NZ512941A - Length polymorphisms in microsatellite (simple sequence repeat - SSR) DNA markers and their use as genetic markers in pine - 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 SSRs were synthesised 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- and 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 that were polymorphic among the species. Also described are the methods of using the identified SSR loci as genetic markers.

Description

New Zealand Paient Spedficaiion for Paient Number 512941 5129 PCTAJSO0/00325 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 repeal (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 inheritance studies in various plant breeding programs.

Background of the Invention Loblolly pine (Pinus taeda L.) is an important, experimental 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 materials (wood, fiber, and chemicals), ecosystem components (wildlife habitat and water and soil conservation), and recreational activities. Most of planting stock originates 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 2 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., 23:1083-1089., 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 an.d MAS is provided l?y 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-Anoll6s 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 polymorphic, 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.

PCT/US0O/OO325 4 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 polymorphic 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 polymorphic DNA markers.

Detailed Description of the Invention All patents, patent applications and references cited in this specification are hereby incorporated 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, for example, a mono-, di-, iri-. 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,)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, i.e., N,N2...Nj, 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)S 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)„ locus refers to the fragment of the chromosome containing this repeat, while (ATC)4 and (ATC)^repeats represent two different alleles of the (ATC)„ 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 polymorphysm.

"Polymorphism" 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 polymorphism. In general, higher informativeness means greater utility. Informativeness is usually defined in terms of either heterozygosity or "Polymorphism Information Content" (PIC) (for PIC PCT/USOO/00325 6 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 PCMJSOO/00325 7 with a complementary strand through base pairing.

Polynucleotides are "hybridizable" to each 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 8 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...Nj)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)„; (ACC)„...(ATC)n; (TTG)n...(TTA)n; (C)n...(ATT)n; (TAT),..(A)„; (ATT)n...(AAT)n; (TTC),..(T)„; and (A)„(AAAC)n(A)n.

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 ED 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 9 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 ID 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: aiignment 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 PCT/USOO/00325 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:I-236 and 367-390.

The present invention also provides probes specific to at least part of the aforesaid SSRs for detecting SSR markers using methods other than polymerase chain reaction, such as for example hybridization with labeJed 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 polymorphic. They may have a PIC of at least 30% (0.3); prei'eraiv . .. ai least (0.7); and most preferably of at least 90% (0.9).

The polynucleotides and primers of the invention may tie 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 n plasmids, pET plasmids (Novagen, Inc., Madison, WI), or pRSET or pREP (In'vitrogen, 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 a.nimal 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 polymorphisms 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 their 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, PCT/USOO/00325 12 H., et al., 1992, Genomics 13: 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, trim eric, 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 to 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 PCT/US00/0032S 13 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 polymorphic 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 polymorphisms.

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 jgenomic DNA with a polymorphic amplified DNA marker containing one or more SSR sequences. The analysis comprises amplification lining ihe 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 polymorphism 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 orT; H = A, C orT; and V = A, C or G.

PCT/USOO/00325 14 The present invention is further described in the following non-limiting examples. Examples Materials and methods Genetic slocks 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), centra! 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 sites during the primer extension step, thus primer extension 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 Hindlll 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 Corp., Madison, WI) or QIAPrep Spin Miniprep columns (Qiagen Inc., Valencia, CA). DNA cycle sequencing reactions were analyzed with either ABI373A or 377 automated DNA analyzers, or with a LICOR 4200-2 automated DNA analyzer.

Duplicated or repeated clone sequences were identified with the cbntig 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 el al. (1996). PCR amplification success and locus polymorphism were evaluated on WO 00/42210 PCT/US00/0032S 16 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).

Polymorphism 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 (RIPPTl 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 polymorphism 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.): WO 00/42210 PCT/USOO/0032S 17 I H = 1 - ^ pii i=l and I i=l PIC =2^ [pipjO-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 zth or jth 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 (RIPPT100 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, polymorphic 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 polymorphism 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 PCT/USQ0/00325 18 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. 2:649-56.) 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 polymorphic marker is still rather low (Table 1).

PCT/US0O/OO325 19 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 motif clones sequenced unique sequences primer pairs primer pairs amplifying a single locus polymorphi polymorphic c single loci per loci sequenced clone (%) AC 605 430 315 121 76 12.6 AAC 58 22 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 1 0 0 AGG 14 12 2 1 0 0 ATC 298 108 53 34 11 3.7 AAAC 117 67 13 6 .1 AAAT 98 59 21 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)n, (AAAT)n, and (AAAC)„ loci had respective conversion frequencies from sequenced clones to single locus polymorphic markers of 12.6%, 8.2% .. and 5.1%. (AGAT)„, 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 polymorphic markers despite their relative abundance in the pine genome reported by Echt and May-Marquardt 1997. The three trinucleotide motifs that did produce polymorphic 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)„ clones sequenced were unique sequences. (AAT)„ SSRs produced similarly few polymorphic 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.

PCT/USOO/00325 Appl. Genet., 90:43-48.).

SSR locus polymorphism For both enrichment methods, the total numbers of clones sequenced, PCR primer pairs designed, and polymorphic 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.

TABLE2 Locos Forward sequence Reverse sequence RIPFT1 GCATGCCA AAAGATCTCAA (SEQ ID NO: 1) RIPPT6 TTTGGACAAGTGGCTTGTTG (SEQ ID NO:3> RIPPT1I GGCTTCTCrCCAAGCTTTTTG (SEQIDNO:5) RIPPT22 CTCAGTTTCATAATCTTTOTCGC (SEQIDNO:7) RIPPT24 GACACCGGATACTGAGGTGG (SEQ ID NO:9) RIPPT31 CCAACCAATGTGGTTCATCA (SEQ IDNO:l I) RIPPT32 TAGCAGGTTACAACCTGGGG (SEQ ID NO: 13) RIPPT33 TTGGAGAACATGCTTGCAAG (SEQ ID NO:IS) RIPPT64 GCAGCGTAATCAGATGGTCA (SEQ ID NO: 17) RIPPT65 CCAACAGCACTTACCCAAAA (SEQ ID NO: 19) RIPPT66 GTTGATAG AGTTTCATGTGGTGC (SEQ ID NO:21) RIPPT67 AGCCCTCCAAGACCAAGATT (SEQ ID NO:23) RIPPT69 TCAAGAATGGGGGATGATTC (SEQIDNO:25) RIPPT71 CTACTCAAAGTGCTTGGGCA (SEQ ID NO:27) RIPFT77 ACACCGGATACTGAGGTGGA (SEQ1DN0:29) R1PPT79 TGATTTGATCCCTCTAGGCG (SEQ ID NO:31) RIPPT80 CACACAACCAA A ATT AAAACATTCA (SEQ ID NO:33) RIPPT89 ACGAAACCCCGAGTTGATAA (SEQIDNO:35) RIPPT101 ATGTTTGATGGGGTCGTCAT (SEQ ID NO:37) R1PPT103 CCCCTTGGTGGAACAACATA (SEQ ID NO:39) RIPPTI04 TGCATTTCATTTTTGCGTGT (SEQIDNO:41) RIPPTI06 ATCAGATTGGTGGATCGGAG (SEQIDNO:43) RIPPT117 GCTTCATGATTTCTCGATCG (SEQ ID NO:45) RIPPTI23 TCGTGTCGAAACATTGGAAA (SEQIDNO:47) AGTGAACTCGGGAGGCTTCT (SEQ ID NO:2) ATGTTTGATTGCATGGGGAT (SEQ ID NO:4) GAATOAGCCTCCCAACTCAA (SEQ ID NO:6) TTTTAGAAAAGAAGGAAATCTTCA (SEQ ID NO:8) CCCGCAACTTCGTAACAGTC (SEQ ID NO: 10) AGGAAAATAGAAGGGAATAAGACC (SEQ ID NO: 12) AGCCCAATTGATGGGAAATT (SEQ ID NO: 14) TGGAGCATTTTCCACAAAAT (SEQ ID NO: 16) CGGAAGGCGAGTTGAAGATA (SEQ ID NO: 18) AGCCTCATGAAAGCCCAGTA (SEQIDNO:20) TGOATGAAOAATTTTOTAGTCAA (SEQ ID NO:22) CCATTTGCAAATACCCCAAC (SEQIDNO:24) TTGCATCCAACAACTOCTTC (SEQ ID NO:26) CCCCTTCCCTTTCTATCTGC (SEQ ID NO:28) GGTTGTAGCCTCCCGTAGGT (SEQIDNO:30) AATCTTGAAAAGAAATTCAATATGAGA (SEQ ID NO:32) CACAAACAAGGGGGTCTCAT (SEQ ID NO:34) TAAGCOCTTGAACATOGTGG (SEQ ID NO:36) CATCATCCCATCAGACAACG (SEQIDNO:38) TTGGA AAATGGCGGAATTTA (SEQIDNO:40) AGGACATGGAGAGTTTACACATG (SEQ ID NO:42) TGACTGATAAGGGTTTCGCC (SEQ ID NO:44) TCTGCGTGGATAAAGGAATTT (SEQIDNO-.46) TATCACCTATAGCCCCGTCG (SEQ ID NO:48) § Nr. loci P=polymorph Expected Size (bp) Null allele segregating ksl IP 263 N IP 295 N IP 171 N IP 250 N IP 151 N IP 261 N IP 188 N IP 181 N IP 258 N IP 142 N IP 114 N IP 227 N IP 150 N 1 246 N 1 175 Y 1 153 N 2 251 Y 1 225 N 1 100 N 1 210 N I 164 N 2 180 N 2 208 N 1 129 N ro s CO e i § VI RIPPT126 TCATACCGAGAGAGGTCTTTG (SEQ ID N0:49) GAGCTTAATTTGTGCCTGCC (SEQIDNO:50) 1 174 N 3 o RIPPT128 CGACCCTAGTCTCTTGTGCA (SEQlDNO:51) TTTTGGACCCTAAGCCAGAG (SEQ ID NO:52) 1 175 Y o 44 RIPPT132 AACCGTGGTGCTCTGATACC (SEQ ID NO:53) TGCAAGTCAAGAGCTAGAGACAA (SEQIDNO:S4) 1 113 N K> ksi RIPPT134 GTTTACATTTTCCTGGGGCA (SEQ ID NO:55) GATTTACAAAAATCCCTGCCA (SEQ ID NO:56) 1 145 N ® R1PPT135 CACGCATGAGCTGAGTCATAA (SEQIDN0:57) TGTGTTTCCCACTATGCTAAGC (SEQ ID NO:58) 1 218 N RIPPT139 ACCAACCGAGGGAGCTAAAT (SEQ ID NO:59) AAAAACGACATTCACTTCAACA (SEQ ID N0:60) 1 121 N RIPPT1S8 GTGTGCCACGGATGTATGAG (SEQIDN0:6I) TTGCTGAAAGGGCCAGTAGT (SEQ ID NO:62) 2 211 N RIPPT159 ATATGGCTTACCTCGGGTCC (SEQ ID NO:63) CATAAACCCATTGGGTCCAG (SEQIDNO:64) 2 131 N RIPPT165 TGGAAGCCACAATTTGTTGA (SEQIDN0:6S) TGGAATAAAACCATGCAACAA (SEQ ID NO:66) 1 220 N RIPPT166 TTTTGAGAATGTCCGTGCG (SEQ ID N0.67) TGATGCATTGCAAAATCATG (SEQ ID NO:68) 1 155 Y R1PPT17I TGATCCTAAGCCTTAGAAACCC (SEQIDNO:69) TTTTGTCACCCATGCATATGA (SEQ1DNO:70) 1 207 N RIPPT179 TGTAGGAGCACAAGCCATTG (SEQIDNO:7I) AACACAGTTGGACCGTTTGA (SEQIDNO.-72) 1 170 N RIPPTI85 TGTTTGCAAATCATGGGGTA (SEQIDNO.-73) CCAGTGT^CATGCCAATTTT (SEQIDNO:74) I 300 N IN) no RIPPTI93 GATCCCTTGTCCCAGAAACA (SEQ ID NO:75) TGTTGATGTTATGCCTGGGT (SEQIDNO:76) 1 163 N R1PPT2I1 GAGGGGGTCTCATACACCAA (SEQ ID NO: 77) ■TGCATAGAGOATGTATTTCTTGGA (SEQ ID NO:78) 1 159 N RIPPT255 TCCTCCTGAGTGGTCCCATA (SEQIDNO:79) ATGGATATGAGGCCTGTTGG (SEQ ID N0:80) 1 123 N RIPPT263 TTGGATTCGACCTGAATCAA (SEQ ID NO:81) TTGGCAGTCTTCGAGGTCTT (SEQ ID NO:82) I 183 N RIPPT274 TGTTCCTCTCAAGTGACCCC (SEQ ID NO:83> CTTCAGCTTCCCACCAGAAG (SEQ ID NO:84) 1 264 N RIPFT287 GGAATGTATTCCCGGTTCCT (SEQIDNO:85) CTCCCGGATATTGAGGAGGT (SEQ ID NO:86) 1 224 N RIPPT293 CGCTACTATTGGCCGAATCT (SEQIDNO:87) CTOTGAGGAAATCCCTGGAA (SEQ ID NO:88) 1 184 N RIPPT298 CTTTTCCCITTCCATGACCA (SEQIDNO:89) GAGTOGAGTAACCAGGTGGC (SEQ ID N0:90) 1 319 N RIPPT305 TCAATCACCAATTATTTGGCT (SEQ ID N0:9I) GGAGTGGATGAAACTATGCCA (SEQ ID NO:92) 1 230 N R1PPT367 CCAATGCATAATGCAACCAC (SEQ ID NO:93) TAGCCATGGTGCTCAGTCTG (SEQ ID NO:94) 1 209 N RIPIT369 GGTTGTTGTGCACGAGCTTA (SEQ ID NO:95) TCAGTGAAGTTCAAGGGAGGTT (SEQ ID NO:96) 1 161 N O H RIPPT376 AGGAATTGGTGATTCATGTGG (SEQ ID NO:97) ATAAAAGAATCGGCCCTGGT (SEQ ID NO:98) 1 189 N £ V) R1PPT388 CACAACACTCAAACATGCTCAA (SEQ ID NO:99) AAGAGGATGTGAGGTCCCAA (SEQ ID NO: 100) 1 203 N i RIPPT467 CTTGGCGACCTTGTCATACA (SEQ ID NO: 101) GGGTCCTTAGGGATCATGGT (SEQ ID NO: 102) 1 178 N o Ui ts> RIPPT496 GTAAGAGTGCCTCGGGTCTG (SEQ ID NO: 103) GGTGGTAGGTAGATCGGCAA (SEQ ID NO: 104) 1 203 N in RIPPT508 GGCACAGGTTGGACATCTCT (SEQ id NO: 105) RIPPT538 AAACACTTGGACTaGATOGO (SEQ ID NO: 107) RIPPT540 TGTTGTCATTAGTGGTAGGATCA (SEQ ID N0:109) RIPPT548 TTTTGTGGTCATTCGTTOQA (SEQIDNO:lll) 5 RIPPT556 TCGTGATTACATTGCTGCCT (SEQ ID NO: 113) RIPPT560 CATTGGAACTTCACCGAAGG (SEQ ID NO: 11s) RIPPT567 GTTGGTGAGGAGACTTGGGA (SEQ ED NO: 117) RIPPT584 GCGAGACAGAAACGGAAAAG (SEQ ID NO: 119) RIPPT609 CAAAATGCAGAGGGGCTTAA (SEQ ID NO: 121) 10 RIPFT619 CAGCTCTCTTAATAGCCTCGG (SEQ ID NO: 123) RIPPT621 GCAAAGGGAAGCAAAGTCAT (SEQ ID NO: 125) RIPPT627 GACAAACAACCCTTGCGTTT (SEQ ID NO: 127) RIPPT629 GGTTGTGCnTCCCAGAGAG (SEQ ID NO: 129) R1PPT630 CGCAAGCTATGATACAACGC (SEQ IDNO:l31) 15 RIPFT644 GTTGTGATCCAAGTCCCCTG (SEQIDNO:133) RIPPT647 TGGCCATCGAACTTGTGTTA (SEQ ID NO: 135) RIPPT649 TAGTCG A ATCGGGCCTGTAC (SEQIDNO.131) R1PPT658 TGCATGCATTACAAATGTCA (SEQ1DN0:139) RIPPT675 ACAGATGTCAAGGCCAAAGG (SEQ IDNO:141) 20 RIPPT683 TGAAACCAATCCTTCTGCAA (SEQ ID NO: 143) RIPPT688 TTCAGTTATGCATTCACGAGC (SEQ ID NO: 145) RIPPT689 GAAACTTTCCCCTACGAGCC (SEQ ID NO: 147) RIPPT690 ATTCCTAGATGGACCTGGGG (SEQ ID NO: 149) RIPFT692 TGGATCGTGATCCTCTGTGA (SEQ ID NO: 151) 25 RIPPT700 TTGCAATTGCGATTAACTGC (SEQ ID NO: 153) rippt767 TGCATAOAAAGTCGCCCTCT (SEQ ID NO: 155) RIPPT789 CATCCCAAGCATCCTCAAGT (SEQ lDNO:157) RIPPT790 TTGTGAATTGTGTCCATGGG (SEQ ID NO: 159) GTGGTGGAAGGGAGATTTCA (SEQ ID NO: 106) TTTGGAGGATGTTTGTTGCA (SEQ ID NO: 108) AAGCGATGTCACTTGTTGAGAA (SEQ ID NO: 110) tCACATGGAAGATTATCTCCAAA (SEQ ID NO: 112) TCCACAACAATGATCGCTTC (SEQ ID NO:l 14) GTGCTATTGGGTCCAGCAAT (SEQ ID NO: 116) AAGAACAATTCCAATATGGATGA (SEQ ID NO: 118) CTCtGCTAGACCGCTCAGCT (SEQ ID NO: 120) CCAGTCCATCGAATCACGTA (SEQ ID NO: 122) GCACATAGCAACGCTGAAGA (SEQ ID NO.124) TTCGTCCTCTTTTGAACGAGT (SEQ ID NO: 126) GACCCATCAAGCCAACATG (SEQ ID NO: 128) GAATGCAAGGTAGCCAGGAG (SEQ ID NO: 130) TGTTGGCTGAGTGTGAAAGC (SEQ ID NO: 132) TGGTCCATTCGGTCCTATTC (SEQ ID NO: 134) CACGACCACCAGTCACCTTA (SEQ ID NO: 136) TTGCTCCrCTGTGTCCTTCA (SEQ ID NO: 138) CGCmTAAATCAACCAAACG (SEQ ID NO: 140) CTGCATTCAAATTACCCGCT (SEQ ID NO: 142) CTGATTCCTCTGGCTTCTCG (SEQ ID NO: 144) GTCCTCCTGGGTTATCCCTC (SEQ ID NO: 146) TTCCCCAAAAGTTCACAGGT (SEQ ID NO: 148) CGACATAAGCCCACCAAATT (SEQ ID NO: 150) GCTTCCATCACATTGGGATT (SEQ ID NO: 152) ATAATGGCATAGCCGAATCG (SEQ ID NO: 154) ATGCATGAGGTAACTTGGGG (SEQ ID NO:156) TCAAAAATGTGGTTTAATGGAAAA (SEQ ID NO: 158) ATCGGTGAGGCTTAAACACG (SEQ ID NO: 160) 90 N 212 N 200 N 207 N 183 N 108 N 152 N 136 N 154 N 191 N 154 N 168 N 157 N 157 N 204 N 214 N 218 N 219 N 172 N 187 N 141 N 158 N 142 N 166 Y 180 N 186 N 170 N 182 N 3 O to ro CO o § CM w Ul RIPPT79I ATGGAAGGATCCACAACCAA (SEQ ID NO: 161) GGGCTTGTTGCTGGTCTATG (SEQ ID NO: 162) 2 168 N 3 o RIPPT792 GGTTOATGATGTCGATGTTGA (SEQ ID NO:163) TTCTTGCAAACACAGCATGTT (SEQ ID NO: 164) 2 202 N © Ja* RIPPT799 TGATCCTAAGCCTTAGAAACCC (SEQ ID NO: 165) TTGTCACCCATGTCATATGATACA (SEQ ID NO: 166) 1 209 N h) K> RIPPT814 AAAAAGAATGAGGCGCACAC (SEQ IDNO:167) CCCGTTTATGGCATTGATTC (SEQ ID NO: 168) 2 100 N © RIPPT815 GAAAACGAACAAGCCATCGT (SEQ ID NO: 169) TGTTTACTTGCATGCATGTGTG (SEQ ID NO: 170) 2 162 N R1PPT841 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 R1PFT852 GTTATCCCCCATGTTGTTGC (SEQ IDNO: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: 179) 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) 1TGATCTGATGTGGGATCAACA (SEQ ID NO: 186) 1 151 N 32 RIPPT947 CCATTGCCCGAGCTAGTTTA (SEQ ID NO: 187) ITATATTGGACCCAAGGCCC (SEQ ID NO: 188) 1 214 N RIPPT958 TGGAGTCTCGAACACTGTGG (SEQ ID NO:189) . AATCATCCCAATGGCA ACAT (SEQ ID NO: 190) 1 111 Y RIPPT960 GCATCCATCTTCAGCATCCT (SEQ ID NO: 191) itTCATACGACACCTTTGAAATG (SEQ ID NO: 192) 1 188 N RIPPT961 CCATT AG 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) i. GGCTTGCAACCAGTTCCATA (SEQ id NO: 198) 1 220 N RIPPT990 GACCTAAAGAGGTTCACGCG (SEQ ID NO: 199) TCAAATCTTGGGTTAGTATGCAGA (SEQ ID N0:200) 1 220 N RIPPT10I3 ATCCCTGTGGGGATOAGTTA (SEQ ID N0:201) TGCCTCTTAAGCATCAAATGTT (SEQ ID N0:202) 1 124 N rippt1023 GAACCCGATGGATnTCAAA (SEQ id n0:203) CAAACTGTAAGCTCAGGAGGA (SEQ ID N0:204) 1 175 N RIPPT1027 CAGTGTTGATTGTGTGCCAG (SEQ ID N0:205) TCTGCCACAATTTGGA AACA (SEQ ID N0:206) 1 220 N REPPT1035 AGCATAATGAGCCCTTCTCO (SEQ ID N0:207) AGAATATGTGTCCCTCCCCC (SEQ ID NO:208) 1 174 N o § o RIPPTI036 TGGTTGTGCGAGATCACAAT (SEQ ID N0:209) TTGAGGGAATTGAAATTGGG (SEQ ID NO:2lO) 1 211 N RIPPTI037 TGCTCAATATAGACCACrroCA (SEQ ID NO:211) AGCCATAATTCAACAAAAGGAA (SEQ ID NO:212) 1 152 N o § rippt1040 tcaaggaattcattggagcc (seq id no-.213) tttggccatatcaaacccat (seq id no:214) 1 192 N I* UI rippt1066 aaagggggtgtttgatggat (seq id no:215) gatcgaaatcagcgaacaca (seq id no:216) 1 175 y RIPPT1072 TTTCATGACCTTOGAGTOGA (SEQ IDNO:217) RIPPTl076 TGTOTAAACCCAGGCTAOGC (SEQ ID NO:219) RIPPT1077 AACATTCTAGCATGCCCCAC (SEQ ID NO:221) RIPPTl 125 GAOCCACACAAACATGCATC (SEQ ID NO:223) RIPPTl 137 CCCATGCAACTGCCTAGAAT (SEQ ID NO:225) R1PPT9058 CCCGCTCCTATTCAAGATCA (SEQ ID NO:227) RIPPT9104 TTCCTATCGTCAGCGTCCAT (SEQ ID NO:229) RIPFT9I38 TGAAACCAATTTTTCCCCTTT (SEQ ID NO:231) RIPPT9238 CCCTGAGACATCCAATCCAT (SEQ ID NO:233) RIPFT9315 GGCTTAGGCATAGAGGGACC (SEQ ID NO;235) ATTGATCCCATTGTTGCTCC (SEQIDNO:218) ATGATTTCACAAAGCCCCTC (SEQ ID N0:220) TTGTGGTGGATGTCTCTCCT (SEQ ID NO:222) TTTCCCAAAAGTTCACGAGG (SEQ ID NO:224) AAGCTCGCACGTGGGATA (SEQ ID NO:226) AGGCGCCTAGAGGCATAATT (SEQ ID NO:228) GTTCACAGGGGTCATGCTTT (SEQ ID NO:230) CCAAGAAAGACAAGGAGCCA (SEQ ID NO:232) ACTTTACATGAGTTGGGCGG (SEQ ID NO:234) AACAAGTTGGAAGCCACCAT (SEQ ID NO:236) 26 Polymorphism 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 5 single bp allelic differences were not detectable some 2 bp allelic differences were possibly missed, the number of polymorphic loci reported may be slightly underestimated. In table 1, the number of polymorphic 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 10 fragment, or fragments, well outside of the expected size range was observed, but did not compromise interpretation of the single locus marker phenotype. SSR loci of P. taeda identified using the primer pairs in Table 2 are represented in Table 3.

TABLE3 SSR loci RIPPTl LOBSEQ3-27-97ATC44IR (SEQ. ID. NO. 237) ATTAATTTTTTTTGAA AAAAAAAGAGTTTTG AG AAAAAGTCTA ATATATACTTGGTGGCATGCCA A AAGATCTCAAAAA TTCCTTTCATATATTTGATTAGACAAGAAAATATATTATATrATAATCGTTTAACnTTTATAATTTTAAAAAATATATTA TAATTAnTTAAGTTTATGATGATGATGATGATGATGGTGGTTAACOTCCACrrGAGACCAAATAATGATCATCGGACCT AAAAAGACAAATTATTTATTTTGATTTAAGATTTTATTCCrATGCTCAAAAAGCrTGCAGAGAAGCCTCCCGAGTTCACT ataattttggcattgtaaaaggntaggaaaggtcattggtggttacaaagggtggtgaaattgaaatctaatgttggtg 25 tttgccggggcttcc RIPPT6 LOBSEQ5-2-97ATC402R (SEQ. ID. NO. 238) ccgagaccaagcaggcttgtaacagggctacaggtgagtggctcctcaagaccggtggtgtcttcaggaactgaacttt catgatgatcatgatgatgatgatgatgactggctagccgtgttccaaataacgagtccacactcgccccccgatgatc 3 0 gattctcgtcgtccgatggacgcgacggacgatacgagatctctgtctaggcgggatcgaacgatcgatggacgagctt gcactaccaaatgtacctgcggtttcatatctcacggtggcttcgacactggtcgncnaaactgactttgttcttctgtg aggtccgcgttggctttaaagcccnctgggcgggncxjntccaaacnttgcatctaaagggcccnttccnccttntagtt aattcctnttncaatccccngggccggcngttttcancgtcgtnatgggaaaacccgngttncccacttnatcncttgc 35 annnatccccttcccactggngtataccaaaaggccgcccnttncctcccnangttggncncctgantggaanggcnn ccttttggggctnancc wo 00/42210 27 RIPPTl I LOBSEQ3-8-97ATC229 (SEQ. ID. NO. 239) GCGCAATTTGTTATTCCTCCTATTTCACAGCTTGATCAAAGTGNTCTAGCTGCACTGCCTGATGCTATACGAGATCAAAT TTTGAAGAAGCAAGCAGGCAGTGCCAACCTGACCTCTGTCGCCAAGCATGAAGAAGNGAAAGAAGNTTTCTCAATGCA AAGTCCATCATCATCATCATCATCTGTGACAATCACACCCAAAAAGCAACGAATAA1TGATCCATTTGAACGAATGCGT GCAGCTTCAATTACACCAACGAAAAAAGGAAAATTGAAAAAAGlTACAATTAATTCAGCrCCTTCTACACCAAGTGGA TCACAAAAACGTTGCAAATGTTAGANAATCATGGGAACCTACATGGTCGCCAGTTGATTCCAAAGTTTTATCCGAACTA CCGATAGAAAT RIPPT22 LOBSEQ5-2-97ATC272 (SEQ. ID. NO. 240) ACAACCACATTAGATCTCAGTTTCATAATCTTTGTCGCAATACTGACCTTCCTAGCCTTTTACGATGTCATAATTATAGT GAGCTCGGGAGGCTTCTCTCCAAGCTTTTTGAGCATAGGAATAAAATCTTAAATCAAACTAAATAATCTGnTCTTTAGG TCTGATAGACCAAAGTTTGGTCTCAGTGGATGTTAACCACCACCACCACCACCATCATCATCATCATATCTTTTGAGAA A ATG AAG ATTTCCTTCTTTTCTA AAAT RIPPT24 LOBSEQAATIO (SEQ. ID. NO. 241) ACGCACTATTAATGAACACAAACAATGTATATGTAGATTACTTGATTTTTCACAATTCTATAAATTTATCTAAATCATTA TATTATTCGCCATACATTATGCAATAAOCATGAGCAGTATCATCATAATAGAATATAAGCACATCATCAACACATCAAC ACAAAATTCATATTGACACCGGATACTGAGGTGGAAACCTAATTTGGGAGAAAACCATTGTTGTTGTTGTCTCTTATTAT TATTATTATTATTATTATTATTATTATTATAAAGAAAAATTCTTCTTACATCTTGCACAATCACAGACTCTTACGAAGTTG CGGGCTCCTACCTACGGGAAGNTACAACCTCTAGAAATTATCCAGCTCCACTGGAANGAAGCTACTACTCCCTAATCAA GTTTACCAGCTCCNACTGAAAGGAAC RIPPT31 LOBSEQAATI8 (SEQ. ID. NO. 242) ATTGTTCTTCTGGATTAATTACACTAGTAATTrnrCAAATCAAAGTTrCAAACCAACCAATGTGGTTCATCATCAAAATA TAGATGAGGGAGGTTGAACTAAGCCATCGAGATTGATAAGAGGACTGGCAATCTGAACATAGATAATGGGTGGAAAAT TAGGAGTAGTTGGATTCCTAtXCTGACTTCTTAGGTGGCTrTGTCCCCCATCTAAAATTTAATTTAATTATTATTATTATT ATTATTATTATTATTATTATTATTATTATTATTATTATTATTACTATTTTGGTCTTATTCCCTTCTATTTTCCTAGCTTAATT TATGATTATTACATATAATTATTTACTTCTAGTTTAACCXnxrrTGCrrTTTTNATTTCTNTCTTTATTTCTTTCCTATnTTT TATNTTCCATATCTCrAGTTAATAATTTATTATTAATTANTTCAAGGATGTnTAATTATANTTCTNNAAGTrTAACATNT CCTAATTTATATTTTATTTCGCAACTCNCAATCCNTTACTAA RIPPT32 LOBSEQAAT49 (SEQ. ID. NO. 243) ACTTGTAGTCAGTTCAAGAGTTAACGCAAGGGAAACCTAGCAGGTTACAACCTGGGGTCAATCGGATTGGTAGCTACCT GTTOGAG ATATTCTGATTTCACATTAAAAAGTGA A AAGTGAGTGTTTTA AGTCTATTATT ATTATTATTATTATTT ATG A CCTTCTCAATTATGCCTAAAACATCTTGTCTATTTCCAGAATCTGAAATTTCCCATCAATTGGGCTATACTACAGACTCC TCTGCATACACCTTCCTCACTTTGAACATCGAGAGTTCAACTACAGAAAATTTGCACCTTCCACTTTGAGAGTTCAACTA CAGAAAATTTGCAGGCTGGCGGTGGAAAACAGTCAAGGTATGCATACGANTCCATGGTGCTGTmTNCCGGAAGAAA TTA RIPPT33 LOBSEQAAT46 (SEQ. ID. NO. 244) ATTACAATTTTCTTCTTACTTAATTGGAGAACATGCTTGCAAGTTGCAATTTGTAAATGAGATTTTTACTCGAGAAATAA AAAGGACTAGGTXiAAGAACATGCTOCAACCATTAGGGAATATAAGGTGGTrGGGTATAATCCTAGTCAATATATTATTA TTATT ATTATTATTATTTTTA ACTG ATTTTGTGG A A A ATGCTCCATTTTTTATACATGTTACTTTTCTCTTA A ATCCACTTA TATAAGTGACTATAAATTGAAGAAACTGTGACTTTACCTAGAT PCT/USOO/00325 28 RIPPT64 LOBSEQ5-6-97AAAC2C4 (SEQ. ID. NO. 245) ACGGGAAGGTGGTGAGGAGCAGCAGCGTAATCAGATGGTCATAGGCGTTGTGCAAACTGCAGGCTCCGGACAAAACCC TAACCCCATCCTGTACGTTGTTTCCTCATCTTTGCTTTTTCCAAATCCAAGCATATATATAACCCAATGAGATGAATAGT TAAAAAACAAAAAAAACAAACAAACAAACAAACAAAAACATCCTAAAAAATAGCCAAAAATGTAAAATCTCGAAATA ATCCnTTGAGGAGAGCTTTTCAATATCTTCAACTCGCCTTCCGGTGTAGTGGTTTAGG RIPPT65 LOBSEQ5-6-97AAAC2F7 (SEQ. ID. NO. 246) ACACGAACGGTCTACTTACACTTGACTGTATTGGATAATATACCTTCTATATATTCAATACTGTCCACCTATAACCAACA GCACTTACCCAAAAAAACCTCAAAAACATTTATATAAACAAACAAACAAACAAAAAAACCCTAAAAACACCTATAAAC ATAAACTAAOTCCAGGAAAAGATTTTAATTTTCTGATTTACTGGGCTTTCATGAGGCTGAGATTCTTCTAAAATTTAGAA CGAAATGCATGATGTATACTTCCATAATGGGAAAGCACTTGGTTTTTTTGGTTGCTTATTTTTGTGCATACCGAATCGTC ATATTTTAATCTTTGCTACTATGGC RIPPT66 LOBSEQ6-5-97AAATE2 (SEQ. ID. NO. 247) ATTATnTTATGTAGGCTTTGATTATATTGGTTCCCCTTAG ACTCCT AT ATATAG AA AGG AGGTCTTCTCATTTGTATCAT CAAAAAATTATTCACTTATGTAATGTAAAGGAGGTTGCCTTCGAAGTGGCTTATTTTGAATTCGTTGATCCATCTCTTCA ATTTGCGAGTTGGTTTCATAACATGACTACCTTGGCACATTATTCCATGGCGTCTATCATGGATATTATTATATTATTTTT TGTTGATAGAGTrTCATGTGGTGCAACATAGTCCTCCTCATTATCTCATTTGAAGAGATAAAAATAAATAAATAAATAA ATAAATAAATAAATTGACTACAAAATTCTTCATCCATGTTAT RIPPT67 LOBSEQ6-5-97AAATC7 (SEQ. ID. NO. 248) ACAAG ACTTCAAAGTTGTCTAACAGCCCTCCAAGACCAAG ATT AATGCCTCA A A ACA AGCCTATT A AAGTTTGCAA ACA AAACGATAAGGATATGTTCACATCGOCTCCAAATAAATAAATAAATAAACAAAATGTA1TAATATATGTCATCTTGGTT TGATGCATAGACACATCGGTTCATAAGGTAATGTCGACTCAATTGTATGTGCACAGCCGTTATAAATACCCTGTTGGGG TATTTGCAAATGGGCAAGAAAGAATTCACATGAAATCTTCGCATAAGGGAGCCATAATGAANGGAGCCACAATCAAGG TTGATGGANCCGfAATCCAGGTCAAAAACCACAGTTAGGGTATTGACTGCCCCGTGTTTTTGGGTTTTANGTGCAATCC ATGG RIPPT69 LOBSEQ6-5-97AAATE9 (SEQ. ID. NO. 249) ATCAAGAATGGGGGATGATTCACCATTTTTGGAGTAAAAGGATAAAAATAAATAAATAAATAAAAATAAAACCTTATT TTCAACTCGTATTTTCAACTTATCTCTTTACGATTTTACTCAAATTTCAAGCAGAAGCAGTTGTTGGATGCAAAATTTAA TTTTACATCCAAAAAATACCCATGTTGCATACnTCAATGGACCCTACTACACAGAAAATGTOAAATACAAGAAATAAT GTG ATTGTAATCATGTTTTTTCATGTATTTCATATTTTTCATGTAGTGGGGTCC ATTG A A AGTATGC A ACATGGGT RIPPT71 AAATI-A1 (SEQ. ID. NO. 2S0) CCATCTTATATTATCCTCCAAAAAAATTTCCTCTCTATACTTTAGGAAAAAAATTATGTATCTACTCAAAGTGCTTGGGC ACATTTGCTAATATAAATATATGGTTAGTGTTATAAAAACAATTTATTTATTTATTTTATTTTTATTTATTTATTTATTTAT TTATTTTCGTGGGAATGAGCATGGCATTCAAGCCATCACGTGGATGCTTGCAAGAAGGAGAGTTGAATTTTTAAGACGT GAAAAATGAGGTGGCTGAGCAGAAGATGTAATTGGGTGATTTGTTGGCAGATAGAAAGGGAAGGGGAACCCTCCATCT CTGCTAAATCTGCTAGAGAAGAGTAAAAGCAATGCAACAAAGGGACATAAACGAACAACTGGAAGCAACTCAGATGT GGACCATGGTTTGGAGGAGGATAAGAAOCAATTGGAGT RIPPT77 AAT3-BS (SEQ. ID. NO. 251) ACGCACTATTAATGAACACCAACA ATGTATATGTAG ATTACTTG ATTnTCACA ATTCTATAA ATTTATCTA AATC ATTA 29 TATTATTCGCCATACATTATGCAATAACCATGAGCAGTATCATCATAATAGAATATAAGCACATCATCAACACATCAAC ACAAAATTCATATTGACACCGGATACTGAGGTQGAAACCTAATTTGGGAGAAAACCATTGTTGTrGTTGTCTCTTATTAT TATTATTATTATTATTATTATTATTATTATAAAGAAAAATTCTTCTTACATCTTGCACAATCACAGACTCTTACGAGGTTG CGGGCTCCTACCTACGGGAGGCTACAACCTCTAAAAATTATCCAGCTCCAACTGGAAAGGAACTACTACTCCCTAATCA ANTTTACCAGCTCCTACTGAAAGGAACTCTTACTCCCTCCCTAAAGGNTCCATCCCCATACCTTTTNGGAAAAANTTCCT AATCTTAAANCCNCTGGNGGCGGTACTATGGATCGAACCGNNCAACTGANCNNACTGNATTTCCNATGTCXXAATACTG GGTANCNGGCNACTGTCCCTTTTAATGTT RIPPT79 AAT5 (SEQ. ID. NO. 252) ACTTTATATAGCATTTAAAAACACAATTTAAATGATGAAAAGTCACACATTGTATATTTAAAAAGACATAAGCACCCTA AATGTGATTTGATCCCTCTAGGCGCATGCTAGATCTGTGATTAAAATAGACCTATAATGGTATATnTCCATTATTATTA TTATTATTATTATTATTATTATrATTATATATCTGGTTAATTTTCACTATTCTCATATTGAATTTCTTTTCAAGATTTCCAA AATATAAAATTAAAATGT RIPPT80 AAT55 (SEQ. ID. NO. 253) ACATCACACAACCAAAATTAAAACATTCATAAAAATTAATTAAATTAAATTATAATTTTATTATTATTATTATrATTATT ACATAAACTAATGAATTAAATATATATACATTCAATAACACAATAATGACTAATTAAATTTTATTTTAAAATAAACCAA (XTATCCACATTCTAATAATAATAATAATAATATCCATTTTTAAGGTCACAGCCAAGCCAGACACCAAATTGGTGTATG AGACCCCCTTGTTTGTGGGACAAGGGACCCACCTCCGCGCAGCGATTTGACTGCTAAGTCTACGAAGGCATACATACTC TTTACCCCCTCTGGU'rrrrGAACCCTGTGACTCCTTAGGAAGGAAACACTTGATCTTACCCTTTAGGG RIPPT89 LOBSEQ8- 19-97TTC3B9 (SEQ. ID. NO. 254) CCTGGAACCCTAATGGTCAAGTGCCCCAGTTGAGAAGTGACATAAGTATAATGATGCATATGTTGTGTATATAGCACTA TCACCTCCAAATTTATACGAAACCCCGAGTTGATAAAGTAAAGTTGGTAGCCCTAGGAGTTGCAACATCTTTGTTGCAA CCTAT'l'i'i'iCriCTACri'CrrCTl'CATCri'L.'ri'CCTCTTCAACACTAGANOTANOGGTCCCACCATGTrCAAGGGCTTAGG.

RIPPTI01 pPTlOl .seq (SEQ. ID. NO. 255) ATCTGGTAATTCTGTTATGATCATGATTATGCTGATGTTTATATATGTATACATGTAGGTGTGTATGTATGTCATGTTTGA TGGGGTCGTCATGG ATCATGATTATTAACACTAACATTTGGTGTGTGTGTGTGTGTGTGTGTGTATGTATGTCGTTGTCT GATGGGATGATGATTGTTTTATGCTAATGATTATATATGTATGTATCTAnTATCrATGTCATTGTTTGACAGGATTGTCA GGGATTGTTCTTGTGGCGGTATTATTGCTTATGCAGGTGAATTGAATGAAGAAGTTCCTGG1TTATGAAGACTCGTGTAG GAAGAGTAATCATG ACAA ATATATGAGGTCA ATATCATGTGGTTATGAGTTGAT ATTATGTGATT ATATTATGTAT ATGT TATGGATATGGTATATCGAGGGATACATCAACGAAATGAAAGAAGTGTGATCTACGACATGCTTGGGAAAAAGCATGG ATAAGACACNANAAGCAAGGTTTATTGGTAAGGATGGGGTGGTTTTAGATGTATGGTAATACTATTTTGCATATGTTGT R1PPT103 pPTI03 jeq (SEQ. ID. NO. 256) CCAATCACCATATAGCCACCTGTCAGCTTCCATCGTTACCACNAAAATAGTCCCCATCTAAGCAGTTTTCCTCAGCTTTG GAATTTGTGTCCCCAAATCAACTCATTTAATTTGATTAAGGTTATAATATTTATAATAATGGAAAATGCAACTGGGATAT TACAAACCTCTCCCCXnTGGTGGAACAACATACTTAGTGATCGATGACACCTATACAACACAAACAAAAAAGATATGCT TAAAAGTCCAAACACAATTGAGATGGAGC7TTATATATAGATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTATAGAGAGAGAGAGATACATCTCACTTTATAAATTCCGCCATTTTCCAATGGCCCXTTTTTGTAGT CTTCTAN PCT/USOO/00325 RIPPTl04 pPTI04.seq (SEQ. ID. NO. 257) GCTAGTAAGAATAACrAATTATGTGATCAAAATTGTGTCATGTAAATGTTCTAGTTTCTACCAAAATTTGAATCTTTATG TCTGCATTTCA'l "1 'I' ri'GCGTGTATA ATTTGTG ATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGCGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTCTACATGTGTAAACTCTCCATG TCCTATGTCANTGTGTCTTTAGACTNTGGATGTGGGT RIPPT106 pPT106.seq (SEQ. ID. NO. 258) cctcttcaatccttaagaaaatoaggattttcccttgaaaaaggagtcttaagccttaaggccaaatcgcaatcggagg TTGCATGAGGGCAGACTTAAGGTTTGGGCCGTGGAACCCCCGATCACTAATCTCTGATCAAATCAGATTGGTGGATCGG AGGCAATCACCTTCAGTGGTCATATATATATATATCTATATATATATATATATGTGTGTGTGTGTGTGTGTGTGCGTGTG TGTGTGTGTGTGTATAAATGGATATTAGCGAGTGGATAGTGTGATCAGTGATTAGCCGAATCGGCGAAACCCTTATCAG TCACCCGGGGCAACATATGTGGGCGATGCTGACGCGATCAGCTGACGCCGAT RIPPTl 17 pPTlI7.seq (SEQ. ID. NO. 259) CTCATATCTTCTGACTAATGCAATATACAAATAGTAGGGG'rrrrrAGCTGTGTAATGTGTTGATTGAATTCTTTCTCTTGT ATGCTTCATGATTTCTCGATCGATTAATTnTTCCACTAATGAGTAGAGTAGATTCAAGTAnTTGTTTTAGTAAATTACA ATTAGTAGTTTGAATCTACAAGGAATACACACACACACACACACACACACATTAACATAACCATGATCACAACACAATT ACCATTGCTCATAAGTTCAAGGCTAAGAGAAATTCCTTTATCCACGCAGAGTATACATAATATTGAGAACGACAGTTCT CAACATAGCCA AGGCATTTGTTACCTCAAGCCCATGT RIPPTl 23 pPT123.seq (SEQ. ID. NO. 260) ATCGGCGGTTCGCAATCGGTATTGGCATCAGCCATGCCATATGGGAAACCCCCGGCCCATGCGATACACGATTGCAATG TCGCAATATCGTGTCGAAACATTGGAAAGGAGCGGGGCCATTATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGATGCGATGGCTAGACCCGACGGGGCTATAGGTGATAGTTGATATATTTTTAAATATGC ccaacaacattttgctatgtatttcgcatatttcgtttgataaatgaaatatgaagggaagcactgatgcaatgaaaac AGGGCCCGAAAGGGTNTGTGAATGAAATCATATATCGCTTCAAGTCTGATATGGGTTGTTTTGCAAGATnTCCAATGT TTTAATGCATTTCTCTGTGTAAACAGAGAATGGTTGTTCCAGGGATTTCAAAGGA RIPPTl 26 pPT126.seq (SEQ. ID. NO. 261) CCTCAANGCTAAGANGGCTTTACTGTAAATCATACCGAGAGAGGTCTTTGTAAAAATCATGTGTGTGTGTGTGTGTGTG TGTGTATATGTGTGTGTGTATOTATGTATATTAATATGGGTTACTCTGTTGGAGTAACTGTACTATTGTGTGGATTGAAC ttatgtttatgttagaatagatgtggcaggcacaaattaagctcaagagggatcaatgctcatatggaagtatataaca tcatcttcatagatatcagagcacacaacagagggagaaaggttacataaccaagattgcagtgtcaagatcttaaga CTGACrGTAAGGTCGAGGCATAACAGAGGAGGAATTTGTAGAATGGGTGGGAGAAATCTAGATTAAGCCGAATCAGAG TGGTGCAACACAAGT RIPPTl28 pPTI28.seq (SEQ. ID. NO. 262) CCAAGGCCTATGTTTTGTGATCGACCCTAGTCTCTTGTGCATGGTATCCTACACTTTTCATGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGTGTGTTTTACATGTTCCITGAGGGGTAAGAAATATTTTTTGGTCGTCGTTTATCATTTCCATAGAG ACATCGAGTTTCTACTCTGGCTTAGGGTCCAAAATTCTAGATAAAGTTATCGTACTAGTTGTTCnTCAAAAATTTTATT TTCACTCTGTAAATAGTCATATGGGTTTATAGGTTGCAATCTCTTGTTCATGAATTCCAATTATTACAAATTGGTAATGA A ATATTTTTGTTCTGGTCAAGGGTCCA AATCTTGGGGTATTGTCTANACGGTATTTTTGTTGGTC AGGTTAG AGTGGTTT ataagttctctaccccctctatagagaatgacaagttgatgagggggtgcaagaatatctacttctcaatgt PCT/US0Q/0032S 31 RIPPTl 32 pPTI 32.seq (SEQ. ID. NO. 263) ACAAGCCTAGTTCCTACAGTTGOTCTCACTAGTGGTGTGATTCCrACTCCTCCCCTCATCCTGCTTCCACTCTGTACACC ATTAACACATTTTTTATTCTCTAAATCrcCCAAACAAGACACAATGTTGAAACCGTGGTGCTCTGATACCXJCTrGTAACA TGCAAAGTCACCAACCAAACACACACACACACACACACACGCACACACACACATACATAATTGTCTCTAGCTCTTGACT 5 TGCACTATCACTTATTTATATTTTTnTAGAGCATTTGAATTAATTGACACACAACTAAATTAATTGACCTAGTCATAGC TAGTCATGGATACACTTTATGTTCCTTATAATGTGGTAAATATAACTTATAAGTGTGAATGCATTAGCGACGAACCCACC TAATATTAATAGCACTAAGGGAACCACGCTATAATTGTTTGGATTAATATTTGGTTGTCATATTATAATATTGGGANGTG ACCTACCTTAAAATGTTTCTCGAAGGGCTCTTTGGTCTCTAGCAATCATACAAAGANG RIPPTl 34 pPTI 34.seq (SEQ. ID. NO. 264) ATCTGCACTTAGTGCTTGGCTATCTGGTCCTTACXAGACTTGGCCATTrrTCITTTTTTTTTCCnTTCTGTTTGTCTTCTA TAGCACAACCTAACTTCCCCCTTTTCTT<XCTTnTCCTAGAAACCTTTCCAGTCAGGAGAGATAGTTAGGGTTGTAATT ACTGCAACTGCmTCCCAACTTCCCATTAAG a ANTTOCCAGCATTTTTCCA aaa AC ATTTCTTATCTTATTA a aga AAT AATAAAAATATTATTAAACrCCAAAACAATTAATAATATCATATTCAATTTTTAACCACAGTAAATTCAAATGTTTACAT 15 TTTCCTGGGGCA1TACACACACACACACACACACACACACACACAT ATATa ATTACAGTATG a AACTGTTTnTCTCTG aaaatcatagaaatcatggcaatattttgataaattatggcagggatttttgtaaatctangttatagttgttaaaattc AAGANTTTOGGTT RIPPT135 pPTl35.seq (SEQ. ID. NO. 265) ATCTTTTCA ATATTTAACATTGAAAAGCATTAAAGAATAGCATTTTGACAACT AAGGGTGAATACCCA AATTCATACAC TCACGCATGAGCTGAGTCATAAGATCTAAATCTAGACTATATTGCTAAAACACTTAGTCATCACTTTTGATTATGATTGT AGAAATTAAACTTTTAATATTGTTACTTTCATTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGCTTAGCATAGTGGGAAACACAAATAGACTTTAGTTTNNGACTT GTTGTATGAATTATCTCTCCATAATATAAATGCAGACAACT R1PPT139 pPT139jeq (SEQ. ID. NO. 266) ATCATTTCACCATTACTGTGAACCAATTGGAAACTTTGCAAAAGGATGTAGGGAGCATCCACACGTCTACGGAGGTCAT TATGACGAGGCTACAAAATTTGGACCGAAAGGTCGAGAACCTCAATGAGAGGGTGGAGATTGTTGTGGTGCCTATCCTC AAAGAAGTTTTCGCACTTGAGGAGGGTGCACeAGTGATCACTTTGAAATGGGACTTCCCTCTCrAGAGCTCACAATGCA "30 aatggagatgcagcaagatgcggagcaacaggaacctaaggtcgcaggtcaattgcgagttgtagaataggaagaag TGGAAGATGAAGTGCAACAACCAACCGAGGGAGCrAAATTGATGGAATTCAAGAGTGGTTTTTTGTTTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTTGAAAGTATGTTGAAGTGAATGTCGTrmTTGGAGAACGC RIPPTl 58 pPT158.geq (SEQ. ID. NO. 267) 35 ACTAGAGGCACACAGTGGGAGTCTCAGATCGGATCCACCGACTACTTAGTAATGTTGCACGAGTTGTCTCGTGCTACCA TGACCACCATGCTGAGTAGTTCCTAGGAATAGCACTTACATATTTTTCGAGGCCTGTGTGCCACGGATGTATGAGGCTT ACGAGCCACTAATGTTATTTTAGACACACATATTATTTTTTGGGGTTCTGATATATCAAACATTCTCATCATATATATAT ATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGGCTTGTTGGGGCCTAACCTAGGTTTAG AAG AGGTTTAGCCA A ACA A ATCCCACACTACTGGCCCTTTCAGCAACAATCCACAGAGCGTGGACTGAAGTCTCACCCGAGGTANTATGGGAGGGTG 40 CTGGAACCAAGTTTCTCCACCCTTGGTATGTCTTGATGTGGTCTGGANGATCGCAAACCATTCTGCACTCCTACACTTCT CTGCACAGAT RIPPTl59 pPT!59.seq (SEQ. ID. NO. 268) ATCAACATTGGCCGTGCCATATGGCTTACCTCGGGTCCACGCGATATTGCGATGCGGCGATCAACATAATGGAGCAGGT 32 ATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGATGTGATACATATATCTGGACCCAATGGGTTTATGAATGCAAATA TATGATACTTCAAOTTCACCATAAGGTCCrCTGATGGGATTTTTCAATGTTTAATGTATTTTTAAAATAGAAAGGGAACC catgtaacagggcatttgtgacaagcgatagcaatttaaaaggcaaacataatggaaaaaaatgtagagttcataatt TTTGAA ATCTG ATAG ACGCGTTTGGGCAA ATTGTT ACAGGCTTTCTGTGTATTTTCC AGTGTA AGCCG AG A ATTACCATC 5 TTroGGATTTCAAGGGCACTGATGGTCCATTCCATTACGGAATAAGTGG R1PPT165 pPT165.seq (SEQ. ID. NO. 269) CCTTGTGGAAGCCACAATTTGTTGAGTATTOGCAATTATTGAAAAAACCCTTTCAAGCTCTTGAATCTGTATTCGTCCTC TTTTGAACGAGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCrCTCTCTCTCTCTCCACACACACACACACAC 10 ACACACATATTCATGGGTATGTTCAACTTTGATGTGTTTGTTTTGTTGCATGGTTTTATTGCAATGACTTTGCTTCCCTTT GCCAAGGTAAAGTTTAAATTGCTTATCCGTGTTTATCATATTTTCTCATGCTAGATTTCTTTACGAGAATCGGGGGTTAA CTAAGGAATTCCTTTTGTCTCATCGCAGGTTAGTTTTGGCAATATGGGCACGTTGAATCCGACAAGTTnTTGGAACXIAA tcanggttccaactcatacactatatacgt RIPPTl66 pPTI66.seq (SEQ. ID. NO. 270) TTOAGATGAGTAGTGTGGGTGGTGGTCTGTTGTAGGGAGCAGAACCCACCACAGTAGACAACTAGGTTTCTTTTTCCGC TATTrTTGAGAGACATGTGTCTCAGATGTATCACGAACACATACTCTATrnTTGACATACACACTATTTTTGAGAATGT CCGTGCGCCACACGATATATATATATACACACACACACACACACACACTATCTATGGTTTATTACTGTCTAAATGCTTGC 20 ATACAATGTrrTGATTATAAATTGCATAAATGTCTATCTAATACATGATTTTGCAATGCATCAGTTAACGTGTTTAATCT TCTTGGAATGTGTTTATATGTGATGAAATCAAAATTTCTTTCCAATAATCTAACACGCAAATACAACCTTAAAATATGAG GGTTTCTGATGGAATAATGCCCGTTAAGCTGCAGAGCGACAAGTTAAGAATCCAGGTTCACAACCAATGCATGAATATC GAGGTTAACAACCTCAGTCAATAGTTCAATGACAGTGCTATCAGCGACGTTGTTTGCGGT RIPPTl71 pPT171.seq (SEQ. ID. NO. 271) ATCTCTCCACAGCTTTAGAGACACATTGTGGGGGTATAGTCCTCTCATTATGAAGGACTCCTATGACATCTTTTTCATGT TCTTTTGTGTTATCACC AACATAG AGG AG ATTA ACTCCTTAGG ATATTTTTXATGGG ATATCTCTTTGCATATGTATTGG ATTATGATCCTAAGCCTTAGAAACCCTTATTTTGACTAATGTATGCAAACTATATrnTGTTGTAAAACTCnTnTGATA GTTTTCATTTAGGTTTCTAATGAATGTATCATATGCATGGGTGACAAAATTAATGTGACAAATGTCAATAfATGACACAA TTAGCCAATGAATAAGCTAATGTTrTAGAGTCTAGTAATATAAATGTATGAAATGGTATAAAATCCCATTCTAGCAGAT GT RIPPT179 pPT179.seq (SEQ. ID. NO. 272) 35 ATCATTTATTTCAAAACATGTAAAAAAAATAAACATGTAGGAGCACAAGCCATTGATTATTTTCTCTATTTTTTAAAGGA AGACATTTAACTTAAACATTTGTAGTCAAAATCAACTGACTTTCAATAGTATTTTTTAATTATTATTATTATTATTATTAT TTATTATTATTACACTTTTTTCTATCA AACGGTCCA ACTGTGTTA AGTGTCTA ATACATATGCCT A ATTTTT ATATGCTA A CATTGCAATACTACTTACTAATTTTGGTTGTGCTATCCATAGAAGCATTTGGTAAGTGTTCATCATGCAATATTTTTACC ATACACGTTATGATTGCATGTCCATnTCAAGCAAGTTTTCAAAAGAGATGGTTATAGATATAATTCTCATATGAGTTGT 40 AGG ATAGTGTTCC AGC AGTTTGCACCCATG ANGG A ACTATCATTTACA ATG A A ACTACA A A ACATGTTGGCATT ATTG A CTTGTTTAACACTACATTA1TAA1TGTTATATGAAAAATT RIPPTl85 pPT185.seq (SEQ. ID. NO. 273) ATCTTTCGATGTCTATTGGTATACTATTTCAACAGAGGGTGTTTGCAAATCATGGGGTAAAATAATAATAATAATAATA wo 00/42210 PCTAJS00/00325 33 aaataagaataaactaacttcaagacaaagttccttaaagagggagagtatgtaatgccccataacaacaacaacaac aataataataaatgacaataaagtaaataaaatatttttaatatataaatagaataataattaacaaagaaaagataa AATAACAATTAACAAGAAAAATACATGAATATAAAAATAAAATAACAAATAACGAAGAAAATATAATAATAAATAAAT AAACTAAAAATTGGCATGGACACTGGTGGGCTCCAGTATTGTAGCAATAATGCTATAACTCCTGGGACTCCCTTCTTTA CnTTATCAACCTGGTAGTTCGTAACAAACTTGGAAGTGACAGTGTTGTATGCACAAGT RIPPTl93 pPT193.seq (SEQ. ID. NO. 274) ataatggccttggtgggcttaatcgttaagaacatgtatttccttctaagaggtcatagattcaaatctagagggaagt AGAGAANGTAAACCTCCACAGACTTTAGTAGTCAAATCACTGTAAGGAGGCGGATCCCTTGTCCCAGAAACAAGGGGT CTCATAAACCAATTTTGTGTCTGGCTTGGCCATGACCTAAAATTAGATTATTATTATTATrATTATTATrATTATTATTCA AAGGGAGAATCTAGCTCTGTTTGTAAAAAGTATCTAACCCAGGCATAACATCAACATAAAACCAACTTGTAGTAGAAGT atgttgaagatttgttttaatata RIPPT211 pPT21 l.seq (SEQ. ID. NO. 275) ACATTATGCAATAATAATATTACACAACCAAAGTAATAATATAACITGAGTGGGCATAATGGTTAAGAGCATGTGTTrc TTCCTAAGAGGTCACATATTCAAAACCAGAGGGGGGTAGAGTATGTATGCCTTCGTAGACTTAGTAGTCAAATCGTTGC GCAGAGGTGGGTCCTGTGTCCCACAAACAAGGGGGAGGGGGTCTCATACACCAATTTGGTGTCTGACTTGGTCGTGACA TTAAAATTGGATAATAATAATAATAATAATAATAATAATAATAATAATATTGTCAACCCACCTAATTAATTATACTTCAT TAGTTGATTCTCCAAGAAATACATCCTCTATGCACATTnTCTAGTTTCATGAGTAAAAAAAGGG RIPPT255 pPT255.seq (SEQ. ID. NO. 276) CTCCTGAGTGGTCCCATACTTAAAAATAAAATAAAATAAAATAAAATAAATAAATAATAAATAAATAATAATAATAAT AATAATAATAAAATAAAAQGOCTCCAACAGGCCTCATATCCATCnTGTTATCAATCAGTGCAOCACTTTAGTCTCCAT GTATGCCCAACATTCTATATGTTAGAATCTTTTTANATTCGTGACAATGTTCACTANATCCCCATATrTCAAATATTTCTT GTGCTAACAACCTCTTGATGATCTTTATTGCCATGGAATGAANAGTGCATCTCTTTTTCAAATTGAATTCATAAAC RIPPT263 pPT263.seq (SEQ. ID. NO. 277) " TATGTGTATTCAATGTTATGGACTAGGAAGGCTTCAACATCACTAGAATACCCAATTTTCTGAAGAAGAAAACGTANTC TANCAATGAATATTGTCAACrAAATTTGGATTGGACCTGAATCAATAGATTGTTCrTTATTCAAGCGAAAAAATAAATA AATAAATAAATAAATAAAAATAAAAATAAAAATTGATGTGCTAGTTGTAGACAAGGACATTAAGATGAAATGGAAAGA GGTCAATATGCATTTGCAGGAACTTTTGGGGAAAACCTCGAANACTGCCAAGTCTATCTGAACATTCACAAtTCCAGCT TATATCGGATTGCATGCAAGCATCCCGTGTTCCCATGTGCAGACATGATACATTQGATCATCTCNCACNCTGATCTTGA GATGATGACNTTGAGAAGTGTCCTTGGGATAGAGATTGTTACCTTCAAGGCATAGGATTATCCNTAGATGTTATCCATA TGTCAAGACCCAGTAGTCACCCATGGAAGACACCCTTCANCATACCCCAACCAACAATGCCAACTCCAGGGGAT RIPPT274 pPT274.seq (SEQ. ID. NO. 278) TAGTGTTCCTCTCAAGTGACCCCXnTTTCCTAAACAAATTTGAAGTATATCCCACATTTACCCTTGCATCCrGCACAAGT ATATCA ACTTCCCT AATTTTATTTATTT ATTTATTTTTATTT ATTTATTT ATTTTTTTCTGTCTTCTCTGGGTGGTTCTGTGT GAGTGGATGTCTCCACTCTCTTCTCCTCAATTTGTGGCTTCTCCACAACTTCCCGAGGCTCATCCTCTAGGGCTAAAATA TCCTCTTCTGGTGGAATCTGAAGCTCCATCTTAAGAGAGCACTAAATTTCTTTCTCCTTATCAAACTTTACATCCCTTTGC ACAACTTCCATTATGAGGGAAGGTAAGT RIPPT287 pPTZ87.seq (SEQ. ID. NO. 279) ATTOCATGTGAGGACnTCAGTGAGAATATnTAAAACTATATCATCTCCCACTTCTTGCAATGAAACCTTTCTAGTTTT 34 CCTrCCTTTTTTTrrGCnTTnTAACCAATTCTTTG ACTAAAACATCGTT ACCTTTCTCT ATGAATTCCTCT ATGGAATGT ATTCCCGGTTCCTTC A A ANATTTTATTTTATTTATTTATTTATTT ATTTTTrCCTTTCA A AC A A ATCCTTTGG AT A ATGCTC ACATTTCTCTTCAGACAAATTCGGCAAATATCTCTCTACCCATTCATTGAGGAGGTCCACTAAAACACTTTCTTCCTTGG AAAAATAATCATAATTATGTCTTATTAATCGAGCAACCTCCTCAATATCCGGGAGGTAAGTAACAATAATTTTCTCATTA CCCCCTCTTCATATTGTG AAGTCTTCCTCTTCACCGGTTGCTTCATTTCCTTC ACATTCTGGGTTTGTTCCATCATCTAT A CATCATTTGGTTTCCTCTTTAGGGT RIPPT293 pPT293.seq (SEQ. ID. NO. 280) ACCTACGGTTGTGAAAAANCGCTACTATTGGCCGAATCTAATAAATAAATAAATAAATAAATAAAGGAAGGTTGTTGG TTTCATCACTAGGTGTTTGGATTGTCGATAGGTAAAAGTGGAGTTCAAGCATCCAAATGGTCTATTGCAACCAATTTCG ATTCTAGAGTGGAAATGGGAGGCCATTTCCAGGGATTTCCTCACAGGTTTGCCAAGAACATCTANATAGCATGATTCCA TCATGGTTGTGGTTGACAGOTTG ACTAAGGTAGCTC ACTTCATAATAGTT A ATTCTACTTATATAGCTAGTTAGGTAGCT CGAGTCGTCGTCAAGGATATAGTTAGGTTACATGGTATTCCTAAGAAGATATTTGACAAAGATACCAAGTTCACTTCCA GGTTTTGGAAGGAATTATTTGCAGGTTTGGGTACAGAATTGCCCTTTAGT RIPPT298 pPT298.seq (SEQ. ID. NO. 281) GGATATCNAGANTCGAGGATCATGCATACTAGCTTGTCCAACGGGGNAGTTGGCGCTAGGGAACCGCAAGACTTGCCA AAACAGCGGAGGCTTTANCCATGAGTGGACCCNGGTGAAGGGGATGATTGCCTTCACAAAATCTATAGGGGAATC GAATATCCCCTTTTCCCTTTCCATGACCAACTACGGAGGTAGCATCGCCGTGAGCTTCAGTGGAAGATGAAGGACGGGC TGGATCACGGGACCATACTTCTAAGACCTTCGGAATGCCAGGACAATCACGAAGAAGATGGTCCTCCTTCCATAAT AG ACAAGG A AACTTAACCTTCTGCGG AGG AGTGGG A AGGANTCANACTTCTA ATGTCTGTGATGGAGCA ACAGTTGTG CATGTAGTCTCTGTTGTGTTGGTAGCCTCTCCCTTAGGAGACTTCTTCTTCTTGGTCTTCTTCrrCTTCTTCTTAGCCACCT GGTTACTCGACTCGAAGACTTTCACCAAGGCGGAGTANGCTTGGCGTTAATCATGGTCATANCTGTTTCCTGTGTTGAA ATTGTTATCCGCTCACAATTCCACACAACATACNAGCCGGAAGCATAAAGTGTNNGCCTGGGGTGOCTAATGAGTCAGC TAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCNTTCGGGAAACCTGTCCTGCCANCTTGCCTTGAATNAATC TGCCNCCCCCCNGGGAAAAGCNGTTTCNTNTTTGGGCGCTCTTC RIPPT305 pPT305jeq (SEQ.- ID. NO. 282) TCAATCACCAATTATTTOGCTNTCTAGGTGTTTTTTTTCATATACCTAGATCGAGTCrTNNGCCnTGAAATTCTTCTTCT TCTIXnTCCirriUI'lACTCTTCTCCTCCTCCTCCTCCTCCTTCATCl'ltrriC'I'rACTCTTCTACATATTTGAAATCAGATT CTTCATCTTCATCTAATTTCTCCCCITCTTTAGACTTTGTTATATGTGGCATAGTTTCATCCACTCCGAC RIPPT367 pPT367.seq (SEQ. ID. NO. 283) CCrCTATTTGAAATGTGATCATCATATTGGACTTATAAGAGGTAACATATAACATACATTTCCAAAACTTTCGTAAGGA GATCAACACTCTCCAAATAATTAGGAATCCCTCCTTCTAAGGTCAATCATAGGCATAAACCATGGATATAAATACAT GATTGATAATATTAAGAWCTTATCAACATTCCATTCTTGTGGATGGTAATTCCATAGATGGGTTGTATAGATGGTTATC ACCCACCCATGTCTATAGACTTGGTGACCCCTGAAGAGCCTCCAATACTCATACCACTCACTGCAATACCTCCAATGCA TAATGCAACCACACATGTGGACATGTGGACATGTGTATGTGTGTGTGTGTGTGTGTTTGTGTGTGTGTGTGTGTGTGTTC TCCTACCTAATGGATGAAGAAGCATTACCCTATGAAAGGTTTCCAGGAAACTCATTAACAAAAGTAACCAACATTTGTA TACCCACAAGTGGAGTGTCAATCAGACTGAGCACCATGGCTATAACCATCCATTCAAGCAAGGGTTTCACCACGTCATC TCAACACTGAGACAACGT RIPPT369 pPT369.seq (SEQ. ID. NO. 284) ATCAGATCATTTTTCAAATACTTAGACTAGATGGGCTAGATGCATGTTGTTAAGCATGCATTGTCATGGTTATGGTTGTT GTGCACGAGCTTATTGTATGAAGTTAGTGTTATCATACATGATGTCGATATGTTGAAGTGAGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTAG ATATCATGCTAATCT AG ATTGATTTTGTAGGTGAG ATAACCTCCCTTGA ACTTCACTG ATGT ATG TTGATTATGGTrATGTTTGGTTrATCTAACAAGGTATATTTTCAGGGAGAAGATCAAATATGACTAGCACAATGGTGGAT TATAGTTCACGTGACTCTCCTTATAGGTCACATGAGGAGCGCAAAGATAAGTAGTGATGTGCCTCATAAATGGGT RIPPT376 pPT376.seq (SEQ. ID. NO. 285) ATCATATAACTGCTCATTGCAATAAGGAATTGGTGATTCATGTGGTAATAAAATAATTGCCCACTACATCTTTGTGACTG CGATTAACCGCATCGATTAACACACACACACACACACACACACACACATATATAT ATTGGTTATGTCTCA ATATA ACCA nTATAATATGATTTAACTTGGTCACATGGGTATACCAGGGCCGATTCTTTTATACTTGTTCTTTTAGCGATTCCACTATG ctattatcacttcttcatcttcctttttatcttttttttgaaatattttcaagtaattcttgaatgaaaaatgaatactac AGGCA ACA AA A ATGCATGTTACA AT AGGTTCCATGCTCATTATT AATGGTCTTTTAGGT A AA AGCATGCCCTTCATGCTT CTAGGGCTGGTTGGGTCTGTGGATTTATTGTCACGAGTCAAAGGACTCAACATCATGTTGATAAGACCCTTCTCAGCAA aatgt RIPPT388 pPT388.seq (SEQ. ID. NO. 286) ATCTTGTCCTTAGGCTCAAATCTGAACACAACACTCAAACATGCTCAATGTCTCTTCTCCrAAAGAACAAATCTGAATA CA ACAC ACACACACACACACACAC ACAG AAACATCTATGTTnTCTTAGTTGGGACCTCACATCCTCTTTTGCATTAAA AGCATCACTTCAAATTTGTGTTTATCACCCCTACCTCCTGACATTCTCTAGGACTATGCTTCAACACTTATTTTTCTCACC ATTCA AGTCCTCAAAGCCrCITGTCACTCATCAACTTTGnTCTTTCAACTATATCCAA ATCCTCTTTTACATTAT ATATC CTTTGTAGCATTTTTTTCCTTATCTCATCACCGTAGC RIPPT467 pPT467.seq (SEQ. ID. NO. 287) ARKAAGCTTACACAACACTGCACACACAACACCAGTACTGCACATTCGAAGACCACACTATCTTTACCAAGCAACCTTG cttctagtgtctttccactatccatacttgtcccaacatgccacaaatcacaatgctttcattctgttgatgtatcc ctcaacatagcatgtccattacataaacataggcatagtgtaaacacataatatcaatcatatccacatgatactaaca TCATAGATCTGTTAGGGATACTCTGCCX:ACTTGATTCCTCACGATACCAAGAACCTCTTCATGCTACTCACATGCATAAG CAATCATTCCTCCACAAGAATGATCCTTGGCGACCTTGTCATACAATGNCATACATACATACATATATACATATAARCA tcatcataaaacaatcatgatccctaatqacccagttagacacacacacacacacacacacacacacacacacacaca cacatatataaacattagcataaaacaaccatgatcoctaaggaccctgtcagacaatgacatgcatatatacatatat acatatatagrcaccagcataataatgatcacaacataattatcagataagcttgcatgcctgcaggt RIPPT496 pPT496.seq (SEQ. ID. NO. 288) CCTCCGTCCGCTTTGAATCCTCTTCCAAGCATTATACAATCATTGCTTAATGCCGAAAGGGGAGTYGGCCACCATrTCTG TAATGTAAGAGTGCCTCGGGTCTGAYCCATCGGATCACCCATATAATTGTGTGTGTGTGTGTGTGTGTGTGTCCCCGCTC CATTGTAACATGTCGATGCGATATTACAATGCGACTTATCGCAATCGCATATCGCATAAGCCCGCAGCCTTCCCATATA ACATGGTTTATTGCCAACGCCGATTGTGATTGCCGATCTACCTACCACCACCGATTGCGATTGTCGATCTGCCAACAATT GCATGTCGCATTAGCCrCAACCTTTTGTTAAGTCCTCCCTTTGGCTTTTTGCGATGCGATAAAGCGGTAATCGCATATCA CAATGTTTT R!PPT508 pPT508.seq (SEQ. ID. NO. 289) GCATGATTTAATTTCCAGTAGTAATCTCCGTTnTTAITGTTTGTTAAAATATATACGTATTTCAATATTCCTTTCCCACT TGAAACATCCTCAACTTAATATCCTGGGTTGGGGTCCXjAGGATCTCCTTGCCAGTAGCAACTTGCAAATTGCAACAACX; 7TCACAANTTCAANATCCATTTAAACAAACCTGTCATACTCATCANAGGACACTTTACAAAATTGCCACTAAGCAACTC PCT/USOO/00325 36 TGGANATGGGTCAGTTTTGCATTATCTATACCAACTAGCATAGACCCGTCTTCACCCTACATCTATTAAGCATTGGAAA gggataaagaaaacaatgtgcaaaccacttggaanttggtgtttaaaaaacccagggttaaaatggatctatgttagt TTCTTTTATTTACTTATTTTTTACACCACTTTTCTTGGCACAGGTTGGACATCTCTrATTCATTTTAAGTTTATGCACATCA cacacacacacacacacacacctatgaaatctccctrccaccacaaaaacttggcgttatcatggtcataactgtttcc tgtgttgaaattgttatccgctcacattccccacaacatacaacccggaaacatnaanttttaaacctggggttgccta ATGA ATT A ACTACTCCCATTAATTGCTTTGCCCCACNGCCCCTTTCCATCC RIPFTS38 pPT538.seq (SEQ. ID. NO. 290) GTAGGTCAGGTCATGCCATGGTAACTCTTCATCTCATGTTTACCCTGATAGGCCAGTGGAATTAGGTAACTTTTGGAGG GTGTGTYACAAATTGGTATCATAGATTCCAAGTTCAAACACTTGGACTGGATGGGCGGGATGTGATTGTTATGCATGCA TGTCAGTCATATGCATGCATTGTAGTAGCTATGGTTGTCATGTATGTATTCAGTGTTACTTTTTG ATTTATCATGCATGAT GTCGATGGGTAGATATGTTGGTGTCATTATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTTGCAACAAACATC CTCCAAAATTTTGTGT RIPPT540 pPT540.seq (SEQ. ID. NO. 291) CCTTAGGACATTCTCTACTATTGTTATCTTAATGTTGTCATTAGTGGTAGGATCATTATTTTTAATTGTATACTTTGTATC TA ATACACACMCACACACACACACACACACACTATGG ATATrGTTCACACG AATCAATATTTATTAATAAGCGGT AAG CTATAG ARG AGGTTTCCTTTACAA AG AAACC WTTTWATTTA ATCATATnTAACATTCTCAACA AGTG ACATCGCTTAT TCTTTATATTATTATTTTTTAGGGTTA RIPPTS48 pPT548.seq (SEQ. ID. NO. 292) ACTAGATGTGTCCTCTAGTCCCATTGAACTACTTTTGTGGTCATTCGTTGGAAATTAATAAAATTATCTTATCTTACCTTT aattactcatttattggcattgaaatattttactatggtcgatgtgtgtgtgtgggtgtgtgtgtgtgtgtgcgagtgag cgcgtgcatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgatttggagataaytcttccatgtwga gtttacaattagtttggaattttgttaagaraaaatctcaacctccttgtctatttttggaatcaattggatctcaatcc AACTATATATGTGTATCATATTTATGAATGAATAATCGmTWGATGTGTGAAAATTAGCTTTATTTGTTGGTATCAAAG ccctatgggtctggggaaacctgggcgttaatcatggtcatagctgtttcctgtgtgaaAttgttatccgctcacaattc CACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCYGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGT TGCGCTCACTGGCCCGCnTCCARTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCCGCCAACCCCCSGGGAAAA. GCGGTTTGCCTTATTGGGCGCTCTCCGCTTCCTCGCTCAATGAATCCCT RIPPT556 pPTSSfi.seq (SEQ. ID. NO. 293) gtctcttaattatgttatgatcgtgattacattgctgcctatatttgtgtgtgtgtgtgtgtgtgtgtgtgtgtatagata tatatatgccaatgtctaatagggctgtcagggatcatgatcttttatcatoatgtttatatgtatatatgtatgtctgt ATGTCATTTTCTGACAAGTATGGAAGCGATCATTGTTGTGGAGGAGTGATTTCTTATGTAGGTGAATGACATGAAGAAG ctcatgttgtcaagtgggaagagtatctcgaatagatctatgaggttagtggcatgtggatatgagtttatattatgtg ATTATACTATCATAGGTAATGTCGAGGGATACATGAATGGAATGAGAAAGGTGTGATTTACAACATGCTAGAAC RIPPTS60 pPT560.seq (SEQ. ID. NO. 294) ATCGCAATATAGCATTGGAACTTCACCGAAGGGCGAAGCTATACACACACACACACACACACACACACACACACACAC A AATAT ATATATATGCG ATCTG ATTGCTGGACCCAATAGCACCTT A ACACT ATATTCAATAAATATITCA A ATCTA AG AT AAAGCCATTATTATGGGATTTTGCAATATTTTAATGTATTTCTAAAATACAAATGGACAAGCCATTTGTGACnTCOACA GTGACTTCTGGGTAGACCCAATAAGAGTATAGACTAC.AnTAAGGACTTTTGAAGTTTGAAAGACTATGTTTOGGTGGA TTTTCACCATnTGAATACATTTCTCCGTGTTAACAAAAAGCAGCTATTCTAGGGATTTTAAAGCCTCGATGCACTCATC 37 CATTTCAGTTTARGTKGCCATCTCCCCTGAATGTGTnTGCAATGAWTOTTTAACGTTTTGAAAGAAGAAnTOATTGGA tagaggccgggaaamaraaagcccaataagttggaaatgaataaggttaagaaatattggctttaaaagcccttgaaa araaaaaaaaaccgaagggttg RIPPT567 pFT367.seq (SEQ. ID. NO. 295) CCTAGTTGACAACGTAATTAGTGTGCCATGAACAATCTATCACATGTGATTTGTATGATCCTCTTGTGGCAGCACCATCC aactatattgcaaaatcaacgtcatcctgacatgtctatcaatacacatgccaacacatgccaactcctccatgaccaa CATGCTACCATAAAGCTCCACCAATGCCATCATCTGATCAACATCAATGCCAATACATCYCAACACAAACCGTCATGAG tgatatcatattattgacatcaatgccaacataatcaatccaatgtcaactcaactagtagcataatgaccatgttgac 10 ATTAATGCAAATACTATAATGTCAACACTATATKCTATAAATCTATATCGATCCCATCAATAGAGGATGCTAAGGGTTA GGTTGGTGAGGAGACTTGGGAGTTCATAGTGATGACACACACACACACACACACACACACACACACATGTTTTGTATTA TrrTCrTAAATTTTCrTTrrCACATTGCTATTnTnTAATATTATACATATCATOCATArTGGAATTCnTCTTAGGAAATT TTGTGTGTGTGTGCATGC RIPPT584 pPT384.seq (SEQ. ID. NO. 296) TKTTACCCXXJAACTCCAGAAAATGCAGAMATTGGGACGGCTCACATGGCTMTGGGGAGGGCTGGAGAAGCCCCAGGA AGGCACAAGCCAAGCAATACAAATCGAAGATGAGCAGTCTGTAGAGGCCGAWATGGAAGAAGAAGACAATGAGGAA GCATAGCAGGCTGCAGAGACAGAAACAGAAGAAGAATACGACTAGGAAGCAGAGCASTCTGCWGAGACAGAAACGGA AAMAGAAGATGTCGAGGAAGAAGAGCAGTCTGCAGAGGCTSAAGTAGAAGAAGAAGAAGAGGAGGCAGAGCAGTCTA 20 AAGAGGAAGOAGACGAGAAAAAAGAAGCTGAGCGGTCTKAGCASGAKGAAGGAATGGAACAACAGGAAGGTAGCCC ATTACCARACCCGTTAGGGGAAGATGAATTGGCCAACATnTGGCCTATATGGGTGAATATGGAAAACCCTG RIPPT609 pPT609.seq (SEQ. ID. NO. 297) GTCGGCAGATCAGAACGGTGAAACAAAATGCAGAGGGGCTTAAACACACACACACACACACACACACATATACGCTCG 25 ATCCAATGGTGCAATCATTGAAATCAACGGATAGCAATCAAAGCATTATAGAGGATTAGCCGATATGTTATAAGTTATA TACGTGATTCGATGGACTGGACTCGCTAACATTACAAGCCATATTGTATGGAfCTTGAGATCCATCAAACACCCCTTTTT AACGTATnTCACAGTTTCATTTGTTAAATGAAATGTGAAGGAGAAAGGCCAATTGTGACTTACGATAGCGACCTACGG GTAGACCCGATAGGAAAATAAAGCATAGTTATGGGAATTrCAAGTCTGfiAATTGTGTnTATGCGGACTTTCCCATGTT TTCAC^GCATTCTCTGTGTAACCAAAGAATGACTGTTACTAAGGGATTCCAAGTGCCGATTTCCCTCATTCGTTGCAAAA 30 ACAAGTGGAAGCTrATCAGTAGCATATTATGGCTTTAACATTTGAAATATGCAAGCTCAAGTTTTATTCTTCATGCTTAA GGGAAAAATTCTTATATACAATATANGTTCAAGATATGCCCTCTTTATTTAAGGCTrATTTAATATGATATAGGTCATTT AAATTTTAGTATTTATCCTTrACACCATTAACATAAC3>JTATTAATTGTGCATGTAACCCATGGATAANTAGATTAA RIPPT619 pPT619.seq (SEQ. ID. NO. 298) 35 ATCACAACAGCTCTCTTAATAGCCTCGGTATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGCGTGCTOCTTCATGGTTT CAATATTGATGAAAGTAAAGTCACAGTpCTTATCTCATTGATCTCAGTATAATGAAAGGAAGCAGTATCACATGCATTG TGATGTTTTAAAGACATTCTCTTCAGCGTTGCTATGTGCTACGCCATAGGCCATAGCACCATAGCTAGGACTACCTTGGG GCTATTGTATTAGGTCTATTTAGAGACATCATGGTGATGAAGTGCAACATAACATGATGGGTGCACTAGTGTAATGAAG TACAGTATAATGATGAGTGTAGCGGTATGAAGGTTAGCAGCACCATGATGG1TGGATGCAGCATGATGACTGGATGCA 40 GCATGGTTGATGTACCGCACTGTTGGGTGCAACATGGTGATGATGAGTGCAGCACAGTGATGATGGACATGGTACGAA GGGTGCAACACAATGATGGGTCCAGCAAAGTGATAGGTATAGCTTOGTTCAAATCTGCTACCTCAGGTOTCATCTTCTT AGAAAAGATCNCTGCTCrTATTTTGTCAAGTXrTAAATGTTGACTTCTGAAGTATATGTT RIPPT62I pPT62I.seq (SEQ. ID. NO. 299) PCT/USOO/00325 38 acgtatatagtgtatgagttggaccttgagtggcttctatgacttgaagaaaacaaaaaacatagataagcacatgtat agttgcgcaaaagaagaggaattatgagtagttggttccaaaaaacttgtcggattcaacgtgcccatattgccaaaac taacctgcgatgagacaaaaggaattccttagttaacccccgattctcgtaaagaaatctagcatgagaaaatatgata aacacxjgataagcaatttaaactttaccttggcaaagggaagcaaagtcattgcaataaaaccatgcaacaaaacaaa cacatcaaagttgaacatacccatgaatatgtgtgtgtgtgtgtgtgtgtgtgtgtgtggagagagagagagagagag AGAGAGAGACTCGTTCAAAAGAGGACGAATACAGATTCTAGAGCTTGAAAAGGGTmTTCAATAATTGCCAATACTCA ACANATTGTGGCTTCCACAAGGG R1PPT627 pPT627.seq (SEQ. ID. NO. 300) GTGTTCTTTTATCTCTGTGATCTAGACGCACACCATATGACTATTACTTACTAGGATTTGGATGAATTCATGTCHjCAAAA AATCACTGCAGTTTGGATGAATTTATGTGTCAAAAAATCACAGTGGTTTTGATGAATTTATGTGACAAACAACCCTTGC GTTTTGGAAAACAATAGTTACAGTTTGAGTGAATTTATATGGAAAATATrCATTGTGGTATGAGTGAGTnTATAGAACT ATATTTTGTGGCTCTTGCGCACGTGCACACACACACACACACACACACACACGCATGTTGGCTTGATGGGTCCTGACCC AGGTTCAGAGTTAATTAGCCAAAAAAGTCCTTGT RIPPT629 pPT629.seq (SEQ. ID. NO. 301) atctccactcttaaaoaoaggaatgaccacactagtagtccactctctcggaaagcxxjtcctagatgatcccattgaaa ATTCCTTTTAAATGAGGAGCAAGGAGCTTAGCTCCCCATTTTAGGAATTCAGCTTTAAGCTCGTCTATGTCCrCTGCnT ACTGCTTGCTAGATTCTTTATTCTCTGCTTGATGTCATCCTCTGTGAATAGTTCCACTGAATTGTTCACTATGGGGGGTAT CTTTCTCATGGACCCACTCATAAAGGAGCCTCACATACTCCAACCATTTGCCACCTATAATACTGTTTTTAGTCTGCTTT ATCCTTTGTTTTAGCTXnTTCCAAAATCCCTTGGGGTTGTGCTTTCCCAGAGAGATTAACTCCTTTCTTCrrcCACACACA CACACACACACACACACACACACATATATATTATACTAATTTTCCATATGTCTTCTTGTTTTCTTGCrCCATTTTCTCACr tttagagatctcctggctaccttgcattcttcatcataccatggatttgttggaaaagt RIPPT630 pPT630.seq (SEQ. ID. NO. 302) acxjcaagctatgatacaacgctgcaatatatttatatatgcagggaaaacaacacacacacacacacacacacacata TATATACATGCAATOGCACCATATATATACACAGCAATAGAGAAGATATTrACACGCTCAGCnTCACACTCAGCCAAC atatatacatacacacagtcaatatatatacacatagtcaatttatataaacgcacaatatgcagatattcacgagtag tagggaatcagaatagtgatgcatgttatagtgatgctctgtctatagggaatcagatattcacgggtagtaggcatgt tatagtgatgctttgtctacataactacagtcaagatctggtgagaacaaatcccgatggatttatagat RIPPT644 pPT644.seq (SEQ. ID. NO. 303) AGTTrCITGTCTTCGGTTAATTTTTGTTGCCTCTCATCTGTCACTCTTACAAOCCrCTAACTTGCATAATCATAAAATAAT agtccaattgtccatgtatctggttacgaggattaaaacaaagcataggttgtgatccaagtcccctgcaacacacaca CACACACACACACACACACACATATAATTAAGGGTTGAAAATAGAGACTGTAAAAGGAATGGCTTATGAGATTTATTC ATAAATCCACTCTTGCCACACTCTAATTATTTTTATTAAATAAATACATCACTAAAGGTCACTCAAGATOCAAGAGAAT aggaccgaatggaccattattagcataatagaaaaattacaacaattggataaatttaatagattacatggcaatcaa AACAAACTCCTATCATAACAATCCCACTCATTAAGGAACCAACTCGTTTAATAAAATATTTTGACCTCTCAGTCAATGG atttt atgaatgctcttgtatcgt RIPPT647 pPT647.seq (SEQ. ID. NO. 304) ACCTACAAAAACCACCATTATGCTGCTCAAAACCACCTTTGCGGATGAAAAACCACCTTGGAAAATTGTGTTCTGGTAT GGCAGGTTCGACACCTGCATAACAATTAAATAAAGTTTGGGCGOCTTGAATACCAAGCTCCACCGCTTGGTCTATTGGC CATCGAACTTGTGTTAAGGCGCTAGTATATATAGTGGGAGGTTGGCGGGTCGAAACCCCCACCCGACACTCCAACATTT PCT/USOO/00325 39 TACATGGAATTGATATGATATGATATGGATAGATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGATATTTTATATGGTT TAATACATATGTTTCAATACACATATTCTGGTAAGGTGACTGGTGGTCGTGGGGTTGACCCCGACCGTCCGACCCTTTTT CGGTGGCCACATCCTGAAACATTAAATATAACCAT ATCCCITCTGAT RIPPT649 pPT649.seq (SEQ. ID. NO. 305) ACATATGTAGTGAGCTTTANGGCTTTAGAGAGTGATTTTCGATCCGATCCAATGGTTCCATATAAATATGTAAGCCTAC AGGCAGAAAATAGGAGGCCACCAATAGTCGAATCGGGCCTGTACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGAG ACCAAGGAGTCTCGGATGTNATCTATATTTTACATTAATATATGCAAGATTATTTCTTGTCTATGTCATGATGCTTGAGA AGTAATTTATGTGTGAATTACTTGTGAATrTGTAGGCTTTGTTGTAGGTTGCAGGTANAATTTTGAAGGACACAGAGGA GCAAAGATGATTCAGAGAAATCATAGAGGGATCATTGAATCCNAGTGAAAGGTAGAGTTTCCTTGGAGAGGGGTTGTG AAGCAAGAATTCGCTAAGGAATCTAGCGCAGAGAAAGCTGAACAATGGAAGTANAAGACAGAGTTTGAATGACGCTGC TGGGAAAGAAATATGATTGGGAGAAATCATATTGATCCATTCGAGGGG RIPPT658 pPT658.seq (SEQ. ID. NO. 306) ATCATAGCTCTTAGGTTCAACAATTGGACTAGATGGGTGGAATGTATTTGTCATGCATGCATGTTAGTTCTATATGTGTT CATTATGCATGCATTACAAATGTCATTGTTGTAAGTAGTTTCATCATGTAAAGGTTGATGATATTGATGTCGAGATGATA TAACGTCCTCTAAACTTCACTAAGGTATGTTGTTTATGTCTACGTTTGGTTGATTTAAAAGCGTATATTTTCATATTGGTA GAAGATCAAGGATGACAAGTAAAATTGTGGACTACAGTTCACATGATTCTCCATACCTATCAAATGAGTAGGATGGTXjA TGAGTAAGCTTCCAACCCOAATGTCCCCGATAGTCAGGCGTGTCACCCATCCTATTGATGTTGTCACCCCTTATTANGAA AGGGGAGTGGTAACGANGANGANATTGTGAGGAACCTCGAGGGGTTGACTCATGGTATCCGGAANTATGCTCAGATAG GANACATGCCCTGACGANACCAGAAGCACACAGTGAGANTCTTAGATCGGATCCACCGACTOCTAAATCGAATTGCAC AAGTTGTCTCATGCTAOCATGANCACTATGCTCAGTAATTCCTANGTCCNACACTTACATATmTT RIPPT675 pPT67S.seq (SEQ. ID. NO. 307) ATCTTGATCTTTTATCTTTCATTGCAATCAAATATAACCATAGCTATCTTTGATATTGTGAAATAATATTTGATGTGGTTG GTGTTTACAATCTCCTCCACACTTATGTTCTATGCATATAAGGAT1TCTGATGGCTTGAAATGTATTCGTATATCCTTCCA AAAACCTACAACGTTGCCTCTGAGATATAAGAATGCAAATCAATGAACXJCATACCAAGGAAGCTATCAAATCXJCCTGA TGGATCCAGATAGGTTTTCATAGTCXjGTGATTGTGTCTATCTTCGACCCTAACAGATGTCAAGGCCAAAGGCAOAGAGC TATGCATTGTTTTCATAACTGTTGTTTTGATATATATATATGTGTGTGTGTGTGTGTGTGTGTGTGCGTGTGGCTTTTAAA TATTGTTTTGGCATATATATGTTGTTCAGCTTTGGCACACCATAGCGGGTAATTTGAATGCAGACATTGAGGGAGCTTTC TG ATTTGCTAACCATTGCGAAAAGG RIPPT683 pPT683.seq (SEQ. ID. NO. 308) GCAAGAGTTTTCCTGTGAAACCAATCCTTCTGCAATCTTTATTTCATCCATCGATATGTGTGTGTGTGTGTGTGTGTGTGT GCTTCAACAGTGCAAGAGCAAGCATTATAAACGAGATAACCTTTTAATTAATATTGATAGAGCTGCAGCAGCAAGAGG AGCCAAGGAGACCAAGAGGGCTGCGAGAAGCCAGAGGAATCAGGGAGTCATGAAGAGTCCTGGTGCAGAGTTATCTA CCATCAGTCGTCAACACTCATAACAGGGATTGCCCAAAAGCCCGTAATATTTACAAATTGCATTTTAAAAAGCGTCGGG CACCAGAGAACGTCTATTAAAAGAATTGCAAGAAGACGAGAATTTTCATGTTGCCCAAGGCCATCAAGCCCAGAGGTA ATTCGAATTGAGAGTCAGGCACACAAGTGGG RIPPT688 pPT688.seq (SEQ. ID. NO. 309) atgagagaaatctcaaacacaacacaacacgcacacacacacacacacacacacacacacacacacacatatgtaag 40 CATATGAACACTTATTTCAOTTATGCATTCACGAOCTAAGTCTAGGCTGAAGTAGCACAAGTTTAGGAGGGATAACACA CACATTTGTTTTGAAAGCGACAAGTCGAATTGGGTTGAATACCTTCAGAAATCTCAGGAGGGATAACCCAGGAGGACCT ACATATATCATAGTAT ATATAAAGACTGCGCATCAGAG RIPPT689 pPT689.seq (SEQ. ID. NO. 310) CTAAGTCCATCGAGAATGAGAAAGAGGACATATTTCCAAAGTAAAAGAACCTTAAAGAATTTGGGGGCTGAGCTCAAG GACTTGGAAGATGAGTTAAATATCTGCAGGGAAGAACTTAGAAACCTCACTTGGGAATAGTGAGAGTTGATTGAGTGG AATGTTTAACTATGAAAATAGTTGTGAGAAATGACATATAAAGATGAATCCTTGAAAAGGAAAAATGGAATTGACAGG AAGAGGTCTCGTGAGGTTGAATAGGAGAAGGGGATATTCTCCTATTGGTGTTGTGGAGAAGAAAATTTGAGAAAGGAC TATCCCCGTAGGAAGAATAAGAAAGAAACTTTCCCCTACGAGCCACAGGGGGAACATGTCNATGATGCAGGATGCCCA NAAANGCTGAGGGGGGACTCCTTCAAGCTCCTTGACACATATATATGTGTGTGTGTGTGTGTGTGTGTGTTTGTTGAAA ACATCACXTGTGAACTTTTGGGGAATGTNAGACTTCNNGGTTGCTGTGAGCCTTNTAAAGAAGTCTTGAACTAATTTTG GTTTATGAATATGCATNTGT RIPPT690 pFT690.seq (SEQ. ID. NO. 311) ACAATTCCCCACATCGTTGTAATCTCCTGCTTTGAGACCTTAATAACAATTCTATTATGGACTAAAACTCAATCGCTCCA TTATGTTTGTGTTGACGTAGTA AG7TGCGTCCATATT ATA ATCCCTACCCAACT ATGTCCA ACCTTCATA AT ATTTATTCC CATACAATAGTATATGATTCCTAGATGGACCTGGGGGTCAAGACAATCAACGCCGCTTTAGACAAGGTTATGGGTAATG CCAAGTAAATTATCATTCCACACACACACACACACACACACACACACACACACATATATAATTTGGTGGGCTTATGTCG ACAATACCAACGTTGTCTATCCCTCTAAGAACAAATCATTATATTATATGTAATGTATAAGATGGGATCAACAATCTAC ATTGACTACCGTCAACATAATGGTGAACCGTGAGTTTCCATCACGGAGG RIPPT692 pPT692.seq (SEQ. ID. NO. 312) ATCTTTGGGGCACAATCACTCACGGATTGAGATACACTGTCAGNAGATGTGAGGTTGCATGGTTATTCTGAATGTTGAT CGAGAAGCAAAAATCGGTTGCTTTGAGCACCATCGANGTTGAATACATAGCTGCTAGTATGGCCTCCTGTGAAGCTGTC TGGTTGAGGAAGCTCTTCAGTGAGTTTTTTGGACATATGTTGGATCGTGATCCTCTGTGACAACCAGAGTCOAATCTGAT TATCAAATAATCTTGTGATTCATGATCACTCCAAGCACACACACACACACACACACACACACACACACACACACACATA TATNTATATATCAACGACCTTCTCNTCAATCCCAATGTGATGGAAGCCTrATTTCTCCTCNTTGT RIPPT700 pPT700.seq (SEQ. ID. NO. 313) ATCATATAACTGCTCATCaCAATCGGGAATCGGTGATTCTTATGGTAATAATGTAATTGCCTGCTGCATCTTTGCAATTG OGATTAACTQCATTGCTTAAAACACACACACACACACACACACACACACACATATATATATTGGTTATGGCTCCATATA ACCATTTATA AGGTGATTTAACTTGGTCACATGGGTATACT AGGGTCA ATTCTTTTATACTTGTGCTTTTAGCG ATTCGG CTATGCCATTATCACTTCTTCATCTTGCnTTATCTTTnTTGAAATATTTTCAAGTGATTATTGAGTGAAAAATGAATAC GATACGTAATAAAAATOCATGTTACAACAGAAGGTATGTCTAATTTCTCTCTAGATTTTGATTTCTGTGAAAATTGTGTA TTTGGGAAGCATAATCGGGTGAGTTTCCCCTCTAGTGCTAAGANGGCGAAACATATA1TANAGCTTGTGCACAGTGATG TGTTTGGACCTATGTCGGTTCCATCACTGGGTAAGTCTATGT RIPPT767 pPT767.seq (SEQ. ID. NO. 314) ACATATGATTCTACAAATATCATTTAAGAACACACACACACACACACACACACATATACCACCATCATGATGTGACAAA TGATGATCCrTCCCCGATTCATGTTATAATCTCCTTGATCCCTGACCAATCTCATCAACATGCATAGAAAGTCGCCCTCT AGTTOATOCATGAGACTTAATTGATGCTACATCGCATTTACACCTCAAAGAATCAAGTGGTAACTGCTCATGTGATGAT CCAGACCCTCGTTCCATACATACATACATACATACATACATACATATATATATAATCTTTAAAAAAACCCCAAGTTACC PCT/USOO/OQ325 41 TCATGCATTTAAAATCnTAAAATTOTTATTTAAAAACATCAACAATTCAAAAAATTGATAAGACAAGTAAATOTAACT ATCCAAGGG'rrri'CriCAAAAAATTTTGATGTAAATTCTTTATTTCAAATATACTCAAOACTAAAAACACAAATTCTGAA GTGCACATTTTGATAATTAAATACATTTnTTAAAACATGTCAAATTCACCAAGTTGAAAGAAACACATGGTCATTAAA AACATACACATATCAAAGAT RIPPT789 pPT789.seq (SEQ. ID. NO. 315) ATCAATTGAAATATACAAAAGAAGATACTGAAAGGAAGGACATTGACCACAAACAAAAATATAAGAAGAATGTAGATT TATTGAACAAGCTATGGCAATAAATAGCCAATTTGAGAGAAAATTATGAAAGAAAAATTTGATAGCTCTAACAAGGAT CAAAGGATGTGTCTGTTTrATTGCCCAAATGATCACATGTCTCCACAGAAGGAAAGATACACTCTTATTCTCAAGAGGA 10 GGGCTTGTAGAATCATCCCAAGCATCCTCAAGTTTCA A AATAGCA ATTTTTAGGTGTTGTATCTATGTAG ACACATTTCA ACATGTCACACACACACACACACACACACACACACACACACATATATATTCTTGTTAACATGCATTATATATCATTATA TTTTCCATTAAACCACATTTTTGAATAAATTTCTAGTCTTTTTTGAGGATTrACACTTATGTCTCCTrCATGGAGTTTTTAT CTGGTTGTGGTCTCATTGGGATCTAGTTACTCTTTTATTTTTGGACAATGTNATGGAATTTTATAAACTGATAGCTTATTG AAGTGAGAAATCGGTGTTATGGAATGT RIPPT790 pPT790.seq (SEQ. ID. no. 316) ACTTTGTATTTATCATTCTnTTCATATGAATGTA A A AGCATGATTTACCTATTTT ATTGTTCAGCA ATAGCCTGTTGTAG ATGGGAATGTTTATAAGTTTTCTAGAATGTTATCATTCCATTTTGTGTGTAGTTCTGTTAAATGTTGCATGATGTGAATGC CTTCACTrCATGTTTACTCGTTAATGCGCTTACTAGCTTTTTTTCCTCATGAGAAATATGTTTAAATTTGTGAATTGTGTC 20 CATGGGAACAGGGTCACCTTCGGGCTCAAACCCTAAATCATTAAAGTTAGTTACTCTGTTGCTATTTTGGTCTCGCACAC ACACACACACACAC ACACACACACACACACAC ACACACACACAC ACATAT AT ATATTGCTTCACATTrCGTGTTTA AGC CTCACCGATTCITGGCCTGTAATTCATTGCTCCGGCTACGGA R1PPT791 pPT791.seq (SEQ. ID. no. 317) actatattt attg atttgaaatattaaga agagcatctg ac aa agaat aa ag aggttag a a a agaga agtangctgag aagtangagggaaagtatgtgctaaagctaaggtagaagccatgacgcanaagcatggtaAaagggacaatatggaa ggatccacaaccaatggatgcaacaaagagaatccatgagaagtggggcgacggaagatgactagtgaccaataaag catacatatatatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtttgtgtaaacactocaaatacatagaccagcaaca agccccnaggcaaagaattagataaaggagcnagaagagcatgaaggctagttgaggaatacnaagggagaagcaga 30 agggaggcgaaatgagaccagagggctaaagaaagagagtagttgtanaaaatgatattattctaagagtgga(Xna caatgttgtgcccnaagcctqctactagggaacaaagaaacnaactatggaagcgaaaagg RIPPT792 pPT792.seq (SEQ. ID. no. 318) atctaataccacagaatggtatcggaactctaaggttcaacactaggactggatgggctgaatgtgtttttcatgcatg 35 cacgttagttgtatatgtgttcattatgaatgcctagtgattttcattaitgtgagtagtgcagtcatgtagaggttgat gatgtcgatgttgagatgattgatatattgatatatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcgtgtgt gtgtgttgtgctaatacatattg attcttgtag atgagatagcctcccttg a acttctcta aggt atgttg agtatgtttg tatttggtttatctaacatgctgtgtttgcaagaagtagaggatcaaagttgacaagtaaaatggtogactatagttca cgtgattctccctaaagataaaatgaacaacatagtgatgagt 40 RIPPT799 pPT799^eq (SEQ. ID. no. 319) atctctccacagctnnanagacanattgtgggggtagagtcctctcattatganagactcctatgacatctttttcatgt tctttcgtgttatcaccaacatanaggagattaactccttangatattttttatgggatatgtctttgcatatgtattgg attatgatcctaagccttagaaacccttatnttgactaatgtatgctaagtatatttgtgttgtaaaactcttttgtgat PCT/USOO/00325 42 atataatgtgtgtgtgtgtgtgtgtgtgtgtanotgcatgtgttncagcctcantattgtacacttgtgaatgttgtctt atgatgttngcattcaggtttctactgaatgtatcatatgacatgggtgacaaanttantgtgacaaatgtccanatat gacatnnttancccatgaataacctaatgtcttagaatcnagtaatataaatgtntgaaanggtggtgaatcccattct agcanangt RIPPT814 pPT814.seq (SEQ. ID. NO. 320) atcaagagaataagtttgcccaaatttgaacaagctgtagcatgaagatcactggtagtagcaacagatgcacacact aaatcaaccttcccttcaattgaatcaacactgaagcactgaatcaatcttggctcaccaagttctactcttactctcca AGTCTCTCCCTCACAACCTTCTTCAACTCTCTCCAAC<XTCrCTCAAGTTTGAATTCTTCrTTCACAATGCGCAAGTAGTG 10 AGGGAAAAAAGAATGAGGCGCACACATACACACAGACACACACACACACACAGAGATATATATATATATATATAGGA ggagcaacgaatcaatgccataaacggggattaactcgcatgccaagagggagagtaacatatgcccttttaggaaat ^ gtaactcatgccattaagaaggt RIPPT8I5 pPT815.seq (SEQ. ID. NO. 321) 15 ACTTCAnTAGGTTTTAACATTTTTCAATAAATTAGCAAAGGAATGAAAACGAACAAGCCATGGTTAATTTACrAGAAA TAACTCTCTCTCTCTCTCTCTCACACACACACACACACATGCATGCAAGTAAACACTAAACTCTCTCTCTCTCTCTCTCT ctctctcacacacacacacacacacgcacacatgcatgcaagtaaacactaaactctctctctctctctctctctctctc TCTCTCACACACACACACACACACACACACACACACACACACACAAGTAAACACTAAATCTACAACCGTAAAATATAT GTCAATAATAATGCTAAGTAGAGAGG RIPPT841 pPT841.seq (SEQ. ID. NO. 322) ATCAAATrCACTGAGATAAAATCGTAATAGATGTTGATTCAGGTGCATGGTGGTGATGTTACAGGITTAGCTTCTTCGTT GGGCACTAAGATGCTTAGAGTTGAAGCCCTAAAGAGGGCTGAGTCCTTCCTCACTTCAAAATAGACATTGCATTGCTCA GATATTCACAAGAGTGATACCACAAGGCTGGGCATTGAAGTCATGCTTTAAACGTGCTTCCCrTGCTTCAGACTTGGCC 25 TTACAGCATAGTGTCATATGCGTCAACTTAGGTTCAGAGGAATCCATAGATCATGCTCAGAACAACGAAACCAACTCTG nTTTATCTCAGAAACTCGTGGATGATTAGAAGCGGCCACACACACACACACACACACACACACACACACATGTATATA CCCAAAGTTTGCATTTGC RIPPT846 pPT846.seq (SEQ. ID. NO. 323) 30 ACCTCATAGATTTTAGACCATATACTAATCCAATGGATGAOAGAGATTATACAATCAACGTATCCGATTTAATTACATG CXICCCCTCCTCCCTGTGACATCACCGAGGGGGGTGTCACATTCATGGTTCCAATGTGGGATTCCACCTACATATATATAT ATATATGTGTCTGTGTGTGTGTGTGTGTGTGTGTGTGTTATAATTATCACGAGATCCACGCTTATCATTATACAACCATA GGTATAATTACTTATAGATTGGGTCCCTTGCTCCACTTAATTACATCCCCCATGTATATGTGGATGTGACAATCACAAGA CCCCTATTTTAGTTATCCATATATGTATATATGACCTTAATATTTGTGGACCCCCTCTTTACCTTTATATGACTTAATGCT 35 AATAGAAACGACATAGTGAGGGCCCACCTTC RIPPT852 pPT852.seq (SEQ. ID. NO. 324) ATCTATGTAGATCGCATACCACATAGAAAATACCCTGGTTTATATTAGAATAAGTTAATTAGAAGATTTGCATGATAGA ATAGGTCATTTAGATTATCATTATTTTATTTGCAAGCGGTTATGAACCGCXXACTCTCATATTAAAACCCACACTAGGAA 40 ATTGGTCCCATTATCCCCAGTCGGAQCCACCACTTTAAACGAAAAAGGAGTTGTCTTAAGGTCCAGATCXKjGTAGTTAT CCCCCATGTTGTTGCAACGATrTTTGAGGCCCCAACAGAACCCTTCCAATCCTTTAACATATTTGGCTTCCTCTCTTAAA GGTCAGATGAGTTTGCACTCTCTAGTCACTTCTATATAAAACTTCNTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTTTATTTCTCTCGTCNAGGNAATGAAAGCATAGTGCTTCTACCCNGAAATTAAGT 43 RIPPT860 pPT860.seq (SEQ. ID. NO. 325) GNAGAATATTGAGCAGACATCATCAACACTAAGTAATGTCTAACAAGGACCCTCAATAATTCTCATATGTTTTTTACTT ATAACCATGAGTCTTGTAGATAGAGAGTGCATCTGTGTGTGTGTGTGTGTGTGTGTGTGAGTGTATGAGTGTTTGAAAG AGTGGTTGTAATCTGTGCTTTGAGAGACTAGAGACTAGAAAGCTCTACTCTTTGAGGCATAACCTGGTATGTAACAATT TTCTTG A ATTAATACTG A AGCCTACTGGGCTGCCTGTCTGAGG ACAGTTTGTTTCG AG ATCATCTA A A AGCG AG ATTTTA AAGCGAGTTGGAGAAGCGAAAGAGTTGCGTGTCCTCGTATTTCTCGAGCTGGTGTGAGGAATTTCAGCATACTCTTCAG AGGTTCAAGTAAGTTATTGACTCCATCATGTTGT RIPPT90S pPT905.seq (SEQ. ID. NO. 326) ATCTAAACTTTTGTGCAAATGTAACACGGATCTCTGGAAACCATGAACTACACACACACACACACACACACACACACA CACATGTTGGAAATAATACTGATAGGGGGGGTGAATCAGTATTATCAAGTCAGATATTCCTTTTCCAAAATCAATTGAT ATACGCTACTTCAGTTAATTAAACCCGCGTATATACGCAACTATTCTGAGGGAAACCAGCGTATACAAAGCAAACTATT G ATATAATTTATCTTTTTG ATGTTG ATTGTTAATCTGTGTTTCCATA ATCA A ATAACCAGTA ACTAG A A ATAGTAAAACA AAGCAGAAAGACAATTGCACACAAGAACACAAGATATAAGGTGGAAACCCAATGTGGGTGAAAACCACCTGCTGCAA TTCGTCTCTATCTTCCTTrAAATTAGAAACTTCTTTTACAATGC RIPPT921 pPT921.seq (SEQ. ID. NO. 327) GCTCAAATAGAGAATATGGAAATTTTCCGCAAACTATGTGACAACCCTAAGTTATAGAAAGGTATGTTGACACCTTTTG AGATCCTATACCTGATGCATGAAAAGTGGGAACCTATGATGACAATATTGTAAGCTAGAAAAAGGATAGTTTGAGGAA AATGAGTATNGGGAGATGTCNTAAAAAATGTCTGACATGTCACAACACACACACNCACACACACACACACNCACATAT ATATTCTACATATAGAAAGAAAGAAAATOGATCAAATAAAATTCCGATAAOCGNGAATGTCNTCGTATGATGTGTTGAC ATGG AAAGTGGCATATGCTATGCCCTAAAT RIPPT932 pPT932.seq (SEQ. ID. NO. 328) ATCTCGTCTGCAAGACCGACTGGATTAGCACAACACACACACACACACACACAAACATAATCAAATCAACATATAGTC ATCATTCCAACCATTGTCAAACTATCAATGTATGAGCATACCCACCACATATCATGACCTCATGCATGGTAACTACTAC CATATCACTGTAGGCATAACAACATGTGTATGAAACGCACACATACCNCTGTGTTATGTGTGTGTGTGTATGTATAGGT GTATATGTGTGTGTGTGTGCGTGTGTNTGTGTGTGTACAACGACACCATCAAATTTCACATGGTGACACGACTAAAACC ATACCCA AG ACCATGCATACACA ACCA ATACATAGACC ATA A ACATA AG A ACACATATGCAACCA ACATG AATGCATG ACCAACACAACACCCATTCAATCCAAGGGGCGAAACATGCTATGATACCACTCTATTACACCCTCTTCCCACATCTACC CAAGTAACCQACTAACCAGGGTAAAAATGTGATGAGAGGCTACTATGATCTAAGTAACCTCCCCATGT RIPPT941 pPT94l.seq (SEQ. ID. NO. 329) CCATCTTTGATAGGATTGTTGTGGAAATTGTGTGGGGTCCAAAGTTGCCCATTCGAGTCGOCAGACATCTGCACAAGTT GTGTGAGTGAGCAATGCCGTAACCCACCAAGCCACCGTTCGAAAAGGAGTCGCTAATTTTCATCATGCATCACGTCGTG TGGGGAAGAAAGAGAACAAATAAAATGTGTATTTTATTTTCATTCATTTGGGATGTAGAAATTCTAGACTACAAACATC GTGATCGCATGATCTCAGGAGCCAAGAAAATTTTATTTTGAACXXAAAGTTGAAGCAAATTCAACXXjTGGACCTGGCGT TGTATCGAATCTCTGTTTGTGGTAGCTTGGATGTCCTTAGTGAACTAAGGCTGCGTAGCAAATCACTGGANGTTGTGTGC GTTAAAAGGTAGTGGAGAAGTGCGTATGATATATATATATGTGTGTGTGTGTGTGTGTGTGTATGTTACAGTGTGAAAA CCAATACATAGAGAAGTCAATTGTTGATCCCACATCAGATCACATCATTAGT RIPPT947 pPT947.seq (SEQ. ID. NO. 330) CX:CACAAACTCTTGGTGTTCCATCTCCTAGGGTTAGGGTTTTGGTTGTTTAGGTTTCCCTTTGTAGTTCAATAATAAAAA 44 TTCCCTTGAGCCTCATTGTAGCACAATTCAAAGGCATGTGGCTGCCACATCACTTCTCAGAGATAAGCTACCATTGCCC GAGCTAGTTTATTTCCATCATGTGTGTAAGCATCTTTTAGTTCTTTGATATTAGTGAAATGAAGTTTTATTCCTACTACAT CTTTGTGTTTATGTTTATGTATGTATnTATTTTGnTCTACATAAGTCCAGATCAATAAACACGCACACACACACACNC ACACACACATITGGTCCA ATGGTTGGGCCTTGGGTCCA AT ATA AC ATTGGC ATGT A ACCATG AATT A ATTTCCACGCTA TGAACCrTGATCACTTGGGGCTTACACATTGTCCATATGACTTTATCTTTGCAGGTAATTAACCAGCCACATGGTGACAT TAGCATTATGTCATCATGCCACATGATGGCTAGTGAAGANGTGCCACATGTCACCTGGAAAACAAGTTGATTGGTCACT CG AGCT ACCATTTGT RIPPT958 pPT9S8.seq (SEQ. ID. NO. 331) ACATTGTANGATTGCAAAATGTGACTTTAGAGGATAGACATCAGTTTTTTCTAACAAGTCCAATCACCAACTATTAGTG ATTTTATACTrATCTTGGAGTCTCGAACACTGTGGGAACACACACACACACACACACACACACACACATATGTTGGCAT TATGGTGTGCAAGGATCCAACTANTTGATGTTGCCATTGGGATGATTGTGTTGGCATTGATGTGAACGATATGTTGGCAT TGGGAAATGATGATTGCCACACTAGTTGATTGGGAACTAGGTCTTCANGCTTATGTTCGTGGAGATGACTTCTTGCGTGT TAGGTAAACTTGACTCAGGATANAGTCNAAGTTGACCANGTTAAACAGTGATGGTTTTGGGGCACATGGTATTGTGCGC CCATAGTATTCTGCCCATGGTATTATATGTAATGGGAGCTTATAAAAGGATGGAAGGACTTCNTTGTCATGTATGCCTG CANGTGAGCGGTGAAOGCTCACTTGGTCAGATTGGCTAGGGTTTTTGGCCANTGCTAATGAA RIPPT960 pFT960.seq (SEQ. ID. NO. 332) ATCAACCCACTATCACAATCTTCCTnTAATTATTCCTATCAAAGGTGTCCCACCACTTGCACCTAAGAAATAACACATT TGAATGAAGATAAGTCCACTTGAAATTATATCTTGTATCTTTCCAACGTAACCTAATGTCCCCTGCATTATGTTTTGATC TTGGTGTCTAACACGACTAGATTGGTCAAACTCGACTTCCACTCCATAGTCTTGTGTCATGCATCCATCTTCAGCATCCr TCACGCAACAATTATGTCCATATGCCCACATACATGGGAATAAATGCACCTATAAAAAAGTAAAAAAATCATAGTGTC ATOTGTCTA ATAACCCAAACACACACAC ACACACAC ACACACAC ACACAC ACACAG ATATATATGTTACATTTCAA AG GTGTCGTATGAAGTTAAAAATGT RIPPT961 pPT961.seq (SEQ. ID. NO. 333) ATCAATCCACGAGGTCGATAAATTAAAATAACAATCCAAAATCATAAATAAGTAGCCCCCAAGAGTCCTCTTAATCTAT ATGAGCTCAAAGTCCATGGAACTATCAACCATTAGACAAGTGCGCATGCACACACACACACACACACACACACACACA CACGCACACACACACACACACACACGCACACACACACACACACACACACACACACACACACACACACATATATATCAA GGAGClTGAGGGAGTATGCCCTCCTCCTCCTCAACATCTTTCTTATTCACTCTCrTTTTCTTCGGTTGGGGAAATTCCTTT TTCAATGCTCTTACCCACAACACCAACAAGATATTATTTTCTCCCCCTCTTCAGCTOCACATAGGTTTTTCCTTTTTGAGG AATCAACCAAGT RIPPT968 pPT968.seq (SEQ. ID. NO.334) ATCTTATGCTCACTTACTTTCTTACCACACTATG A A ACCCAACCTA AA ATCAATC ATACT AACAGA ATGCTCTACTACCA AGCTAAATGAATCTTCATGAGGAATACCCTTAATAGGTCACTCTAAAGTATGGGTTGTTACCCCAACACCCCTTCCTAA GGCACACTTCG ATTA A AG AATCATA AGCACCTTG A ATCTTCTTA AC ACT AATTACAA A ACTAGG ATC A A ACCCA ACCTA ATACATATCATAAACTACTATAGATATCTACGACAAAACCACGTAGTGTTTCATACAATCCAACCTAAGCATGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGGATNACTACTTCCNAAGCTACATCCCATTCACTTCTCANGGAAG ATAAAAATATCNAAACCAACTTAATACCTACCATTTACTAATATAGATATATGCCACAAAGCCACATGCTNTTTTATAA AAACCACTCTAAAAAATTCAAAACCAACTTAGCAAGCTTNTCTATCGAGTATTANGGGGCGGGATTGANAGGCCAGTC CGATCTATGCTCTGCAGGGAATATCTTGAAACGTATTTCCTACCGACCACCCGAGCGTTCNGATCCCGAANTCAATGAA AACTNAATCA PCT/I) S00/0032S 45 RIPPT984 pPT984.scq (SEQ. ID. NO. 335) ACCACTTATA ACATACCTT A ACCACCTGCTAGTrTATTCCAACCTCTTGTTGGGTCCCA ATTTCGTGTTAG A AACCCCTG GGAAACCTGTGACCTGAAAATTCCCCTTTCTATTAAAAATTTGTCTGATGAAATGCACATCTAACTGCATGCTAAAATGT AATACATGTGCATATGTATGTTCTAACATGATTATCTTATTTCAATTAACTTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG 5 TGTGTGTTGTNOCANTGGAATAAAGACAAATACTGCTCTTTTAATTTATGGAACTGGTTGCAAGCCTTTGACGTNTAACC TANCCAAAGACCATGTTGTTCATTTCCTTCAAGANCAAGCCACCCCCGAGTTGGGGCTTTGAGTCCATGGGGAATCAAC AATAACTCTCCTAAAACCTANC RIPPT990 pPT990.seq (SEQ. ID. NO. 336) 10 ATCACTGGAAAGCTCTTAATGAGCTAAACACGATGGTAA'rrrrrrrri'AAAGTnTGATGAGTTTGGAAAAAAGATGATT TTTGCAACCCCATCTTCATAATAGATTGCGAGGGTAAGTTTAAGTTATAAGTAACACCCTCCACAACCCAAGAATAACC CAAGCGGGCGGGAGCGACTATTACAAAATGGTATTAGAGAATGGTTCAACACTAGACCTAAAGAGGTTCACX3CGCACA CGCACACACACACACACACACACACACACACACACACACACACACACACACACTTAGGATAAACATGTTNGCTGCTAT TTCCAATATTGTATACATATG AGGGTTGATTTTA AGTTAC AA ATGATAT AATTGTTG ACTTCNCN AA AT ATTG ATA AGTT 15 ATTTTATCCTTTAATCTGCATACTAACCCAAGATTTGATGATTTAGCTGGTTTCACATCNACATGGTTATTGAAAACATG CCNAGATGATGGGTNNATATGTTTATNTACATTTTTAnTGATGAAGTGTTATGTGTGTGTGATAACACCTCAGAAGACA CCANTGAAACGTCGAAAAAATTCCNAAATGAA RIPPT1013 pPT1013.scq (SEQ. ID. NO. 337) 20 ACATCCCTGTGGGGATGAGTTATGTGTCTGTGTGTGTGTGTGTGTGTGTGTGTGTAAAAGCNAAANGCAAANATGTGTG TAAGGACTTCTANAGGTANACATANAACATTTGATGCTTAAGAGGCATAACNAATCACATGCTATANCAAACCACATG CTAACAAAGATTAGTAACATATTAAGGAATAAACAGAACTCAATACATGTCATGGTCAAGCCNTGANAGGTTGGATCA AACTNGAAANATAGTGGGTTTTGAOAC RIPPTl 023 pPT1023.seq (SEQ. ID. NO. 338) ATCACGCTTGATCACTTAGCCTACTANGTTTGAGTTACTCGTCrCTTCCAATCCCTAATATTCTACCCTTATXGCTTAAGT AATATGTGAATTACTTGGGCCTTTCTACCCTCAGAGCATGGTCGCAATGGTTTTATTGTCCAAGGCnTATTACGACTTA GTGTCTGAGCCGTTATGTTATTGAGACTTAGCCTCCACTTGGTGCATATGCACATAAATATGCATAAGGCTTGAACCCG atggattttcaaatgaagtcagcctagtcttggttgtagtggttttgttcccaatttgacctaagaatagttttatgagg 30 ccccttggcctccaacctacacacacacacacacacacacacacacacacacacatataattgttaatcctcctgagct tacagtttgtaaaccaagg .

R1PPT1027 pPTI 027Jeq (SEQ. ID. NO. 339) ccttagattctaatcgtactgtgtataatacaaaagcatgtgttacagtgttgattgtgtgccagtatgtatttcatata 3 5 tatgtgtgtgtgtgtgtttgtgtgtgtgtgtgtgtgtgtgtgtta atggcttg aantcctgtgtttataagtgtattg att gaccatcgatcactgtagcatcgatggtgatccaaagatatttgagttaactgttgtatggagtgatggcatgcttaaa cagagtgtttccaaattgtggcagatctgtgttttttaatgcagagatccatcanaatgatcaanattgatttaaggaa gaaatggacaagaacacatagaaaccgtcagatctggaagatcaatgttccanatcaaatcgcatggagcanaacctt tttatcacatcggcaaaaatcccntgggtgat 40 R1PPT1035 pPT1035.seq (SEQ. ID. NO. 340) gctcaacagttttgtaagtgtcgaggcatatctttgctatggtcttagtcattgggatttcaaaaacaaaactaccccat TTTCATTTTTGTCTGCA ITI'I'I CCGTAATCTTGGTGTGCTAAACCAAGGGCATGCTGGTTTTGGAAACTTTATCCATATTT GGTAACACAAACTCTCAAGAAAATACTGATTAAGGATGTCTAATACATAATGGAAGGTAACCCAAGACTCAGTGAACT PCT/USOO/0032S 46 AGCTTTGATTTTGAAACCATAATGAGCCCTTCTCGTTACTCATAATTTATAAATTGGCAGGTGTGTGTGTGTGTGTGTGT gtgcgtgtacacattaccatgcaaaggaacgccgatgactttaaatggaggctaaggtgtttaacanaggggagtgtt gtgttaaatgggggggagggacacatattcttgatatggaagtgtgtgacacctcttcnagattttgtgaggaacaatg aatctgaacaatgtcntgangataaactcgacnataaaaaatgccacaatgtaataaaatgcattggt RIPPTl 036 pPT1036.seq (SEQ. ID. NO. 341) ccttgttatatctagcttcatcgaangtggtcgttacctccttaccctcgaatacctagactcaaggtgttaattaaaag GTAATTTAGTTAGAAACATAACCAAGCTAGCAAGAGGAGTAGCATCACTAGATATCCGAGACTTTGAACCTTGGATrAA CGAGAAACACACATAATTCTATA'l'rrn'ATGATACAAAGGTn'CrrriLl 1GCAGGTCA ATAG AGTGCATGGTTGTGCG A GATCACAATATGTTTGCAACTATGCTAGATTAGTAGGAAGTTTTGAGATCATCGACAACrGTGTGTGTGTGTGTGTGTGT gtgtgtgtgtgtgtgtgtctgtgtgtgtgtgtgtgtgtgcattgtccantcaattgggtttattttaggtggttagttgta GTGATTGGGATCCTCCCCACCCAATTTCAATTCCCTCAAGTGACATGATTAGCTTTGATATAATGGTTTAGACCCTTGTT GTTGAGAATGAATANTCCCTAAGATTAACGGGAT RIPPT1037 pPT1037.seq (SEQ. ID. NO. 342) ACAAATGCTCAATATAGACCACTTGCATCATATTTGTTTGTTGATCTAGATGAATGATGGTGCXjcgcgcgcacacacac ACACACACACACACACATGTATGAGATAGTTAACAANGTTGATGACAGGTTGATTATTCCTTTTGTTGAATTATGGCTA TTTGATTGAGAGAATTTTGTTGAAGGATTTTAGTANTAAGATGTGTTGTTGAGTTAGAGTTGTATGAATGCTTAAGATTG AGAGTATGGATGAGGACAAAGTATGCTACTGCCAAATCATATGATGTTAACATTGATGTCTAAGTATATGTTGATGATG GAGAGTGATTTTGTGGTTTAATGCTGCTAGATnTGTATTGCAGCAAGTTTTTGGAGTCTCGGTATAAAAGATAAGGGA AAAGAGAA RIPPTl040 pPTI040.seq (SEQ. ID. NO. 343) ACATACATGTCATGTAGGCITGAGGTGGATATGTOCAAAAGTCATGTCTCCTCAAGGAATTCATTGGAGCCACATGTGA ATATATATGTGTGTGTGTGTGTGTGTGTGTGAGGTTATTAGACCATCCAAGAGAAACATTTCATAGAGAGATCATCTCC AATGGTAGCCACAACACAAGAAGGAAGGGGTCGAGGAGAACAAGGGAAAAT1TGAGGTATGTTTTATGGGTTTGATAT GGCCAAAGTGTAGAAGAGGATAGCCAAGTGAATTATGGGCAGTAAGGTGCATATTTTAGQGTGTAATTCTCCTTGTnT GGAGGGCCACAAGCTCrGTGGGACATGGTAGATACCGTGGGATTTTTTTGGGATTACTGGAGGGGTCATAAGG RIPPT1066 pPT1066.teq (SEQ. ID. NO. 344) ATCCTTAGTAACAGTTGTTCTCTGTTTACACAGAGAACATTGTGAAAACATGGGAAATTTOGTAGAAAACATAGCTOCT AGACTTGAAATTCTCATAACACCACTTTATAGTGCCATCGGGTCCGTCTATTGGTTGTCGTCGCTTATTGCAATGGCTCT TTCTCCTTCACATTTCGTTTAAGAAATGAAATTGTGAAAATACATTAAAAAOGGGGTGTTTGATGGATCTCGAGATCCA TATAATATGGCTTGTAATATTATCGCGTCCAATCCATCGGATCACCCATATATATATATGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGNGCATGCATGTCTATCCCTGCTTTGNTGTGTTCGCTGATTTCGATCCGNCAATATCGGNGAT CATGGTATCGNATCAGCATC RIPPTl 072 pPT1072.seq (SEQ. ID. NO. 345) ATCACCTAGTCTGCCCCTAGTGTGATG'ni'L I' IATCTCCAAAG AGTCTTCCTTGTAAACGAGACTCACAAAGTGAATTTT TTCACTCTTTATTTTACTAATTTGAAGTTTTCATGACCTrGGAGTGGATTCACACACACACACACACACACACACACACA TATTTTTTTACAAAATGTTAATATTTTATGTATTTTTTGGTTGACTAGTCCAGTTTTGATGACATTGTTGAGGAAGTTGTG ACTGTTATTGCGAAATATAACATCGCAGTGCAAAGTTCACTAAGCACTCTAGAATAGGAGCAACAATGGGATCAATCA CCAGTGTAAGCGTAGCAACCATTCCCCCGCTATTCTOTGGTTGGATATTATCOCCACAAGGGAGTCTTCCCTAGTAGAA TTT AGGG ACTATATA ATGTTA AGCCCTTA ATA AGCCTCATGTTACCCATA A ACCTCTT ATTAAGCCCTAG ATATTG AGTG PCT/USOO/00325 Al attacttatctattggt atattggtatgtaggctataacccctcatggt RIPPT1076 pPT1076.seq (SEQ. ID. NO. 346) gctagcatgtaaatgtgtaaacccaggctaggctgaggcacatttaagcataggagggataacactcgtttgtatcttt atacataatatgtgcgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgcaggctacagtt ngaggggctttgtgaaatcatatgtatgacaagccaaattttggagtaagaccccaaattcataccaataaccactaca tctaatacttggactaantccntcgaaaaaccactgggttcaagccttatgcatatctatgtgcatatgcatcaatcgg gggtngagtctctgttggcattatggcatatgttngatgctatggctctggtgttgtcctngatggcaactcaacttggt gacaaatcccagatcaggcacccagaaagaagaaaaagtcatatacgcnggttcatctanacaaagacgtgt RIPPTI077 pPTI077.seq (SEQ. ID. NO. 347) ATCTCTCTTCGTTGGGCATATTTCCTACCATA A ATCACAGCCCA AGTCTATGACCTACTGTA ACATTCT AGCATGCOCCA CATATTTTTATCAAACACAGGTCTCGCAATACATCTAATTACAGATTAAGGAATTGGATTACATTTTGCCATGAAGTGG AAAATTTTACTTTGTTCACCGCACAATAGTCATATTCAAATTCTTACCTTCCTTTTGTGTGTGTGTCTGTGTGTGTGTGTG 15 TGTGTATATATATATATAAAGAGGAGAGACATCCACCACAAAGAAACTATTGGATTTCCTCTTAGAACTTAAAAAAAAA AACATTAACAATTTCAATTAAGGACAAAGAGAAATAATTTTTCTTTTTTGCCACACCTATTGAAAATAGAAACAAGAAA ATGCTAAAAATAGAAGTGCTAAAAATAACACTTCTATAAATCGCAATTTGGGT RIPPTl 125 pPTI I26.seq (SEQ. ID. NO. 348) 20 CCTTGTGAGGCTAAAAAGGAGAAGAGGAATTTTTTTTTATGGTGTTGTGGAGAAGAGAATTTGAGAAAGGATTGTCCCT GAAGGAAGAAGATGAAATAAGTTAGCCACTATGAGCCACACAAACATGCATCAACGATGCAGGATGTTGAGAANGAG GACGGGGCTTCCCCTCAAGCTCCTTCACACACACACACACACACACACACACACACACACACACACACACACACACAC ACACACACACACACACACATATACATATTTGTGTGTGTGTGTGTCGAAAATATCCCTCGTGAACTTTTGGGAAATGTAA GACTTCNAGGTTATTGTGAGCCTTGTGAANAANTCTTGATGTGATTTTGGTATATGATTATGCGTATGT RIPPTl 137 pPTI 137 jeq (SEQ. ID. NO. 349) ACAATCACrcCTGTATTAATTAGAAGAGTCAAAATTCTCTTAAGCAATGTATCTATCTATCTATCTATCTATCTATCTAT ATATATATATCCAAAAATTCCTCAGCAATTGATCCCTACAAATGAGGCATGAGGGTGAAGCTTATCACAATGCACAAAG ^ AGAAGGCAAGATTTACCTTGGGAAAACCCACTAAGGGGAAAAAACTAACAACCTTTTCAATAGAGXAATGCTTTTGTTC AACAAGGGACACACCTAGACCCTTCTAGTCATTAAATAGTTCACACTTGGTCAAACCATCTAAGTCAAGCCTCCCAATC TAATCCAACACTTGGCATTTACAGATCTACCCAAAAnTCAACCCTCTTGCAACTGCTAGATTCCCAAAATTTCGGCCCC ATGCAACTGCCTAGAATTrCAGCACCTACAATGGAAACTGCTCATAAATATGTGAACTATCGATAGATAGATAGATAGA GATAGATAGATAGATAGATAGATAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGTATCCCACGTG CGAGCTTGAGAGAGAGAGAGAAATAGAGATGCCANT RIPPT9058 k>bseq3-11 -97clone249 (SEQ. ID. NO. 350) CCCGCTCCTATTCAAGATCAAAAGAAGGATATGTTAAAGGTCAAGTGCTTCATTTGTCAAAACnTGGACACTATGCAT CAACAACCAATGTTGTTGTTGATGATGATGATGATGATGAGCATCCACCTCAAAAGAAGTCAAAGGAATTCTTCCTTTA GGAGCTACTACCAATAATGCTTGAGGGTAATTATGCCTCTAGGCGCCTGATCGGTTTCCAAGATTCTCTTATAGGTTTTT 40 AGGGARGGAGTTGACAATCCTCAACAATTTCTTCCTTAATCAACAGATAAGTATGTCATATTTTTCTCTTGATTATT RIPPT9104 lobseq3-7-97ATCclonc71 (SEQ. ID. NO. 351) CCTTTCTGAATGACAAAAGGGCTTCTACGNCXrrTCCTATCGTCAGCGTCCATGGATGATGATGATGATGNAAATTCAGG NTGCTCCACAGTGCCTTCATCGTCCTTCAAAAGAGATTGAAGAGAAGCACTTCCACCAGTCGTGCGCTCATACAGATGA 48 TCATGGTGTAAAGCATGACCCCTGTGAACATGNGAGTAATGATATGGCTTCATGGAATAACTAATAGTTGCCAGGAGAA GAAGCCACACTAGTATGATTATCGACACAGCACTTTTCTCCTTTCTCTCTTTATCTCAATTCGAAAGAANTCNAAGANTG GCCTGTGATTNCCTTGTTTGC3GGGCNCC RIPPT9138 lobseq5-2-97ATC 187 (SEQ. ID. NO. 352) ATTGAAACCAATTTTTCCCCTTnTAATGTAAAAAATATCAAAATAAATAACAAATTATATAATAATTCATTATAAAACA ATATAAATTTTAACATATATATGAACTTTGAAATAAACCTTAATGGTGATGATGATGATGATGGTGGTGGTTAACGTCC ACTGAGACCAAATTGGTCTATCGGACCTATAATTTCTTAGTTTTTACTTATCTGGCTCCTTGTCTTTCTTGGCGAGATTAG TGTraTAGCTTTCrrTTTTTTCTCTACAGTCTITCCAACTTCCTTTATCTTGCATCTCCCTCCACTCCrCTCGTTCCrTGGGA AATTGGTGTCTNACTTCCCAANCCATNGTGTTTCATCCTTTGAAGG RIPPT9238 lobseq8-19-97TTC2DI (SEQ. ID. NO. 353) CCCTGAGACATCCAATCCATGTGTTTTTCXACCATTTATTTATCATTTTATTTTCCTTCTTCTTCATAATTTGATTAGTCTT CITCTTCTTCTrCTTCTCCGCCCAACTCATGTAAAGTATCATCTAAGAGCACTACATTATCACCATCAtXATCATTTAAG GTTGAAGGTTTTTCTAGTTTTTCTATATTTTATTTAGAGTCTAACTATTCAAGGGTGGTAAGAACCAACTCTTCAACTGTT GACACCATTATCCTANTGGGGGTGGTTTGGTTCTNATTCACAAAATACGGAAAAGTTTCTATTCTGGATCCTTTAGA RIPPT9315 lobseq12-4-97ACI-G8 (SEQ. ID. NO. 354) CCTCTTCCTAGTAGGGGGATTACTTGGAGGAGGATCCGAGGGGTCCATAGGGTCCATGCGATCATCATCAACCTCCTTT AAACATATCCATCGAAAGTTACATCCTTGATAAACTCGATCTTCCTTTTGCTATATGTGTGTGTGTGTGTGTGTGTGTGT GATTTAGAGTTTTCACAATATCCAACAAACATATCTTTCTTTATGGTGGCTTCCAACTTGTTCCTCTTGTCCTTCNGCACA TGGAAATATACGOG AC AACCA AAT ATCAT PCT/USOO/00325 49 * The PIC and H values arc presented in Table 4 for the 18 polymorphic loci amplified from the first 89 primer pairs that were synthesized.

Table 4 Allelic diversity data for SSR loci in 20 Pinus taeda trees RIPPT SSR repeat in cloned allele # alleles allele size PIC H locus ranee, bp 1 (A)«...(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), (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-..(ATDl9 225-267 0.859 0.916 32 (TAT)7...(A)s 6 173-189 0.586 0.653 33 (TAT), (SEQ ED NO:357) 3 169-178 0.410 0.484 64 (A)6C(A)S(AAAC), (A)s (SEQ ID NO:358) 11 233-261 0.751 0.818 65 (AAAC)S(A)7 (SEQ ED NO:359) 9 130-139 0.816 0.879 66 (AAAT), (SEQ ID N0:360) 3 98-110 0.577 0.685 67 (AAAT)4 (SEQ ID NO.-36J) 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),, (SEQ ED NO:364) 154-180, 0.581 0.691 - null 79 (ATT),j (SEQ ID NO:365) 8 130-161 0.723 0.765 80 (ATT),...(AAI)6 4 247-263, 0.438 0.525 null 89 (TTC)10...(T)S 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 35 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 40 PCR amplified region. The REPPT71 locus had no repeats other than (AAAT)„. 50 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, polymorphism among species was scored from high resolution agarose gels, so the number of polymorphic 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 (bo) RIPPTl* (ATC) 260 _i (+r (+) - (+) (+) y RIPPT2 (ATC) 185 - <+) (+) (+) (+) (+) y -R1PPT4 (ATC) 145 - - - - D RIPPT6* (ATC) 290 . 3' 1 1(+) K+) 2 y RIPPT7 (ATC) 105 1 4 1 2 1 2 y RIPPT9 (ATC) 120 1 1(+) 1 1 (+) 1 y RIPPTl 1* (ATC) 170 1 (+) (+) 1(+) (+) 1(+) y . RIPPTl 3 (ATC) 105 1 3 1 3 y RIPFT16CATC) 220 t 1 1 I 1 1 n RIPPT19 (ATC) 105 1 1 1 I n RIPPT20(ATQ no 1 1 1 1 - 1 n RIPPT21 (ATC) 185 1 1 1 K+) 1 n RIPPT22* (ACC) 245 1 1 1 - 1 y RIPPT24* (AAT) 150 1 1 1 - y RIPPT26 (AAT) 190 1 1 1 1 1 n RIPPT27 (AAT) 130 1 1 - 1 y RIPPT29 (AAT) 210 1 (+) K+) K+) y RIPPT30 (AAT) 215 1 H+) 1 K+) 1<+) K+) y RIPPT3I* (AAT) 245 1 1 1 2 (+) y RIPPT32* (AAT) 180 I 1 1 1 I 1 y RIPPT33* (AAT) 170 1 1 1 - 1 y RIPPT35 (AAC) 270 1 1<+) K+) 2 1 D RIPPT37 (AAC) 110 I 1 I 2 1 y RIPPT38 (AAC) 270 1 I 1 1 1 1 n RIPPT40 (AAC) 190 1 1 1 1 I I n RIPPT42 (AAC) 230 1 1 1 I 1 1 y RIPPT43 (AAC) 145 I 1 1 1 1 1 y RIPPT44 (AAC) 210 1 I i 1 K+) 1 n RIPPT5I (AAG) 260 I 1 1 1 1 I n RIPPT52 (AAC) 1 1 1 1 - 1 n PCT/US00/D0325 51 RIPPT54 (AAG) 175 1 1 1 1 1 1 a R1PPT56 (AAG) 390 - - - - 2 2 y RIPPT58 (AAG) 235 1 1 1 1 - - y RIPPT64* 345 - 1 2 1 (+) I y (AAAC) RIPPT65* 135 1 - - y (AAAC) R1PPT66* 105 1 1 1 1 1 - y (AAAT) RIPPT67* 220 - 1 1 1 2 1 ' y (AAAT) RIPPT69* 145 - 1 1 1 2 1 y (AAAT) RIPPT71* 240 t 3 1 1 2 1 y (AAAT) R1PPT74 (AAT) 130 1 1 2 (+) - - n RIPPT75 (AAT) 205 1 1 1 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 1 - 1 y RIPPT80* (AAT) 250 2(+) 3 (+) I 1 (+) y RIPPT86 (ACQ 260 1 2 - (+) - - y RIPPT88 (AAG) 235 1 1<+) - (+) (+) (+) y RIPPT89* (AAG) 225 1 K+) - 1 (+) K+) y RIPPT90 (AAC) 150 2 1 1 1 (+) l(+> v bp = approximate allele size for P. taeda tree 7-S6.

PAS = Polymorphic Among Species * = an asterix indicates that the locus was polymoiphic 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.' 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 pairs tested # amplifying single locus 566 118 498 119 566 566 168 47 127 38 84 138

Claims (4)

1. WO 00/42210

   PCT/USO0/00325

   52

   The 54 unique loci that were polymorphic in P. taeda were also polymorphic 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 monomorphic in P. taeda, but were polymorphic among species (Table 7). Loci that are monomorphic within a species but polymorphic 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 N0:370)

   (ATC)e

   125

   GAGATGCCCCTAGGTTCTCC (SEQ ID NO:371)

   RIPPT27

   TCCACAGCCATCACCACTTA (SEQ ID NO:372)

   (ATT)6(GAT)6

   132

   TGGGTCCGATAGACCAATGT (SEQ ID NO:373)

   RIPPT29

   TAAGGTTTCACCAAGGGCTG (SEQ ID NO:374)

   (ATT)1S

   189

   TCATGGGGTCAATTCTCCTC (SEQ ID NO:375)

   RIPPT30

   ATGGATGGAAAATTTCTATAGCC (SEQ ID NO:376)

   (ATT),3

   236

   ATGTTTCC A ATT A AAGGATTTCC (SEQ ID NO:377)

   RIFFT58

   GCCTTGCAAAGTGACCTCTC (SEQ ID NO:378)

   (AGG)4

   240

   TCCATGACAACCCAGTTCAA (SEQ ID NO:379)

   RIFPT81

   GAGAACGCGCGACTGTATTA (SEQ ID N0:380)

   (ATT)4...

   178

   TTTCCCATCTGGTTCATGTG (SEQ ID NO:381)

   (ATT)S...(ATT)

   RIPPT86

   CCAATTCTTTGAAGTATTATAG (SEQ ID NO:382)

   (ATG)5(GTG)7

   262

   GATCGCGAAGCTAAGACACC (SEQ ID NO:383)

   REPPT90

   TCGATCACAGTGTTGGCATT (SEQ ID NO:384)

   (TTG),

   150

   GCCAAGCCCATTCAGTTTTA (SEQ ID NO:385)

   RIPPT314 AGAGGTTGCAGGAAGCAAAA (SEQ ID NO:386)

   (GAA)«

   142

   ATTGGTTTCTCCATCGTTGC (SEQ ID NO:387)

   RIPPT914 AGGCGAAGCTTATGGAACAA (SEQ ID NO:388)

   (GAT),

   143

   I

   TGTTTCCCGATCCTCTGTTC (SEQ ID NO:389)

   1

   2

   1

   2

   1

   2

   I

   1

   2

   1

   2

   3

   1

   2

   1

   2

   3

   >

   1

   2

   1

   2

   3

   1

   2

   1

   2

   3

   1

   2

   WO 00/42210

   PCT/US00/00325

   53

   WHAT IS CLAIMED IS:

   I 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 genomicDNA is from a pine.

   10 The polynucleotide of claim 8 wherein said genomic DNA is from a species of the Pinus subgenus.

   II 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

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   3 of: (A)n...(ATG)n; (ATG)n...(C)n; (CAT)n...(A)n; (ACC)n...(ATC)n; (TTG)n...(TTA)n;

   4 (C)n...(ATT)n; (TAT)„...(A)n; (ATT)„...(AAT)n; (TTC)n...(T)n; and (A)n(AAAC)n(A)n.

   1 15 An isolated SSR locus comprising a SSR motif, wherein said motif is

   2 selected from the group consisting of AC, AAC, AAG, AAT, ACC, ACG, AGG, ATC,

   3 AAAC, AAAT, and AGAT, and all complements and permutations of said motif.

   1 16 The isolated SSR locus of claim 15, wherein said SSR motif forms a

   2 compound repeat, which may be perfect or otherwise, selected from the group consisting

   3 of: (A)„...(ATG)n; (ATG)n...(C)„; (CAT)n...(A)„; (ACC)n...(ATC)D; (TTG)n...(TTA)II;

   4 (C)n...(ATT)„; (TAT)„...(A)n; (ATT)n...(AAT)n; (TTC)n...CT)n; and (A)n(AAAC)n(A)n.

   •jl 17 The isolated SSR locus of claim 15, wherein said locus is polymorphic in

   2 at least one pine species.

   1 18 The isolated SSR locus of claim 15, wherein said locus is polymorphic in

   2 at least one species of the Pinus subgenus.

   1 19 The isolated SSR locus of claim 15, wherein said locus is polymorphic in

   2 at least one species selected from the group of P. taeda, P. caribaea, P. ponderosa, P.

   3 radiata, P. resinosa, P. strobus, and P. sylvestris.

   1 20 The isolated SSR locus of claim 15, wherein said locus is RIPPTl,
2. 2 RIPPT6, RIPPTl 1, RIPPT22, RJPPT24, RDPPT31, REPPT32, RIPPT33, RIPPT64,
3. 3 RIPPT65, RIPPT66, RIPPT67, RIPPT69, RIPPT71, RIPPT77, RIPPT79, RIPPT80, and
4. 4 RIPPT89.

   1 21 An isolated polynucleotide comprising the nucleotide sequence selected

   2 from the group consisting of SEQ ID NOS:"237-354.

   1 22 The isolated polynucleotide of claim 21, wherein said polynucleotide is a

   2 maximum of about 500 nucleotides long.

   1 23 An isolated polynucleotide that hybridizes under stringent conditions to the

   2 polynucleotide of claim 21.

   1 24 The isolated polynucleotide of claim 23, wherein said polynucleotide is a

   2 maximum of about 500 nucleotides long.

   1 25 An isolated polynucleotide having at least 85% homology to the

   2 polynucleotide of claim 21.

   1 26 An isolated polynucleotide having at least 85% identity to the

   2 polynucleotide of claim 21.

   1 27 A method of detecting the presence of a SSR locus comprising a SSR

   2 motif of the sequence (N,, N2...Ni)n, wherein: N represents nucleotides A, T, C or G; i

   3

   4

   5

   6

   7

   8

   1

   2

   3

   4

   5

   I

   2

   3

   1

   2

   3

   4

   1

   1

   1

   2

   3

   4

   5

   6

   1

   2

   1

   2

   1

   2

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   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)n...(ATG)n; (ATG)n...(C)n; (CAT)....^),,; (ACC)n...(ATC)n; (TTG)n...(TTA)n; (C)n...(ATT)n; (TAT)n...(A)n; (ATT)n...(AAT)n; (TTC)n...(T)n; and (A)n(AAAC)n(A)n.

   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 (Nt, N2...Nj)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.

   END OF CLAIMS