WO1999053095A2 - Biallelic markers - Google Patents

Abstract

The invention provides nucleic acid segments of the human genome including polymorphic sites. Allele-specific primers and probes hybridizing to regions flanking these sites are also provided. The nucleic acids, primers and probes are used in applications such as forensics, paternity testing, medicine and genetic analysis.

Description

BIALLELIC MARKERS

RELATED APPLICTIONS

This application claims priority to U.S. Application No. 09/057,871 filed April 9, 1998, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution, generating variant forms of progenitor sequences

(Gusella, Ann . Rev. Biochem . 55, 831-854 (1986)). The variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms.

Several different types of polymorphism have been reported. A restriction fragment length polymorphism

(RFLP) Is a variation in DNA sequence that alters the length of a restriction fragment (Botstein et al . , Am. J. Hum. Genet . 32, 314-331 (1980)). The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment. RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; W090/11369; Donis-Keller , Cell 51, 319-337 (1987); Lander et al . , Genetics 121, 85-99 (1989)). When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait.

Other polymorphisms take the form of short tandem repeats (STRs) that include tandem di-, tri- and tetra- nucleotide repeated motifs. These tandem repeats are also referred to as variable number tandem repeat (VNTR) polymorphisms. VNTRs have been used in identity and paternity analysis (US 5,075,217; Armour et al . , FEBS Lett . 307, 113-115 (1992); Horn et al . , W0 91/14003; Jeffreys, EP 370,719), and in a large number of genetic mapping studies .

Other polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPs, STRs and VNTRs . Some single nucleotide polymorphisms occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Examples of genes, in which polymorphisms within coding sequences give rise to genetic disease include β-globin (sickle cell anemia) and CFTR (cystic fibrosis) . Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing) . Other single nucleotide polymorphisms have no phenotypic effects .

Single nucleotide polymorphisms can be used in the same manner as RFLPs and VNTRs, but offer several advantages . Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. The greater frequency and uniformity of single nucleotide polymorphisms means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms . The different forms of characterized single nucleotide polymorphisms are often easier to distinguish than other types of polymorphism (e.g., by use of assays employing allele-specific hybridization probes or primers) .

Only a small percentage of the total repository of polymorphisms in humans and other organisms has been identified. The limited number of polymorphisms identified to date is due to the large amount of work required for their detection by conventional methods. For example, a conventional approach to identifying polymorphisms might be to sequence the same stretch of DNA in a population of individuals by dideoxy sequencing. In this type of approach, the amount of work increases in proportion to both the length of sequence and the number of individuals in a population and becomes impractical for large stretches of DNA or large numbers of persons.

SUMMARY OF THE INVENTION

The invention provides nucleic acid sequences comprising nucleic acid segments of from about 10 to about 200 bases as shown in the Table, column 7, including a polymorphic site. Complements of these segments are also included. The segments can be DNA or RNA, and can be double- or single-stranded. Segments can be, for example, 10-20, 10-50 or 10-100 bases long. Preferred segments include a biallelic polymorphic site. The base occupying the polymorphic site in the segments can be the reference (Table, column 3) or an alternative base (Table, column 4) . The invention further provides allele-specific oligonucleotides that hybridize to a segment of a fragment shown in the Table, column 7, or its complement. These oligonucleotides can be probes or primers. Also provided are isolated nucleic acids comprising a sequence shown in the Table, column 7, or the complement thereto, in which the polymorphic site within the sequence is occupied by a base other than the reference base shown in the Table, column 3.

The invention further provides a method of analyzing a nucleic acid from an individual. The method determines which base is present at any one of the polymorphic sites shown in the Table. Optionally, a set of bases occupying a set of the polymorphic sites shown in the Table is determined. This type of analysis can be performed on a number of individuals, who are tested for the presence of a disease phenotype. The presence or absence of disease phenotype is then correlated with a base or set of bases present at the polymorphic sites in the individuals tested.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

An oligonucleotide can be DNA or RNA, and single- or double-stranded. Oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means. The oligonucleotides of the present invention can comprise all of an oligonucleotide sequence presented in column 7 of the Table or a segment of such an oligonucleotide which includes a polymorphic site. Oligonucleotides can be all of a nucleic acid segment as represented in column 7 of the Table; a nucleic acid sequence which comprises a nucleic acid segment represented in column 7 of the Table and additional nucleic acids (present at either or both ends of a nucleic acid segment of column 7); or a portion (fragment) of a nucleic acid segment represented in column 7 of the Table which includes a polymorphic site. Preferred oligonucleotides of the invention include segments of DNA, or their complements, which include any one of the polymorphic sites shown in the Table. The segments can be between 5 and 250 bases, and, in specific embodiments, are between 5-10, 5-20, 10-20, 10- 50, 20-50 or 10-100 bases. For example, segments of the invention can be 5 , 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 nucleotides in length. The polymorphic site can occur within any position of the segment. The segments can be from any of the allelic forms of DNA shown in the Table. In a particular embodiment, the nucleotide at the polymorphic site is different from the nucleotide in a corresponding reference or wild-type allele.

Hybridization probes are oligonucleotides which bind in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al . , Science 254, 1497-1500 (1991) .

As used herein, the term primer refers to a single- stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions ( e . g. , in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template. The term primer site refers to the area of the target DNA to which a primer hybridizes. The term primer pair refers to a set of primers including a 5 ' (upstream) primer that hybridizes with the 5 ' end of the DNA sequence to be amplified and a 3 ' (downstream) primer that hybridizes with the complement of the 3 ' end of the sequence to be amplified.

As used herein, linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome. It can be measured by percent recombination between the two genes, alleles, loci or genetic markers. As used herein, polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymorphic locus may be as small as one base pair. Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu . The first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic or biallelic polymorphism has two forms. A triallelic polymorphism has three forms. A single nucleotide polymorphism occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations) .

A single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymorphic site. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine or vice versa. Single nucleotide polymorphisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele. Typically the polymorphic site is occupied by a base other than the reference base. For example, where the reference allele contains the base "T" at the polymorphic site, the altered allele can contain a "C", "G" or "A" at the polymorphic site. Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM NaPhosphate", 5 mM EDTA, pH 7.4) and a temperature of 25- 30°C, or equivalent conditions, are suitable for allele- specific probe hybridizations. Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleotide sequence and the primer or probe used.

The term "isolated" is used herein to indicate that the material in question exists in a physical milieu distinct from that in which it occurs in nature. For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances) , buffer system or reagent mix. In other circumstance, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC . Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present.

I. Novel Polymorphisms of the Invention

The novel polymorphisms of the invention are listed in the Table. The first column of the Table lists the names assigned to the fragments in which the polymorphisms occur. The fragments are all human genomic fragments . The sequence of one allelic form of each of the fragments (arbitrarily referred to as the prototypical or reference form) has been previously published. These sequences are listed at http://www-genome.wi.mit.edu/ (all STS ' s (sequence tag sites)); http://shgc.stanford.edu (Stanford STS's); and http://ww.tigr.org/ (TIGR STS's). The Web sites also list primers for amplification of the fragments, and the genomic location of fragments. Some fragments are expressed sequence tags, and some are random genomic fragments. All information in the websites concerning the fragments listed in the Table is incorporated by reference in its entirety for all purposes.

The second column lists the position in the fragment in which a polymorphic site has been found. Positions are numbered consecutively with the first base of the fragment sequence as listed in one of the above databases being assigned the number one. The third column lists the base occupying the polymorphic site in the sequence in the data base. This base is arbitrarily designated the reference or prototypical form, but it is not necessarily the most frequently occurring form. The fourth column in the Table lists the alternative base(s) at the polymorphic site. The fifth column of the Table lists a 5 ' (upstream or forward) primer that hybridizes with the 5' end of the DNA sequence to be amplified.

The sixth column of the Table lists a 3' (downstream or reverse) primer that hybridizes with the complement of the 3' end of the sequence to be amplified. The seventh column of the Table lists a number of bases of sequence on either side of the polymorphic site in each fragment. The indicated sequences can be either DNA or RNA. In the latter, the T's shown in the Table are replaced by U's. The base occupying the polymorphic site is indicated in EUPAC-IUB ambiguity code.

II. Analysis of Polymorphisms A. Preparation of Samples

Polymorphisms are detected in a target nucleic acid from an individual being analyzed. For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. For assay of cDNA or mRNA, the tissue sample must be obtained from an organ in which the target nucleic acid is expressed. For example, if the target nucleic acid is a cytochrome P450, the liver is a suitable source . Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols : A Guide to Methods and

Applications (eds. Innis, et al . , Academic Press, San Diego, CA, 1990); Mattila et al . , Nucleic Acids Res . 19, 4967 (1991); Eckert et al . , PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al . , IRL Press, Oxford); and U.S. Patent 4,683,202.

Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren et al . , Science 241, 1077 (1988), transcription amplification (Kwoh et al . , Proc . Natl . Acad. Sci . USA 86, 1173 (1989)), and self-sustained sequence replication (Guatelli et al . , Proc . Nat . Acad. Sci . USA, 87, 1874 (1990)) and nucleic acid based sequence amplification (NASBA) . The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively. B. Detection of Polymorphisms in Target DNA There are two distinct types of analysis of target DNA for detecting polymorphisms. The first type of analysis, sometimes referred to as de novo characterization, is carried out to identify polymorphic sites not previously characterized (i.e., to identify new polymorphisms) . This analysis compares target sequences in different individuals to identify points of variation, i.e., polymorphic sites. By analyzing groups of individuals representing the greatest ethnic diversity among humans and greatest breed and species variety in plants and animals, patterns characteristic of the most common alleles/haplotypes of the locus can be identified, and the frequencies of such alleles/haplotypes in the population can be determined. Additional allelic frequencies can be determined for subpopulations characterized by criteria such as geography, race, or gender. The de novo identification of polymorphisms of the invention is described in the Examples section. The second type of analysis determines which form(s) of a characterized (known) polymorphism are present in individuals under test. There are a variety of suitable procedures, which are discussed in turn.

1. Allele-Specific Probes

The design and use of allele-specific probes for analyzing polymorphisms is described by e.g., Saiki et al . , Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms.

Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence.

2. Tiling Arrays

The polymorphisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described in WO 95/11995. One form of such arrays is described in the Examples section in connection with de novo identification of polymorphisms. The same array or a different array can be used for analysis of characterized polymorphisms. WO 95/11995 also describes subarrays that are optimized for detection of a variant form of a precharacterized polymorphism. Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence. The second group of probes is designed by the same principles as described in the Examples, except that the probes exhibit complementarity to the second reference sequence. The inclusion of a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (e.g., two or more mutations within 9 to 21 bases) .

3. Allele-Specific Primers

An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res . 17, 2427-2448 (1989) . This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single- base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is included in the 3 ' -most position of the oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).

4. Direct-Sequencing

The direct analysis of the sequence of polymorphisms of the present invention can be accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al . , Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al . , Recombinant DNA Laboratory Manual , (Acad. Press, 1988) ) .

5. Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed. , PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7.

6. Single-Strand Conformation Polymorphism Analysis Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al . , Proc . Nat . Acad. Sci . 86, 2766-2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences. III. Methods of Use

After determining polymorphic form(s) present in an individual at one or more polymorphic sites, this information can be used in a number of methods .

A. Forensics

Determination of which polymorphic forms occupy a set of polymorphic sites in an individual identifies a set of polymorphic forms that distinguishes the individual. See generally National Research Council, The Evaluation of Forensic DNA Evidence (Eds. Pollard et al . , National Academy Press, DC, 1996) . The more sites that are analyzed, the lower the probability that the set of polymorphic forms in one individual is the same as that in an unrelated individual. Preferably, if multiple sites are analyzed, the sites are unlinked.

Thus, polymorphisms of the invention are often used in conjunction with polymorphisms in distal genes. Preferred polymorphisms for use in forensics are biallelic because the population frequencies of two polymorphic forms can usually be determined with greater accuracy than those of multiple polymorphic forms at multi-allelic loci.

The capacity to identify a distinguishing or unique set of forensic markers in an individual is useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining • whether the set of polymorphic forms occupying selected polymorphic sites is the same in the suspect and the sample. If the set of polymorphic markers does not match between a suspect and a sample, it can be concluded (barring experimental error) that the suspect was not the source of the sample. If the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene. If frequencies of the polymorphic forms at the loci tested have been determined (e.g., by analysis of a suitable population of individuals) , one can perform a statistical analysis to determine the probability that a match of suspect and crime scene sample would occur by chance . p(ID) is the probability that two random individuals have the same polymorphic or allelic form at a given polymorphic site. In biallelic loci, four genotypes are possible: AA, AB, BA, and BB. If alleles A and B occur in a haploid genome of the organism with frequencies x and y, the probability of each genotype in a diploid organism is (see WO 95/12607) :

Homozygote: p(AA)= x²

Homozygote: p(BB)= y² = (1-x)²

Single Heterozygote : p(AB)= p(BA)= xy = x(l-x)

Both Heterozygotes: p (AB+BA) = 2xy = 2x(l-x)

The probability of identity at one locus (i.e, the probability that two individuals, picked at random from a population will have identical polymorphic forms at a given locus) is given by the equation: p(ID) = (x²)² + (2xy)² + (y²)². These calculations can be extended for any number of polymorphic forms at a given locus. For example, the probability of identity p(ID) for a 3-allele system where the alleles have the frequencies in the population of x, y and z, respectively, is equal to the sum of the squares of the genotype frequencies: p(ID) = x⁴ + (2xy)² + (2yz)² + (2xz)² + z⁴ + y⁴ In a locus of n alleles, the appropriate binomial expansion is used to calculate p(ID) and p(exc) .

The cumulative probability of identity (cum p(ID)) for each of multiple unlinked loci is determined by multiplying the probabilities provided by each locus. cum p(ID) = p(IDl)p(ID2)p(ID3) .... p(IDn)

The cumulative probability of non-identity for n loci (i.e. the probability that two random individuals will be different at 1 or more loci) is given by the equation: cum p(nonΙD) = 1-cum p(ID) .

If several polymorphic loci are tested, the cumulative probability of non-identity for random individuals becomes very high (e.g., one billion to one) . Such probabilities can be taken into account together with other evidence in determining the guilt or innocence of the suspect.

B. Paternity Testing

The object of paternity testing is usually to determine whether a male is the father of a child. In most cases, the mother of the child is known and thus, the mother's contribution to the child's genotype can be traced. Paternity testing investigates whether the part of the child's genotype not attributable to the mother is consistent with that of the putative father.

Paternity testing can be performed by analyzing sets of polymorphisms in the putative father and the child.

If the set of polymorphisms in the child attributable to the father does not match the set of polymorphisms of the putative father, it can be concluded, barring experimental error, that the putative father is not the real father. If the set of polymorphisms in the child attributable to the father does match the set of polymorphisms of the putative father, a statistical calculation can be performed to determine the probability of coincidental match.

The probability of parentage exclusion (representing the probability that a random male will have a polymorphic form at a given polymorphic site that makes him incompatible as the father) is given by the equation (see WO 95/12607) : p(exc) = xy(l-xy) where x and y are the population frequencies of alleles A and B of a biallelic polymorphic site.

(At a triallelic site p(exc) = xy(l-xy) + yz(l- yz) + xz(l-xz)+ 3xyz (1-xyz) ) ) , where x, y and z and the respective population frequencies of alleles A, B and C) .

The probability of non-exclusion is p(non-exc) = l-p(exc)

The cumulative probability of non-exclusion (representing the value obtained when n loci are used) is thus: cum p(non-exc) = p (non-excl) p (non-exc2 )p (non- exc3 ) .... p(non-excn)

The cumulative probability of exclusion for n loci (representing the probability that a random male will be excluded) cum p(exc) = 1 - cum p(non-exc) .

If several polymorphic loci are included in the analysis, the cumulative probability of exclusion of a random male is very high. This probability can be taken into account in assessing the liability of a putative father whose polymorphic marker set matches the child's polymorphic marker set attributable to his/her father. C. Correlation of Polymorphisms with Phenotypic Traits

The polymorphisms of the invention may contribute to the phenotype of an organism in different ways. Some polymorphisms occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both beneficial and detrimental, depending on the circumstances. For example, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. Other polymorphisms occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymorphism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymorphisms in different genes. Further, some polymorphisms predispose an individual to a distinct mutation that is causally related to a certain phenotype.

Phenotypic traits include diseases that have known but hitherto unmapped genetic components (e.g., agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, osteogenesis imperfecta, and acute intermittent porphyria) . Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, diseases of the nervous system, and infection by pathogenic microorganisms. Some examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependent and non- independent) , systemic lupus erythematosus and Graves disease. Some examples of cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, skin, stomach and uterus. Phenotypic traits also include characteristics such as longevity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments .

Correlation is performed for a population of individuals who have been tested for the presence or absence of a phenotypic trait of interest and for polymorphic markers sets. To perform such analysis, the presence or absence of a set of polymorphisms (i.e. a polymorphic set) is determined for a set of the individuals, some of whom exhibit a particular trait, and some of which exhibit lack of the trait. The alleles of each polymorphism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest. Correlation can be performed by standard statistical methods such as a κ-squared test and statistically significant correlations between polymorphic form(s) and phenotypic characteristics are noted. For example, it might be found that the presence of allele Al at polymorphism A correlates with heart disease. As a further example, it might be found that the combined presence of allele Al at polymorphism A and allele Bl at polymorphism B correlates with increased milk production of a farm animal . Such correlations can be exploited in several ways. In the case of a strong correlation between a set of one or more polymorphic forms and a disease for which treatment is available, detection of the polymorphic form set in a human or animal patient may justify immediate administration of treatment, or at least the institution of regular monitoring of the patient. Detection of a polymorphic form correlated with serious disease in a couple contemplating a family may also be valuable to the couple in their reproductive decisions. For example, the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymorphism from her husband to her offspring. In the case of a weaker, but still statistically significant correlation between a polymorphic set and human disease, immediate therapeutic intervention or monitoring may not be justified. Nevertheless, the patient can be motivated to begin simple life-style changes (e.g., diet, exercise) that can be accomplished at little cost to the patient but confer potential benefits in reducing the risk of conditions to which the patient may have increased susceptibility by virtue of variant alleles. Identification of a polymorphic set in a patient correlated with enhanced receptiveness to one of several treatment regimes for a disease indicates that this treatment regime should be followed.

For animals and plants, correlations between characteristics and phenotype are useful for breeding for desired characteristics. For example, Beitz et al . , US 5,292,639 discuss use of bovine mitochondrial polymorphisms in a breeding program to improve milk production in cows. To evaluate the effect of mtDNA D- loop sequence polymorphism on milk production, each cow was assigned a value of 1 if variant or 0 if wildtype with respect to a prototypical mitochondrial DNA sequence at each of 17 locations considered. Each production trait was analyzed individually with the following animal model:

Y__kp„= μ + S_X + P_: + X_k + β_x + ... β₁₇ + PE_n + a_n +e_p where Y_llknp is the milk, fat, fat percentage, SNF, SNF percentage, energy concentration, or lactation energy record; μ is an overall mean; YS₁ is the effect common to all cows calving in year-season; X_k is the effect common to cows in either the high or average selection line; β_x to β₁₇ are the binomial regressions of production record on mtDNA D-loop sequence polymorphisms; PE_n is permanent environmental effect common to all records of cow n; a_n is effect of animal n and is composed of the additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect; and e_p is a random residual. It was found that eleven of seventeen polymorphisms tested influenced at least one production trait. Bovines having the best polymorphic forms for milk production at these eleven loci are used as parents for breeding the next generation of the herd.

D. Genetic Mapping of Phenotypic Traits The previous section concerns identifying correlations between phenotypic traits and polymorphisms that directly or indirectly contribute to those traits. The present section describes identification of a physical linkage between a genetic locus associated with a trait of interest and polymorphic markers that are not associated with the trait, but are in physical proximity with the genetic locus responsible for the trait and co- segregate with it. Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait. See Lander et al . , Proc . Natl . Acad. Sci . (USA) 83, 7353-7357 (1986); Lander et al . , Proc . Natl . Acad . Sci . (USA) 84, 2363-2367 (1987); Donis-Keller et al . , Cell 51, 319-337 (1987); Lander et al . , Genetics 121, 185-199 (1989)). Genes localized by linkage can be cloned by a process known as directional cloning. See Wainwright, Med. J. Australia 159, 170-174 (1993); Collins, Nature Genetics 1, 3-6 (1992). Linkage studies are typically performed on members of a family. Available members of the family are characterized for the presence or absence of a phenotypic trait and for a set of polymorphic markers. The distribution of polymorphic markers in an informative meiosis is then analyzed to determine which polymorphic markers co-segregate with a phenotypic trait. See, e . g. , Kerem et al . , Science 245, 1073-1080 (1989); Monaco et al . , Nature 316, 842 (1985); Yamoka et al . , Neurology 40, 222-226 (1990); Rossiter et al . , FASEB Journal 5, 21-27 (1991) .

Linkage is analyzed by calculation of LOD (log of the odds) values. A lod value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction θ, versus the situation in which the two are not linked, and thus segregating independently (Thompson & Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company, Philadelphia, 1991); Strachan, "Mapping the human genome" in The Human Genome (BIOS Scientific Publishers Ltd, Oxford) , Chapter

4) . A series of likelihood ratios are calculated at various recombination fractions (θ) , ranging from θ = 0.0 (coincident loci) to θ = 0.50 (unlinked) . Thus, the likelihood at a given value of θ is: probability of data if loci linked at θ to probability of data if loci unlinked. The computed likelihoods are usually expressed as the log₁₀ of this ratio (i.e., a lod score) . For example, a lod score of 3 indicates 1000:1 odds against an apparent observed linkage being a coincidence. The use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of lod scores for differing values of θ (e.g., LIPED, MLINK (Lathrop, Proc . Nat . Acad. Sci . (USA) 81, 3443-3446 (1984)). For any particular lod score, a recombination fraction may be determined from mathematical tables. See Smith et al . , Mathematical tables for research workers in human genetics (Churchill, London, 1961); Smith, Ann . Hum . Genet . 32, 127-150 (1968) . The value of θ at which the lod score is the highest is considered to be the best estimate of the recombination fraction.

Positive lod score values suggest that the two loci are linked, whereas negative values suggest that linkage is less likely (at that value of θ) than the possibility that the two loci are unlinked. By convention, a combined lod score of +3 or greater (equivalent to greater than 1000:1 odds in favor of linkage) is considered definitive evidence that two loci are linked.

Similarly, by convention, a negative lod score of -2 or less is taken as definitive evidence against linkage of the two loci being compared. Negative linkage data are useful in excluding a chromosome or a segment thereof from consideration. The search focuses on the remaining non-excluded chromosomal locations. IV. Modified Polypeptides and Gene Sequences

The invention further provides variant forms of nucleic acids and corresponding proteins. The nucleic acids comprise one of the sequences described in the Table, column 8, in which the polymorphic position is occupied by one of the alternative bases for that position. Some nucleic acids encode full-length variant forms of proteins. Similarly, variant proteins have the prototypical amino acid sequences encoded by nucleic acid sequences shown in the Table, column 8, (read so as to be in-frame with the full-length coding sequence of which it is a component) except at an amino acid encoded by a codon including one of the polymorphic positions shown in the Table. That position is occupied by the amino acid coded by the corresponding codon in any of the alternative forms shown in the Table.

Variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter. Usually, the promoter is a eukaryotic promoter for expression in a mammalian cell. The transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host. The selection of an appropriate promoter, for example trp, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected. Commercially available expression vectors can be used. Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.

The means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra . A wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli , yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e . g. , mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post- translational modification, and the like.

The protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology Volume 104, Academic Press, New York (1984) ; Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer-Verlag, New York (1987); and Deutscher (ed) , Guide to Protein

Purification, Methods in Enzymology, Vol. 182 (1990). If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If not secreted, the protein can be isolated from a lysate of the host cells.

The invention further provides transgenic nonhuman animals capable of expressing an exogenous variant gene and/or having one or both alleles of an endogenous variant gene inactivated. Expression of an exogenous variant gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinjecting the construct into a zygote. See Hogan et al . , "Manipulating the Mouse Embryo, A Laboratory Manual," Cold Spring Harbor Laboratory. Inactivation of endogenous variant genes can be achieved by forming a transgene in which a cloned variant gene is inactivated by insertion of a positive selection marker. See Capecchi, Science 244, 1288-1292 (1989) . The transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous variant gene. Mice and other rodents are preferred animals. Such animals provide useful drug screening systems . In addition to substantially full-length polypeptides expressed by variant genes, the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides. Biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding. Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.

Polyclonal and/or monoclonal antibodies that specifically bind to variant gene products but not to corresponding prototypical gene products are also provided. Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof . Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies , A Laboratory Manual , Cold Spring Harbor Press, New York (1988) ; Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986) . Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.

V. Kits

The invention further provides kits comprising at least one allele-specific oligonucleotide as described above. Often, the kits contain one or more pairs of allele-specific oligonucleotides hybridizing to different forms of a polymorphism. In some kits, the allele-specific oligonucleotides are provided immobilized to a substrate. For example, the same substrate can comprise allele-specific oligonucleotide probes for detecting at least 10, 100 or all of the polymorphisms shown in the Table. Optional additional components of the kit include, for example, restriction enzymes, reverse-transcriptase or poly erase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin) , and the appropriate buffers for reverse transcription, PCR, or hybridization reactions. Usually, the kit also contains instructions for carrying out the methods.

The following Examples are offered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachings of all references cited herein are hereby incorporated herein by reference.

EXAMPLES The polymorphisms shown in the Table were identified by resequencing of target sequences from three to ten unrelated individuals of diverse ethnic and geographic backgrounds by hybridization to probes immobilized to microfabricated arrays or conventional sequencing. The strategy and principles for design and use of such arrays are generally described in WO 95/11995. The strategy provides arrays of probes for analysis of target sequences showing a high degree of sequence identity to the reference sequences of the fragments shown in the Table, column 1. The reference sequences were sequence-tagged sites (STSs) developed in the course of the Human Genome Project ( see, e . g. , Science 270, 1945-1954 (1995); Nature 380, 152-154 (1996)). Most STS's ranged from 100 bp to 300 bp in size.

A typical probe array used in this analysis has two groups of four sets of probes that respectively tile both strands of a reference sequence. A first probe set comprises a plurality of probes exhibiting perfect complementarily with one of the reference sequences. Each probe in the first probe set has an interrogation position that corresponds to a nucleotide in the reference sequence. That is, the interrogation position is aligned with the corresponding nucleotide in the reference sequence, when the probe and reference sequence are aligned to maximize complementarily between the two. For each probe in the first set, there are three corresponding probes from three additional probe sets. Thus, there are four probes corresponding to each nucleotide in the reference sequence. The probes from the three additional probe sets are identical to the corresponding probe from the first probe set except at the interrogation position, which occurs in the same position in each of the four corresponding probes from the four probe sets, and is occupied by a different nucleotide in the four probe sets . In the present analysis, probes were 25 nucleotides long. Arrays tiled for multiple different references sequences were included on the same substrate.

Multiple target sequences from an individual were amplified from human genomic DNA using primers for the fragments indicated in the listed Web sites. The amplified target sequences were fluorescently labelled during or after PCR. The labelled target sequences were hybridized with a substrate bearing immobilized arrays of probes. The amount of lable bound to probes was measured. Analysis of the pattern of label revealed the nature and position of differences between the target and reference sequence. For example, comparison of the intensities of four corresponding probes reveals the identity of a corresponding nucleotide in the target sequences aligned with the interrogation position of the probes. The corresponding nucleotide is the complement of the nucleotide occupying the interrogation position of the probe showing the highest intensity (see WO 95/11995) . The existence of a polymorphism is also manifested by differences in normalized hybridization intensities of probes flanking the polymorphism when the probes hybridized to corresponding targets from different individuals. For example, relative loss of hybridization intensity in a "footprint" of probes flanking a polymorphism signals a difference between the target and reference (i.e., a polymorphism) (see EP 717,113). Additionally, hybridization intensities for corresponding targets from different individuals can be classified into groups or clusters suggested by the data, not defined a priori , such that isolates in a give cluster tend to be similar and isolates in different clusters tend to be dissimilar. Hybridizations to samples from different individuals were performed separately. The Table summarizes the data obtained for target sequences in comparison with a reference sequence for the individuals tested.

From the foregoing, it is apparent that the invention includes a number of general uses that can be expressed concisely as follows. The invention provides for the use of any of the nucleic acid segments described above in the diagnosis or monitoring of diseases, such as cancer, inflammation, heart disease, diseases of the CNS, and susceptibility to infection by microorganisms. The invention further provides for the use of any of the nucleic acid segments in the manufacture of a medicament for the treatment or prophylaxis of such diseases. The invention further provides for the use of any of the DNA segments as a pharmaceutical.

All publications and patent applications cited above are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application were specifically and individually indicated to be so incorporated by reference .

ω 0⁾ ω β O

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4-t SNP Forward SNP Reverse 0⁾

Marker Name a Primer Primer Sequence

WIAF-i,^" GGCAGGGAAGGAAAATCCTAGGGNCAGCATTGGGGAGGGGGGGACTCTC [C/ ] AAATT

KST291092 49 TATTGGGCAACAGGCTGCAGGTGAGGGGGCTGACAGGAGGAGGGT

TATTGCAGCTGAAATACTATTTTCGTTAAGTCTCGGACACTTAGACCCACTGATCCTGTT IAF-176, ACTCTGCTTGTCTCTGGTNTGCAGGGAATCA [C/T] TTTGCTGGATTAGAGGAAAGGTGC EST226740b 91 CGCCGTCTGTTTCCATGACTT

TGACTTGTTGCAGTGAAAGAGCCACACACCAGAGGAACCATGAGGCATCTCACCAAATGT AGGAGATTGATTATTATACATTGTTGGGGAAGGTGGTGTTTACGTGACATTTAAATGATC

WIAF-3038, AGTGTCTTACTAGGCTCACAG [C/T] AAGGCAAAGCTGAATGTGAAGGGGCAATGTCAGG J WIR-1410 201 TCTGGAATGCAAAGTG

CTGTTGTACATCCTTOCCAACGTGTOTTTTAAA^ ϊ^ATTTC^TCC^T^YCAATA

WIAF-3648, AAAGGGTCATTAAAAACAAAACAAAATTGTGAAAAAA [A/G] AGAAATAAGAATGTGTCT stSG28751 97 CTGTTGCACAACTGCATTCTATCCTT

GATCTGTTCCCCATATTTGGCACCCGAGACAACACTTAGGACACTATAGTGTGTGCCAAT GGTTACTGGAAGGCTTAGGACATTTATCCTTCTGTTGTCCCCACCTCACTGGGAACCATT TCACTTATCATCTCTCTTGTTTGGGATCAGCAATCCTAACTATGGAGCTCTTCCACTCAG GGTACCACTCGCACTTATGGGAGTTAAGTAATATAATAGACCATTGACTGGGAAATCAAA AGTCATATTCTTATGATGGAAAGTGGGAACAAAAGGCTAGTACACGAGATGTTATTTCAG

WIAF-2688, CCTTGGTCCAACACCCTATTGCTCCCCACCTTAAAATTGCTATGTCTATGTACAATCTTT WTR-866f 369 A AAGGAGTT [A/G] CTTATTGGGGCAGTTTCAAACTCAGAAAT

TGCCAGATCAAATCAAACCACCTTTTATAGAAAAGGAGCCAAAGCGTTGACTGTGAGCTT TAGTCACGATCATTTTCCTCTAAGCCCATACAAACCTTTCTCAGAAACTGCACAAAGGAG

WIAF-3261, ATGAACTGAAA [T/A] TATCCCAGGACACAGGGAAGGAAGGGCAAACCCTGGGAAAAGGA st-SGI 5935b 131 AGGCAAATGAGCTATACCTTTGATGGGGCTCAGCTAG

TGAGCAAGAATGATGGAATGAAGAGGAAAGATGATGGTACTTGAGAGTATCATC [C/T] G AAATATGTAGAAGAGAAATAAGCCCCAAGTAACAAACTCATGGCAAACCAAACTTTCCTC

WTAF-3042, CCTTTTCCTGCCAACAACGTCATTGAACCATTATTGCCATTCATTATCAGATGCCTACAC WIR-1415 54 TGCACTGGCCTGAGGGATATGGAGAT

CO

0

Co O

CAACAACTTTCTGTTTGACCCAAGGTCTCCAGATACTGGATAATGGGCTTTTTCAAAAAA TACCTATAGGGCCGGGCGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAG GGGGGCGCATCACGAGGTCAGGAGATTAAGACCATACCTGGCTAACACAGTGAAACCCCG TCTCTACTAAAAATACAAAAAATTAGCCGGGCATGGTGGCGGGCGCCTGTAGTCCCAGCT ACTGGGGAGGCTGAGGCAGAAGAATGGCGTGAACCCGGGAGGTGGAGCTTGCAGTGAGCT

WTAF-3141, GAGATCGTGCCACTGCACTCCA [G/A] CCTGGGCGACAGAGTGAGAGTCTGTCTCAGACA WIR-1704d 322 AACAAACAAACAAATAAATGCCTGTAAACAGACTGCCTGGTGCTCT

CCTTTGAGGAGTTAAGCTAATATTGAATTTTTCTCATTATATAACCCTTTGATTTA [T/G ]TCACTTACCCTCAGCCACTATTCCTCCCTCTGTCCCTTTATATCAGTTGTTTCCAGGTT TGGGAGTGACATTAGGTTTGTCTGCTGGGCTGGCCTAGACTGCAGGCAGCAATAGTATTC TAGCATGTCTTCCCTCAGTCTAGTCTTGATCATAGAGGGTAGGTTATATAGGTAAGGAAC TAGTGGGGGCTATCAGACCACCAGGCTATATAACTCTACTTACTGTTAATCCTAACTTTT

WIAF-3142, CAGATGAAATGAATACTTGAGAATTCTTACATAAAGGTGTAAAAATATAGTTATGGTTTT WIR-1275b 56 TCGCTTAGGGATAATTCCTGTTTCTGGCACTTTTATTTACATCCC

CCTTTGAGGAGTTAAGCTAATATTGAATTTTTCTCATTATATAACCCTTTGATTTATTCA CTT [A/G] CCCTCAGCCACTATTCCTCCCTCTGTCCCTTTATATCAGTTGTTTCCAGGTT TGGGAGTGACATTAGGTTTGTCTGCTGGGCTGGCCTAGACTGCAGGCAGCAATAGTATTC TAGCATGTCTTCCCTCAGTCTAGTCTTGATCATAGAGGGTAGGTTATATAGGTAAGGAAC TAGTGGGGGCTATCAGACCACCAGGCTATATAACTCTACTTACTGTTAATCCTAACTTTT

WIAF-3147, CAGATGAAATGAATACTTGAGAATTCTTACATAAAGGTGTAAAAATATAGTTATGGTTTT WIR-1275C 63 TCGCTTAGGGATAATTCCTGTTTCTGGCACTTTTATTTACATCCC

CCTTTGAGGAGTTAAGCTAATATTGAATTTTTCTCATTATATAACCCTTTGATTTATTCA CTTACCCTCAGCCACTATTCCTCCCTCTGTCCCTTTATATCAGT[T/C]GTTTCCAGGTT TGGGAGTGACATTAGGTTTGTCTGCTGGGCTGGCCTAGACTGCAGGCAGCAATAGTATTC TAGCATGTCTTCCCTCAGTCTAGTCTTGATCATAGAGGGTAGGTTATATAGGTAAGGAAC TAGTGGGGGCTATCAGACCACCAGGCTATATAACTCTACTTACTGTTAATCCTAACTTTT

WIAF-3154, CAGATGAAATGAATACTTGAGAATTCTTACATAAAGGTGTAAAAATATAGTTATGGTTTT WIR-1275g 104 TCGCTTAGGGATAATTCCTGTTTCTGGCACTTTTATTTACATCCC

CATGTGCTGCATGAAGAGCTAATTTAAAAAAGCAAAGTAAGACTAATTATTTAAAATAAA AATGCCACAAATTTCATTTTCTCCTTCTAAGTATTACAATGGAGTTTATTCTCTGCCTAA AAAGTGGAAGAA [A/C , T] TTGAGTGAATGATAATTTTGTAATTTAGGATAAGATCCAAG TTATTTTCCCCAACTCTTGTTTCCCCCATAAAGTTAGGCATGAGGAGGAGCACTCATTAA

WIAF-4037, TGGAAGAAATTG GGATCTTATCCT AGGCAGAAGACGGAAAAGTGTTTTTAAAATGGTGAATTTAAGTGGTAAGGATTTTCTCTT 7963 132 C,T AGTGA AAATTACAAA ACTCTGTTTATTTTTAAATGATCATCATAATCCTTTGCTTACTATTTATGCAGCTT

0

AAGTACCTTGATTTGAAAGAGCTGTGACCAACCTCAGTGGCATGTCCAACTACCACGCTG CAGAAGTCTGTNAGGCCGTGGTCCTCAGCCCTTTGCACAGATTAAAACCCCAATCCCAGC

WIAF-1528, CGCACCCCAGACCAATCAATCAGGATCTCTGGAGATGGGGCCCAGGCATCTATATATATG stSG9569b_ 191 T TATATATATAT [A/T] TTTTTTTAATTCCTCAGATGATTCCAATGTACAGC WΪAF-3782, TGCGTACGTCAGAGCTGCCTCCGAAGTGGTAAAATGTGCTGCGAGAAGTGGAGCCGCGTG stSG26432b_ 79 T GCANAAATGTTTCTCTTCA [C/T] TGAGGAGCGGGAGGATTGTAAGATACTG

AAAATATACAGTGTGCCGGAGCCCTATGGCCTCTTTTACTCTTTGGCACACATTTTCTGA

WIAF-3233, AGCAACATAAGCCAAAT [C/G] TTTGTGTTTATCTTACACGAAAGAGACCAGTATCTTTC WTR-1635a 77 TTTTGGCTTGGGTGGCTCACAACTTTTGTTCTTTAAATGAGTGGATATCCAAGGAAAAA

AAAATATACAGTGTGCCGGAGCCCTATGGCCTCTTTTACTCTTTGGCACACATTTTCTGA

WTAF-3239, AGCAACATAAGCCAAATCTTTGTGTTTATCTTACACGAAAGAGACCAGTATCTTTCTTTT WTR-1635d 127 GGCTTGG [G/T] TGGCTCACAACTTTTGTTCTTTAAATGAGTGGATATCCAAGGAAAAA

TCTTGACAAAGTCACTGCATCTCTCAAAGCAGCCCAAGCCCCAGGACTCTCTCCGTGCCT TATAGATTCTCATAAACTCTTCCTCCCCTCTATCCTTCAAGCCTAGGG [G/A] TGGTGAA

WIAF-3245, AATTTCATCACTAACACAGGGTACCTGTCACCATCGTTTGTTATTTCCTGCATACCCTAC WIR-1279b 108 TCACACCTTTATCACTTTGTCTTTT

TAGTGAAGTTAAACAAACATTGGGATACCNACACAGACAACTTGAGACAGTTTTTTTAAT

O

WIAF-136, CTTTTAATCAGTGTAGCTATGTGCTGCTGCTGCTCAAGAGCTGGATCCATACAGGTTGTG EST198420b 122 TG [C/T] CATCTGTCCTCTTGAGGTTCAGGAGAT

CAAATCCTGAGAGAATTTAAAACCCCAGATTTGCCATACAAGAGCTCCTGAAAGGAGTGC TAAATATGGAAACACTCTTACCATCTAATTCAAAAA [T/C] GCACTTAAGTACATAGACC AGTGACACTATAAAGCAACCACATAAAAAAGTCTGCATAATAACCAGCTAACAACACAGT GACAGAATCTAATCCATACATATCATACTAACTTTTAATGTAAATGGGCTAAAAGCCCAA

WIAF-3053, TTAAAAGGCAGAGTGTCAAGCTGGATAAAGAAGCAAGAGCCAAAGCTATGCTGTCTTTAA WTR-1563a 96 GAGATTCATCTCACATGAAATGACACTCAGGCTCAATAAATAAATAAAAGGGGA

GCTCAGCTAGCTTTGAAATTGGCTGATGAAAAAATATACATAAAAGGGTAAAATTCACAC ATACAGCAAACAAAAATGCACAAAGCCTGCTTCGTAACTTTTTTTTCTGGAATTGTTTTT

WIAF-2554, CACTTTGCCTTTTTCTGCCAAAACAATAATCAAAGAACTCTTGCTTTAACCTATTCCTGT A002V42 50 T C ACAAAG WΪAF-1539, CTAGTCCTTATTCCCATGCAGGACTAACTGCAGGCAGCACATTTGTATCTNGCTTAAGTT stSG1437 71 G T TTGACTTTGGA [G/T] TCAAGTTTTTATTACAGGTGTCATGATTGCCAA

C

Cπ 00

Cπ vo

σ

ι_ ι_o

J

en

σ

oo

<_n

VD

GCAGTCAGTACTTAAGAGTCTAGAGAACAAAGAGGTGAACCAGCTGAAAGTCTCGGGGAA

WIAF-1661, GCTTACATGTGTTGTTAGGCCTGTCCCATCATTGGAGTGCACTGGCC [A/G] TCCCTCAG A004B36 ATTTGTCTGGGCTGGCCTGAGTGGTCACCGCTTTGAGAAAGCTGTTA _

GGTCTTCCATCCTTTCTTAGGTTACAGTAAATGAGAGTTTGTGCCAG [A/C] AGTGAGAA AACTAGAGAAAGGGTGCCACCTGTGCTTTCTACCCCAAACCAAAGATGAAGGGGCTCAGA

WIAF-2813 , CAAGAGTGCCTGATTCCAGGGCTCAACAGTGAGGGTTCCTTCCCTGCCTGCATTCAGCTT WTR-807c TGTCTGCAGAATGGTGGAAAAGAA _ WTAF-149, CTTTCAGGAGTCTATACCCTG [A/C] TCTGGGTCGNCTCTGTATTGGGGGTTGCTAACCA EST115878 GTGCAAT

ACGAGTCCCTGCTGTGACTTG [G/A] GTGCCATCAGTGTAGAGCTATGTGTGTCCACACC AGTCGTGAAAGGCCCATGGATTCATGCCTGATGCTGATAGGACAAGGCAGTGGAATAGCC

WIAF-3375, ACAAACGTGAATTTACCATCTGGCTGTTGTAAAAATCATTGTCTGGAGGACCTAGAGAGA stSG15857 AAGTGTTGCATAACTTCCGGG _

CAGATTTTAAAAAATGTTCAGTGTGCACAATTGTTACTAC. AGATCTTTGTCAGAATAAT

WIAF-3750, CTTTCTGTCACTGAATAACTGACCCATAAAACAATAGAGCATATTGACCTAGTGTGCAGG stSG29781a GTAAGCAATGAG

TTTTAAAAAATGTTCAGTGTGCACAATTGTTACTACCAGATCTTTGTCA [G/A] AATAAT t

WIAF-3753, CTTTCTGTCACTGAATAACTGACCCATAAAACAATAGAGCATATTGACCTAGTGTGCAGG stSG29781b GTAAGCAATGAG

AAAAATCCTGTCAGATTGTGTTGAATCAATAGATTGTTTTGGGGGAGAATTGCTATGTAA

WIAF-3379, AAATTGGAGTCCTTCAAC [G/A] CATGACAATGGAATGTCTCTTTAGTTACTΓAGGTCTC WIR-2087a TACTTTCTCTCTGCAGTGTTTTGTTGCTTGTTTG

AAAAATCCTGTCAGATTGTGTTGAATCAATAGATTGTTTTGGGGGAGAATTGCTATGTAA

WIAF-3381, AAATTGGAGTCCTTCAACGCATGACA [A/G] TGGAATGTCTCTTTAGTTACTTAGGTCTC WlR-2087b TACTTTCTCTCTGCAGTGTTTTGTTGCTTGTTTG

AAAAATCCTGTCAGATTGTGTTGAATCAATAGATTGTTTTGGGGGAGAATTGCTATGTAA

WTAF-3383, AAATTGGAGTCCTTCAACGCATGACAATGGAATGTCT [C/G] TTTAGTTACTTAGGTCTC WIR-2087c_ TACTTTCTCTCTGCAGTGTTTTGTTGCTTGTTTG

ATGTTCTAACAAAAGACCAATGACATTTATAAGGGAGAATAAAATAAGCAACAAATTTAA

WIAF-152, AGCG [A/C] AACTAGGNTACTTTATGCTGTCTGCATTTGTATAGTGGATTTTGGTGCTAA ES1333606 GATGCAAGTCTGAAC

CATGTAGGGCTCGACTCCATCTTTAGAAAGTGAGTTCACTGAAGGTGAGTTAAGGACAAG GCTCTGAAGAGCGGCCTGACTTGCTTTCTTTCCTTGTGTTTGTGGAGGGGTGTAGACAAA

WIAF-3398, GACACTAAGTGAAGATGCAGCCCCCAAGGCTGT [A/G] TTGCCTAAAAACCGTTTCTGGC stSG15867

TTCTTCACAGGAATATTCCTCGTGGGTCTAGAAGGA

e

^4

0

vo

00 o

0

o

vo O

vo to

v

C

vo

o

Cπ

vo

ON

vo

o

00

GCTGCNCTGAATACTATAGACAGTTGTAACACACTGGCAGGGATTTGC [G/A] TATCTAA

WlAF-1682, ACATAGAAAAGGTACAGTAAAAAATACGGTATTATGGGGCCACCATCATATGTTTGGTCT stSGl 0082a 48 GTCGTGGACTAAAATGTCATTAT

GCTGCNCTGAATACTATAGACAGTTGTAACACACTGGCAGGGATTTGCGTATCTAAAC [A

WIAF-1683, /C ] TAGAAAAGGTACAGTAAAAAATACGGTATTATGGGGCCACCATCATAT GTTTGGTCT SLSG10082b GTCGTGGACTAAAATGTCATTAT _ _ _ _

CCCAAGAATGGAGGAGTTCAAGCGCAGTCTTGATGACTGTGGCAACACTGTAGACAGCAA TTGTTGTTTTGGCTGTCCAGTATCTAAGCACTCTTCTTATTTAGGGGAATCCCAATATGA

WIAF-2683, GTTAAGGGGGAGCCCC [G/A] CTAAAGAAGCTGATGGGGCAGATGTTCrGNCTTTCAGCT MR842a 136_ TCCTGACAGATGATGCCCAGCATATG

CCCAAGAATGGAGGAGTTCAAGCGCAGTCTTGATGACTGTGGCAACACTGTAGACAGCAA TTGTTGTTTTGGCTGTCCAGTATCTAAGCACTCTTCTTATTTAGGGGAATCCCAATATGA

WIAF-2685, GTTAAGGGGGAGCCCCGCTAAAGAA [G/A] CTGATGGGGCAGATGTTCTGNCTTTCAGCT MR842b 145 TCCTGACAGATGATGCCCAGCATATG

GGTGCCATCTCTAACTTGCCTCCGTATCTTCTTAAAAGGCTTTCAGG [A/C ] GACAGTTC GTTATTTCCTCCTGGAGCCTGACAAGGCAGGGGGCTCTCTTTCACACTCCCACTTAGGGT

WIAF-2846, GCAGCTTCAGGCTATAAAACTTCTTGCTTCAGCGTGGCGAGGGCTCTTCAGCAACAGGAT o vo WTR-818b 47 A C GTTCCACCACTCACGTTGCAGTCATC _ WIAF-3943, TAAAACATTCTTAGCTTTTCTTGCAGTTTGFTCCNAAAAGATTTGATTGGGCACAAGAGG EST350474 69 A G AACGAAATT [A/G] TTAATAAAATAAAAGCTTATTTTTGTTTTGCTG T

TTGGAAAAGAAGGGGTTCTGTATCCAGAGGGGGTTCCATACCCACATCGTCGGTGGTG[G

WIAF-3951, /A] GCAGTGTGGCCACATGCCCGGATGGCAAAGCGGNCTATCTAGCAGGCAGCTAT CTCC EST386227a GCCTGGTTTTTGTGCTT

GATCCAATTTGTAGCTTCCTGCCTGGCTTCAGAGAGCCCAGCAACCTTCTAGGCCTGCTT

WIAF-3801, TCCAGACTTCTGAGATAGCCTGGGATGAGCAATCCTGTTA [C/T] AGTACATCTGGACCT stSG26818 100 TCCCTACCTGGGCTCTG

TGACACTTTCCCTGAGGTCCTTCCTACTTCTCACTAACATAGAGATGGCATCTGCCACCA TTTATAGTTTCATCCCAAAGGACAAAAACACTAGAAAAACAACAGAATGTAGCCTACTCT

WIAF-63, AAGGNCTAAAAGGAAATGTAATTCAAAATTCCTCTTCTTATGTG [C/T] ATACATTGATT MR14813b_ 164_ T TTTTTTTCTTTAACTTAGTGTTTCCAACAGTCAAATGACTC WIAF-3822, GCCTCTT [C/T] CCCTGGAAGCTGCACATCACGCAGAAGGACAACTACAGGGTCTACAAC stSG26825 67__ T ACGATGCC

ACTGATGGCCAGGACTCACTCAGACCAATTAAATCAGGATTATGGGCATTTTTAAAAAAA GCTTCTCAGGTGAT [C/T] CTAAAGTATAACTAGATTTGAGAACCAGAAGACTCAGACTG

WIAF-3285, TGAGCTTCTTGAGGGTAGAAATTGTGACTGCTTGTTTATACAGCTCAGGACTCAATGCTG SLSG15972 74 GGACACCGTGGTGCTTCATAATCACCGGGGΓGAT

o o

o

C

o

e

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00

v

TGAAAGGTGAACCTAAGGCAGAAATTTATAGCCAATATGAAGAGGAAGTTAGAAGTTGAG AGATATTAAAAGGGGATAACCCGGC [G/A] TACTTGGTGATTGTCTGATCAAAGAGGAAG AATAAAGAACAGGTGGGGTTAATTGTAAGTTACCGTTCTGATTTCTGACTTTTGTGGATG ATTGGAAGGTGGTGTCATTCGCTGAGAGAATGAATACAGGGAGCTAGGTCTATTTCGAAA

WIAF-3223, AGAAAGGTTGAGTAAGTTTGACACAGTTTGACTTTAACATGTCAGTGAAAGTTGAAGGTA WIR-1441a ACAAAGTTTCATTTGCAGTTAGAGGTGTCTCAATAGAGCGGAAGTATCTGCATTGA

TGAAAGGTGAACCTAAGGCAGAAATTTATAGCCAATATGAAGAGGAAGTTAGAAGTTGAG AGATATTAAAAGGGGATAACCCGGCGTACTTGGTGATTGTCTGATCAAAGAGGAAGAATA AAGAACAGGTGGGGTTAATTGTAAGTTACCGTTCTGATTTCTGACTTTTGTGGATGATTG GAAGGTGGTGTCATTCGCTGAGAGAATGAATACAGGGAGCTAG [G/T] TCTATTTCGAAA

WIAF-3224, AGAAAGGTTGAGTAAGTTTGACACAGTTTGACTTTAACATGTCAGTGAAAGTTGAAGGTA WIR-1441b 223 ACAAAGTTTCATTTGCAGTTAGAGGTGTCTCAATAGAGCGGAAGTATCTGCATTGA

TGAAAGGTGAACCTAAGGCAGAAATTTATAGCCAATATGAAGAGGAAGTTAGAAGTTGAG^" AGATATTAAAAGGGGATAACCCGGCGTACTTGGTGATTGTCTGATCAAAGAGGAAGAATA AAGAACAGGTGGGGTTAATTGTAAGTTACCGTTCTGATTTCTGACTTTTGTGGATGATTG GAAGGTGGTGTCATTCGCTGAGAGAATGAATACAGGGAGCTAGGTCTATTTCGAAAAGAA

WIAF-3228, AGGTTGAGTAAGTTTGACACAGTTT [G/C]ACTTTAACATGTCAGTGAAAGTTGAAGGTA WIR-1441C 265 ACAAAGTTTCATTTGCAGTTAGAGGTGTCTCAATAGAGCGGAAGTATCTGCATTGA

AGCAGAGGGGGAATTTGGCAATATCTAAATATTCGAGCCAGCGGTTCAGTGGTACCAGTG TGCAACAGCTGATCTACCAGCCTGGGCATTGAGTCCTGT [C/T] GTCTAGGCTTTGTGTA

WIAF-3023, CAGTGCTTGGGCCAAGCTGGGGCTTGGAGGAGCTTTACAGACTCTATTGGCCCCCACACA WIR-603 99 TGCTGGGCCCCTGAAGCTGGGTGTC

ACTGCTCTTGAGGTCTTGGCCACACACCCACACCCAGCACCCCAACTCACCCCCTGCACC CTTGCCTTTCTGTCTGGAGTTGGCCCCACTCCAAG [A/C] GGCCCCCAGATCCTCCCCTC

WIAF-3025, TGCGTTTATCACTCTGGTTTCTATTCCTTCACCCCTCAGCCTTCTATAAAAAGCCAACAG WIR-602 95 TAATGACCCTCAAGCAGGTACAGTTCCCA

ATTCAACAAATACTTTTCAGTAGCCTATTATGTAATTAAATTAAGTTTAACAAACATTTC TTTAATGAACCATACACATGACTGTAGATATAATAAAGAGTACAACC [T/C] GATCCCTG CTCTTGATGAGCTCACAGTTTAGCAAGAGGAACGTATAACAAGTCAAAACAACTAATCCT

WIAF-3028, AATAGTTGTTATGAAAGAGCAGTGGAGATACAAAGAGGAAGTAGTCACCTCAAACTGGAA WIR-604 107 AAGCAATGGTAGGTAAAGAGACAGGAAGGTATTCCAGGT

TGTAAACACCACATTTATTTGTCTGAGGCTTGCAAACCTCTGGTAAGAAGNACAGAACGC AGGGCTCTCCTTCATGCTGCCCTGGGCCCCAGCTCGCCAGGCATGCAGGAC [A/G] GAGC

WIAF-150, TGGCAGATGAGTCAGAAATCTTTGGGAGCAGCGCCAGGGAAGCAGCAGGCCCTGCTCCTT HSC0MG122b 111 A CCCATGCCCAATCCCGCCAGCACGGGCCTGGACTGCAGCCAGGAAGGTG

AGCAAACCCATGAGTTTTGGACATTCTGATATAAAAATAGGT ATT [C/T] GCCTTTTTA CAGTGACCACTTAATTATAGAAGCAGTTACTAACACAAGTGTATTTCAAATAATCATGAG

WIAF-3369, TTATGAAAACACATTGGAAAAATTAAATAACCCAAGAGTTTCTCAGCACAGGAAAAACAT stSG15609a 46 GGCCTGGNCCTCTCTGCATGTGCACTGTGCTGCCGTGACAA

AGCAAACCCATGAGTTTTGGACATTCTGATATAAAAATAGGTTATTCGCCTTTTTACAGT GACC [A/T] CTTAATTATAGAAGCAGTTACTAACACAAGTGTATTTCAAATAATCATGAG

WIAF-3371, TTATGAAAACACATTGGAAAAATTAAATAACCCAAGAGTTTCTCAGCACAGGAAAAACAT stSG15609b 64 GGCCTGGNCCTCTCTGCATGTGCACTGTGCTGCCGTGACAA

AGCAAACCCATGAGTTTTGGACATTCTGATATAAAAATAGGTTATTCGCCTTTTTACAGT GACCACTTAATTATAGAAGCAGTTACTAACACAAGTGTATTTCA [A/C] ATAATCATGAG

WIAF-3373, TTATGAAAACACATTGGAAAAATTAAATAACCCAAGAGTTTCTCAGCACAGGAAAAACAT stSG15609c 104 GGCCTGGNCCTCTCTGCATGTGCACTGTGCTGCCGTGACAA

CTCCAGACAATGCACAGCATATACATGATTTATGAAATTCTTACAGTAATTCTGCAGCCA

WIAF-3746, TGGGGCATTTCGACAATATTATTGTCTTCAGAACAATCAG [T/C] ACTACACTGCTTCCC stSG24766 100 ATTAAAACCCA

GTGCAGCTTTAGAACCTGGGAGGCGCTGGCGTTTTTCTATTA [G/A] AGTGTCCTGCAG

WIAF-3749, CTAGAGACACTGAGAAGATCTGATGTTCTAGTTTGAGGGTTGTGCGGCTGAAGACTCAGG to ;tSG29870b 43 GAGGACCTGATGTTGT o

GTGCAGCTTTAGAACCTGGGAGGCGCTGGCGTTTTTCTATTATGAGTGTCCTGCAGCTAG

WIAF-3752, AGACACTGAGAAGATCTGATGTTCTAGTTTGAGGGTTGTGC [G/C] GCTGAAGACTCAGG stSG29870c 101 GAGGACCTGATGTTGT

CACTTTTGATAGATACTGAGTTTTTGCTCATTTGCTACATGAA [A/G] CAGAGGCAGAGT

WIAF-1580, ATTCTGTGTGGGGTTTGGGACAGGAACACTGACCCCTGAAGTCGAGCCTGGGGGGTCTAA stSG3277a 43 CCATAGTGGGTCATTTGTCCNAGCCTGTTTTATGGGAAGGAACT

ATAGTGGAACATCGAACGCCTTTATGCCAGTGTGGCACTTAAGGAAAAGTAACTATGACT

WIAF-1665, AGAA [G/A] AGGCAGAGGAGTGGGTTGAGATTCTGAATTGGGTTCGAAGCAACTTCAGAG stSG8917 64 GCCATAGAGCAAGAACTTCTCGACTCCCAGATGCAGAGGAACCATCA

AAATTTATTCCCATAGTTCTGGAGGC [C/T] AGGAAGTCCAATATCAGTTTTATCGGTAT

WIAF-169, CTGTAGGGCCATGCTCCTGCTGGAAGAGGCTCTGGGGAAGAATCTGTNCTTTGCCTCCTC EST120235a 26 CAGCTTCTGGTG

TGCTTCTAACTTTGCTAGTCCTTGACTGCTCATTCTAAATCTCACTAAAATATTTAACTT TGTCCTGGCATTTATCAAGACAATAGTGCC [T/C] GCGTCCAGTGGTAAAATAAATNCTT

WIAF-158, TTTTTTCCAAAAAAGATGCAAATCTTTTCCTTATATGGATCAATACAGTAGTCAATCTTT MR5689a 90 T GTATTGGTTGATTAGAACTCCTGGAATGTA

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GCCTATAGCCATTACCATGACCACCTTGTTAGTATATCTGGACCATGTCCCACCAGACCC ATAACCTGAGAGAATACTGGCCCTTTGTTGTCAACTAAAATTCCAAGACTGTTTTCCTCT CACTAACTAATTCAGTCTGAAATTTCTCAAGGATGTAATCTGAGGGTTGACTATGACTCT TCCTCCTCCTTCAAACCCAGTATTTTCTTTTCCTACTTCTGTGATTTTCTTTTGGAACCC CTCTTTTATCTGCCTACTCCATTTCTTCACCACTGCCTCACTTGAACCCATCCTGATTAT

WTAF-3200, TAATTGGAACTGTAGAATGAGAATGCTGATTCTTATTCTGGTTCTGTCAA [T/A] ATTTT WTR-1869 350 GCAAACCTTTACCTAATTGTGCTACCTTATTGGTCCCTGAAAACAGGA WIAF-31 , CTCCCAGTCTCTCTCTCTGCA [T/G] AATCTGGTCAAGCAGACGAGAAAAATCAGTTTGT MRI 1901 21 CTCTTTT NCTTTCCAAMTTAAGATTAATGGGAGAAAAGCATTT

TTTTTCTGCAAATAATCTGTGCTAAATTGAGGTTAAAATTATTGTTTTGAAGTTGAAACT

AGTAGCTTAAATGTATTTACCGTAGTTTTTAATGGTAGCTAAGTACCATAGTGCAATTAG

GTCTTGCTTAGAGA [C/A] ATGAATCAATATGAAAATATTTTATTTTCATTTAATTTAAA

AGGGATTAAATGGAGAAGTAAATGGATGTTTGTTACTACCTTCTATTTTCTTAAAAAAGT

AATAGCCTCACCATTATACATTGATTAGGTTACAGTCTAATCAAAGTAAACAGAAACACT

WIAF-2990, GAGAAACAGACAAACAGGTCTGGGTTCTCTGACTAGGAGTTCCGCTTCTGAGATACAGTC WTR-258a 134 TTCTCAGTTTATGCTTGCTCTGTTCTGATACCAGTCGGGGTTGT

TTTTTCTGCAAATAATCTGTGCTAAATTGAGGTTAAAATTATTGTTTTGAAGTTGAAACT CO

AGTAGCTTAAATGTATTTACCGTAGTTTTTAATGGTAGCTAAGTACCATAGTGCAATTAG CO

GTCTTGCTTAGAGACATGAATCAATATGAAAATATTTTATTT [C/T] ATTTAATTTAAA

AGGGATTAAATGGAGAAGTAAATGGATGTTTGTTACTACCTTCTATTTTCTTAAAAAAGT

AATAGCCTCACCATTATACATTGATTAGGTTACAGTCTAATCAAAGTAAACAGAAACACT

WIAF-2992, GAGAAACAGACAAACAGGTCTGGGTTCTCTGACTAGGAGTTCCGCTTCTGAGATACAGTC WTR-258b 163 TTCTCAGTTTATGCTTGCTCTGTTCTGATACCAGTCGGGGTTGT

TGACAGTTTTAATTCTATCATACACAAAGACCTCCCATCc cTTG

CCACTGTTTGCCTGGGGTTCCATCTCAAGTTAGTTATGCAACTAATCAATCAATCTAAAA

WIAF-3580, AAAATTTTTAAAGCTTCTTTTCAATCTACTTTTTGCTCAGNAAGGAATGTCTTCCCAACA stSG16412a 39 TCTTTCAATGCA

AGTTTTAATTCTATCATACACAAAGACCTCCCATCCTC [T/C] TGTTCCATCCACCTCTT CCACTGTTTGCCTGGGGTTCCATCTCAAGTTAGTTATGCAACTAATCAATCAATCTAAAA

WIAF-3583, AAAATTTTTAAAGCTTCTTTTCAATCTACTTTTTGCTCAGNAAGGAATGTCTTCCCAACA stSG16412b 98 TCTTTCAATGCA

TGGCCTGTAAAGGTTTCTGCTGAGAAATCTGCT^

WIAF-3584, GTTACTGCATGCTTTTTTTCTT [G/A] CTGTTTTTTGAATTCTCTCTTTGTCTTTGATGT stSG21940 182 TAGATAGTTTGACTATAATGTACCATGGAGAATGCCTTTTTGCATTTCATCTGCCTGGGG

GTAGA∞AACCTCGCGCACCGCCTCTCTGGTNCTGGGAATCTGCTGAAC

WIAF-124, C [C/T] TGGGGTCCCTCGAACGCCCCAGCTAAGAAGGGCGGGGCCTTGCCAGGGCGCGAC EST301673b 61 AACATGACGTTCAAGGTCTTCCTGTGGCTTCTGT

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ATCAGCCAATGTCGTTGACAAGGCATTGTTCTTTTATCTCAAGAAATTTAAATTTGTCTC AGTTGTTCTGTGCATGCTAAAACGAAATACTCCATGGCCTTTCCATGTACACCACACACC GTCCTTAATTCATCTGTGATG [C/G] TATAGCCTAGATTTCTGCAGGCCCAGGTTGTTCT GTGTCCTATAGAGAGTCATGGTCCCATCCCCTGGAACCATGACTTCCAGCAGATGAAATG TACGGATCCCATGGATGTCCTTTGCATGCCTGAACTTCCAGCAACATTTGACATGCTTAT

WIAF-3229, CTTGTCTTCATGAAACATGTTTCCTTTGTCTGTGTGACACCATGTTTTTCTGGTTTTCCT WIR-1740b 141 CCTACCTCAATGACTATTTTTTCCCTCAATCTCTTGTGCGCCTCCT

ATCAGCCAATGTCGTTGACAAGGCATTGTTCTTTTATCTCAAGAAATTTAAATTTGTCTC AGTTGTTCTGTGCATGCTAAAACGAAATACTCCATGGCCTTTCCATGTACACCACACACC GTCCTTAATTCATCTGTGATGCTATAGCCTAGATTTCTGCAGGCCCAGGTTGTTCTGTGT CCTATAGAGAGTCA[T/C]GGTCCCATCCCCTGGAACCATGACTTCCAGCAGATGAAATG TACGGATCCCATGGATGTCCTTTGCATGCCTGAACTTCCAGCAACATTTGACATGCTTAT

WIAF-3231, CTTGTCTTCATGAAACATGTTTCCTTTGTCTGTGTGACACCATGTTTTTCTGGTTTTCCT WTR-1740c 194 CCTACCTCAATGACTATTTTTTCCCTCAATCTCTTGTGCGCCTCCT

GGGTGGGAGTGCAGAGTTTAACTCATAAGGCACATTGTGGGGACTCCTGTCAAGCTTAGC CAAATCTCTTA [G/C] GGTCAAGGTGACTGCAGCTGACATCTAGGTCACACTTACCCATT CCTCAAGCTCTCTCTGTTTCAAGAAAATGAAAGGGTGAGGAGATTGGATTAATTATCTCT AAGCTTCCTTAAATTCCTAAGATTTTAAAATATTCTCTGTTTAACTGTGTCACTAGGAGT 4>- 00 ACAGAAACCACCCTACCGTATAAAGAAATCAAACTCTGCAACCATACATCAATACTGAGC

WIAF-3234, CTTCCATTGCATATGGTGGACGGTACACCAAGTATTACCCTGGGAGCATCTGCTGTTTTG WTR-1384a 71 ATGCTCGTAACTGCAATAATAACCATTGTATGTGTAATTGGAGCCAGGTTGATGAA

GGGTGGGAGTGCAGAGTTTAACTCATAAGGCACATTGTGGGGACTCCTGTCAAGCTTAGC CAAATCTCTTAGGGTCAAGGTGACTGCAGCTGACATCTAGGTCACACTTACCCATTCCTC AAGCTCTCTCTGTTTCAAGAAAATGAAAGGGTGAGGAGATTGGATTAATTATCTCTAAGC TTC [C/T] TTAAATTCCTAAGATTTTAAAATATTCTCTGTTTAACTGTGTCACTAGGAGT ACAGAAACCACCCTACCGTATAAAGAAATCAAACTCTGCAACCATACATCAATACTGAGC

WIAF-3237, CTTCCATTGCATATGGTGGACGGTACACCAAGTATTACCCTGGGAGCATCTGCTGTTTTG WIR 1384b 183 ATGCTCGTAACTGCAATAATAACCATTGTATGTGTAATTGGAGCCAGGTTGATGAA

TCTGGAGGTGAATGGCATGGGAATCTGGAGAGGAAGTTCAA [T/C] GGGCCCAGGGCTGA NAGTTTGAGGAAATATTCCTGCTGCTCAAGAGCCTGGAACTCTCACCTGCCCCACATCTA

WIAF-2675, TTAGTCTTTCTGGACAATGATGGAAGTTTCTACGTCTCCCTTTTCTATCCCTGTTACTTC MR6498 41 TTTTAAAAGTCTTTTTCCTCCTAGGATTAGTCCAGGCTATCCTCACCCAGG

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TCTCTACATTCTATGGACAACCTCCATGCCTTTGCACATGCTGATCCCTCCTCCTGGAAT TCCTTTCCTACTTGTCCTCATGTACAATTTTCTGCTCGTCCTTCA [A/T] GGGGCAGCTT

WIAT-443, GCAAGCCTCCCTTTAGACACCTCTACAGGTACAGCCGACCATGCCCTACCTCCATGGCAC

HSC15C072b, TGCCAGGGGACCCTTATAGGCCTCTGTCTTTAAACCTGTAATGGTATATTAATCCTTGGΓ

WI-9651b GTTTGAATGTCTCTCTCTCCCCTGCCAGCTCAAGAACCTGTTGAG __

CTCTCCTTTTTTCTTCAGATCTGCTCCTGGGTTTTAATTTCGGAGGTCA[G/A] TTGTTC TACCTCACTGAGAGGGAACAGAAGGATATTGCTTCCTTTTGCAGCAGTGTAATAAAGTCA ATTAAAAACTTCCCAGGATTTCTTTGGACCCAGGAAACAGCCATGTGGGTCCTTTCTGTG

WIAF-1135, CACTATGAACGCTTCTTTCCCAGGACAGAAAATGTGTAGTCTACCTTTATTTTTTATTAA 7314c, WI- CAAAACTTGTTTTTTAAAAAGATGATTGCTGGTCTTAACTTTAGGTAACTCTGCTGTGCT 7314c GGAGATCATCTTTAAGGGCAAAGGAGTTGGATT _ ___ _

TCAGAAGCAGACATGGCATCTGTTCCTTGCTTGCTTGTTGGTTGTGTACCTTTCACGAGA CCTGAATTTTAGAATTGCCCAGTGCTGCCAGAGTGAGTGAGTGTAATTCTCCTTTCAGGT

WIAF-1834, AAAGATAGGCTATCTCAACACTGCTGAGTGATTCATAAACATATCAACCA [G/A] TAGCA STS-U31525a, TTAACCCATTTTATTTCCTGTCCTTAGTGTCTGAAGATGCTCACCAGTTTTCTGTGTACA WT-19179a GTAAGGCAGCATGCTAAAATGCT _ __

AGCTTTGCTTGAAAATTTGGTACTTACTACCTTTGCAATTCTCTTTATTTATTATTAT A t CTTTTATTTTTCC [G/A] TAAGTTATTGGGGTACAGGAGGTATTTGGTTATATAAGTTCT 4

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WIAI 2512, TTAGTGGCGATTTGTGTGATTTTGGTGCACCCATTACCCAAGGAGTATACACTGCACCAT STS R53533b, ACTCGGTCTTTTATCCCTCGCCCCTCTCCCACTTTTCCCCTCAAGTCCCAAAAGRCCATT WT 21961b GTATCATTCTTATGCCTTG _

AT AAATGTCAAGGTTTCATGTTT_CATTTTCTTATAT^AGT_^^T TATATATAC TTTTTTTTGAGATAATTATTCTAGATTCCAGGCTTTCTTCTAGATGTAAGTNCCTAAAGC

WIAF-1227, TTATAGTTTACATTGATATCTAGACATATATCTTAAACAGTCTCCAAATTTNCTTTAATT

HSC0ZA102b, AATCA [A/+] AGTATGTTAATGTCACTTGGAATTCTACATGGAAAAGCCAACAAAATAAC

WI-9497b TAAAACTTGACTAATGAAGATCAGCGTCACTAATAAAAG

AGCTTTGCTTGAAAATTTGGTACTTACTACCTTTGC_.ATTCTCTTTATT_ATT_.TT_.TTA CTTTTATTTTTCCGTAAGTTATTGGGGTACAGGAGGTATTTGGTTATATAAGTTCTTTAG

WIAF-2513, TGGCGATTTGTGTGATTTTGGTGCACCCATTACCCAAGGAGTATACACTGCACCATACTC

STS-R53533c, GGTCTTTTATCCCTCGCCCC [T/G] CTCCCACTTTTCCCCTCAAGTCCCAAAAG CCATT

WI-21961C GTATCATTCTTATGCCTTG

TATTGCTGCTTGTCACTGCCTGACATTCACGGCAGAGGCAAGGCTGCTGCAGCCTCC [C/ G] CTGGCTGTGCACATTCCCTCCTGCTCCCCAGAGACTGCCTCCGCCATCCCACAGATGA

WIAF-2462, TGGATCTTCAGTGGGTTCTCTTGGGCTCTAGGTCCTGGAGAATGTTGTGAGGGGTTTATT

STS- l2959a, TTTTTTTAATAGTGTTCATAAAGAAATACATAGTATTCTTCTTCTCAAGACGTGGGGGGA

WI-19067a

AATTATCTCATTATCGAGGCCCTGCTATGCTGTGTGTCTGGGCGTGTTGTATG

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CTCTTTCTCAGCACATTGATGGGCAACTAGAATTACAGCAGTTTCAAACTCTACCATGGA TAATGCAAACAAACCGAAGCTACATGCCAATGATAGGTGCAAAGAATATTGGCAAAAGGT GCTTT [A/C] CCTTGAGCCATTATTTGTGTCAGAGAACAAAAGAAACAGAATCAATATAT

WIAF-1212, AAATTCAAAGACTATCTGCAGCTAGTGTGTTTCTTCTTTACACACATATACACACAGACA 7338m2b, WI - TCAGAAAATTCTGTTGAGAGCAGGTTCATTAAATTTGTAAGATGGCATATTCTAAAGCCT 7338b 125 GTGCTACCAGTACTAAGAGGGGAAGACTGGCAAT

CTCTTTCTCAGCACATTGATGGGCAACTAGAATTA_AGCAGTT_CAAACT_:TACCATGGA TAATGCAAACAAACCGAAGCTACATGCCAATGATAGGTGCAAAGAATATTGGCAAAAGGT GCTTTACCTTGAGCCATTATTTGTGTCAGAGAACAAAAGAAACAGAATCAATATATAAAT

WIAF-1213, TCAAAGACTATCTGCAGCTAGTGTGTTTCTTCTTTACACAC [A/G] TATACACACAGACA 7338m2c, WI- TCAGAAAATTCTGTTGAGAGCAGGTTCATTAAATTTGTAAGATGGCATATTCTAAAGCCT 7338c 221 GTGCTACCAGTACTAAGAGGGGAAGACTGGCAAT

TGTGAAACTCCACTTGAAGCCAAAGAAAGAAACTCACACTTAAAACACATGCCAGTTGGG AAGGTCTGAAAACTCAGTGCATAATAGGAACACTTGAGACTAATGAAAGAGAGAGTTGAG ACCAATCTTTATTTGTACTGGCCAAATACTGAATAAACAGTTGAAGGAAAGACATTGGAA

WIAF-313, AAAGCTTTTGAGGATAATGTTACTAGACTTTATGCCATGGTGCTTT [C/T] AGTTTAATG

UTR-01034, CTGTGTCTCTGTCAGATAAACTCTCAAATAATTAAAAAGGACTGTATTGTTGAACAGAGG to

WT 7070 226 GACAATTGTTTTACTTTTCTTTGGTTAATTTTGTTTTGGCCAGA _ Cn

AGGTTCTGGACTTGATGCTGGGAAACAVTTGGGTNCTGGAGAATTCCTATTTTGAGTNTT

TCACAGATCAGTAGAGCCAAATGGGAAAGGTATCCTAGTCCATCCCTTTATTAGGAACTT

WIAF-4038, TCCTGATCTATTGGGAACTTCCTCCTAATAGATCAGGAAAATCCACCTCATTTAATCATG MH598, WI- GACAACNNAAAAGGAATA [T/C] GATCCCGCATGCAACATTTATTCAGTGAAAACATGAT 884 198 GAAAATGAACATAATGGTACTACTGAAAATGNGAGCACACCAGAAAAATTATAAATTAA

AACTATGGCAGTGGTCCTGGTTATAGTAGTAGAGGCGGGTATGGTGGTGGTGGACCAGGA

TATGGAAACCAAGGTGGTGGATATGGTGGCGGTGTTGGAGGATATGATGGTTACAATGAA

GGAGGAAATTTTGACGGTAGTAACTATGGTGGTGGTGGGAACTATAATGATTTTGGAAAT

WIAF-1110, TA [C/T] AGTGGACAACAGCAATCAAATTATGGACACATGAAAGGGGGCAGTTTTGGTGG 7301m5b, WI- AAGAAGCTCGGGCAGTCCCTATGGTGGTGGTTATGGATCTGGTGGTGGAAGTGGTGGATA 7301b 182 TGG AGCAGAAGGTTCTAAAAACAGCAGGAAAAGGGCTACAG

AACTATGGCAGTGGTCCTGGTTATAGTAGTAGAGGCGGGTATGGTGGTGGTGGACCAGGA

TATGGAAACCAAGGTGGTGGATATGGTGGCGGTGTTGGAGGATATGATGGTTACAATGAA

GGAGGAAATTTTGACGGTAGTAACTATGGTGGTGGTGGGAACTATAATGATTTTGGAAAT

WIAF-1111, TACAGTGGACAACAGCAATCAAATTATGGAC [A/C] CATGAAAGGGGGCAGTTTTGGTGG 7301 5c, WI- AAGAAGCTCGGGCAGTCCCTATGGTGGTGGTTATGGATCTGGTGGTGGAAGTGGTGGATA 7301c 1211 TGGTAGCAGAAGGTTCTAAAAACAGCAGGAAAAGGGCTACAG

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WIAF -2267, CATAGAAAGGAGTCTTTGAGTATTGTACAGTTTTGAAAATTCTCTTTGAGATAATTGATT

TTGR TCATATTCTGTGGCTTTCAACCTCCATTTACCTCTTGTCATTCCAACATCTTTATAGAGA

A005I.24a, AAT [A/G] AAAACCCAATTTCTCTTTCACCATTT GTTTGATTATCATCTGGATTTTCAC

TTGR- TCAAGATGCAGCTCCTAAGATTATTGTTATGTTAAATTCATAAACTCCTTCACCTTTAAT

A005D24a 123 AATTAAGGAAACAATACCAGTGTTGATAAAGACAATACCAGTGTTGATAAAGATA

AGGGAATTGTGTTGCTCCTGGAGG [A/G]AGCCCAGGCATCATTAAACAAGCCAGTAGGT CACCTGGCTTCCGTGGACCAATTCATCTTTCAGACAAGCTTTAGAGAAATGGACTCAGGG AAGAGACTCACATGCTTTGGTTAGTATCTGTGTTTCCGGTGGGTGTAATAGGGGATTAGC

W1AF-214, CCCAGAAGGGACTGAGCTAAACAGTGTTATTATGGGAAAGGAAATGGCATTGCTGCTTTC

UTR-03180a, AACCAGCGACTAATGCAATCCATTCCTCTCTTGTTTATAGTAATCTAAGGGTTGAGCAGΓ

WI-7227a 24 TAAAACGGCTTCAGGATAGAAAGCTGTTTCCCACCTGTTTCGT _

WIAF-389, CATAGAAAGGAGTCTTTGAGTATTGTACAGTTTTCAAAATTCTCTTTGAGAT^ATTGATT

ΓIGR- TCATATTCTGTGGCTTTCAACCTCCATTTACCTCTTGTCATTCCAACATCTTTATAGAGA

A005D24b, AATAAAAACCCAATTTCT [C/T] TTTCACCATTTAGTTTGATTATCATCTGGATTTTCAC

T1GR- TCAAGATGCAGCTCCTAAGATTATTGTTATGTTAAATTCATAAACTCCTTCACCTTTAAT

A005D24b 138 AATTAAGGAAACAATACCAGTGTTGATAAAGACAATACCAGTGTTGATAAAGATA __

AGGGAATTGTGTTGCTCCTGGAGGAAGCCCAGGCATCATTAAACAAGCCAGTAGGTCACC to

TGGCTTCCGTGGACCAATTCATCTTTCAGACAA [G/T] CTTTAGAGAAATGGACTCAGGG c

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AAGAGACTCACATGCTTTGGTTAGTATCTGTGTTTCCGGTGGGTGTAATAGGGGATTAGC

WTAF-212, CCCAGAAGGGACTGAGCTAAACAGTGTTATTATGGGAAAGGAAATGGCATTGCTGCTTTC

UTR-03180b, AACCAGCGACTAATGCAATCCATTCCTCTCTTGTTTATAGTAATCTAAGGGTTGAGCAGT

WI-7227b 93 TAAAACGGCTTCAGGATAGAAAGCTGTTTCCCACCTGTTTCGT

TGGAGAACATTCAATCTTGCCGTCACTATTCATCAATGAAGATTATG/ATCACTGAG^~ATC CAGAGAGGCTGGATGACTTGCTCAAGTTCACCAGCATGGTAGTGGCAAAGAGAGGTCCAG AGTCCTGGCCCTTGATGCCCAGCTCAGTGCCACAAAGCTCAGTAGGAGGGATGTTCCAGT

WIAF-1352, GGATGAGGGCCACCAGGAAGCACAGGTCCAAGGCTGGTCCCACACTTATCAGCAGCAACA UTR-02921 , ACTGTCAGTTCATCCTGCATGGGAAAAATGTTGGAATGGGAGTCTGAAATGGGGCTACTG WI 7690 45 TTTCAGTCCTAACGTGCTGTGTGACATTGGGAC _ _

AGGGAATTGTGTTGCTCCTGGAGGAAGCCCAGGCATCATTAAACA_^GCCAGTAGGTCACC TGGCTTCCGTGGACCAATTCATCTTTCAGACAAGCTTTAGAGAAATGGACTCAGGGAAGA GACTCACATGCTTTGGTTAGTATCTGTGTTTCCGGTGGGTGTAATAGGGGATTAGCCCCA

WTAF-213, GAAGGGACTGAGCTAAACAGTGTTATTATGGGAAAGGAAATGGCATTGCTGCTTTCAACC

UTR-03180C, AGCGACTAATGCAATCCATTCCTCTCTTGTTTATAGTAATCTAAGGGTTGA [G/A] CAGT

WT-7227c 291 TAAAACGGCTTCAGGATAGAAAGCTGTTTCCCACCTGTTTCGT

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ACAAGGCGACTTGAAGAGGACGCAGGCTTCCAGAGGACAAACCCCAATACAGGAGAAGCA CAAGACAGAGAAGGGCCAATGGGGTCATCCCCTCCCTAACGAGACTCTCTGTGCTGGGGG TGCTAATTACATGGCAGGAAGAATGGGGCCTCTAAGGGGAGTGTGGGGΓCTGTCTCTCCC

WTAF-4076, TTTTTTCCATCTTTTTCCTCTCTCGCTTTCTTTCTTACACAGAAACAT [A/G] CACATAC 7619m4o, WI- CGAGAAACCTATTTCTCAGACCCCTTTTTCTCCTCTGTCTTTCTCTCTCCCTCTCCCACA 7619o 228 CCTCACACACACATACTCCCACTTGCAACTATTCTGTTTC _ __

ACAAGGCGACTTGAAGAGGACGCAGGCTTCCAGAGGACAAACCCCAATACAGGAGAAGCA CAAGACAGAGAAGGGCCAATGGGGTCATCCCCTCCCTAACGAGACTCTCTGTGCTGGGGG TGCTAATTACATGGCAGGAAGAATGGGGCC [T/C] CTAAGGGGAGTGTGGGGTCTGTCTC

WIAF-4077, TCCCTTTTTTCCATCTTTTTCCTCTCTCGCTTTCTTTCTTACACAGAAACATACACATAC

7619 4p, WI- CGAGAAACCTATTTCTCAGACCCCTTTTTCTCCTCTGΓCTTTCTCTCTCCCTCTCCCACA

7619p 150 CCTCACACACACATACTCCCACTTGCAACTATTCTGTTTC _

ACAAGGCGACTTGAAGAGGACGCAGGCTTCCAGAGGACAAACCCCAATACAGGAGAAGCA

CAAGACAGAGAAGGGCCAATGGGGTCATCCCCTCCCTAACGAGACT [C/G] CTGTGCTG

GGGGTGCTAATTACATGGCAGGAAGAATGGGGCCTCTAAGGGGAGTGTGGGGTCTGTCTC

WTAF 4078, TCCCTTTTTTCCATCTTTTTCCTCTCTCGCTTTCTTTCTTACACAGAAACATACACATAC 7619m4q, WT- CGAGAAACCTATTTCTCAGACCCCTTTTTCTCCTCTGTCTTTCTCTCTCCCTCTCCCACA t 7619q 106 CCTCACACACACATACTCCCACTTGCAACTATTCTGTTTC

GCAGGAAATAGTCACTCATCCCACTCCACATAAGGGGTTTAGTA [A/G] GAGAAGTCTGT

CTGTCTGATGATGGATAGGGGGCAAATCTTTTTCCCCTTTCTGTTAATAGTCATCACATT

TCTATGCCAAACAGGAACGATCCATAACTTTAGTCTTAATGTACACATTGCATTTTGATA

WTAF-4079, AAATTAATTTTGTTGTTTCCTTTGAGGTTGATCGTTGTGTTGTTTTGCTGCACTTTTTAC 7830ml, WI- TTTTTTGCGTGTGGAGCTGTATTCCCGAGACAACGAAGCGTTGGGATACTTCATTAAATG 7830 44 TAGCGACTGTCAACAGCGTGCAGGTTTTCTGTTTCTGTGTTGTGGGGTCAA

AGCCATACAATGCATTGCAAAGAAACAAAGCAGCTGTACAGGAGTGGGGACGC [G/A] TC

AGTGTACAATACATTCATGTCCAGGATAAGGAGCATACACCAGGATTTATACACGGTGGC

W1AF-2509, AGCGGCTATAGGCACGATGATACAAAATATAAAGTATATTTCCATCTATATAAATACACA

STS-T57230a, GCTGGGGTGGGGAAGGATGCTGGGTGATCTTGTTTCCCCCGCAGAGGGCCTGGGAGGCAG

WI 20270a 53 GGNGGGTGGTGGGAAGGGATTTCTT __

AGCCATACAATGCATTGCAAAGAAACAAAGCAGCTGTACAGGAGTGGGGACGCGTCAGTG TACAATACATTCATGTCCAGGATAAGGAGCA [T/G] ACACCAGGATTTATACACGGTGGC

WTAF-2510, AGCGGCTATAGGCACGATGATACAAAATATAAAGTATATTTCCATCTATATAAATACACA

STS-T57230b, GCTGGGGTGGGGAAGGATGCTGGGTGATCTTGTTTCCCCCGCAGAGGGCCTGGGAGGCAG

WT 20270b 91 GGNGGGTGGTGGGAAGGGATTTCTT

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GCGTCTACAGACAGCTCACCA1TTTTGTCCTGTATCTGTAAACACTTTTTGTTCTTAGTC TTTTTCTTGTAAAATTGATGTTCTTTAAAATCGTTAATGTATAACAGGGCTTATGTTTCA GTTTGTT [T/C] CCGTTCTGTTTTAAACAGAAAATAAAAGGAGTGTAAGCTCCTTTTCT CATTTCAAAGTTGCTACCAGTGTATGCAGTAATTAGAACAAAGAAGAAACATTCAGTAGA ACATTTTATTGCCTAGTTGACAACATTGCTTGAATGCTGGTGGTTCCTATCCCTTTGACA CTACACAATTTTCTAATATGTGTTAATGCTATGTGACAAAACGCCCTGATTCCTAGTGCC AAAGGTTCAACTTAATGTATATACCTGAAAACCCATGCATTTGTGCTCNNNNNNNNNNNA TGGTGCTTGAAGTAAAACAGCCCATCCTCTGCAAGTCCATCTATGTTGTTCTTAGGCATT

WIAF-1144, CTATCTTTGCTCAAATTGTTGAAGGATGGTGATTTGTTTCATGGTTTTTGTATTTGAGTC UTR-03390, TAATGCACGTTCTAACATGATAGAGGCAATGCATTATTGTGTAGCCACGGTTTTCTGGAA WI-7238 128 AAGTTGATATTTTAGGAATTGTATTTCAGATCTTAAATAAAATTTGTTTCTAAATTTC

GACTTCATGCTCATGAACAAGCATTTGTCTTAATTTACAGACATTAAGAACAAGCTTTCC [A/G]CTCCCACTTCCCTCCCACTATCACCTCAACCTCTTCATCCACTTTAAAGAGGTTT

WIAF-2065 , CTTTAGGTCCTCTGCATATCATGGAAGCCAACTACTCTATTAACGCTTTCCCAATGATGC MR2981 b , WI - AGCCCAGTTCTGCATACAGTTTGTACAGAAATGCTATATTTATGGAAACAGCTGAAAAAT 2868b 60 GAAATATCGATATACCCCTAACAGTCATTTCTACAAAGGT

TTAATTTACTGATTCCAGCAAGACCAAATCATTGTATCAG^TTATTTTTAAGTTTTATCC t GTAGTTTTGATAAAAGATTTTCCTATTCCTTGGTTCTGTCAGAGAACCTAATAAGTGCTA 0

C CTTTGCCATTAAGGCAGACTAGGGTTCATGTCTTTTTACCCTTTNNNNNNNNNTTGTAAA

WT AF- 1354 , AGTCTAGTTACCTACTTTTTCTTTGATTTTCGACGTTTGACTAGCCATCTCAAGCAA [C/ 7 773mlb , WI - G] TTTCGACGTTTGACTAGCCATCTCAAGCAAGTTTAATCAAAGATCATCTCACGCTGAT 7 773b 237 CATTGGATCCTACTCAACAAAAGGAAGGGTGGTCAGA _ __

ATCTTTGCTCCCTGCAAGAAATCAGCCATAAGAAAGCACTATTAATACTCTGCAGTGATT AGAAGGGGTGGGGTGGCGGGAATCC [T/C] ATTTATCAGACTCTGTAATTGAATATAAAT GTTTTACTCAGAGGAGCTGCAAATTGCCTGCAAAAATGAAATCCAATGAGCACTAGAATA

WTAF-1324, TTTAAAACATCATTACTGCCATCTTTATCATGAAGCACATCAATTACAAGCTGTAGACCA 7870b, WI- CCTAATATCAATTTGTAGGTAATGTTCCTGAAAATTGCAATACATTTCAATTATACTAAA 7870b 85 CCTCACAAAGTAGAGGAATCCATGTAAATTGCAAATAAACCAC _

TAGAAGGATATGCTGATCAAAAAAAGGGGACATATTCAAGGAGTNTCCCTGGGT [C/T] A ACCCTTTATTCAGTCTCTGCCACATGTCTAGTAACTGTGAGTGATGGGTGCATCAGTATA

WIAF-4177, ATCCTGAGCCTCCCAAGGTACAGCCTTTCACTACTATTCATCATATTGGCTAAGGTATTC WI-1732, WI- ATCATATTGGCTAAGGTATTCACCAACAGGGCTCATTTTCTATCAGACCTACAAGAAACC 1732 114 TACAGTGGCTATA

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GTGTAATTTGGTGGCTTTGCAACTTTTCCCACAGTAACCTTTAGAATNTNAAAGGTGGAA

GGTAAGGATGAGGAAGAAGAGGGNGTAAGAAACAAAAGATGTCTATGTTGAAGAAGTATC

WlAr-1174, CTTAGGATATTCTGATACATG [A/G] TAATGACCCTCCATGACTCTGGTACCTCATCATT 597c, WI- ACCAATGTGAGAATTATTAACTTGATCTAATATTCTTCACAACTAATATACCTGAGAGAA 597c 141 ATAAGTCTATTTAATTTTTAATTTTGGGGGGAATATACACA

ATATTACAACTTGCTTTTTAGCTGATCTTCCATCCTCAAATGACTCTTTTTTCTTTATAT

GTTAACATATATAAAATGGCAACTGATAGTCAATTTTGATTTTTATTCAGGAACTATCTG

AAATCTGCTCAGAGCCTATGTGCATAGATGAAACNNNNNNN [A/T] AAAAAAAGTTATTT

AACAGTAATCTATTTACTAATTATAGTACCTATCTTTAAAGTATAGTACATTTTACATAT

GTAAATGGTATGTTTCAATAATTTAAGAACTCTGAAACAATCTACATATACTTATTACCC

AGTACAGNNNNNNNNCCCCTGAAAAGCTGTGTATAAAATTATGGTGAATAAACTTTTATG

TTTCCATTTCAAAGACCAGGGTGGAGAGGAATAAGAGACTAAGTATATGCTTCAAGTTTT

AAATTAATACCTCAAGTATTAAATAAATATTCCAAGTTTGTGGGAATGGGAGATTAAAAT

GCATGTTTGAGAATAGAGAAATTTTCTTCTTGGTTTCATTGCAAAGAGTAAAACAAACAT

WIAF- 1 1 97 , GTTAAAACATCAACTGAAGGGTTGGGTTAGGAACATTTACCCTGAAAAAAATATGAGGAT UT R- 01990 , GCATCATAAAATGTAAATATTTTCCTACCATGTTGGGGGGGCACAAATTTTAAAACTGGC WT - 7 1 53 161 ATCTTTACAAGTTTCTTCTTTATAAACACCCAAACAAAATCAAGTTTTATAAAG to

00

CATCTATCCCTGAGGCAGAAAATACAGAACACCCTGTGGCTGCCTGAACGGAGGAAGGAT cn GGGGGCGGGGAGACATCGGTCAATGTATCAAAGCATCTCTCTGCCTGAAAGACCTCTCCT

WIAF-4093, GAAAGACATGAGCTATTAGGAGCTCTGGCAAGGGCTTTGTCTTATCCTCCTTGCTATCCC PB876b, WI- TGATGACTGGGCAAAACAGTAGCTGCCCTGATTCCATGAGACAGAAAGGGGTGACTTATT 276b 25 TAATCCCAGAGCCACG

GCATGAAAAGAACTCCAATCAGACTTTATTCAATAAAGCAGCTTTTCATGAATGCTTCAG GTCAGTGTATGATCAGCTCAGCTTCCAGTATCAACTTGAGTACCTC [A/G] TTATGGATA

WIAF-1643, TTTATGCTAGGAATGACAACAGTAAGGGCATTGCAAAATCCAAAGTCATCTAATATTAAA STS-R42732a, CCATATTTTACATAATTTGTAGGGACAGTATACTAATACTCTACAATAAATAAGGGTTTA WT 21627a 106 AAAATGTGTTGCTTAGCCCTTGG

TCCAATTTTCAGAAACATGTTCCATGTTTATTGTGATAAGCACTAG [A/G] TATTATAGT

CTCATGTTTTTAATTTATGAATAACGTCTGATTCATTTGATTTTGTATTTACAGAAGATG

WIAr-1953, TCAGGGCTATCTCATTCAGTTATTAATAAATGGATCAGAGTAGTAAGTCAAGAATAAGTG

STS-T15424a, CATAATGTGGTTTAAATTTTAAAAAATACTCAGAATGAGGTAGTATTTTAATTTTTAATT

WT-1964la 46 C^TCCACCCACCTTGCTCTTCTCTGTTCCATAATAGGCCTTCCCTGTGGAC

GCATGAAAAGAACTCCAATCAGACTTTATTCAATAA_.GCAGCTTΓTC__TGAATGCTTCAG

GTCAGTGTATGATCAGCTCAGCTTCCAGTATCAACTTGAGTACCTCATTATGGATATTTA

WIAI-1644, TGCTAGGAATGACAACAGTAAGGGCATTGCAAA [A/G] TCCAAAGTCATCTAATATTAAA

STS-R42732b, CCATATTTTACATAATTTGTAGGGACAGTATACTAATACTCTACAATAAATAAGGGTTTA

WI-21627b 153 A AAAATGTGTTGCTTAGCCCTTGG

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CCACCAGGATCCCAGCCCAAGCGGCCCCTCCCGCCCCTTCCCACTCGCAGCAGACGCCGG GGACAGAGGCCTGCCCGGGCGCGCCAGCCCCGGCCCTGGGCTCGGAGGCTGCCCCCGGCC CCCTGGTCTCTGGTCCGGACACTCCTAGAGAACGCAGCCCTAGAGCCTGCCTGGAGCGTT TCTAGCAAGTGAGAGAGATGGGAGCTCCTCTCCTGGAGGATGCAGGTGGAACTCAGTCAT TAGACTCCTCCTCCAAAGGCCCCCTACGCCAATCAAGGGCAAAAAGTCTACATACTTTCA TCCTGACTCTGCCCCCTGCTGGCTCTTCTGCCCAATTGGAGGAAAGCAACCGGTGGATCC TCAAACAACACTGGTGTGACCTGAGGGCAGAAAGGTTCTGCCCGGGAAGGTCACCAGCAC

WIAF-1207, CAACACCACGGTAGTGCCTGAAATTTCACCATTGCTGTCAAGTTCCTTTGGGTTAAGCAT

UTR-04684d, TACCACTCAGGCATTTGACTGAAGATGCAGCTCACTACCCTATTCTCTCTTTACGCTTAG

WI-7252d 540 [T/C]TATCAGCTTTTTAAAGTGGGTTATTCTGGAGTTTTT

GTTTGGAGAGCACACCTATCTTAGTGGTTCCCCACCGAAGTGGACTGGCCCCTGGGTCAG TCTGGTGGGAGGACGGTGCAACCCAAGGACTGAGGGACTCTGAAGCCTCTGGGAAATGAG AAGGCAGCCACCAGCGAATGCTAGGTCTCGGACTAAGCCTACCTGCTCTCCAAGTCTCAG TGGCTTCATCTGTCAAGTGGGACTCTGTCACACCAGCCATTCTTATCTCTCTGTGCTGTG GAAGCAACAGGAATCAAGAGACTGCCCTCCTTGTCCACCCACCTATGTGCCAACTGTTGT AACTAGGCTCAGAGATGTGCACCCATGGGCTCTGACAGAAAGCAGATCCTCACCCTGCTA CACATACAGGATTTGAACTCAGATCTGTCTGATAGGAATGTGAAAGCACGGACTCTTACT 4 GCTAACTTTTGTGTATCGTAACCAGCCAGATCCTCTTGGTTATTTGTTTACCACTTGTAT TATTAATGCCATTATCCCTGAATTCCCCTTGCCACCCCACCCTCCCTGGAGTGTGGCTGA GGAGGCCTCCATCTCATGTATCATCTGGATAGGAGCCTGCTGGTCACAGCCTCCTCTGTC TGCCCTTCACCCCAGTGGCCACTCAGCTTCCTACCCACACCTCTGCCAGAAGATCCCCTC AGGACTGCAACAGGCTTGTGCAACAATAAATGTTGGCTTGGNNNNNNNNNNNN

CCACCAGGATCCCAGCCCAAGCGGCCCCTCCCGCCCCTTCCCACTCGCAGCAGACGCCGG GGACAGAGGCCTGCCCGGGCGCGCCAGCCCCGGCCCTGGGCTCGGAGGCTGCCCCCGGCC CCCTGGTCTCTGGTCCGGACACTCCTAGAGAACGCAGCCCTAGAGCCTGCCTGGAGCGTT TCTAGCAAGTGAGAGAGATGGGAGCTCCTCTCCTGGAGGATGCAGGTGGAACTCAGTCAT TAGACTCCTCCTCCAAAGGCCCCCTACGCCAATCAAGGGCAAAAAGTCTACATACTTTCA TCCTGACTCTGCCCCCTGCTGGCTCTTCTGCCCAATTGGAGGAAAGCAACCGGTGGATCC TCAAACAACACTGGTGTGACCTGAGGGCAGAAAGGTTCTGCCCGGGAAGGTCACCAGCAC

WTAF-1208, CAACACCACGGTAGTGCCTGAAATTTCACCATTGCTGTCAAGTTCCTTTGGGTTAAGCAT

UTR-04684e, TACCACTCAGGCATTTGACTGAAGATGCAGCTCACTACCCTATTCTCTCTTTACGCTTAG

WT-7252e 552 T TTATCAGCTTT [T/C] AAAGTGGGTTATTCTGGAGTTTTTGTTTGGAGAGCACA

CCTATCTTAGTGGTTCCCCACCGAAGTGGACTGGCCCCTGGGTCAGTCTGGTGGGAGGAC GGTGCAACCCAAGGACTGAGGGACTCTGAAGCCTCTGGGAAATGAGAAGGCAGCCACCAG CGAATGCTAGGTCTCGGACTAAGCCTACCTGCTCTCCAAGTCTCAGTGGCTTCATCTGTC AAGTGGGACTCTGTCACACCAGCCATTCTTATCTCTCTGTGCTGTGGAAGCAACAGGAAT CAAGAGACTGCCCTCCTTGTCCACCCACCTATGTGCCAACTGTTGTAACTAGGCTCAGAG ATGTGCACCCATGGGCTCTGACAGAAAGCAGATCCTCACCCTGCTACACATACAGGATTT GAACTCAGATCTGTCTGATAGGAATGTGAAAGCACGGACTCTTACTGCTAACTTTTGTGT ATCGTAACCAGCCAGATCCTCTTGGTTATTTGTTTACCACTTGTATTATTAATGCCATTA TCCCTGAATTCCCCTTGCCACCCCACCCTCCCTGGAGTGTGGCTGAGGAGGCCTCCATCT CATGTATCATCTGGATAGGAGCCTGCTGGTCACAGCCTCCTCTGTCTGCCCTTCACCCCA GTGGCCACTCAGCTTCCTACCCACACCTCTGCCAGAAGATCCCCTCAGGACTGCAACAGG CTTGTGCAACAATAAATGTTGGCTTGGNNNNNNNNNNNN

CCACCAGGATCCCAGCCCAAGCGGCCCCTCCCGCCCCTTCCCACTCGCAGCAGACGCCGG GGACAGAGGCCTGCCCGGGCGCGCCAGCCCCGGCCCTGGGCTCGGAGGCTGCCCCCGGCC CCCTGGTCTCTGGTCCGGACACTCCTAGAGAACGCAGCCCTAGAGCCTGCCTGGAGCGTT TCTAGCAAGTGAGAGAGATGGGAGCTCCTCTCCTGGAGGATGCAGGTGGAACTCAGTCAT C TAGACTCCTCCTCCAAAGGCCCCCTACGCCAATCAAGGGCAAAAAGTCTACATACTTTCA C TCCTGACTCTGCCCCCTGCTGGCTCTTCTGCCCAATTGGAGGAAAGCAACCGGTGGATCC TCAAACAACACTGGTGTGACCTGAGGGCAGAAAGGTTCTGCCCGGGAAGGTCACCAGCAC

WIAF-1209, CAACACCACGGTAGTGCCTGAAATTTCACCATTGCTGTCAAGTTCCTTTGGGTTAAGCAT UTR-04684f , TACCACTCAGGCATTTGACTGAAGATGCAGCTCACTACCC [T/C] ATTCTCTCTTTACGC WI-7252f 520 TTAGTTATCAGCTTTTTAAAGTGGGTTATTCTGGAGTTTTT

GTTTGGAGAGCACACCTATCTTAGTGGTTCCCCACCGAAGTGGACTGGCCCCTGGGTCAG TCTGGTGGGAGGACGGTGCAACCCAAGGACTGAGGGACTCTGAAGCCTCTGGGAAATGAG AAGGCAGCCACCAGCGAATGCTAGGTCTCGGACTAAGCCTACCTGCTCTCCAAGTCTCAG TGGCTTCATCTGTCAAGTGGGACTCTGTCACACCAGCCATTCTTATCTCTCTGTGCTGTG GAAGCAACAGGAATCAAGAGACTGCCCTCCTTGTCCACCCACCTATGTGCCAACTGTTGT AACTAGGCTCAGAGATGTGCACCCATGGGCTCTGACAGAAAGCAGATCCTCACCCTGCTA CACATACAGGATTTGAACTCAGATCTGTCTGATAGGAATGTGAAAGCACGGACTCTTACT GCTAACTTTTGTGTATCGTAACCAGCCAGATCCTCTTGGTTATTTGTTTACCACTTGTAT TATTAATGCCATTATCCCTGAATTCCCCTTGCCACCCCACCCTCCCTGGAGTGTGGCTGA GGAGGCCTCCATCTCATGTATCATCTGGATAGGAGCCTGCTGGTCACAGCCTCCTCTGTC TGCCCTTCACCCCAGTGGCCACTCAGCTTCCTACCCACACCTCTGCCAGAAGATCCCCTC AGGACTGCAACAGGCTTGTGCAACAATAAATGTTGGCTTGGNNNNNNNNNNNN

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AACCCCTGAAATCGGAAGGGACTTCCTCTTTCTCTCCTTCTTCCCTGTTTTAAATTATAA GATGTCATCCCCTTGTGTCAGAGACAGACCCCTTGGCTTTTGCTTGGCAGAGAGGACCCC ACTGGACTGGGTTTTGTCTCTGCATCTCATTGTAGAGCTTGGTGGCTGAGCTTGGCCCTA TT£AGATAAATAGAGTTCCAAATAAGGATTTGTTCACATGCATCATAACCATTCCCATTG GTTCTCCTAAAACATGAAAAT [T/G] ATCTCCCTTAGTAATCCCCCTTGCCAAATTCCAT GTCCCTGTATAATTCTACAGGATGGGGACACTAATGAAGATACGGTTGCTTCACCTTGGA GCCTGAACATGACATTTCTAAGTGGGGTGCATCCCCCAGCACTGATGTTGTTACTGATTC

WIAF-1371, TCCTGTCAGAGATCTGGGAGGTCTCCACTGAGGATGTGAGCCTGATTATCCTATAGGCAG UTR-05629, ACGTGGGGAGGGTGGAGGGGTGACAGTGGAGGAAAATCCATGGATATCCACGCAGCAGCC WI-7981 261 CCTCTTTAACCTCATCTACAAGCA _ _ _ _

TTTGCCCTGTGGATTCCAGCATTTGCCATTCCTGGAATCAAGGAATCCTGAGTCTGGGCA ATGAAACCAAAGCCAGGAGTTGACGCATCCTGCAGTTGGGCCAGCTGTCGCATCTCAGCG GGGCGCACATGTTATCCACAAGCAATGGACCTTTGGGGAAGGGGGAGTTTTTAGTTTGTT TTACAAATTTTTCCTGCAAAAGTGGAATCACTGTATTTTCATTTTAATTTATATTTGAAA TTTTATTTAGTTCTTGAGTAGATCTGCTTCTTCATCTTGACATGTAATGAATGGTCAGTT GTACGTAATGTATTTATATGTTAATTTGTTATGTATATAGATGTGCAAGTCTTGTCAGAA O TTGGCCTCAGTGTAGTTAAAGGGCAGAAGGGGAAGATACTGACTAGTCATAGAAATACCT ' CO CATTCGCCTGTGGGAAGAGAAGGGAAGCCTCTTCAGGGTGAGTGAATGGCAAAGCGGTTG CTTCTCCG

AATCTTAACAGCCTTTTGATGCCAAAGCCACTTTCAGTCTTAATTCTTTTTGGAGCCTAA GATCAGTGCAACCCTCCAAGGCTCCCCAGTATCTGGCACATCTTTCCCTTTTC [A/G] TC

WIAF-4117, TCCGTTTGTGTGTTTGGCCAAATAATATCTCCCCCAGGGACGTCCTCTTTCTAATCCCTG WT-867, WI- AAACCTGAGAAAATGTTATCTTATGCAGTGCTATGGTTTGAATGTGTCCCCCACAAAGCA 167 113 CACATTAGAAACTTAATCCCCAGTGCAACA

TGTCCTTGCTTATGCCTGCCTCTTTCGCTTGGCAGGATGATGCTGTCATTAGTATTTCAC AAGAAGTAGCTTCAGAGGGTAACTTAACAGAGT [G/A] TCAGATCTATCTTGTCAATCCC AACGTTTTACATAAAATAAGAGATCCTTTAGTGCACCCAGTGACTGACATTAGCAGCATC

WIAF-439, TTTAACACAGCCGTGTGTTCAAATGTACAGTGGTCCTTTTCAGAGTTGGACTTCTAGACT

UTR-02799, CACCTGTTCTCACTCCCTGTTTTAATTCAACCCAGCCATGCAATGCCAAATAATAGAATT

WI-7069 93 GCTCCCTACCAGCTGAACAGGGAGGAGTC

TGGATCCGAATTTTAGTGTGATTTGGCAGGCvATGCGGGG AACATGTT-^AGTCTTT-A ACTTGCACAGAATTGCCAGATTAGCGATTGTTTGACTTGTCCAATTAATGAAATGTGGAA

WIAF-440, AAAAAAAAAGGGTGGTAACTGTTAAGCCTGCTGCAATGTTTAGACACGAGGGTGGGGGTG MR529, WI- GGGAGGTGGAATACCAAGGGAGGCGACACAGAATTTCCTTGCCTTTTGGTTTTCTCATAC 1819 51 TCAGTATTTCTGCCGTGGC

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4

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the claims.

Claims

CLAIMS WE CLAIM:

1. A nucleic acid segment shown in column 7 of the Table, or a portion thereof which includes a polymorphic site, or the complement of the segment or portion thereof.

2. The nucleic acid segment of claim 1 that is DNA.

3. The nucleic acid segment of claim 1 that is RNA.

4. The segment of claim 1 that is less than 100 bases.

5. The segment of claim 1 that is less than 50 bases.

6. The segment of claim 1 that is less than 20 bases.

7. The segment of claim 1, wherein the polymorphic site is biallelic.

8. The segment of claim 1, wherein the polymorphic form occupying the polymorphic site is the reference base for the fragment listed in the Table, column 3.

9. The segment of claim 1, wherein the polymorphic form occupying the polymorphic site is an alternative form for the fragment listed in the Table, column 4.

10. An allele-specific oligonucleotide that hybridizes to a segment of a fragment shown in the Table, column 7 or its complement.

11. The allele-specific oligonucleotide of claim 10 that is a probe.

12. The allele-specific oligonucleotide of claim 10, wherein a central position of the probe aligns with the polymorphic site of the fragment.

13. The allele-specific oligonucleotide of claim 10 that is a primer.

14. The allele-specific oligonucleotide of claim 13, wherein the 3 ' end of the primer aligns with the polymorphic site of the fragment.

15. The allele-specific oligonucleotide of Claim 10, which is selected from the group consisting of the nucleotide sequences of the Table, column 5.

16. The allele-specific oligonucleotide of Claim 10, which is selected from the group consisting of the nucleotide sequences of the Table, column 6.

17. An isolated nucleic acid comprising a sequence of the Table, column 7 or the complement thereof, wherein the polymorphic site within the sequence or complement is occupied by a base other than the reference base shown in the Table, column 3.

18. A method of analyzing a nucleic acid, comprising obtaining the nucleic acid from an individual; and determining a base occupying any one of the polymorphic sites shown in the Table.

19. The method of claim 18, wherein the determining comprises determining a set of bases occupying a set of the polymorphic sites shown in the Table.

20. The method of claim 18, wherein the nucleic acid is obtained from a plurality of individuals, and a base occupying one of the polymorphic positions is determined in each of the individuals, and the method further comprising testing each individual for the presence of a disease phenotype, and correlating the presence of the disease phenotype with the base.