACAGAAAATTTGCACCTTCCACTTTGAGAGTTCAACTA CAGAAAATTTGCAGGCTGGCGGTGGAAAACAGTCAAGGTATGCATACGANTCCATGGTGCTGTTTTTNCCGGAAGAAA

TTA

RIPPT33 LOBSEQAAT46 (SEQ. ID. NO. 244)

ATTACAATTTTCTTCTTACTTAATTGGAGAACATGCTTGCAAGTTGCAATTTGTAAATGAGATTTTTACTCGAGAAATAA AAAGGACTAGGTGAAGAACATGCTCCAACCATTAGGGAATATAAGGTGGTTGGGTATAATCCTAGTGAATATATTATTA TTATTATTATTATTATTTTTAAC GATrTTGTGGAAAATGCTCCATTTTTTATACATGTTACTTT^ TATAAGTGACTATAAATTGAAGAAACTGTGACTTTACCTAGAT RIPPT64 LOBSEQ5-6-97AAAC2C4 (SEQ. ID. NO. 245)

ACGGGAAGGTGGTGAGGAGCAGCAGCGTAATCAGATGGTCATAGGCGTTGTGCAAACTGCAGGCTCCGGACAAAACCC TAACCCCATCCTGTACGTTGTTTCCTCATCIRRG ITTTTCCAAATCCAAGCATATATATAACCCAATGAGATGAATAGT TAAAAAACAAAAAAAACAAACAAACAAACAAACAAAAACATCCTAAAAAATAGCCAAAAATGTAAAATCTCGAAATA ATCCTTTTGAGGAGAGCΓΠ CAATATCTΓCAACTCGCCTTCCGGTGTAGTGGTTTAGG

RIPPT65 LOBSEQ5-6-97AAAC2F7 (SEQ. ID. NO. 246)

ACACGAACGGTC ACTTACACTTGACTGTATTGGATAATATACCTTCTATATATTCAATACTGTCCACCTATAACCAACA

GCACTTACCCAAAAAAACCTCAAAAACATTTATATAAACAAACAAACAAACAAAAAAACCCTAAAAACACCTATAAAC ATAAACTAAGTCCAGGAAAAGATTTTAATTTTCTGATTTACTGGGCTTTCATGAGGCTGAGATTCRTCTAAAATTTAGAA

CGAAATGCATGATGTATACI CCATAATGGGAAAGCACT GGTTTTTTTGGTTGCRTATTTTTGTGCATACCGAATCGTC

ATATTTTAATCTTTGCTACTATGGC

RIPPT66 LOBSEQ6-5-97AAATE2 (SEQ. ID. NO. 247) ATTATTTTTATGTAGGCTTTGATTATATTGGTTCCCCTTAGACTCCTATATATAGAAAGGAGGTCTTGTCATTTGTATCAT

CAAAAAATΓATTCACΠ ATGTAATGTAAAGGAGGTTGCCTTCGAAGTGGCΓTATTTTGAATTCGTTGATCCATCTCTTCA ATTTGCGAGTTGGTTTCATAACATGACTACCTTGGCACATTATTCCATGGCGTCTATCATGGATATTATTATATTATTTTT TGTTGATAGAGTTTCATGTGGTGCAACATAGTCCTCCTCATTATCTCATTTGAAGAGATAAAAATAAATAAATAAATAA ATAAATAAATAAATTGACTACAAAATTCTTCATCCATGTTAT

RIPPT67 LOBSEQ6-5-97AAATC7 (SEQ. ID. NO. 248)

ACAAGAC ΓCAAAGTTGTCΓAACAGCCC CCAAGACCAAGATTAATGCCTCAAAACAAGCCTATTAAAGTTTGCAAACA AAACGATAAGGATATGTTCACATCGGCTCCAAATAAATAAATAAATAAACAAAATGTATTAATATATGTCATGTTGGTT TGATGCATAGACACATCGGTTCATAAGGTAATGTCGACTCAATTGTATGTGCACAGCCGTTATAAATACCCTGTTGGGG TATTTGCAAATGGGCAAGAAAGAATTCACATGAAATCTTCGCATAAGGGAGCCATAATGAANGGAGCCACAATCAAGG

TTGATGGANCCGTAATCCAGGTCAAAAACCACAGTTAGGGTATTGACTGCCCCGTGTTTTTGGGTTTTANGTGCAATCC ATGG

RIPPT69 LOBSEQ6-5-97AAATE9 (SEQ. ID. NO. 249) ATCAAGAATGGGGGATGATTCACCATTTTTGGAGTAAAAGGATAAAAATAAATAAATAAATAAAAATAAAACCTTATT

TTCAACTCGTATTTTCAACΓΓATC CTTTACGATTTTAC CAAATTTCAAGCAGAAGCAGTTGTTGGATGCAAAAT^ TTTTACATCCAAAAAATACCCATGTTGCATACTTTCAATGGACCCTACTACACAGAAAATGTGAAATACAAGAAATAAT GTGATTGTAATCATGTTTΠTCATGTATTTCATATTTTTCATGTAGTGGGGTCCATTGAAAGTATGCAACATGGGT

RIPPT71 AAAT1 -A 1 (SEQ. ID. NO. 250)

CCATCTTATATTATCCTCCAAAAAAATTTCCTCTCTATACI TAGGAAAAAAATTATGTATCTACTCAAAGTGCTTGGGC ACATTΓGCTAATATAAATATATGGTTAGTGTTATAAAAACAATTTATTTATTTATTTTATTTTTATTTATTTATTTA TTATTTTCGTGGGAATGAGCATGGCATTCAAGCCATCACGTGGATGCTRGCAAGAAGGAGAGTTGAATTTTTAAGACGT GAAAAATGAGGTGGCTGAGCAGAAGATGTAATTGGGTGATTTGTTGGCAGATAGAAAGGGAAGGGGAACCCTCCATCT CTGCTAAATCTGCTAGAGAAGAGTAAAAGCAATGCAACAAAGGGACATAAACGAACAACTGGAAGCAACTCAGATGT

GGACCATGGTTTGGAGGAGGATAAGAACCAATTGGAGT

RIPPT77 AAT3-B5 (SEQ. ID. NO. 251 ) ACGCACTATTAATGAACACCAACAATGTATATGTAGATTACΓΓGATTTTTCACAATTCTATAAATTTATCTAAATCATTA TATTATTCGCCATACATTATGCAATAACCATGAGCAGTATCATCATAATAGAATATAAGCACATCATCAACACATCAAC ACAAAATTCATATTGACACCGGATACTGAGGTGGAAACCTAATTTGGGAGAAAACCATTGTTGTTGTTGTCTCTTATTAT TATTATTATTATTATΓATTATTATTATTATAAAGAAAAATTCT CTTACATCTTGCACAATCACAGACTCTTACGAGGTTG CGGGCTCCTACCTACGGGAGGCRACAACCTCTAAAAATTATCCAGCTCCAACTGGAAAGGAACTACTACTCCCTAATCA ANTTTACCAGCTCCTACTGAAAGGAACTCTTACTCCCTCCCTAAAGGNTCCATCCCCATACCRITTNGGAAAAANTTCCT

AATCTTAAANCCNCTGGNGGCGGTACTATGGATCGAACCGNNCAACTGANCNNACTGNATTTCCNATGTCCCAATACTG GGTANCNGGCNACTGTCCCTTTTAATGTT

RIPPT79 AAT5 (SEQ. ID. NO. 252) ACTTTATATAGCATTTAAAAACACAATTTAAATGATGAAAAGTCACACATTGTATATTTAAAAAGACATAAGCACCCTA

GATTCATTAGGATCTATCAAGCCGTATGAGGGACACAATTTTTTTT1 CTAATTTTGCACTCTAAAACAGGTTTGCACCC AATGTGATTTGATCCC C AGGCGCATGCTAGATCTGTGATTAAAATAGACCTATAATGGTATATTTTCCATTATTATTA

TTATTATTATTATTATTATTATTATTATATATCTGGTTAATTTTCACTATTCTCATATTGAATTTCTTTTCAAGATTTCCAA AATATAAAATTAAAATGT

RIPPT80 AAT55 (SEQ. ID. NO. 253)

ACATCACACAACCAAAATTAAAACATTCATAAAAATTAATTAAATTAAATTATAATTTTATTATTATTATTATTATTATT ACATAAACTAATGAATTAAATATATATACATTCAATAACACAATAATGACTAATTAAATTTTATTTTAAAATAAACCAA CCTATCCACATTC AATAATAATAATAATAATATCCATTTTTAAGGTCACAGCCAAGCCAGACACCAAATTGGTGTATG AGACCCCCT GTTTGTGGGACAAGGGACCCACCTCCGCGCAGCGATTTGACTGCTAAGTCTACGAAGGCATACATACTC

TTTACCCCCTCTGGGTTTTGAACCCTGTGACTCCTTAGGAAGGAAACACI GATCTTACCCTTTAGGG

RIPPT89 LOBSEQ8-19-97TTC3B9 (SEQ. ID. NO. 254)

CCTGGAACCCTAATGGTCAAGTGCCCCAGTTGAGAAGTGACATAAGTATAATGATGCATATGTTGTGTATATAGCACTA TCACCTCCAAATRRATACGAAACCCCGAGTTGATAAAGTAAAGTTGGTAGCCCTAGGAGTTGCAACATCTTTGTTGCAA

TACΓGAGGTTCATCACITTTCTTCTTCTTCΠTCITCΓTCTTCTTCTTCCT^^ CCTATTTTTCTRCTACRTCTTCRTCATCRTCTTCCTCITCAACACRAGANGTANGGGTCCCACCAT^

RIPPT101 pPT101.seq (SEQ. ID. NO.255) ATCΓGGTAATΓC GTTATGATCATGATTATGCΓGATGTTTATATATGTATACATGTAGGTGTGTATGTATGTCATGTTTGA

TGGGGTCGTCATGGATCATGATTATTAACACTAACATTTGGTGTGTGTGTGTGTGTGTGTGTGTATGTATGTCGTTGTCT GATGGGATGATGATTGTTTTATGCTAATGATTATATATGTATGTATCTATTTATCTATGTCATTGTTTGACAGGATTGTCA GGGATTGTTCI GTGGCGGTATTATTGCTTATGCAGGTGAATTGAATGAAGAAGTTCCTGGTTTATGAAGACTCGTGTAG GAAGAGTAATCATGACAAATATATGAGGTCAATATCATGTGGTTATGAGTTGATATTATGTGATTATATTATGTATATGT TATGGATATGGTATATCGAGGGATACATCAACGAAATGAAAGAAGTGTGATCTACGACATGCTTGGGAAAAAGCATGG

ATAAGACACNANAAGCAAGGTTTATTGGTAAGGATGGGGTGGTTI AGATGTATGGTAATACΓATTΓTGCATATGTTGT

RIPPT103 pPT103.seq (SEQ. ID. NO. 256)

CCAATCACCATATAGCCACCTGTCAGCTTCCATCGTTACCACNAAAATAGTCCCCATCTAAGCAGTTTTCCTCAGCTTTG GAATTTGTGTCCCCAAATCAACΓCATTTAATTTGATTAAGGTTATAATATTTATAATAATGGAAAATGCAACTGGGATAT

TACAAACCTCTCCCCCTTGGTGGAACAACATACTTAGTGATCGATGACACCTATACAACACAAACAAAAAAGATATGCT TAAAAGTCCAAACACAATTGAGATGGAGCTTTATATATAGATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTATAGAGAGAGAGAGATACATCTCACTTTATAAATTCCGCCATTTTCCAATGGCCCCTTTTTGTAGT CTTCTAN RIPPT104 pPT104.seq (SEQ. ID. NO. 257)

GC AGTAAGAATAACTAATTATGTGATCAAAATTGTGTCATGTAAATGTTCTAGTTTCTACCAAAATTTGAATCTTTATG TCTGCATTTCATTTTTGCGTGTATAATTTGTGATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGCGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCTACATGTGTAAACTCTCCATG TCCTATGTCANTGTGTCTTTAGACTNTGGATGTGGGT

RIPPT106 pPT106.seq (SEQ. ID. NO. 258)

CCTCTTCAATCCRTAAGAAAATGAGGATTTTCCCTTGAAAAAGGAGTCTTAAGCCTTAAGGCCAAATCGCAATCGGAGG TΓGCATGAGGGCAGACTTAAGGTTTGGGCCGTGGAACCCCCGATCACTAATCTCTGATCAAATCAGATTGGTGGATCGG AGGCAATCACCTTCAGTGGTCATATATATATATATCTATATATATATATATATGTGTGTGTGTGTGTGTGTGTGCGTGTG

TGTGTGTGTGTGTATAAATGGATATTAGCGAGTGGATAGTGTGATCAGTGATTAGCCGAATCGGCGAAACCCTTATCAG TCACCCGGGGCAACATATGTGGGCGATGCTGACGCGATCAGCTGACGCCGAT

RIPPTl 17 pPT1 17.seq (SEQ. ID. NO. 259) CTCATATC TCTGAC AATGCAATATACAAATAGTAGGGGTTTrTAGCTGTGTAATGTGTTGATrGAATTCTTTCTCTTGT

ATGCTTCATGATTTCTCGATCGATTAATTTTTTCCACTAATGAGTAGAGTAGATTCAAGTATTTTGTTTTAGTAAATTACA ATTAGTAGTTTGAATCTACAAGGAATACACACACACACACACACACACACATTAACATAACCATGATCACAACACAATT ACCATTGCTCATAAGTTCAAGGCTAAGAGAAATTCCTTTATCCACGCAGAGTATACATAATATTGAGAACGACAGTTCT CAACATAGCCAAGGCATTTGTTACCTCAAGCCCATGT

RIPPTl 23 pPT123.seq (SEQ. ID. NO. 260)

ATCGGCGGTTCGCAATCGGTATTGGCATCAGCCATGCCATATGGGAAACCCCCGGCCCATGCGATACACGATTGCAATG TCGCAATATCGTGTCGAAACATTGGAAAGGAGCGGGGCCATTATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGATGCGATGGCTAGACCCGACGGGGCTATAGGTGATAGTTGATATATTTTTAAATATGC CCAACAACATTTTGCTATGTATTTCGCATATTTCGTTTGATAAATGAAATATGAAGGGAAGCACTGATGCAATGAAAAC

AGGGCCCGAAAGGGT^π'GTGAATGAAATCATATATCGCTTCAAGTCTGATATGGGTTGTTTTGCAAGATTTrCCAATGT TTTAATGCATTTCTCTGTGTAAACAGAGAATGGTTGTTCCAGGGATTTCAAAGGA

RIPPT126 pPT126.seq (SEQ. ID. NO. 261) CCΓCAANGCTAAGANGC<ΠTTACTGTAAATCATACCGAGAGAGGTCTTTGTAAAAATCATGTGTGTGTGTGTGTGTGTG

TGTGTATATGTGTGTGTGTATGTATGTATATTAATATGGGTTACTCTGTTCGAGTAACTGTACTATTGTGTGGATTGAAC TTATGTTTATGTTAGAATAGATGTGGCAGGCACAAATTAAGCTCAAGAGGGATCAATGCTCATATGGAAGTATATAACA TCATCTTCATAGATATCAGAGCACACAACAGAGGGAGAAAGGTTACATAACCAAGATTGCAGTGTCAAGATCTTAAGA CTGACTGTAAGGTCGAGGCATAACAGAGGAGGAATTTGTAGAATGGGTGGGAGAAATCTAGATTAAGCCGAATCAGAG TGGTGCAACACAAGT

RIPPTl 28 pPT128.seq (SEQ. ID. NO. 262)

CCAAGGCCTATGTTTTGTGATCGACCCTAGTCTCTTGTGCATGGTATCCTACACTTTTCATGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGTGTGTTTTACATGRRCCTTGAGGGGTAAGAAATATTTTTTGGTCGTCGTTTATCATTTCCATAGAG ACATCGAGTTΓCTACTCTGGCΠ AGGGTCCAAAATTCTAGATAAAGTTATCGTACTAGTTGTTCTTTCAAAAATTTTATT

TTCACTCTGTAAATAGTCATATGGGTTTATAGGTTGCAATCTCTRRGTTCATGAATTCCAATTATTACAAATTGGTAATGA AATATTTTTGTTCTGGTCAAGGGTCCAAATCTTGGGGTATTGTCTANACGGTATTTTTGTTGGTCAGGTTAGAGTGGTTT ATAAGTTCTCTACCCCCTCTATAGAGAATGACAAGTTGATGAGGGGGTGCAAGAATATCTACTTCTCAATGT RIPPTl 32 pPT132.seq (SEQ. ID. NO. 263)

ACAAGCCTAGTTCCTACAGTTGGTCTCACTAGTGGTGTGATTCCΓACTCCTCCCCTCATCCTGCTTCCACTCTGTACACC ATTAACACATTTTTTATTCTCTAAATCTCCCAAACAAGACACAATGTTGAAACCGTGGTGCTCTGATACCGCTTGTAACA TGCAAAGTCACCAACCAAACACACACACACACACACACACGCACACACACACATACATAATTGTCTCTAGCTCTTGACT TGCACTATCACTTATTTATATI ΓTTTTAGAGCATTTGAATTAATTGACACACAACTAAATTAATTGACCTAGTCATAGC

TAGTCATGGATACACT TATGTTCCTTATAATGTGGTAAATATAACTTATAAGTGTGAATGCATTAGCGACGAACCCACC TAATATTAATAGCACRAAGGGAACCACGCTATAATTGTTTGGATTAATATTTGGTTGTCATATTATAATATTGGGANGTG ACCΓACCI AAAATGTTTCTCGAAGGGCTCTTTGGTCTCTAGCAATCATACAAAGANG

RIPPTl 34 pPT134.seq (SEQ. ID. NO. 264)

ATCTGCACTTAGTGCTTGGCTATCTGGTCCπACCAGACTTGGCCATIT

TAGCACAACCTAACΓTCCCCCI 'I'CI 'CCCTTTTTCCTAGAAACCTTTCCAGTCAGGAGAGATAGTTAGGGTTGTAATT

ACTGCAACTGCTTTTCCCAACTTCCCATTAAGAA ΠTGCCAGCATTTTTCCAAAAACATTTCTTATCTTATTAAAGAAAT

AATAAAAATATTATTAAAC CCAAAACAATTAATAATATCATATTCAATTTTTAACCACAGTAAATTCAAATGTTTACAT TTTCCTGGGGCATTACACACACACACACACACACACACACACACATATATAATTACAGTATGAAACTGTTTTTTCTCTG

AAAATCATAGAAATCATGGCAATATTTTGATAAATTATGGCAGGGATTΓTTGTAAATCTANGTTATAGTTGTTAAAATTC AAGANTTTGGGTT

RIPPTl 35 pPT135.seq (SEQ. ID. NO. 265) ATCTTTTCAATARRTAACATTGAAAAGCATTAAAGAATAGCATTTTGACAACTAAGGGTGAATACCCAAATTCATACAC

TCACGCATGAGCTGAGTCATAAGATCTAAATCTAGACTATATΓGCTAAAACACTTAGTCATCACTTTTGATTATGATTGT AGAAATTAAACTTTTAATATTGTTACTTTCATTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGCTTAGCATAGTGGGAAACACAAATAGACΓTTAGTTTNNGACTT GTTGTATGAATTATCTCTCCATAATATAAATGCAGACAACT

RIPPT139 pPT139.seq (SEQ. ID. NO. 266)

ATCATTTCACCATTACTGTGAACCAATTGGAAACTTTGCAAAAGGATGTAGGGAGCATCCACACGTCTACGGAGGTCAT TATGACGAGGCTACAAAATTTGGACCGAAAGGTCGAGAACCTCAATGAGAGGGTGGAGATTGTTGTGGTGCCTATCCTC AAAGAAGTTTTCGCACRRGAGGAGGGTGCACCAGTGATCACTRRGAAATGGGACRRCCCTCTCTAGAGCTCACAATGCA AATGGAGATGCAGCAAGATGCGGAGCAACAGGAACCTAAGGTCGCAGGTCAATTGCGAGTTGTAGAATAGGAAGAAG

TGGAAGATGAAGTGCAACAACCAACCGAGGGAGCΓAAATTGATGCAATTCAAGAGTGGTTTTTTGTΓTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTTGAAAGTATGTTGAAGTGAATGTCGTTTTTTTGGAGAACGC

RIPPT158 pPT158.seq (SEQ. ID. NO. 267) ACTAGAGGCACACAGTGGGAGTCTCAGATCGGATCCACCGACTACTTAGTAATGTTGCACGAGTTGTCTCGTGCTACCA

TGACCACCATGCTGAGTAGTTCCTAGGAATAGCACTTACATATTTTTCGAGGCCTGTGTGCCACGGATGTATGAGGCTT ACGAGCCACTAATGTTATTTTAGACACACATATTATTTTTTGGGGTTCTGATATATCAAACATTCTCATCATATATATAT ATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGGCTTGTTGGGGCCTAACCTAGGTTTAGAAGAGGTTTAGCCAAACAA ATCCCACACTACTGGCCCTTTCAGCAACAATCCACAGAGCGTGGACTGAAGTCTCACCCGAGGTANTATGGGAGGGTG CTGGAACCAAGTTTCTCCACCCTrGGTATGTCTTGATGTGGTCTGGANGATCGCAAACCATTC GCACTCCTACACi CT

CTGCACAGAT

RIPPT159 pPT159.seq (SEQ. ID. NO. 268) ATCAACATTGGCCGTGCCATATGGCTTACCTCGGGTCCACGCGATATTGCGATGCGGCGATCAACATAATGGAGCAGGT ATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGATGTGATACATATATCTGGACCCAATGGGTTTATGAATGCAAATA TATGATACTTCAAGTΓCACCATAAGGTCCTCΓGATGGGATTTTTCAATGTTTAATGTATTTTTAAAATAGAAAGGGAACC CATGTAACAGGGCATTTGTGACAAGCGATAGCAATTTAAAAGGCAAACATAATGGAAAAAAATGTAGAGTTCATAATT TTTGAAATCΓGATAGACGCGTTTGGGCAAATTGTTACAGGCTTTCTGTGTATTTTCCAGTGTAAGCCGAGAATTACCATC TRTGGGATTTCAAGGGCACTGATGGTCCATTCCATTACGGAATAAGTGG

RIPPT165 pPT165.seq (SEQ. ID. NO. 269) CCTTGTGGAAGCCACAATTTGTTGAGTATTGGCAATTATTGAAAAAACCCirrCAAGCTCTTGAATCTGTATTCGTCCTC ττττGAACGAGTCT^,CTCT^,cτcrcτcτcτcrcrcrcτcτcτcτcτcτcπ'crrcτcτc^ ACACACATATTCATGGGTATGTTCAACTTTGATGTGTTTGTTTTGTTC

GCCAAGGTAAAGTTTAAATTGCITATCCGTGTTTATCATATTTTCTCATGCTAGATTTCITTACGAGAATCGGGGGTTAA CTAAGGAATTCCTTTTGTCTCATCGCAGGTTAGTTTTGGCAATATGGGCACGTTGAATCCGACAAGTTI TTGGAACCAA CTACTCATAATTCC CTTCTTTCGCGCAACTATACATGTGCTTATCTATGTTTTTTGTTTTCT^ TCANGGTTCCAACTCATACACTATATACGT

RIPPT166 pPT166.seq (SEQ. ID. NO. 270)

TTGAGATGAGTAGTGTGGGTGGTGGTCTGTTGTAGGGAGCAGAACCCACCACAGTAGACAACrAGGTTTCTTTTTCCGC TATTTI GAGAGACATGTGTCTCAGATGTATCACGAACACATACTCn'ATTrTTTGACATACACACTATTTTTGAGAATGT CCGTGCGCCACACGATATATATATATACACACACACACACACACACACTATCTATGGTTTATTACTGTCTAAATGCTTGC ATACAATGTTTTGATTATAAATTGCATAAATGTCTATCTAATACATGATTTTGCAATGCATCAGTTAACGTGTTTAATCT

TCTTGGAATGTGTTTATATGTGATGAAATCAAAATTTCTTTCCAATAATCTAACACGCAAATACAACCTTAAAATATGAG GGTTTCTGATGGAATAATGCCCGTTAAGCTGCAGAGCGACAAGTTAAGAATCCAGGTTCACAACCAATGCATGAATATC GAGGTTAACAACCTCAGTCAATAGTTCAATGACAGTGCTATCAGCGACGTTGTTTGCGGT

RIPPTl 71 pPT171.seq (SEQ. ID. NO. 271)

ATCTCΓCCACAGCΓTTAGAGACACATTGTGGGGGTATAGTCCΓCTCATTATGAAGGACTCCTATGACATCΓΓTTTCATGT TCTTITGTGTTATCACCAACATAGAGGAGATTAACRCCTTAGGATATTTTTRATGGGATATGTCRTTGCATATGTATTGG ATTATGATCCTAAGCCTTAGAAACCCRRATTTTGACTAATGTATGCAAACT^

TATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCATGTGTTTTAGCTTCAATATTGTTCACTTGTGAATGTTGTCTTATGAT GTTTTCATTTAGGTTTCTAATGAATGTATCATATGCATGGGTGACAAAATTAATGTGACAAATGTCAATATATGACACAA

TTAGCCAATGAATAAGCTAATGTTTTAGAGTCTAGTAATATAAATGTATGAAATGGTATAAAATCCCATTCTAGCAGAT

GT

RIPPT179 pPT179.seq (SEQ. ID. NO. 272) ATCATTTATTTCAAAACATGTAAAAAAAATAAACATGTAGGAGCACAAGCCATTGATTATTTTCTCTATTTTTTAAAGGA

AGACATTTAACTTAAACATTrGTAGTCAAAATCAACTGACTTTCAATAGTATTTTTTAATTATTATTATTATTATTAπ TTATTATTATTACACTTTTTTCTATCAAACGGTCCAACTGTGTTAAGTGTCTAATACATATGCCTAATTTTTATATGCTAA CATTGCAATACTACTTACTAATTTTGGTTGTGC ATCCATAGAAGCATTTGGTAAGTGTTCATCATGCAATATTTTTACC ATACACGTTATGATTGCATGTCCATTTTCAAGCAAGTTTTCAAAAGAGATGGTTATAGATATAATTCTCATATGAGTTGT AGGATAGTGTTCCAGCAGTTTGCACCCATGANGGAACTATCATTTACAATGAAACTACAAAACATGTTGGCATTATTGA

CTTGTTTAACACTACATTATTAATTGTTATATGAAAAATT

RIPPT185 pPT185.seq (SEQ. ID. NO. 273) ATCITTCGATGTCTATTGGTATACTATTTCAACAGAGGGTGTTTGCAAATCATGGGGTAAAATAATAATAATAATAATA AAATAAGAATAAACTAACTTCAAGACAAAGTTCCTTAAAGAGGGAGAGTATGTAATGCCCCATAACAACAACAACAAC AATAATAATAAATGACAATAAAGTAAATAAAATATTTTTAATATATAAATAGAATAATAATTAACAAAGAAAAGATAA AATAACAATTAACAAGAAAAATACATGAATATAAAAATAAAATAACAAATAACGAAGAAAATATAATAATAAATAAAT AAACTAAAAATTGGCATGGACACTGGTGGGCTCCAGTATTGTAGCAATAATGCTATAACTCCTGGGACTCCCTTCTTTA CTTTTATCAACCTGGTAGTTCGTAACAAACTTGGAAGTGACAGTGTTGTATGCACAAGT

RIPPT193 pPT193.seq (SEQ. ID. NO. 274)

ATAATGGCCTTGGTGGGCTTAATGGTTAAGAACATGTATTTCCTTGTAAGAGGTCATAGATTCAAATCTAGAGGGAAGT AGAGAANGTAAACCTCCACAGACTTTAGTAGTCAAATCACTGTAAGGAGGCGGATCCCTTGTCCCAGAAACAAGGGGT C CATAAACCAATTTTGTGTCTGGCΠTGGCCATGACCTAAAATTAGATTATTATTATTATTATTATTATTATTATTATTCA

AAGGGAGAATCTAGCTCTGTTTGTAAAAAGTATCTAACCCAGGCATAACATCAACATAAAACCAACTTGTAGTAGAAGT

ATGTTGAAGATTTGTTTTAATATA

RIPPT211 pPT211.seq (SEQ. ID. NO. 275) ACATTATGCAATAATAATATTACACAACCAAAGTAATAATATAACTTGAGTGGGCATAATGGTTAAGAGCATGTGTTTC

TTCCTAAGAGGTCACATATRCAAAACCAGAGGGGGGTAGAGTATGTATGCCTTCGTAGACTTAGTAGTCAAATCGTTGC GCAGAGGTGGGTCCTGTGTCCCACAAACAAGGGGGAGGGGGTCTCATACACCAATTTGGTGTCTGACTTGGTCGTGACA TTAAAATTGGATAATAATAATAATAATAATAATAATAATAATAATAATATTGTCAACCCACCTAATTAATTATACTTCAT TAGTTGATTCRCCAAGAAATACATCCTCTATGCACATTTTTCTAGTTTCATGAGTAAAAAAAGGG

RIPPT255 pPT255.seq (SEQ. ID. NO. 276)

CTCCTGAGTGGTCCCATACTTAAAAATAAAATAAAATAAAATAAAATAAATAAATAATAAATAAATAATAATAATAAT AATAATAATAAAATAAAAGGGCTCCAACAGGCCTCATATCCATCTTTGTTATCAATCAGTGCACCACTTTAGTCTCCAT GTATGCCCAACATTCΓATATGTTAGAATCTTTTΓANATTCGTGACAATGTTCACTANATCCCCATATTTCAAATATTTCTT GTGCTAACAACCTCT GATGATCTT ATTGCCATGGAATGAANAGTGCATCTCTTTTTCAAATTGAATTCATAAAC

RIPPT263 pPT263.seq (SEQ. ID. NO. 277)

TATGTGTATTCAATGTTATGGACTAGGAAGGCTTCAACATCACTAGAATACCCAATTTTCTGAAGAAGAAAACGTANTC TANCAATGAATATTGTCAACΓAAATTTGGATTGGACCTGAATCAATAGATTGTTCΓΓTATTCAAGCGAAAAAATAAATA AATAAATAAATAAATAAAAATAAAAATAAAAATTGATGTGCTAGTTGTAGACAAGGACATTAAGATGAAATGGAAAGA

GGTCAATATGCATTTGCAGGAACT TTGGGGAAAACCTCGAANACTGCCAAGTCTATCTGAACATTCACAATTCCAGCT TATATCGGATTGCATGCAAGCATCCCGTGTTCCCATGTGCAGACATGATACATTGGATCATCTCNCACNCTGATCTTGA GATGATGAC Π GAGAAGTGTCCTTGGGATAGAGATTGTTACCTTCAAGGCATAGGATTATCCNTAGATGTTATCCATA TGTCAAGACCCAGTAGTCACCCATGGAAGACACCCTTCANCATACCCCAACCAACAATGCCAACTCCAGGGGAT

RIPPT274 pPT274.seq (SEQ. ID. NO. 278)

TAGTGTTCCTCrCAAGTGACCCCCrTTTCCrAAACAAATTTGAAGTATATCCCACATTTACCCTTGCATCCTGCACAAGT ATATCAACITCCCTAATTTTATTTATTTATITATTTTTATT^

GAGTGGATGTCTCCACTCTCTTCTCCTCAATTTGTGGCTrcrCCACAACTTCCCGAGGCπ'CATCCTCTAGGGCTAAAATA TCCTCTTCTGGTGGAATCTGAAGCTCCATCTTAAGAGAGCACTAAATTTCTTT^

ACAACTTCCATTATGAGGGAAGGTAAGT

RIPPT287 pPT287.seq (SEQ. ID. NO. 279) ATTCCATGTGAGGACT TCAGTGAGAATATTTTAAAACTATATCATCTCCCACTTCrrGCAATGAAACC TTCTAGTTTT CCITCCITI M^'I ^'I GCI ^'I AACCAATTCTTTGACTAAAACATCGTΓACCTTTCTCT^'ATGAATTCCTCTATGGAATGT ATTCCCGGrrCCTTCAAANATTTTATTTrATTTATTTATTrATrrA

ACATTTCTCTTCAGACAAATTCGGCAAATATCTCTCrACCCATTCATrGAGGAGGTCCACTAAAACACTTTCTTCCTTGG AAAAATAATCATAATTATGTCrTATTAATCGAGCAACCTCCTCAATATCCGGGAGGTAAGTAACAATAATTTTCTCATTA CCCCCTC TCATATTGTGAAGTCTTCCTCTTCACCGGTTGCTTCATTTCCTTCACATTCTGGGTTTGTTCCATCATCTATA

CATCATTTGGTTTCCTCTTTAGGGT

RIPPT293 pPT293.seq (SEQ. ID. NO. 280)

ACCTACGGTTGTGAAAAANCGCTACTATTGGCCGAATCTAATAAATAAATAAATAAATAAATAAAGGAAGGTTGTTGG TTTCATCACTAGGTGTTTGGATTGTCGATAGGTAAAAGTGGAGTTCAAGCATCCAAATGGTCTATTGCAACCAATTTCG

ATTC AGAGTGGAAATGGGAGGCCATTrCCAGGGATTTCCTCACAGGTTTGCCAAGAACATCTANATAGCATGATTCCA

TCATGGTTGTGGTTGACAGGTTGACTAAGGTAGCTCAC1 CATAATAGTTAATTCTACTTATATAGCTAGTTAGGTAGCT

CGAGTCGTCGTCAAGGATATAGTTAGGTTACATGGTATTCCTAAGAAGATATTTGACAAAGATACCAAGTTCACTTCCA

GGTTTTGGAAGGAATTATTTGCAGGTTTGGGTACAGAATTGCCCTTTAGT

RIPPT298 pPT298.seq (SEQ. ID. NO. 281)

GGATATCNAGANTCGAGGATCATGCATACTAGCTTGTCCAACGGGGNAGTTGGCGCTAGGGAACCGCAAGACTTGCCA

AAACAGCGGAGGCTTTANCCATGAGTGGACCCNGGTGAAGGGGATGATTGCCTTCACAAAATCTATAGGGGAATC

GAATATCCCC TTTCCCTTTCCATGACCAACTACGGAGGTAGCATCGCCGTGAGCTTCAGTGGAAGATGAAGGACGGGC TGGATCACGGGACCATACTTCTAAGACCTTCGGAATGCCAGGACΛATCACGAAGAAGATGGTCCTCCTTCCATAAT

AGACAAGGAAACTTAACCTTCTGCGGAGGAGTGGGAAGGANTCANACTTCTAATGTCTGTGATGGAGCAACAGTTGTG CATGTAGTCTCTGTTGTGTTGGTAGCCTCTCCCTTAGGAGACTTC^

GGTTACTCGACTCGAAGACTTTCACCAAGGCGGAGTANGCTTGGCGTTAATCATGGTCATANCTGTTTCCTGTGTTGAA ATTGTTATCCGCTCACAATTCCACACAACATACNAGCCGGAAGCATAAAGTGTNNGCCTGGGGTGCCTAATGAGTGAGC TAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTΓΓCCNTTCGGGAAACCTGTCCTGCCANCTTGCCTTGAATNAATC

TGCCNCCCCCCNGGGAAAAGCNGTTTCNTNTTTGGGCGCTCTTC

RIPPT305 pPT305.seq (SEQ. ID. NO. 282) TCAATCACCAATTATTTGGCrNTCTAGGTGTTTTTTTTCATATACCTAGATCGAGTCT NGCCI^

CTrCATCTTCATCTAATTTC CCCCTTCrTTAGACTTrGTrATATGTGGCATAGTTTCATCCACTCCGAC

RIPPT367 pPT367.seq (SEQ. ID. NO. 283) CCrCTATTTGAAATGTGATCATCATATTGGACTTATAAGAGGTAACATATAACATACATTTCCAAAACTTTCGTAAGGA GATCAACACTCTCCAAATAATTAGGAATCCCTCCTTCTAAGGTCAATCATAGGCATAAACCATGGATATAAATACAT

GATTGATAATATTAAGAWCrTATCAACATTCCATTCTTGTGGATGGTAATTCCATAGATGGGTTGTATAGATGGTTATC ACCCACCCATGTCTATAGACTTGGTGACCCCTGAAGAGCCTCCAATACTCATACCACTCACTGCAATACCTCCAATGCA TAATGCAACCACACATGTGGACATGTGGACATGTGTATGTGTGTUTGTGTGTGTGTTTGTGTGTGTGTGTGTGTGTGTTC TCCrACCTAATGGATGAAGAAGCATTACCCTATGAAAGGTTTCCAGGAAACTCATTAACAAAAGTAACCAACATTTGTA TACCCACAAGTGGAGTGTCAATCAGACTGAGCACCATGGCTATAACCATCCATTCAAGCAAGGGTTTCACCACGTCATC

TCAACACTGAGACAACGT

R1PPT369 pPT369.seq (SEQ. ID. NO. 284) ATCAGATCATTTTTCAAATACI AGACTAGATGGGCTAGATGCATGTTGTTAAGCATGCATTGTCATGGTTATGGTTGTT GTGCACGAGCTTATTGTATGAAGTTAGTGTTATCATACATGATGTCGATATGTTGAAGTGAGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTAGATATCATGCTAATCTAGArrGATTTTGTAGGTGAGATAACCTCCCTTGAACTTCACTGATGTATG TTGATTATGGTTATGTTTGGTTTATCTAACAAGGTATATTTTCAGGGAGAAGATCAAATATGACTAGCACAATGGTGGAT TATAGTTCACGTGACTCTCCTTATAGGTCACATGAGGAGCGCAAAGATAAGTAGTGATGTGCCTCATAAATGGGT

RIPPT376 pPT376.seq (SEQ. ID. NO. 285)

ATCATATAACTGCTCATTGCAATAAGGAARRGGTGATTCATGTGGTAATAAAATAATTGCCCACTACATCTTTGTGACTG CGATTAACCGCATCGATTAACACACACACACACACACACACACACACATATATATATTGGTTATGTCTCAATATAACCA TTTATAATATGATTTAACIΓGGTCACATGGGTATACCAGGGCCGATTCTTTTATACΓTGTTCTTTTAGCGATTCCACTATG CCATTATCACTTCTTCATCI CCITTTTATCRI 'RRRIM'GAAATATTTTCAAGTAATTCTRGAATGAAAAATGAATACTAC

AGGCAACAAAAATGCATGTΓACAATAGGTTCCATGCTCATTATTAATGGTCTI TAGGTAAAAGCATGCCCTTCATGCTT CTAGGGCTGGTTGGGTCTGTGGATTTATTGTCACGAGTCAAAGGACTCAACATCATGTTGATAAGACCCTTCTCAGCAA

AATGT

RIPPT388 pPT388.seq (SEQ. ID. NO. 286)

ATCTTGTCCTTAGGCTCAAATCTGAACACAACACTCAAACATGCTCAATGTCTCTTCTCCTAAAGAACAAATCTGAATA AAGCACTCAAATATGCTCAATGTCTCTTCΓCCTTACTCTATAACAACCTCTTTTTCTTTAATTCAATCATTGTT^ CAACACACACACACACACACACACACAGAAACATCTATGTITTTCTTAGTTGGGACCTCACATCCTCΠTTGCATTAAA AGCATCACTTCAAATTTGTGTTTATCACCCCTACCTCCTGACATTCΓCTAGGACTATGCTTCAACACTTATTTTTCTCACC ATTCAAGTCCTCAAAGCCTCTTGTCACTCATCAACRRRGTTTCTTTCAACTATATCCAAATCCTCTTTTACAT^

CTTTGTAGCATΠTTITCCTTATCTCATCACCGTAGC

RIPPT467 pPT467.seq (SEQ. ID. NO. 287)

ARKAAGCTTACACAACACTGCACACACAACACCAGTACTGCACATTCGAAGACCACACTATCTTTACCAAGCAACCTTG CRRCTAGTGTCRITCCACTATCCATACTTGTCCCAACATGCCACAAATCACAATGCTTTCATTCTGTTGATGTATCC

CTCAACATAGCATGTCCATTACATAAACATAGGCATAGTGTAAACACATAATATCAATCATATCCACATGATACTAACA TCATAGATCTGTTAGGGATACTCTGCCCACTTGATTCCTCACGATACCAAGAACCTCTTCATGCTACTCACATGCATAAG CAATCATTCCTCCACAAGAATGATCCTTGGCGACCTTGTCATACAATGNCATACATACATACATATATACATATAARCA TCATCATAAAACAATCATGATCCCTAATGACCCAGTTAGACACACACACACACACACACACACACACACACACACACA CACATATATAAACATTAGCATAAAACAACCATGATCCCTAAGGACCCTGTCAGACAATGACATGCATATATACATATAT

ACATATATAGRCACCAGCATAATAATGATCACAACATAATTATCAGATAAGCTTGCATGCCTGCAGGT

RIPPT496 pPT496.seq (SEQ. ID. NO. 288)

CCTCCGTCCGCTTTGAATCCTCITCCAAGCATTATACAATCATTGCTTAATGCCGAAAGGGGAGTYGGCCACCATTTCTG TAATGTAAGAGTGCCTCGGGTCTGAYCCATCGGATCACCCATATAATTGTGTGTGTGTGTGTGTGTGTGTGTCCCCGCTC

CATTGTAACATGTCGATGCGATATTACAATGCGACTTATCGCAATCGCATATCGCATAAGCCCGCAGCCTTCCCATATA ACATGGTTTATTGCCAACGCCGATTGTGATTGCCGATCTACCTACCACCACCGATTGCGATTGTCGATCTGCCAACAATT GCATGTCGCATTAGCCTCAACCTTTTGTTAAGTCCTCCCrTTGGCrrTTTGCGATGCGATAAAGCGGTAATCGCATATCA CAATGTTTT

R1PPT508 pPT508.seq (SEQ. ID. NO. 289)

GCATGATTTAATTTCCAGTAGTAATCTCCGTI TΓΓATTGTTTGTTAAAATATATACGTATTTCAATATTCCΓTTCCCACT TGAAACATCCTCAACTTAATATCCTGGGTTGGGGTCCGAGGATCTCCTTGCCAGTAGCAACTTGCAAATTGCAACAACC TTCACAANTTCAANATCCATTTAAACAAACCTGTCATACTCATCANAGGACACTTTACAAAATTGCCACTAAGCAACTC TGGANATGGGTCAGTTTTGCATTATCTATACCAACTAGCATAGACCCGTCTTCACCCTACATCTATTAAGCATTGGAAA GGGATAAAGAAAACAATGTGCAAACCACTTGGAANTTGGTGTTTAAAAAACCCAGGGTTAAAATGGATCTATGTTAGT TTCTTTTATTTACI ATTTTTTACACCACTTTTC^

CACACACACACACACACACACCTATGAAATCT-CCC TCCACCACAAAAACRRGGCGTTATCATGGTCATAACTGTRTCC TGTGTTGAAATTGTTATCCGCTCACATTCCCCACAACATACAACCCGGAAACATNAANTTTTAAACCTGGGGTTGCCTA

ATGAATTAACTACTCCCATTAATTGCΓTTGCCCCACNGCCCCTTTCCATCC

RIPPT538 pPT538.seq (SEQ. ID. NO. 290)

GTAGGTCAGGTCATGCCATGGTAACTCTTCATCTCATGTTTACCCΓGATAGGCCAGTGGAATTAGGTAACTTTTGGAGG GTGTGTYACAAATTGGTATCATAGATTCCAAGTTCAAACACTTGGACTGGATGGGCGGGATGTGATTGTTATGCATGCA

TGTCAGTCATATGCATGCATTGTAGTAGCRATGGTTGTCATGTATGTATTCAGTGTTACTTTTTGATTTATCATGCATGAT GTCGATGGGTAGATATGTTGGTGTCATTATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTTGCAACAAACATC CTCCAAAATTTTGTGT

RIPPT540 pPT540.seq (SEQ. ID. NO. 291)

CCTTAGGACATTCTCT'ACTATTGTTATCRRAATGTTGTCATTAGTGGTAGGATCATTATTTTTAATTGTATACTTTGT^ TAATACACACMCACACACACACACACACACACTATGGATATTGTTCACACGAATCAATATTTATTAATAAGCGGTAAG CTATAGARGAGGTTTCCT ΓACAAAGAAACCWTTTWATTTAATCATATTTTAACATTCTCAACAAGTGACATCGCTTAT TCRTRATATTATTATTTTTTAGGGTTA

RIPPT548 pPT548.seq (SEQ. ID. NO. 292)

ACTAGATGTGTCCΓCTAGTCCCATTGAACTACI ITGTGGTCATTCGTTGGAAATTAATAAAATTATCTTATCΓTACCTTT AATTACTCATRTATTGGCATTGAAATATTTTACTATGGTCGATGTGTGTGTGTGGGTGTGTGTGTGTGTGTGCGAGTGAG CGCGTGCATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGATTTGGAGATAAYTCTTCCATGTWGA GTTTACAATTAGTTTGGAATTTTGTTAAGARAAAATCTCAACCRRCC TGTCTATTTTTGGAATCAATTGGATCTCAATCC

AACTATATATGTGTATCATATTTATGAATGAATAATCGTTRTWGATGTGTGAAAATTAGCTITATTTGTTGGTATCAAAG CCCTATGGGTCTGGGGAAACCΓGGGCGTTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTC CACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCYGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGT TGCGCTCACTGGCCCGCΓTTCCARTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCCGCCAACCCCCSGGGAAAA GCGGTTTGCCTTATTGGGCGCTCTCCGCTTCCTCGCTCAATGAATCCCT

RIPPT556 pPT556.seq (SEQ. ID. NO. 293)

GTCTCTTAATTATGTTATGATCGTGATTACATTGCTGCCTATATTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATAGATA TATATATGCCAATGTC AATAGGGCTGTCAGGGATCATGATCΓΓΓTATCATGATGTTTATATGTATATATGTATGTCTGT ATGTCATTTTCTGACAAGTATGGAAGCGATCATTGTTGTGGAGGAGTGATTTCTTATGTAGGTGAATGACATGAAGAAG

CTCATGTTGTCAAGTGGGAAGAGTATCTCGAATAGATCTATGAGGTTAGTGGCATGTGGATATGAGTTTATATTATGTG ATTATACTATCATAGGTAATGTCGAGGGATACATGAATGGAATGAGAAAGGTGTGATTTACAACATGCTAGAAC

RIPPT560 pPT560.seq (SEQ. ID. NO. 294) ATCGCAATATAGCATTGGAACTTCACCGAAGGGCGAAGCTATACACACACACACACACACACACACACACACACACAC

AAATATATATATATGCGATCTGATTGCTGGACCCAATAGCACCTTAACACTATATTCAATAAATATTTCAAATCTAAGAT AAAGCCATTATTATGGGATTTTGCAATATTTTAATGTATTTCTAAAATACAAATGGACAAGCCATTTGTGACTTTCGACA GTGACΓTCTGGGTAGACCCAATAAGAGTATAGACTAGATTTAAGΩACTTTTGAAGTTTGAAAGACTATGTTTCGGTGGA TTTTCACCATTTTGAATACATTTCTCCGTGTTAACAAAAAGCAGCTATTCTAGGGATTTTAAAGCCTCGATGCACTCATC CATTTCAGTTTARGT GCCATCRCCCCTGAATGTGTTTTGCAATGAWTGTTTAACGTTTTGAAAGAAGAATTTGATTGGA TAGAGGCCGGGAAAMARAAAGCCCAATAAGTTGGAAATGAATAAGGTTAAGAAATATTGGCTTTAAAAGCCCTTGAAA ARAAAAAAAAACCGAAGGGTTG

RIPPT567 pPT567.seq (SEQ. ID. NO. 295)

CCTAGTTGACAACGTAATTAGTGTGCCATGAACAATCTATCACATGTGATTTGTATGATCCTCTTGTGGCAGCACCATCC AACTATATTGCAAAATCAACGTCATCCTGACATGTCTATCAATACACATGCCAACACATGCCAACTCCTCCATGACCAA CATGCTACCATAAAGCTCCACCAATGCCATCATCTGATCAACATCAATGCCAATACATCYCAACACAAACCGTCATGAG TGATATCATATTATTGACATCAATGCCAACATAATCAATCCAATGTCAACTCAACTAGTAGCATAATGACCATGTTGAC ATTAATGCAAATACTATAATGTCAACACTATAT CTATAAATCTATATCGATCCCATCAATAGAGGATGCTAAGGGTTA

GGTTGGTGAGGAGACTTGGGAGTTCATAGTGATGACACACACACACACACACACACACACACACACATGTTTTGTATTA TTTTCI AAATTTTCTTITTCACATTGCTATTTTTTTTAATATTATACATATCATC TTGTGTGTGTGTGCATGC

RIPPT584 pPT584.seq (SEQ. ID. NO. 296)

TKTTACCCCGAACTCCAGAAAATGCAGAMATTGGGACGGCTCACATGGCTMTGGGGAGGGCTGGAGAAGCCCCAGGA AGGCACAAGCCAAGCAATACAAATCGAAGATGAGCAGTCTGTAGAGGCCGAWATGGAAGAAGAAGACAATGAGGAA GCATAGCAGGCTGCAGAGACAGAAACAGAAGAAGAATACGACTAGGAAGCAGAGCASTCTGCWGAGACAGAAACGGA AAMAGAAGATGTCGAGGAAGAAGAGCAGTCTGCAGAGGCTSAAGTAGAAGAAGAAGAAGAGGAGGCAGAGCAGTCTA AAGAGGAAGGAGACGAGAAAAAAGAAGCTGAGCGGTCTKAGCASGAKGAAGGAATGGAACAACAGGAAGGTAGCCC

ATTACCARACCCGTTAGGGGAAGATGAATTGGCCAACATTTTGGCCTATATGGGTGAATATGGAAAACCCTG

RIPPT609 pPT609.seq (SEQ. ID. NO. 297)

GTCGGCAGATCAGAACGGTGAAACAAAATGCAGAGGGGCTTAAACACACACACACACACACACACACATATACGCTCG ATCCAATGGTGCAATCATTGAAATCAACGGATAGCAATCAAAGCATTATAGAGGATTAGCCGATATGTTATAAGTTATA

TACGTGATTCGATGGACTGGAC CGCTAACATTACAAGCCATATTGTATGGATCTTGAGATCCATCAAACACCCCTTTTT AACGTATTTTCACAGTTTCATTTGTTAAATGAAATGTGAAGGAGAAAGGCCAATTGTGACTTACGATAGCGACCTACGG GTAGACCCGATAGGAAAATAAAGCATAGTTATGGGAATTTCAAGTCΓGGAATTGTGTTTΓATGCGGACTTTCCCATGTT TTCACAGCATTCTC GTGTAACCAAAGAATGA RGTTACTAAGGGATTCCAAGTGCCGATTTCCCTCATTCGTTGCAAAA ACAAGTGGAAGCTTATCAGTAGCATATTATGGCTTTAACATTTGAAATATGCAAGCT'CAAGTRI ATTCTTCATGCRTAA

GGGAAAAATTCTTATATACAATATANGTTCAAGATATGCCCTCTTTATTTAAGGCTTATTTAATATGATATAGGTCATTT AAATTTTAGTATTTATCCTTTACACCATTAACATAACNTATTAATTGTGCATGTAACCCATGGATAANTAGATTAA

RIPPT619 pPT619.seq (SEQ. ID. NO. 298) ATCACAACAGCTCTCTTAATAGCCTCGGTATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGCTCCTTCATGGTTT

CAATATTGATGAAAGTAAAGTCACAGTCCTTATCTCATTGATCTCAGTATAATGAAAGGAAGCAGTATCACATGCATTG TGATGTTTTAAAGACATTCRCTTCAGCGTTGCTATGTGCTACGCCATAGGCCATAGCACCATAGCTAGGACTACCTTGGG GCTATTGTATTAGGTCTATTTAGAGACATCATGGTGATGAAGTGCAACATAACATGATGGGTGCACTAGTGTAATGAAG TACAGTATAATGATGAGTGTAGCGGTATGAAGGTTAGCAGCACCATGATGGTTGGATGCAGCATGATGACTGGATGCA GCATGGTTGATGTACCGCACTGTTGGGTGCAACATGGTGATGATGAGTGCAGCACAGTGATGATGGACATGGTACGAA

GGGTGCAACACAATGATGGGTGCAGCAAAGTGATAGGTATAGCΓTGGTTCAAATCTGCTACCTCAGGTGTCATCTTCTT AGAAAAGATCNCΓGCTCTTATTTTGTCAAGTCTAAATGTTGACTTCTGAAGTATATGTT

RIPPT621 pPT621.seq (SEQ. ID. NO. 299) ACGTATATAGTGTATGAGTTGGACCTTGAGTGGCTTCTATGACTTGAAGAAAACAAAAAACATAGATAAGCACATGTAT AGTTGCGCAAAAGAAGAGGAATTATGAGTAGTTGGTTCCAAAAAACTTGTCGGATTCAACGTGCCCATATTGCCAAAAC TAACCTGCGATGAGACAAAAGGAATTCCTTAGTTAACCCCCGATTCTCGTAAAGAAATCTAGCATGAGAAAATATGATA AACACGGATAAGCAATTTAAACTTTACCTTGGCAAAGGGAAGCAAAGTCATTGCAATAAAACCATGCAACAAAACAAA CACATCAAAGTTGAACATACCCATGAATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGAGAGAGAGAGAGAGAGAG

AGAGAGAGACRCGTTCAAAAGAGGACGAATACAGATTCTAGAGCTRGAAAAGGGTTTTTTCAATAATTGCCAATACTCA ACANATTGTGGCTTCCACAAGGG

RIPPT627 pPT627.seq (SEQ. ID. NO. 300) GTGTTCTTTTATCrCTGTGATCTAGACGCACACCATATGACTATTACTTACTAGGATTTGGATGAATTCATGTGGCAAAA

AATCACTGCAGTTTGGATGAATTTATGTGTCAAAAAATCACAGTGGTTTTGATGAATTTATGTGACAAACAACCCTTGC

GTTTTGGAAAACAATAGTTACAGTTΓGAGTGAATTTATATGGAAAATATTCATTGTGGTATGAGTGAGTTTTATAGAACT ATATTTTGTGGCTCTTGCGCACGTGCACACACACACACACACACACACACACGCATGTTGGCTTGATGGGTCCTGACCC AGGTTCAGAGTTAATTAGCCAAAAAAGTCCTTGT

RIPPT629 pPT629.seq (SEQ. ID. NO. 301)

ATCTCCACTCTTAAAGAGAGGAATGACCACACTAGTAGTCCACTCTCTCGGAAAGCCGTCCTAGATGATCCCATTGAAA ATTCCTTTTAAATGAGGAGCAAGGAGCTTAGCTCCCCATTTTAGGAATTCAGCTTTAAGCTCGTCTATGTCCTCTGCTT^ ACTGCΠ-GCTAGATTCΠTATTCTCTGCTTGATGTCATCCTCTGTGAATAGTTCCACTGAATTGTTCACTATGGGGGGTAT CITTCTCATGGACCCACTCATAAAGGAGCCTCACATACTCCAACCATTTGCCACCTATAATACT'GTTTTTAGTCTGCTTT

ATCCTTTGTTTTAGCTCTTTCCAAAATCCCITGGGGTTGTGCTTTCCCAGAGAGATTAACTCCTTTC^ CACACACACACACACACACACACATATATATTATACTAATT1 CCATATGTCTTCTTGTTTTCTTGCTCCATTTTCTCACT TTTAGAGATCTCCTGGCTACCTTGCATTCTTCATCATACCATGGATTTGTTGGAAAAGT

RIPPT630 pPT630.seq (SEQ. ID. NO. 302)

ACGCAAGCTATGATACAACGCTGCAATATATTTATATATGCAGGGAAAACAACACACACACACACACACACACACATA TATATACATGCAATGGCACCATATATATACACAGCAATAGAGAAGATATTTACACGCTCAGCTTTCACACTCAGCCAAC ATATATACATACACACAGTCAATATATATACACATAGTCAATTTATATAAACGCACAATATGCAGATATTCACGAGTAG TAGGGAATCAGAATAGTGATGCATGTTATAGTGATGCTCTGTCTATAGGGAATCAGATATTCACGGGTAGTAGGCATGT TATAGTGATGCTTTGTCTACATAACTACAGTCAAGATCTGGTGAGAACAAATCCCGATGGATTTATAGAT

RIPPT644 pPT644.seq (SEQ. ID. NO. 303)

AGTTTCTTGTCTTCGGTTAATTTTTGTTGCCTCTCATCTGTCACTCTTACAAOCCTCTAACn GCATAATCATAAA AGTCCAATTGTCCATGTATCTGGTrACGAGGATTAAAACAAAGCATAGGTTGTGATCCAAGTCCCCTGCAACACACACA CACACACACACACACACACACATATAATTAAGGGTTGAAAATAGAGACTGTAAAAGGAATGGCTTATGAGATTTATTC

ATAAATCCACTCTTGCCACACTCTAATTATTTTTATTAAATAAATACATCACTAAAGGTCACTCAAGATCCAAGAGAAT AGGACCGAATGGACCATTATTAGCATAATAGAAAAATTACAACAATTGGATAAATTTAATAGATTACATGGCAATCAA AACAAACTCCTATCATAACAATCCCACTCATTAAGGAACCAA CCTTTAATAAAATATTTTGACCTCTCAGTCAATGG ATTTTATGAATGCTCTTGTATCGT

RIPPT647 pPT647.seq (SEQ. ID. NO. 304)

ACCTACAAAAACCACCATTATGCTGCTCAAAACCACCTTTGCGGATGAAAAACCACCTTGGAAAATTGTGTTCTGGTAT GGCAGGTTCGACACCTGCATAACAATTAAATAAAGTTTGGGCGGCΓTGAATACCAAGCTCCACCGCTTGGTCTATTGGC CATCGAACTTGTGTTAAGGCGCTAGTATATATAGTGGGAGGTTGGCGGGTCGAAACCCCCACCCGACACTCCAACATTT TACATGGAATTGATATGATATGATATGGATAGATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGATATTTTATATGGTT TAATACATATGTTTCAATACACATATTCTGGTAAGGTGACΓGGTGGTCGTGGGGTTGACCCCGACCGTCCGACCCΓTTTT CGGTGGCCACATCCTGAAACATTAAATATAACCATATCCCTTCTGAT

RIPPT649 pPT649.seq (SEQ. ID. NO. 305)

ACATATGTAGTGAGCTTTANGGCTTTAGAGAGTGATTTTCGATCCGATCCAATGGTTCCATATAAATATGTAAGCCTAC AGGCAGAAAATAGGAGGCCACCAATAGTCGAATCGGGCCTGTACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGAG ACCAAGGAGTCTCGGATGTNATCTATATTTTACATTAATATATGCAAGATTATTTCTTGTCTATGTCATGATGCTTGAGA AGTAATTTATGTGTGAATTACTTGTGAATTTGTAGGCTTTGTTGTAGGTTGCAGGTANAATTTTGAAGGACACAGAGGA GCAAAGATGATTCAGAGAAATCATAGAGGGATCATTGAATCCNAGTGAAAGGTAGAGTTTCCTTGGAGAGGGGTTGTG

AAGCAAGAATTCGCTAAGGAATCTAGCGCAGAGAAAGCTGAACAATGGAAGTANAAGACAGAGTTTGAATGACGCTGC TGGGAAAGAAATATGATTGGGAGAAATCATATTGATCCATTCGAGGGG

RIPPT658 pPT658.seq (SEQ. ID. NO. 306) ATCATAGCTCTTAGGTTCAACAATTGGACTAGATGGGTGGAATGTATTTGTCATGCATGCATGTTAGTTCTATATGTGTT

CATTATGCATGCATTACAAATGTCATTGTTGTAAGTAGTTTCATCATGTAAAGGTTGATGATATTGATGTCGAGATGATA GATATGTTGATTTAATTATATATGTGTGTGTGTGTGTGTGTGTGTGTGTTTTGCTAATCTAGATTGATCTTGCAAATAGGA TAACGTCCTCTAAACTTCACTAAGGTATGTTGTTΓATGTCTACGTTTGGTΓGATTTAAAAGCGTATATTTTCATATTGGTA GAAGATCAAGGATGACAAGTAAAATTGTGGACTACAGTTCACATGATTCTCCATACCTATCAAATGAGTAGGATGGTGA TGAGTAAGCTTCCAACCCGAATGTCCCCGATAGTCAGGCGTGTCACCCATCCTATTGATGTTGTCACCCCTTATTANGAA

AGGGGAGTGGTAACGANGANGANATTGTGAGGAACCTCGAGGGGTTGACTCATGGTATCCCGAANTATGCTCAGATAG GANACATGCCCTGACGANACCAGAAGCACACAGTGAGANTCTTAGATCGGATCCACCGACTGCTAAATCGAATTGCAC AAGTTGTCTCATGCΓACCATGANCACTATGCTCAGTAATTCCTANGTCCNACACΓTACATATTTTTT

RIPPT675 pPT675.seq (SEQ. ID. NO. 307)

ATCTrGATCTTTTATCTTTCATTGCAATCAAATATAACCATAGCTATCrTTGATATTGTGAAATAATATTTGATGTGGTTG GTGTTTACAATCTCCTCCACACTTATGTTC ATGCATATAAGGATlTCrGATGGCrTGAAATGTATTCGTATATCCTTCCA AAAACCTACAACGTTGCCTCTGAGATATAAGAATGCAAATCAATGAACGCATACCAAGGAAGCTATCAAATCGCCTGA TGGATCCAGATAGGTTTTCATAGTCGGTGATTGTGTCTATCTTCGACCCTAACAGATGTCAAGGCCAAAGGCAGAGAGC TATGCATTGTTTrCATAACrGTTGTTTTGATATATATATATGTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGGCTTTTAAA

TATTGTTTTGGCATATATATGTTGTTCAGCTTTGGCACACCATAGCGGGTAATTTGAATGCAGACATTGAGGGAGCTTTC TGATTTGCTAACCATTGCGAAAAGG

RIPPT683 pPT683.seq (SEQ. ID. NO. 308) GCAAGAGTTTTCCTGTGAAACCAATCCTTCTGCAATCTTTATTTCATCCATCGATATGTGTGTGTGTGTGTGTGTGTGTGT

GCTTCAACAGTGCAAGAGCAAGCATΓATAAACGAGATAACCTTTTAATTAATATTGATAGAGCTGCAGCAGCAAGAGG AGCCAAGGAGACCAAGAGGGCTGCGAGAAGCCAGAGGAATCAGGGAGTCATGAAGAGTCCTGGTGCAGAGTTATCTA TCTATACAATATCACCGAGCCTGCATAGCACTTAGTGAGTTTTCΓTTTTGGTGAAGTCTTTCATTTAACGAAACCCAGGA CCATCAGTCGTCAACACΓCATAACAGGGATTGCCCAAAAGCCCGTAATATTTACAAATTGCATTTTAAAAAGCGTCGGG CACCAGAGAACGTCTATTAAAAGAATTGCAAGAAGACGAGAATTTTCATGTTGCCCAAGGCCATCAAGCCCAGAGGTA

ATTCGAATTGAGAGTCAGGCACACAAGTGGG

RIPPT688 pPT688.seq (SEQ. ID. NO. 309)

ATGAGAGAAATCTCAAACACAACACAACACGCACACACACACACACACACACACACACACACACACACATATGTAAG CATATGAACACTTATTTCAGTTATGCATTCACGAGCTAAGTCTAGGCTGAAGTAGCACAAGTTTAGGAGGGATAACACA CACATTTGTTTTGAAAGCGACAAGTCGAATTGGGTTGAATACCTTCAGAAATCTCAGGAGGGATAACCCAGGAGGACCT ACATATATCATAGTATATATAAAGACTGCGCATCAGAG

RIPPT689 pPT689.seq (SEQ. ID. NO. 310)

CTAAGTCCATCGAGAATGAGAAAGAGGACATATTTCCAAAGTAAAAGAACCTTAAAGAATTTGGGGGCTGAGCTCAAG GACTTGGAAGATGAGTTAAATATCTGCAGGGAAGAACTTAGAAACCTCACTTGGGAATAGTGAGAGTTGATTGAGTGG AATGTTTAACTATGAAAATAGTTGTGAGAAATGACATATAAAGATGAATCCTTGAAAAGGAAAAATGGAATTGACAGG AAGAGGTCTCGTGAGGTTGAATAGGAGAAGGGGATATTCTCCTATTGGTGTTGTGGAGAAGAAAATTTGAGAAAGGAC TATCCCCGTAGGAAGAATAAGAAAGAAACTTTCCCCTACGAGCCACAGGGGGAACATGTCNATGATGCAGGATGCCCA

NAAANGC GAGGGGGGACΓCCTTCAAGCTCCTTGACACATATATATGTGTGTGTGTGTGTGTGTGTGTGTTTGTTGAAA ACATCACC GTGAACΓTTTGGGGAATGTNAGACΓTCNNGGTTGCTGTGAGCCTTNTAAAGAAGTCTTGAACΓAATTTTG GTTTATGAATATGCATNTGT

RIPPT690 pPT690.seq (SEQ. ID. NO. 31 1)

ACAATTCCCCACATCGTTGTAATCTCCTGCTTTGAGACCTTAATAACAATTCTATTATGGACTAAAACTCAATCGCTCCA TTATGTTTGTGTTGACGTAGTAAGTTGCGTCCATATTATAATCCCTACCCAACTATGTCCAACCTTCATAATATTTATTCC CATACAATAGTATATGATTCCTAGATGGACCTGGGGGTCAAGACAATCAACGCCGCTTTAGACAAGGTTATGGGTAATG CCAAGTAAATTATCATTCCACACACACACACACACACACACACACACACACACATATATAATTTGGTGGGCTTATGTCG ACAATACCAACGTTGTCTATCCCTCTAAGAACAAATCATTATATTATATGTAATGTATAAGATGGGATCAACAATCTAC

ATTGACTACCGTCAACATAATGGTGAACCGTGAGTTTCCATCACGGAGG

RIPPT692 pPT692.seq (SEQ. ID. NO. 312)

ATCTTTGGGGCACAATCACTCACGGATTGAGATACACTGTCAGNAGATGTGAGGTTGCATGGTTATTCTGAATGTTGAT TGGGTTGGAAGTGTAGTGGATCGCAAGAGCACΠ ATGGATGTTGTTTTTCTTTGGGCTCTGCTTTGATATCTTGGATGAG

CGAGAAGCAAAAATCGGTTGCTTTGAGCACCATCGANGTTGAATACATAGCTGCTAGTATGGCCTCCTGTGAAGCTGTC TGGTTGAGGAAGCTCTTCAGTGAGTTTTTTGGACATATGTTGGATCGTGATCCTCTGTGACAACCAGAGTGGAATCTGAT TATCAAATAATCTTGTGATTCATGATCACTCCAAGCACACACACACACACACACACACACACACACACACACACACATA TATNTATATATCAACGACCTTCTC^ΠRCAATCCCAATGTGATGGAAGCCRTATTTCTCCRC^R^TGT

RIPPT700 pPT700.seq (SEQ. ID. NO. 313)

ATCATATAACTGC CATCGCAATCGGGAATCGGTGATTCTTATGGTAATAATGTAATTGCCTGCTGCATCTTTGCAATTG CGATTAACTGCATTGCTTAAAACACACACACACACACACACACACACACACATATATATATTGGTTATGGCTCCATATA ACCATTTATAAGGTGATTTAACT GGTCACATGGGTATACTAGGGTCAATTCTTTTATACTTGTGCRTTTAGCGATTCGG CTATGCCATTATCACTTCTTCATCTTGCΠTTTATCI 'I GAAATATTTTCAAGTGATTATTGAGTGAAAAATGAATAC

GATACGTAATAAAAATGCATGTTACAACAGAAGGTATGTCTAATTRCT(^CTAGATTTTGATTTCTGTGAAAATTGTGTA RRTGGGAAGCATAATCGGGTGAGTTTCCCCTCTAGTGCTAAGANGGCGAAACATATATTANAGCTTGTGCACAGTGATG TGTTTGGACCTATGTCGGTTCCATCACTGGGTAAGTCTATGT

RIPPT767 pPT767.seq (SEQ. ID. NO. 314)

ACATATGATTCTACAAATATCATTTAAGAACACACACACACACACACACACACATATACCACCATCATGATGTGACAAA TGATGATCCTTCCCCGATTCATGTTATAATCTCCTTGATCCCTGACCAATCTCATCAACATGCATAGAAAGTCGCCCTCT AGTTGATGCATGAGACTTAATTGATGCTACATCGCATTTACACCTCAAAGAATCAAGTGGTAACTGCTCATGTGATGAT CCAGACCCTCGTTCCATACATACATACATACATACATACATACATATATATATAATCTTTAAAAAAACCCCAAGTTACC TCATGCATTTAAAATCITTAAAATTGTTATTTAAAAACATCAAGAATTCAAAAAATTGATAAGACAAGTAAATGTAACT ATCCAAGGGTTTTCΓTCAAAAAATTTTGATGTAAATTCΓTTATTTCAAATATACTCAAGACTAAAAACACAAATTCTGAA GTGCACATTTTGATAATTAAATACATTTTTTTAAAACATGTCAAATTCACCAAGTTGAAAGAAACACATGGTCATTAAA AACATACACATATCAAAGAT

RIPPT789 pPT789.seq (SEQ. ID. NO. 315)

ATCAATTGAAATATACAAAAGAAGATACTGAAAGGAAGGACATTGACCACAAACAAAAATATAAGAAGAATGTAGATT TATTGAACAAGCTATGGCAATAAATAGCCAATTTGAGAGAAAATTATGAAAGAAAAATTTGATAGCTCTAACAAGGAT CAAAGGATGTGTCrGTTTTATTGCCCAAATGATCACATGTCTCCACAGAAGGAAAGATACACTCTTATTCTCAAGAGGA GGGCTTGTAGAATCATCCCAAGCATCCTCAAGTTTCAAAATAGCAATTTTTAGGTGTTGTATCTATGTAGACACATTTCA

ACATGTCACACACACACACACACACACACACACACACACACATATATATTCTTGTTAACATGCATTATATATCATTATA TTTTCCATTAAACCACATTTTTGAATAAATTTCTAGTCTTTTTTGAGGATTTACACI ATGTCTCCTTC CTGGTTGTGGTCrCATTGGGATCTAGTTACTCTTTTATTTTTC AAGTGAGAAATCGGTGTTATGGAATGT

RIPPT790 pPT790.seq (SEQ. ID. NO. 316)

ACTTTGTATTTATCATTCTTTTTCATATGAATGTAAAAGCATGATTTACCTATTTTATTGTTCAGCAATAGCCTGTTGTAG ATGGGAATGTRTATAAGTTTTCTAGAATGTTATCATTCCATTTTGTGTGTAGTTCTGTTAAATGTTGCATGATGTGAATGC CTTCACTTCATGTTTACTCGTTAATGCGCI ACTAGCRI 'RI CCTCATGAGAAATATGTTTAAATTTGTGAATTGTGTC CATGGGAACAGGGTCACCΓΓCGGGCTCAAACCCTAAATCATTAAAGTTAGTTACTCTGTTGCΓATTTTGGTCTCGCACAC

ACACACACACACACACACACACACACACACACACACACACACACACATATATATATTGCTTCACATTTCGTGTTTAAGC

CTCACCGATTCTTGGCCTGTAATTCATTGCTCCGGCTACGGA

RIPPT791 pPT791.seq (SEQ. ID. NO. 317) ACTATATTTATTGATTTGAAATATTAAGAAGAGCATCTGACAAAGAATAAAGAGGTTAGAAAAGAGAAGTANGCTGAG

AAGTANGAGGGAAAGTATGTGCTAAAGCTAAGGTAGAAGCCATGACGCANAAGCATGGTAAAAGGGACAATATGGAA GGATCCACAACCAATGGATGCAACAAAGAGAATCCATGAGAAGTGGGGCGACGGAAGATGACTAGTGACCAATAAAG CATACATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTGTGTAAACACTCCAAATACATAGACCAGCAACA AGCCCCNAGGCAAAGAATTAGATAAAGGAGCNAGAAGAGCATGAAGGCTAGTTGAGGAATACNAAGGGAGAAGCAGA AGGGAGGCGAAATGAGACCAGAGGGCTAAAGAAAGAGAGTAGTTGTANAAAATGATATTATTCTAAGAGTGGACCNA

CAATGTTGTGCCCNAAGCCTGCTACTAGGGAACAAAGAAACNAACTATGGAAGCGAAAAGG

RIPPT792 pPT792.seq (SEQ. ID. NO. 318) ATCTAATACCACAGAATGGTATCGGAACTCTAAGGTTCAACACTAGGAC GGATGGGCTGAATGTGTTTTTCATGCATG CACGTTAGTTGTATATGTGTTCATΓATGAATGCCTAGTGATTTTCATTATTGTGAGTAGTGCAGTCATGTAGAGGTTGAT

GATGTCGATGTTGAGATGATTGATATATTGATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGT GTGTGTTGTGCΓAATACATATTGATCTTGTAGATGAGATAGCCTCCCTTGAACTTCTCTAAGGTATGTTGAGTATGTTTG TATTTGGTTTATCTAACATGCTGTGTTTGCAAGAAGTAGAGGATCAAAGTTGACAAGTAAAATGGTGGACTATAGTTCA CGTGATTCTCCCTAAAGATAAAATGAACAACATΛGTGATGAGT

RIPPT799 pPT799.seq (SEQ. ID. NO. 319)

ATCTCTCCACAGCTNNANAGACANATTGTGGGGGTAGAGTCCTCTCATTATGANAGACTCCTATGACATCTTTTTCATGT

TCTTTCGTGTTATCACCAACATANAGGAGATRAACTCC TANGATATTTTTTATGGGATATGTCRRRGCATATGTATTGG

ATTATGATCCTAAGCCTTAGAAACCCI ATNTTGACTAATGTATGCTAAGTATATTTGTGTTGTAAAACΓCTTTTGTGAT ATATAATGTGTGTGTGTGTGTGTGTGTGTGTANGTGCATGTGTTNCAGCCTCANTATTGTACACTTGTGAATGTTGTCTT ATGATGTTNGCATTCAGGTTTCTACTGAATGTATCATATGACATGGGTGACAAANTTANTGTGACAAATGTCCANATAT GACATNNTTANCCCATGAATAACCTAATGTCTTAGAATCNAGTAATATAAATGTNTGAAANGGTGGTGAATCCCATTCT AGCANANGT

RIPPT814 pPT814.seq (SEQ. ID. NO. 320)

ATCAAGAGAATAAGTTTGCCCAAATTTGAACAAGCTGTAGCATGAAGATCACTGGTAGTAGCAACAGATGCACACACT AAATCAACCTTCCCTTCAATTGAATCAACACTGAAGCACTGAATCAATCTTGGCTCACCAAGTTCTACTCTTACTCTCCA AGTCTCTCCCTCACAACCrTCTrCAACTCTCTCCAACCCπ'CrCTCAAGTTTGAATTCrTCrTTCACAATGC AGGGAAAAAAGAATGAGGCGCACACATACACACACACACACACACACACACAGAGATATATATATATATATATAGGA

GGAGCAACGAATCAATGCCATAAACGGGGATTAACTCGCATGCCAAGAGGGAGAGTAACATATGCCCTTTTAGGAAAT GTAACTCATGCCATTAAGAAGGT

RIPPT815 pPT815.seq (SEQ. ID. NO. 321) ACTTCATT AGGTTTTAACATTTTTCAATAAATTAGCAAAGGAATGAAAACGAACAAGCCATGGTTAATTTACTAGAAA

TAA<^CTCTCTCTCTCrCTCTCACACACACACACACACATGCATGCAAGTAAACACTAAACrCTCTCrCTCTCTCTCTCT CTCTCTCACACACACACACACACACGCACACATGCATGCAAGTAAACACTAAACTCTCrC CT'C CTCrCTCTCTCTCTC TCTCTCACACACACACACACACACACACACACACACACACACACAAGTAAACACTAAATCTACAACCGTAAAATATAT GTCAATAATAATGCTAAGTAGAGAGG

RIPPT841 pPT841.seq (SEQ. ID. NO. 322)

ATCAAATTCACTGAGATAAAATCGTAATAGATGTTGATTCAGGTGCATGGTGGTGATGTTACAGGTTΓAGCTTCTTCGTT GGGCACTAAGATGCTTAGAGTTGAAGCCCΓAAAGAGGGCTGAGTCCTTCCTCACTTCAAAATAGACATTGCATTGCTCA GATATTCACAAGAGTGATACCACAAGGCTGGGCATTGAAGTCATGCTTTAAACGTGCTTCCCRTGCTTCAGACTTGGCC TTACAGCATAGTGTCATATGCGTCAACTTAGGTTCAGAGGAATCCATAGATCATGCTCAGAACAACGAAACCAACTCTG

TTTTTATCTCAGAAACTCGTGGATGATTAGAAGCGGCCACACACACACACACACACACACACACACACACATGTATATA CCCAAAGTTTGCATTTGC

RIPPT846 pPT846.seq (SEQ. ID. NO. 323) ACCTCATAGATTTrAGACCATATACTAATCCAATGGATGAGAGAGATTATACAATCAACGTATCCGATTTAATTACATG

CCCCCCTCCTCCCTGTGACATCACCGAGGGGGGTGTCACATTCATGGTTCCAATGTGGGATTCCACCTACATATATATAT ATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTATAATTATCACGAGATCCACGCTTATCATTATACAACCATA GGTATAATTACTTATAGATTGGGTCCCTTGCTCCACTTAATTACATCCCCCATGTATATGTGGATGTGACAATCACAAGA CCCCTATTTTAGTTATCCATATATGTATATATGACCTTAATATTTGTGGACCCCCTCrTTACC TTATATGACTTAATGCT AATAGAAACGACATAGTGAGGGCCCACCTTC

RIPPT852 pPT852.seq (SEQ. ID. NO. 324)

ATCTATGTAGATCGCATACCACATAGAAAATACCCTGGTTTATATTAGAATAAGTTAATTAGAAGATTTGCATGATAGA ATAGGTCATTTAGATTATCATTATTTTATTTGCAAGCGGTTATGAACCGCCCACTCTCATATTAAAACCCACACTAGGAA ATTGGTCCCATTATCCCCAGTCGGAGCCACCACTTTAAACGAAAAAGGAGTTGTCTTAAGGTCCAGATCGGGTAGTTAT

CCCCCATGTTGTTGCAACGATTTTTGAGGCCCCAACAGAACCCTTCCAATCCTTTAACATATTTGGCI CCTCπ'CTTAAA GGTCAGATGAGTTTGCACTCTCTAGTCACTTCTATATAAAACTTCNTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTTTATTTCTCTCGTCNAGGNAATGAAAGCATAGTGCTTCTACCCNGAAATTAAGT RIPPT860 pPT860.seq (SEQ. ID. NO. 325)

GNAGAATATTGAGCAGACATCATCAACACTAAGTAATGTCTAACAAGGACCCTCAATAATTCTCATATGRRTTTTACTT ATAACCATGAGTCTTGTAGATAGAGAGTGCATCTGTGTGTGTGTGTGTGTGTGTGTGTGAGTGTATGAGTGTTTGAAAG AGTGGTTGTAATCTGTGCRRRGAGAGACTAGAGACTAGAAAGCTCRACTCTTTGAGGCATAACCTGGTATGTAACAATT TTCTΓGAATTAATA<^GAAGCCTACTGGGCTGCCTGTCΓGAGGACAGTTTGTTTCGAGATCATCTAAAAGCGAGATTTTA

AAGCGAGTTGGAGAAGCGAAAGAGTTGCGTGTCCTCGTATTTCTCGAGCTGGTGTGAGGAATTTCAGCATACTCTTCAG AGGTTCAAGTAAGTTATTGACTCCATCATGTTGT

RIPPT905 pPT905.seq (SEQ. ID. NO. 326) ATCTAAACΓTTTGTGCAAATGTAACACGGATCTCTGGAAACCATGAACTACACACACACACACACACACACACACACA

CACATGTTGGAAATAATACTGATAGGGGGGGTGAATCAGTATTATCAAGTCAGATATTCCTTTTCCAAAATCAATTGAT ATACGCTACTTCAGTTAATTAAACCCGCGTATATACGCAACTATTCTGAGGGAAACCAGCGTATACAAAGCAAACTATT GATATAATTTATC TTTTGATGTTGATTGTTAATCTGTGTTTCCATAATCAAATAACCAGTAACTAGAAATAGTAAAACA AAGCAGAAAGACAATTGCACACAAGAACACAAGATATAAGGTGGAAACCCAATGTGGGTGAAAACCACCTGCTGCAA TTCGTCTCTATCΓTCCTTTAAATTAGAAACTTCTTTTACAATGC

RIPPT921 pPT921.seq (SEQ. ID. NO. 327)

GCTCAAATAGAGAATATGGAAATTTTCCGCAAACΓATGTGACAACCCTAAGTTATAGAAAGGTATGTTGACACCTTTTG AGATCCTATACCTGATGCATGAAAAGTGGGAACCTATGATGACAATATTGTAAGCTAGAAAAAGGATAGTTTGAGGAA ACATGAATACATCΓAACAATGAAAGTGTATGTTAACATCΓTAAGGATTTTGTTTTCCTCATAATCACCTAAAGAATTTCT

AATGAGTATNGGGAGATGTCNTAAAAAATGTCTGACATGTCACAACACACACACNCACACACACACACACNCACATAT ATATTCTACATATAGAAAGAAAGAAAATCGATCAAATAAAATTCCGATAACCGNGAATGTCNTCGTATGATGTGTTGAC ATGGAAAGTGGCATATGCTATGCCCTAAAT

RIPPT932 pPT932.seq (SEQ. ID. NO. 328)

ATCTCGTCTGCAAGACCGACTGGATTAGCACAACACACACACACACACACACAAACATAATCAAATCAACATATAGTC ATCATTCCAACCATTGTCAAACTATCAATGTATGAGCATACCCACCACATATCATGACCTCATGCATGGTAACTACTAC CATATCACTGTACGCATAACAACATGTGTATGAAACGCACACATACCNCTGTGTTATGTGTGTGTGTGTATGTATAGGT GTATATGTGTGTGTGTGTGCGTGTGTNTGTGTGTGTACAACGACACCATCAAATTTCACATGGTGACACGACTAAAACC ATACCCAAGACCATGCATACACAACCAATACATAGACCATAAACATAAGAACACATATGCAACCAACATGAATGCATG

ACCAACACAACACCCATTCAATCCAAGGGGCGAAACATGCTATGATACCACTCTATTACACCCTCTTCCCACATCTACC CAAGTAACCGACTAACCAGGGTAAAAATGTGATGAGAGGCTACTATGATCTAAGTAACCTCCCCATGT

RIPPT941 pPT941.seq (SEQ. ID. NO. 329) CCATCTTTGATAGGATTGTTGTGGAAATTGTGTGGGGTCCAAAGTTGCCCATTCGAGTCGCCAGACATCTGCACAAGTT

GTGTGAGTGAGCAATGCCGTAACCCACCAAGCCACCGTTCGAAAAGGAGTCGCTAATTTTCATCATGCATCACGTCGTG TGGGGAAGAAAGAGAACAAATAAAATGTGTATTTTATTTTCATTCATTTGGGATGTAGAAATTCTAGACTACAAACATC GTGATCGCATGATCTCAGGAGCCAAGAAAATTTTATTTTGAACCCAAAGTTGAAGCAAATTCAACCGTGGACCTGGCGT TGTATCGAATCΓCTGTΓΓGTGGTAGCTTGGATGTCCTTAGTGAACTAAGGCTGCGTAGCAAATCACTGGANGTTGTGTGC GTTAAAAGGTAGTGGAGAAGTGCGTATGATATATATATATGTGTGTGTGTGTGTGTGTGTGTATGTTACAGTGTGAAAA

CCAATACATAGAGAAGTCAATTGTTGATCCCACATCAGATCACATCATTAGT

RIPPT947 pPT947.seq (SEQ. ID. NO. 330) CCCACAAACrCTTGGTGTTCCATCTCCTAGGGTTAGGGTTTTGGTTGTTTAGGTTTCCCrTTGTAGTTCAATAATAAAAA TTCCCRRGAGCCTCATTGTAGCACAATTCAAAGGCATGTGGCTGCCACATCACTTCTCAGAGATAAGCTACCATTGCCC GAGCTAGTTΓATTTCCATCATGTGTGTAAGCATCΓΓΓTAGTTCTΓTGATATTAGTGAAATGAAGTTTTATΓCCTACT CTTTGTGTTTATGTTRATGTATGTATTTTATTTTGTTTCTACATAAGTCCAGATCAATAAACACGCACACACACACACNC ACACACACATTTGGTCCAATGGTTGGGCCTTGGGTCCAATATAACATTGGCATGTAACCATGAATTAATTTCCACGCTA TGAACCITGATCACRTGGGGCITACACATTGTCCATATGACTTTATCΠTGCAGGTAATTAACCAGCCACATGGTGACAT

TAGCATTATGTCATCATGCCACATGATGGCTAGTGAAGANGTGCCACATGTCACCTGGAAAACAAGTTGATTGGTCACT CGAGCTACCATTTGT

RIPPT958 pPT958.seq (SEQ. ID. NO. 331) ACATTGTANGATTGCAAAATGTGACTTTAGAGGATAGACATGAGTTTTTTCTAACAAGTCCAATCACCAACTATTAGTG

ATTTTATACTTATCTTGGAGTCTCGAACACTGTGGGAACACACACACACACACACACACACACACACATATGTTGGCAT TATGGTGTGCAAGGATCCAACTANTTGATGTTGCCATTGGGATGATTGTGTTGGCATTGATGTGAACGATATGTTGGCAT TGGGAAATGATGATTGCCACACTAGTTGATTGGGAACTAGGTCTTCANGCTTATGTTCGTGGAGATGACTTCTTGCGTGT TAGGTAAACTTGACTCAGGATANAGTCNAAGTTGACCANGTTAAACAGTGATGGTTTTGGGGCACATGGTATTGTGCGC CCATAGTATTCTGCCCATGGTATTATATGTAATGGGAGCTTATAAAAGGATGGAAGGACTTCNTTGTCATGTATGCCTG

CANGTGAGCGGTGAACGCTCACTTGGTCAGATTGGCTAGGGTTTTTGGCCANTGCTAATGAA

RIPPT960 pPT960.seq (SEQ. ID. NO. 332)

ATCAACCCACTATCACAATCTΓCCTTΓΓAATTATTCCTATCAAAGGTGTCCCACCACTTGCACCTAAGAAATAACACATT TGAATGAAGATAAGTCCACTRGAAATTATATCTTGTATCRRRCCAACGTAACCTAATGTCCCCTGCATTATGTTTTGATC

TTGGTGTCTAACACGACRAGATTGGTCAAACT'CGACTTCCACTCCATAGTCTTGTGTCATGCATCCATCTTCAGCATCCT TCACGCAACAATTATGTCCATATGCCCACATACATGGGAATAAATGCACCTATAAAAAAGTAAAAAAATCATAGTGTC ATGTGTCTAATAACCCAAACACACACACACACACACACACACACACACACACACAGATATATATGTTACATTTCAAAG GTGTCGTATGAAGTTAAAAATGT

RIPPT961 pPT961.seq (SEQ. ID. NO. 333)

ATCAATCCACGAGGTCGATAAATTAAAATAACAATCCAAAATCATAAATAAGTAGCCCCCAAGAGTCCTCTTAATCTAT ATGAGCTCAAAGTCCATGGAACTATCAACCATTAGACAAGTGCGCATGCACACACACACACACACACACACACACACA CACGCACACACACACACACACACACGCACACACACACACACACACACACACACACACACACACACACATATATATCAA GGAGCTTGAGGG AGTATGCCCTCCTCCTCCTC A ACATCTTrCTTATTCACTCTCTT TTCTTCGGTTGGGG AA ATTCCTTT

TTCAATGCTCTTACCCACAACACCAACAAGATATTATITTCTCCCC^ AATCAACCAAGT

RIPPT968 pPT968.seq (SEQ. ID. NO.334) ATCTTATGCTCACTTACTTTCTTACCACACTATGAAACCCAACCTAAAATCAATCATACTAACAGAATGCTCTACTACCA

AGCTAAATGAATCTTCATGAGGAATACCCTTAATAGGTCACTCTAAAGTATGGGTTGTTACCCCAACACCCCTTCCTAA GGCACACTTCGATTAAAGAATCATAAGCACCTTGAATCTTCTTAACACTAATTACAAAACTAGGATCAAACCCAACCTA ATACATATCATAAACTACTATAGATATCTACGACAAAACCACGTAGTGTTTCATACAATCCAACCTAAGCATGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGATNACTACRTCCNAAGCTACATCCCATTCACTTCTCANGGAAG ATAAAAATATCNAAACCAACΓTAATACCTACCATTTACTAATATAGATATATGCCACAAAGCCACATGCTNTTTTATAA

AAACCACTCTAAAAAATTCAAAACCAACTTAGCAAGCTTNTCTATCGAGTATTANGGGGCGGGATTGANAGGCCAGTC CGATCTATGCTCTGCAGGGAATATCTTGAAACGTATTTCCTACCGACCACCCGAGCGTTCNGATCCCGAANTCAATGAA AACTNAATCA RIPPT984 pPT984.seq (SEQ. ID. NO. 335)

ACCAC TATAACATACCTTAACCACCTGCTAGTTTATTCCAACCTCΓTGTTGGGTCCCAATTTCGTGTTAGAAACCCCTG GGAAACCTGTGACCTGAAAATTCCCCΠTCRATTAAAAATTTGTCTGATGAAATGCACATCTAACTGCATGCTAAAATGT AATACATGTGCATATGTATGTTCTAACATGATTATCTTATTTCAATTAACTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTTGTNGCANTGGAATAAAGACAAATACΓGCTCTTTTAATTTATGGAACTGGTTGCAAGCCTTTGACGTNTAACC

TANCCAAAGACCATGTTGTTCATTTCCTTCAAGANCAAGCCACCCCCGAGTTGGGGCTTTGAGTCCATGGGGAATCAAC AATAACTCTCCTAAAACCTANC

RIPPT990 pPT990.seq (SEQ. ID. NO. 336) ATCACTGGAAAGCTCTTAATGAGCTAAACACGATGGTAATTTTTTTTTAAAGTI TGATGAGTTTGGAAAAAAGATGATT

TTTGCAACCCCATCTTCATAATAGATTGCGAGGGTAAGTTTAAGTTATAAGTAACACCCTCCACAACCCAAGAATAACC CAAGCGGGCGGGAGCGACTATTACAAAATGGTATTAGAGAATGGTTCAACACTAGACCTAAAGAGGTTCACGCGCACA CGCACACACACACACACACACACACACACACACACACACACACACACACACACTTAGGATAAACATGTTNGCTGCTAT TTCCAATATTGTATACATATGAGGGTTGATTTTAAGTTACAAATGATATAATTGTTGACTTCNCNAAATATTGATAAGTT ATTTTATCCTTTAATCTGCATACTAACCCAAGATTTGATGATTTAGCTGGTTTCACATCNACATGGTTATTGAAAACATG

CCNAGATGATGGGTNNATATGTTTATNTACATTTTTATTTGATGAAGTGTTATGTGTGTGTGATAACACCTCAGAAGACA CCANTGAAACGTCGAAAAAATTCCNAAATGAA

RIPPT1013 pPT1013.seq (SEQ. ID. NO. 337) ACATCCCTGTGGGGATGAGTTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAAAGCNAAANGCAAANATGTGTG

TAAGGACTTCTANAGGTANACATANAACATTTGATGCTTAAGAGGCATAACNAATCACATGCTATANCAAACCACATG CTAACAAAGATTAGTAACATATTAAGGAATAAACAGAACTCAATACATGTCATGGTCAAGCCNTGANAGGTTGGATCA AACTNGAAANATAGTGGGTTTTGAGAC

RIPPT1023 pPT1023.seq (SEQ. ID. NO. 338)

ATCACGCTTGATCACrrAGCCTACTANGTTTGAGTTACTCGTCTCTrCCAATCCCTAATATTCTACCCrTATAGCrrTAAGT AATATGTGAATTACITGGGCCTTTCrACCCTCAGAGCATGGTCGCAATGGTTTTATTGTCCAAGGCTTrATTACGACTTA GTGTCTGAGCCGTTATGTTATTGAGACTTAGCCTCCACTTGGTGCATATGCACATAAATATGCATAAGGCTTGAACCCG ATGGATTTTCAAATGAAGTCAGCCTAGTCTTGGTTGTAGTGGTTTTGTTCCCAATTTGACCTAAGAATAGTTTTATGAGG CCCCTTGGCCTCCAACCTACACACACACACACACACACACACACACACACACACATATAATTGTTAATCCTCCTGAGCT

TACAGTTTGTAAACCAAGG

RIPPT1027 pPT1027.seq (SEQ. ID. NO. 339)

CCTTAGATTCTAATCGTA(^GTGTATAATACAAAAGCATGTGTTACAGTGTTGATTGTGTGCCAGTATGTATTTCATATA TATGTGTGTGTGTGTGTTΓGTGTGTGTGTGTGTGTGTGTGTGTTAATGGCTTGAANTCCTGTGTTTATAAGTGTATTGATT

GACCATCGATCACTGTAGCATCGATGGTGATCCAAAGATATTTGAGTTAACTGTTGTATGGAGTGATGGCATGCTTAAA CAGAGTGTTTCCAAATTGTGGCAGATCTGTGTTTTTTAATGCAGAGATCCATCANAATGATCAANATTGATTTAAGGAA GAAATGGACAAGAACACATAGAAACCGTCAGATCTGGAAGATCAATGTTCCANATCAAATCGCATGGAGCANAACCTT TTTATCACATCGGCAAAAATCCCNTGGGTGAT

RIPPT1035 pPT1035.seq (SEQ. ID. NO. 340)

GCTCAACAGTITrGTAAGTGTCGAGGCATATCTTTGC ATGGTCTTAGTCATTGGGATTTCAAAAACAAAACTACCCCAT TTTCATTTTTGTCrGCAl'l 'ICCGTAATCTTGGTGTGCTAAACCAAGGGCATGCTGGTTTTGGAAACTTTATCCATATTT GGTAACACAAACTCTCAAGAAAATACTGATTAAGGATGTCTAATACATAATGGAAGGTAACCCAAGACTCAGTGAACT AGC I GATTTTGAAAGCATAATGAGCCC TC CGTTACTCATAATTTATAAATTGGCAGGTGTGTGTGTGTGTGTGTGT GTGCGTGTACACATTACCATGCAAAGGAACGCCGATGACTTTAAATGGAGGCTAAGGTGTTTAACANAGGGGAGTGTT GTGTTAAATGGGGGGGAGGGACACATATTC TGATATGGAAGTGTGTGACACCTCTTCNAGATTTTGTGAGGAACAATG AATCTGAACAATGTCNTGANGATAAACTCGACNATAAAAAATGCCACAATGTAATAAAATGCATTGGT

RIPPT1036 pPTI036.seq (SEQ. ID. NO. 341)

CCITGTTATATCTAGCTTCATCGAANGTGGTCGTTACCΓCCTTACCCTCGAATACCTAGACTCAAGGTGTTAATTAAAAG GTAATRRAGTTAGAAACATAACCAAGCTAGCAAGAGGAGTAGCATCACTAGATATCCGAGACTTTGAACCTTGGATTAA CGAGAAACACACATAATTCTATATTTTTATGATACAAAGGTTTCTΓTTCTTGCAGGTCAATAGAGTGCATGGTTGTGCGA GATCACAATATGTTTGCAACTATGCTAGATTAGTAGGAAGTTTTGAGATCATCGACAACCGTGTGTGTGTGTGTGTGTGT

GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCATTGTCCANTCAATTGGGTTTATTTTAGGTGGTTAGTTGTA GTGATTGGGATCCTCCCCACCCAATRTCAATTCCCTCAAGTGACATGATTAGCITTGATATAATGGTTTAGACCCTTGTT GTTGAGAATGAATANTCCCTAAGATTAACGGGAT

RIPPT1037 pPT1037.seq (SEQ. ID. NO. 342)

ACAAATGCTCAATATAGACCACΓTGCATCATATTTGTTTGTTGATCTAGATGAATGATGGTGCGCGCGCGCACACACAC ACACACACACACACACATGTATGAGATAGTTAACAANGTTGATGACAGGTTGATTATTCCTTTTGTTGAATTATGGCTA TTTGATTGAGAGAATTTTGTTGAAGGATTTTAGTA RRAAGATGTGTTGTTGAGTTAGAGTTGTATGAATGCTTAAGATTG AGAGTATGGATGAGGACAAAGTATGCTACTGCCAAATCATATGATGTTAACATTGATGTCTAAGTATATGTTGATGATG GAGAGTGATTTTGTGGTTTAATGCTGCTAGATTTTGTATTGCAGCAAGTTTTTGGAGTCTCGGTATAAAAGATAAGGGA

AAAGAGAA

RIPPTl 040 pPT1040.seq (SEQ. ID. NO. 343)

ACATACATGTCATGTAGGCTTGAGGTGGATATGTCCAAAAGTCATGTCTCCTCAAGGAATTCATTGGAGCCACATGTGA ATATATATGTGTGTGTGTGTGTGTGTGTGTGAGGTTATTAGACCATCCAAGAGAAACATTTCATAGAGAGATCATCTCC

AATGGTAGCCACAACACAAGAAGGAAGGGGTCGAGGAGAACAAGGGAAAATTTGAGGTATGTTTTATGGGTTTGATAT GGCCAAAGTGTAGAAGAGGATAGCCAAGTGAATTATGGGCAGTAAGGTGCATATTTTAGGGTGTAATTCTCCTTGTTTT GGAGGGCCACAAGCTCΓGTGGGACATGGTAGATACCGTGGGATTTTTTTGGGATTACTGGAGGGGTCATAAGG

RIPPT1066 pPT1066.seq (SEQ. ID. NO. 344)

ATCCTTAGTAACAGTTGTTCTCTGTTTACACAGAGAACATTGTGAAAACATGGGAAATTTCGTAGAAAACATAGCTCCT AGACTTGAAATTCTCATAACACCACRRTATAGTGCCATCGGGTCCGTCTATTGGTTGTCGTCGCTTATTGCAATGGCTCT TTCTCCTTCACATTTCGTTTAAGAAATGAAATTGTGAAAATACATTAAAAAGGGGGTGTTTGATGGATCTCGAGATCCA TATAATATGGCTTGTAATATTATCGGGTCCAATCCATCGGATCACCCATATATATATATGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGNGCATGCATGTCTATCCCTGCTTTGNTGTGTTCGCTGATTTCGATCCGNCAATATCGGNGAT

CATGGTATCGNATCAGCATC

RIPPT1072 pPT1072.seq (SEQ. ID. NO. 345)

ATCACCTAGTCTGCCCCTAGTGTGATGTTTCI ATCTCCAAAGAGTCTTCCTTGTAAACGAGACTCACAAAGTGAATTTT TTCACTCΓTTATTTTACΓAATTTGAAGTTTTCATGACCTTGGAGTGGATTCACACACACACACACACACACACACACACA

TATTTTTTTACAAAATGTTAATATTTTATGTATTTTTTGGTTGACTAGTCCAGTTTTGATGACATTGTTGAGGAAGTTGTG ACTGTTATTGCGAAATATAACATCGCAGTGCAAAGTTCACTAAGCACTCTAGAATAGGAGCAACAATGGGATCAATCA CCAGTGTAAGCGTAGCAACCATTCCCCCGCTATTCTGTGGTTGGATATTATCCCCACAAGGGAGTCTTCCCTAGTAGAA TTΓAGGGACTATATAATGTTAAGCCCTTAATAAGC<^CATGTTACCCATAAACCTCTTATTAAGCCCTAGATATTGAGTG ATTACTTATCTATTGGTATATTGGTATGTAGGCTATAACCCCTCATGGT

RIPPT1076 pPT1076.seq (SEQ. ID NO. 346)

GCTAGCATGTAAATGTGTAAACCCAGGCTAGGCTGAGGCACATRTAAGCATAGGAGGGATAACACRCGTTTGTATCTTT

ATACATAATATGTGCGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCAGGCTACAGTT

NGAGGGGCITTGTGAAATCATATGTATGACAAGCCAAATTTTGGAGTAAGACCCCAAATTCATACCAATAACCACTACA

TCTAATACTTGGACTAANTCCNTCGAAAAACCACTGGGTTCAAGCCTTATGCATATCTATGTGCATATGCATCAATCGG

GGGTNGAGTCTC GTTGGCATTATGGCATATGTTNGATGCTATGGCTCTGGTGTTGTCCΓNGATGGCAACTCAACTTGGT

GACAAATCCCAGATCAGGCACCCAGAAAGAAGAAAAAGTCATATACGCNGGTTCATCTANACAAAGACGTGT

RIPPT1077 pPT1077.seq (SEQ. ID. NO. 347)

ATCrCTCTrCGTTGGGCATATTTCCTACCATAAATCACAGCCCAAGTCTATGACCTACTGTAACATTCTAGCATGCCCCA CATATTTTTATCAAACACAGGTCTCGCAATACATCTAATTACAGATTAAGGAATTGGATTACATTTTGCCATGAAGTGG AAAATTTTAC I GTTCACCGCACAATAGTCATATrCAAATTCπ ACCTTCCTTTTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTATATATATATATAAAGAGGAGAGACATCCACCACAAAGAAACTATTGGATTTCCTCTTAGAACTTAAAAAAAAA

AACATTAACAATTTCAATTAAGGACAAAGAGAAATAATTTTTCTTTTTTGCCACACCTATTGAAAATAGAAACAAGAAA ATGCTAAAAATAGAAGTGCTAAAAATAACACTTCTATAAATCGCAATTTGGGT

RIPPTl 125 pPTl 126.seq (SEQ. ID. NO. 348) CCTTGTGAGGCTAAAAAGGAGAAGAGGAATTTTTTTTTATGGTGTTGTGGAGAAGAGAATTTGAGAAAGGATTGTCCCT

GAAGGAAGAAGATGAAATAAGTTAGCCACTATGAGCCACACAAACATGCATCAACGATGCAGGATGTTGAGAANGAG GACGGGGCTTCCCCTCAAGCTCCTTCACACACACACACACACACACACACACACACACACACACACACACACACACAC ACACACACACACACACACATATACATATTTGTGTGTGTGTGTGTCGAAAATATCCCTCGTGAACTTTTGGGAAATGTAA GACTTCNAGGTTATTGTGAGCCTΓGTGAANAANTCTTGATGTGATTTTGGTATATGATTATGCGTATGT

RIPPTl 137 pPT1137.seq (SEQ. ID. NO. 349)

ACAATCACTCCTGTATTAATTAGAAGAGTCAAAATTCΓCTTAAGCAATGTATCTATCTATCTATCTATCTATCTATCTAT ATATATATATCCAAAAATTCCTCAGCAATTGATCCCTACAAATGAGGCATGAGGGTGAAGCTTATCACAATGCACAAAG AGAAGGCAAGATTTACCRTGGGAAAACCCACTAAC<K GAAAAAACTAACAACCTTTTCAATAGAGAAATGCRRTTGTTC AACAAGGGACACACCTAGACCCTTCTAGTCATTAAATAGTTCACACTTGGTCAAACCATCTAAGTCAAGCCTCCCAATC

TAATCCAACACTTGGCATITACAGATCTACCCAAAATRTCAACCCTCTTGCAACTGCTAGATTCCCAAAATTTCGGCCCC ATGCAACTGCCTAGAATTTCAGCACCTACAATGGAAACTGCTCATAAATATGTGAACTATCGATAGATAGATAGATAGA GATAGATAGATAGATAGATAGATAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGTATCCCACGTG CGAGCTTGAGAGAGAGAGAGAAATAGAGATGCCANT

RIPPT9058 lobseq3-l l-97clone249 (SEQ. ID. NO. 350)

CCCGCTCCTATTCAAGATCAAAAGAAGGATATGTTAAAGGTCAAGTGCTTCATTTGTCAAAACTTTGGACACTATGCAT CAACAACCAATGTTGTTGTTGATGATGATGATGATGATGAGCATCCACCTCAAAAGAAGTCAAAGGAATTCTTCCTTTA GGAGCTACTACCAATAATGC TGAGGGTAATTATGCCTCTAGGCGCCrGATCGGTTTCCAAGATr(^CrTATAGGTTTTT AGGGARGGAGTTGACAATCCTCAACAATTTCTTCCTTAATCAACAGATAAGTATGTCATATTTTTCTCTTGATTATT

RIPPT9I04 lobseq3-7-97ATCclone71 (SEQ ID NO. 351)

CCTTTCTGAATGACAAAAGGGCTTCTACGNCCTTCCTATCGTCAGCGTCCATGGATGATGATGATGATGNAAATTCAGG

NTGCTCCACAGTGCCTTCATCGTCCTTCAAAAGAGATTGAAGAGAAGCACTTCCACCAGTCGTGCGCTCATACAGATGA TCATGGTGTAAAGCATGACCCCTGTGAACATGNGAGTAATGATATGGCTTCATGGAATAACTAATAGTTGCCAGGAGAA GAAGCCACACTAGTATGATTATCGACACAGCACTTI CTCCTTTCTCTCTTTATCTCAATTCGAAAGAANTCNAAGANTG GCCTGTGATTNCCTTGTTTGCGGGCNCC

RIPPT9138 lobseq5-2-97ATC187 (SEQ. ID. NO. 352)

ATTGAAACCAATTTTTCCCCTTTTTAATGTAAAAAATATCAAAATAAATAACAAATTATATAATAATTCATTATAAAACA ATATAAATRRRAACATATATATGAACRTTGAAATAAACCTTAATGGTGATGATGATGATGATGGTGGTGGTTAACGTCC ACTGAGACCAAATTGGTCTATCGGACCTATAATRRCTTAG L 'L U ACTTATCTGGCTCCTTGTCTTTCTTGGCGAGATTAG TGTTGTAGCTTTCTTTTTTTTCTCTACAGTCTTCCAACTTCCTTTATCTTGCATCTCCCTCCAC^ AATTGGTGTCTΉACΓTCCCAANCCATNGTGTTTCATCCTTTGAAGG

RIPPT9238 lobseq8-19-97TTC2Dl (SEQ. ID. NO. 353)

CCCTGAGACATCCAATCCATGTGTTTTTCTACCATTTATTTATCATTTTAT

CTTCRRCTTCTTCRTCTCCGCCCAACRCATGTAAAGTATCATCTAAGAGCACTACATTATCACCATCACCATCATTTAAG GTTGAAGGTTTTTCTAGTTΓTTCTATATTTΓATTT^

GACACCATTATCCTANTGGGGGTGGTRRGGTTCTNATTCACAAAATACGGAAAAGTTTCTATTCTGGATCCTTTAGA

RIPPT9315 lobseq 12-4-97 AC 1-G8 (SEQ. ID. NO. 354)

CCΓCTTCCTAGTAGGGGGATTACTTGGAGGAGGATCCGAGGGGTCCATAGGGTCCATGCGATCATCATCAACCTCCTTT TTCGGCΓΓAGGCATAGAGGGACCCTCTAGTAGGTCCATAGCATTATCTTTTTTCTAGTAGGTCTCTAGCTAATCCAAAGG

AAACATATCCATCGAAAGTTACATCCTTGATAAACTCGATCTTCCTTTTGCTATATGTGTGTGTGTGTGTGTGTGTGTGT GATTTAGAGTTTTCACAATATCCAACAAACATATCTTTCTTTATGGTGGCΠ'CCAACTTGTTCCTCTTGTCCRTC TGGAAATATACGGGACAACCAAATATCAT

The PIC and H values are presented in Table 4 for the 18 polymoφhic loci amplified from the first 89 primer pairs that were synthesized.

Table 4 Allelic diversity data for SSR loci in 20 Pinus taeda trees

REPPT SSR repeat in cloned allele # alleles allele size PIC H locus range, bp

1 (A)₆...(ATG)₇ 4 199-260 0.297 0.331

6 (ATG)₆...(C)₆ 17 273-315 0.892 0.949

11 (CAT)₆...(A)₆ 3 156-169 0.427 0.542

22 (ACC)₆(ATC)₄ (SEQ ID NO:355) 3 243-249 0.368 0.426

24 (TTG)₄(TC)₂ (TTA)_I2 (SEQ ID NO:356) 3 146-152 0.282 0.320

31 (C)₅...(ATT)₁₉ 15 225-267 0.859 0.916

32 (TAT)₇...(A)₅ 6 173-189 0.586 0.653

33 (TAT)₈ (SEQ ID NO:357) 3 169-178 0.410 0.484

64 (A)₆C(A)₅(AAAC)₅ (A)₅ (SEQ ID NO:358) 11 233-261 0.751 0.818

65 (AAAC)₅(A)₇ (SEQ ID NO:359) 9 130-139 0.816 0.879

66 (AAAT)₈ (SEQ ID NO:360) 3 98-110 0.577 0.685

67 (AAAT)₄ (SEQ ID NO:361) 3 217-225 0.340 0.392

69 (AAAT)₄ (SEQ ID NO:362) 2 140-148 0.319 0.420

71 (AAAT),, (SEQ ID NO:363) 14 219-260 0.853 0.917

77 (ATT)_u (SEQ ID NO:364) 5 154-180, 0.581 0.691 null

79 (ATT),₂ (SEQ ID NO:365) 8 130-161 0.723 0.765

80 (ATT)₇...(AAT)₆ 4 247-263, 0.438 0.525 null

89 (TTC),₀...(T)₅ 3 220-223 0.327 0.386 mean 6.4 0.547 0.617 null - PCR amplification could not be observed in one or more samples from trees that were assumed to be homozygous for a "null" allele.

Referring to Table 4, most loci had stepwise allele size differences, i.e., the minimum size differences were multiples of the unit length of the major repeat motif.

Loci RIPPTl, RIPPT6, RIPPT32, RIPPT64, RIPPT65, RIPPT71, and RIPPT80, however, had minimum size differences among some alleles of a single base pair. For all these loci, except RIPPT71, the lbp allele size differences may have originated in short, mutable mononucleotide stretches found near or adjacent to the target SSR and included in the PCR amplified region. The RIPPT71 locus had no repeats other than (AAAT)_n. SSR markers in other pines

The results of testing P. taeda SSR primer pairs for amplification of marker loci in other pine species are presented in Tables 5 and 6. Table 5 includes individual species results for RIPPTl through RIPPT90, while Table 6 is a summary of success of amplification of single loci patterns in other species for all RIPPT primer pairs. As described above, polymoφhism among species was scored from high resolution agarose gels, so the number of polymoφhic SSR loci amplified among species may have been underestimated. Details of the agarose gel marker phenotypes are given only for the 49 primer pairs that amplified single loci in P. taeda among RIPPTl through RIPPT90 (Table 5).

Table 5

SSR marker phenotypes among various pine species, using primer pairs that amplified single loci in P. taeda.

Locus marker P. P. P. P. P. P.

(SSR motif) size caribaea ponderosa radiata resinosa strobus sylvestris PAS (bp)

RIPPTl* (ATC) 260 -< (+r (+) - (+) (+) y

RIPPT2 (ATC) 185 (+) (+) (+) (+) (+) y

RIPPT4 (ATC) 145 - - - n

RIPPT6* (ATC) 290 3 ' K+) K+) 2 y

RIPPT7 (ATC) 105 1 4 2 1 2 y

RIPPT9 (ATC) 120 1 K+) (+) y

RIPPTl 1* (ATC) 170 1 (+) (+) K+) (+) K+) y

RIPPT13 (ATC) 105 2 2 1 y

RIPPT16 (ATC) 220 1 1 n

RIPPTl 9 (ATC) 105 1 I 2 n

RIPPT20 (ATC) 110 I - n

RIPPT21 (ATC) 185 K+) n

RIPPT22* (ACC) 245 - y

RIPPT24* (AAT) 150 1 - y

RIPPT26 (AAT) 190 1 1 n

RIPPT27 (AAT) 130 1 1 y

RIPPT29 (AAT) 210 (+) K+) K+) y

RIPPT30 (AAT) 215 1 K+) K+) K+) K+) y

RIPPT31 * (AAT) 245 1 (+) y

RIPPT32* (AAT) 180 1 1 y

RIPPT33* (AAT) 170 - y

RIPPT35 (AAC) 270 K+) K+) 2 n

RIPPT37 (AAC) 1 10 1 1 y

RIPPT38 (AAC) 270 1 1 n

RIPPT40 (AAC) 190 1 I n

RIPPT42 (AAC) 230 1 1 y

RIPPT43 (AAC) 145 i 1 y

RIPPT44 (AAC) 210 1 K+) n

RIPPT51 (AAG) 260 1 1 n

RIPPT52 (AAG) - n R1PPT54 (AAG) 175 1 1 1 1 1 1 n

RIPPT56 (AAG) 390 - - - - 2 2 y

RIPPT58 (AAG) 235 1 1 1 1 - - y

RIPPT64* 345 - 1 2 1 (+) I y

(AAAC)

RIPPT65* 135 - 1 - 1 - - y

(AAAC)

RIPPT66* 105 I 1 1 I 1 - y

(AAAT)

RIPPT67* 220 - 1 1 1 2 1 y

(AAAT)

RIPPT69* 145 - 1 1 1 2 1 y

(AAAT)

RIPPT71* 240 1 3 1 1 2 1 y

(AAAT)

RIPPT74 (AAT) 130 1 1 2 (+) - - n

RIPPT75 (AAT) 205 1 1 I K+) - 1 y

RIPPT77 (AAT) 175 1 3 1 3(+) 3(4) 2 y

RIPPT78 (AAT) 220 1 1 1 2 - 1 y

RIPPT79* (AAT) 155 2 1 1 I - 1 y

RIPPT80* (AAT) 250 2(+) 3 (+) 1 1 (+) y

RIPPT86 (ACC) 260 1 2 - (+) - - y

RIPPT88 (AAG) 235 1 K+) - (+) (+) (+) y

RIPPT89* (AAG) 225 1 K+) - 1 (+) K+) y

RIPPT90 (AAC) 150 2 1 1 1 (+) H+) y bp = approximate allele size for P. taeda tree 7-56. PAS = Polymorphic Among Species

* = an asterix indicates that the locus was polymorphic in P. taeda (-) = a dash indicates that no PCR amplification was observed.

(+) = indicateds that one or more fragments were amplified outside of the expected size range, which is ±100 bp from the marker size in P. taeda.¹ integers indicate the number of PCR fragments observed in the expected size range.

Table 6 Summary of P. taeda SSR primer pairs in other pine species

P. P. P. P. P. P. caribaea ponderosa radiata resinosa strobus sylvestris

# primer 566 118 498 119 566 566 pairs tested

# 168 47 127 38 84 138 amplifying single locus The 54 unique loci that were polymoφhic in P. taeda were also polymoφhic among species, but the primer pairs did not always amplify just one locus in the other pine species. The frequency of PCR amplification was lowest in P. strobus (eastern white pine). This was expected, because of the species examined is the most distantly related to P. taeda, and is the only species examined from the Strobus subgenus. All other species, including P. taeda, are classified in the Pinus subgenus (Little and Critchfield 1969, Subdivisions of the genus Pinus (Pines) (USDA Forest Service, Misc. Pub. 1144).

For all single RIPPT loci, 12 were monomoφhic in P. taeda, but were polymoφhic among species (Table 7). Loci that are monomoφhic within a species but polymoφhic between species may be useful as species-specific markers.

Table 7

Marker information for SSR loci that were monomorphic within P. taeda, but polymorphic among seven pine species.

Locus forward and reverse primer sequences SSR sequence allele size P. taeda 7-56

RIPPT2 CCCTAGGGAAAGGTTTCCAC (SEQ ID NO:366) (ATG)₇ 188

GGTCCCATAGACCAATTTGG (SEQ ID NO:367) RIPPT7 GATCAATCATCAAATTCATCACC (SEQ ID NO:368) (CAT)₆ 113

GTTGCAGATGAGGCTAAGGC (SEQ ID NO:369) RIPPT9 CCAATTTGGTCTCAGTGGATG (SEQ ID NO:370) (ATC)₆ 125

GAGATGCCCCTAGGTTCTCC (SEQ ID NO:371) RIPPT27 TCCACAGCCATCACCACTTA (SEQ ID NO:372) (ATT)₆(GAT)₆ 132

TGGGTCCGATAGACCAATGT (SEQ ID NO:373) RDPPT29 TAAGGTTTCACCAAGGGCTG (SEQ ID NO:374) (ATT),₅ 189

TCATGGGGTCAATTCTCCTC (SEQ ID NO:375) RIPPT30 ATGGATGGAAAATTTCTATAGCC (SEQ ID NO:376) (ATT),₃ 236

ATGTTTCCAATTAAAGGATTTCC (SEQ ID NO:377) RIPPT58 GCCTTGCAAAGTGACCTCTC (SEQ ID NO:378) (AGG)₄ 240

TCCATGACAACCCAGTTCAA (SEQ ID NO:379) RIPPT81 GAGAACGCGCGACTGTATTA (SEQ ID NO:380) (ATT)₄... 178

TTTCCCATCTGGTTCATGTG (SEQ ID NO:381) (ATT)j...(ATT)

RIPPT86 CCAATTCTTTGAAGTATTATAG (SEQ ID NO:382) (ATG)₅(GTG)₇ 262 GATCGCGAAGCTAAGACACC (SEQ ID NO:383)

RIPPT90 TCGATCACAGTGTTGGCATT (SEQ ID NO:384) (TTG)₇ 150 GCCAAGCCCATTCAGTTTTA (SEQ ID NO:385)

RIPPT314 AGAGGTTGCAGGAAGCAAAA (SEQ ID NO:386) (GAA)₄ 142 ATTGGTTTCTCCATCGTTGC (SEQ ID NO:387)

RIPPT914 AGGCGAAGCTTATGGAACAA (SEQ ID NO:388) (GAT)₄ 143

J TGTTTCCCGATCCTCTGTTC (SEQ ID NO:389)

Claims

WHAT IS CLAIMED IS:

1 An isolated polynucleotide consisting of the nucleotide sequence selected from the group consisting of SEQ ID NOS: 237-354. 2 An isolated polynucleotide that hybridizes under stringent conditions to the polynucleotide of claim 1. 3 The isolated polynucleotide of claim 2, wherein said polynucleotide is a maximum of about 500 nucleotides long. 4 An isolated polynucleotide having at least about 85% homology to the polynucleotide of claim 1. 5 An isolated polynucleotide having at least about 85% identity to the polynucleotide of claim 1. 6 An oligonucleotide primer adapted for detection of a SSR marker comprising the nucleotide sequence selected from the group consisting of SEQ ID NOS: 1- 236. 7 The oligonucleotide primer of claim 6 having a maximum of about 40 nucleotides. 8 An isolated polynucleotide comprising a SSR motif and having the property of being amplifiable from a genomic DNA using PCR and any primer pair disclosed in Tables 2 and 7. 9 The polynucleotide of claim 8 wherein said genomic DNA is from a pine. 10 The polynucleotide of claim 8 wherein said genomic DNA is from a species of the Pinus subgenus. 11 The polynucleotide of claim 9, wherein said pine is selected from the group of P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, and P. sylvestris. 12 The isolated polynucleotide of claim 8, wherein said polynucleotide is a maximum of about 500 nucleotides long. 13 The isolated polynucleotide of claim 8, wherein said SSR motif is selected from the group consisting of AC, AAC, AAG, AAT, ACC, ACG, AGG, ATC, AAAC, AAAT, AGAT, and all complements and permutations of said motif. 14 The isolated polynucleotide of claim 8, wherein said SSR motif forms a compound repeat, which may be perfect or otherwise, selected from the group consisting of: (A)_n...(ATG)_n; (ATG)_n...(C)_n; (CAT)_n...(A)_n; (ACC)_n...(ATC)_n; (TTG)_n...(TTA)_n; (C),..(ATT)_n; (TAT)_n...(A)_n; ( ATT),.. (A AT). ; (TTC),..(T)_n; and (A)_n(AAAC)_π(A), 15 An isolated SSR locus comprising a SSR motif, wherein said motif is selected from the group consisting of AC, AAC, AAG, AAT, ACC, ACG, AGG, ATC, AAAC, AAAT, and AGAT, and all complements and permutations of said motif.

16 The isolated SSR locus of claim 15, wherein said SSR motif forms a compound repeat, which may be perfect or otherwise, selected from the group consisting of: (A),..(ATG)_n; (ATG),..(C)_n; (CAT),..(A)_n; (ACC),..(ATC)_n; (TTG),..(TTA)_n; (C),..(ATT)_n; (TAT),..(A)_n; (ATT),..(AAT)_n; (TTC),..(T)_n; and (A)_n(AAAC)_n(A), 17 The isolated SSR locus of claim 15, wherein said locus is polymoφhic in at least one pine species. 18 The isolated SSR locus of claim 15, wherein said locus is polymoφhic in at least one species of the Pinus subgenus. 19 The isolated SSR locus of claim 15, wherein said locus is polymoφhic in at least one species selected from the group of P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, and P. sylvestris. 20 The isolated SSR locus of claim 15, wherein said locus is RIPPTl, RIPPT6, RIPPTl 1, RIPPT22, RIPPT24, RIPPT31, RIPPT32, RIPPT33, RIPPT64, RIPPT65, RIPPT66, RIPPT67, RIPPT69, RIPPT71 , PJPPT77, RIPPT79, RIPPT80, and RIPPT89. 21 An isolated polynucleotide comprising the nucleotide sequence selected from the group consisting of SEQ ID NOS: 237-354. 22 The isolated polynucleotide of claim 21, wherein said polynucleotide is a maximum of about 500 nucleotides long. 23 An isolated polynucleotide that hybridizes under stringent conditions to the polynucleotide of claim 21. 24 The isolated polynucleotide of claim 23, wherein said polynucleotide is a maximum of about 500 nucleotides long. 25 An isolated polynucleotide having at least 85% homology to the polynucleotide of claim 21. 26 An isolated polynucleotide having at least 85% identity to the polynucleotide of claim 21. 27 A method of detecting the presence of a SSR locus comprising a SSR motif of the sequence (N„ N₂...N_j)_n, wherein: N represents nucleotides A, T, C or G; i represents the total number of the nucleotides in the SSR motif; and n represents the number of times the SSR motif is repeated in the SSR locus; said method comprising the steps of: (i) isolating genomic DNA from a subject; (ii) analysing the isolated genomic DNA for the presence of said SSR motif by using the polynucleotide of claim 1 or an oligonucleotide primer of claim 6. 28 The method of claim 27, wherein said step (ii) comprises: (a) amplifying DNA molecules from the genomic DNA by polymerase chain reaction; (b) resolving the amplified DNA molecules by electrophoresis; (c) detecting the amplified DNA molecule. 29 The method of claim 27, wherein said SSR motifs is selected from the group consisting of AC, AAC, AAG, AAT, ACC, ACG, AGG, ATC, AAAC, AAAT, AGAT, and all complements and permutations of said motif. 30 The method of claim 27, wherein said SSR motif forms a compound repeat, which may be perfect or otherwise, selected from the group consisting of: (A),..(ATG)_n; (ATG)_n...(C)_n; (CAT),..(A)_n; (ACC),..(ATC)_n; (TTG),..(TTA)_n; (C)_n...(ATT)_n; (TAT)_n...(A)_n; (ATT)_n...(AAT)_n; (TTC),..(T)_n; and (A)_n(AAAC)_n(A), 31 The method of claim 27, wherein said subject is a plant. 32 The method of claim 31, wherein said plant is a pine. 33 The method of claim 32, wherein said pine is selected from the group of P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, and P. sylvestris. 34 A method of genetic characterization of an individual comprising determining the presence of a SSR locus, said locus comprising a SSR motif of the sequence (N„ N₂...Ni)_n, wherein N represents nucleotides A, T, C, or G, i represents the number of the last nucleotide in the SSR motif, and n represents the number of repeats of the SSR motif present in the SSR locus; said method comprising the step of comparing the SSR locus of said individual with at least one of the polynucleotides of claim 1. 35 The method of claim 34, wherein said genetic characterization is a genetic mapping study. 36 The method of claim 34, wherein said genetic characterization is a population genetics study. 37 The method of claim 34, wherein said genetic characterization is an inheritance study of a commercially important trait in a plant breeding program.