EP0944741A2 - Vegetal sequences including a polymorphic site and their uses - Google Patents

Abstract

A nucleic acid segment comprising at least 10 contiguous nucleotides from a vegetal sequence including a polymorphic site; or the complement of the segment.

Description

VEGETAL SEQUENCES INCLUDING A POLYMORPHIC SITE AND THEIR USES

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) means a variation in DNA sequence that alters the length of a restriction fragment as described in 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; WO 90/11369; 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 Let t . 307, 113-115 (1992); Horn et al . , WO 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 proteincoding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein. Other single nucleotide polymorphisms occur in noncoding regions . Some of these polymorphisms may also result in defective or variant 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. Also, the different forms of characterised single nucleotide polymorphisms are often easier to distinguish that other types of polymorphism (e.g., by use of assays employing allele-specific hybridization probes or primers) . Despite the increased amount of nucleotide sequence data being generated in recent years, only a minute proportion of the total repository of polymorphisms has so far been identified. The paucity of polymorphisms hitherto identified 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 oligonucleotides in a population of individuals by didoxy 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 subjects.

SUMMARY OF THE INVENTION

The invention provides nucleic acid segments containing at least 10, 15 or 20 contiguous bases from a vegetal fragment including a polymorphic site notably a single nucleotide polymorphism (SNP). In a particular embodiment, a vegetal fragment does not belong to the Cruciferae family .

The segments can be DNA or RNA, and can be double- or single-stranded. Some segments are 10-20 or 10-50 bases long. Preferred segments include a diallelic polymorphic site. In a preferred embodiment, the invention concerns nucleic acid segments from a fragment shown in Table I (corn) . The Invention further provides allele-specific oligonucleotides that hybridizes to a segment of a vegetal fragment, for example fragment in Table I. These oligonucleotides can be probes or primers . Also provided are isolated nucleic acid" comprising a sequence of Table I or the complement thereto, in which the polymorphic site within the sequence is occupied by a base other than the reference base shown in Table I .

The invention further provides a method of analyzing a nucleic acid from a subject. The method determines which base or bases is/are present at any one of the polymorphic vegetal sites for example of those of Table I. Optionally, a set of bases occupying a set of the polymorphic sites shown in Table I is determined. This type of analysis can be performed on a plurality of subjects who are tested for the presence of a phenotype. The presence or absence of phenotype can then be correlated with a base or set of bases present at the polymorphic sites in the subjects tested.

DEFINITIONS

A nucleic acid, such an oligonucleotide , 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. Preferred nucleic acids of the invention include segments of DNA, or their complements including any one of the polymorphic sites shown in Table I. The segments are usually between 5 and 100 bases, and often between 5-10, 5-20, 10-20, 10-50, 20-50 or 20-100 bases. 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 Table I. Methods of synthesizing oligonucleotides are found in, for example, Oligonucleotide Synthesis : A Practical ApproacA (Gait, ed., IRL Press, Oxford, 1984).

Hybridization probes are oligonucleotides capable of binding 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) .

The term primer refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., 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 means 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.

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, and can be measured by percent recombination between the two genes, alleles, loci or genetic markers.

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 a" small as one base pair. Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatelli tes , dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu . The first identified allelic form is arbitrarily designated as a 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 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 1QSS 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.

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 are suitable for allele-specific probe hybridizations. Nucleic acids of the invention are often in isolated form. An isolated nucleic acid means an object species that is the predominant species present (i.B., on a molar basis it is more abundant than any other individual species in the composition) . Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present. Most preferably, the object species is purified to essential homogeneity (contaminant specie" cannot be detected in the composition by conventional detection methods) .

DESCRIPTION OF THE PRESENT INVENTION I . Novel Polymorphisms of the Invention The present application provides for example oligonucleotides containing polymorphic sequences isolated from graminae species for example maize. The invention also includes various methods for using those novel oligonucleotides to identify, distinguish, and determine the relatedness of individual strains or pools of nucleic acids from plants .

EXAMPLES

Example 1. Maize DNA extraction ϊ

DNA was extracted from maize lines as described in Rogers and Bendich (1988 Plant Mol Biol Manual A6 : 1- 10) with modification described in Murigneux et al (1993 theo Appl Genet 86 : 837-842) . PCR amplification was done on six maize lines representing a wide range of genetic variability and including both european flint material and US dent germplasm. Those six maize lines have been choosen to maximize the genetic variability of cultivated maize. Doing so, optimize the chance of finding polymorphism in the allelic sequences. For example Gl, an european flint line and G3 , an US Corn Belt Stiff Stalk line, are completly unrelated. Their genetic distance (coefficient of dissimilarity) calculated with our standard approach (89 RFLP probe/enzyme combinations and Nei-li distance) is 0.69. This value is close to the maximum distance between two cultivated maize lines .

Among the 15 genetic distance between couple of these 6 lines : 8 are superior to 0.6, 6 superior to 0.5 and only one inferior to 0.5. This shows that the choice of the lines avoided as much as it was possible the potential redudancy (or similarity) of allele at the locus sequenced. With the same effort of sequencing we should therefore have collected the maximum number of polyphomism. Genotypes :

Gl=flint line

G2=flint line

G3=Dent line

G4=Dent line G5=Dent line

G6=Dent line

Example 2. Choice of the markers

The markers have been chosen with the following criteria.

1. Selection of markers that give a single band in southern hybridization. This is to avoid as much as possible the problems of duplicated sequences (very frequent in plants) . If the same (or nearly the same) sequence occurs at several position in the genome (locus 1 and 2) and if the primers used to type the SNP found on locus 1 allow amplification of the sequence at the locus 2, the results of hybridization on the chips will be the addition of two markers pattern and therefore impossible to use.

2. Distribution on the genome : most of the genetic analysis in plant aim to characterize the whole genome (genetic variability evaluation, mapping quantitative trait-locus, back-cross assisted selection). The second criteria was therefore to choose markers nicely distributed over the 10 chromosomes (see Table A hereunder for map position) . 3. Selection of gene coding for enzymes involved in the Carbone metabolism. Wxl , Ael, Sh2 , Brel, Btl, Ssu, Bt2 are involved in sugar-starch metabolism. Such a choice will allow to have a very fast characterization of the allelic variability (possibly linked to efficiency) of gene involved in this metabolism.

The following markers have been used : see Table A.

LEGEND OF TABLE A Probe = name of the marker

COD = in-house code.

MAP Pos = map position, given by the bin location of the

University of Missouri map (Maize Genetic Newsletter n°69

1995). Examples of reading the "MAP Pos" and "Prim" columns : 1.01-1.02 means that it is the core probe that delimit the bins 1 and 2 on chromosome 1

5.01 means that it is located in the bin 5.01 (on chromosome

5)

4 means that it is located on chromosome 4 SOIF is the forward primer for probe 1

S01R is the reverse primer for probe 1

Genbank/ EMBL = Genbank/ EMBL number TABLE A

Csnpld (33 markers )

PROBE COD Map Pos PRIM SEQUENCES OLIGOS Genbank/EMBL

UMC157 SOI 1.01-1.02 S01F CGCACGCACATTAGCTTTCG G10822 S01R TGCAACCGAACAGGATCTGC G10823

U C76 S02 1.02-1.03 S02F ATTATTCGGCGTCCAGCCCC G10865 S02R TTACCAGCGGTGAGAGCTGC G10866

UMC67 S03 1.05-1.06 Ξ03F CGTTCGTGTGGCATCAATCG G10864 S03R CGACATCATCATCGGCAACC G13173

UMC161 S06 1.10-1.11 S06F CAGACCTTGGTTGGAGGCAAC G10824 S06R TCGCTCCCCTTCTTCCTTCC G10825

UMC53 S08 2.01-20.2 S08F CGGACGTGATGCAAGTTTCG G10851 S08R AGCGGCTCAAGCTCTCCATC G10852

UMC131 S10 2.04-2.05 S10F2 TCCTTGGCACTCACGCTACC G10816 S10R2 AGCATGGGGGGCAACAACTC G10817

UMC49 S12 2.08-2.09 S12F CAGAGAGCCGTCTCGAATCG G10845 S12R TTGATACTGCCGTCTGCCG G10846

UMC102 S14 3.04-3.05 S14F TGCTGTGCTGTCACATGGCG G10801 S14R CTGGGTCGTCGTGCTTTGAG G10802

UMC63 S16 3.08-3.09 S16F2 ACGCCCTGACAGAACCATCG G10857 S16R TTGCTCACTCGTGGTCGTGG G10857

Adh2 S17 4.03 S17F2 TGCCTGCTGCATCTCTAGCC X02915 S17R2 CAAGCCCGAAAATCGCCAC X02915

UMC66 S19 4.06-4.07 S19F TGGAGTGTCCAAAGACCGACC G10862 S19R ACCAAAACGGGTGGTCTGCC G10863

UMC90 S22 5.01 S22F GCAGGTGAACAATGCTGCCC G10870 S22R CCAAAAGGCGGAGAACCGAC G10871

Ael S23 5.05 S23F TCGCTGGGGTTTTAGCATTG L08065 S23R CACTCGAACTCTGTTCAAGGCTTG L08065

UMC59 S26 6.01-6.02 S26F TCCAAAGCGAAAGCCTGATG Gl0853 S26R TACGATGGCCGTGACCCTTC Gl0854

UMC65 S27 6.03-6.04 S27F TTCCAGCTTTCCTCGGCACC G10860 S27R AGCAGCAAGAGCAGAGCGTG G10861

UMC21 S28 6.04-6.05 S28F TGCAGATGTGCCTTTCCTGTG Gl0830 S28R CAGTGGATTCGCTCCCTTCTC Gl0831

UMC132 S29 6.06-6.07 S29F CGCAC GAGGCAGATGCAGC G10824 S29R CGCTAGGCAGAGGTTCGAGC G10819

UMC254 S33 7.03-7.04 S33F CCGGGCGC AAAGAATGTG G10832 S33R AAGAAACCAGCACCAGCGGG Gl0833

UMC80 S34 7.04 S34F TCGCCTTTATCGGTGCAATG G10867 S34R TGGAGC AGCATGGAGATCG G10868

BNL9-11 S38 8.01-8.02 S38F2 CGAGGGAATGTCATCAACCC G10778 S38R2 ACCAAAGCTCCTCAGCCAAG G10779

U C109 S42 9.00-9.01 S42F GCACCGTCGTTTACCTCAAGC G13177 S42R TAGCCATCATCAGCGGCGTG G10807

Wxl S43 9.02-9.03 S43F CGTGCTACCTCAAGAGCAAC X03935 S43R ACTTCACGGCGATGTACTTG X03935

UMC95 S44 9.04-9.05 S44F CACTCGGAAGTCGGAATCGC G10872 S44R ACCTTCGCAGTGTTGCGGAC G10872

CSU61 S45 9.05-9.06 S45F TCTCCACGAATCCCACCGTC T12691 S45R AAGGGAGGGAATCCTCTACCG Tl2691

UMC130 S48 10.02-10.03 S48F AAGGGGGAAGAAGGTCATC G10814 Ξ48R CGATGGCAACAACTACCAGTAG G10815

CSU109 S53 2.09 S53F GCTTTCGGTTCCGGATAGCG T12721 S53R ACTGGGCCATCTCCGACCAG T12721

UAZ77 S56 5.04 S56F2 GCAACCAACTGCAACATCGC T18762 S56R2 GAAGGAGCTCAAGGCCAAGG T18762 Shi S57 9.01 S57F TGCTGTTATTGCGTGCCGTG X02382 S57R2 AAGGTGGCACCAAGGCGTTC X02382

Sh2 S63 3 .09 S63F TTCTTCACTGCACCCCGATG M81603 S63R CTGCTCACTCTGCAATGCCC 81603

Brel S65 6 S65F AGCAGCAGATCAGGCACACC U17897 S65R TTGAAGTTCGTTTCGGGCAC U17897

Btl S66 5 S66F GGCAAGGATCGGAGTTGCTC M79333 S66R TAGCGTGGAGGACGTTCTGG M79333

Ssu S67 S67F GCAAGCAAGCAAGCAGCGAG D00170 S67R GACCCGAAGCAAAACCGAAC D00170

Bt2 S71 4 S71F TGCCGAAAAAGGTGGCATTC Seq (Bae et al

1990)

S71R GCCCCCAATGTCGATTCAAC

Example 3. PCR amplification

PCR amplification was done with primer designed using the DNA sequences of the markers listed above. The sequences for all markers/genes were available on Genbank/ EMBL.

Forward and reverse primers are given in the table A hereabove .

PCR condition were as followed

For each reaction in 30 microliters : DNA :60 ng; Taq DNA polymerase (Amersham) : 0.9 unit; Buffer lOx : 3 microliter; dNTP ' s : 0.2 mM each; MgC12 : 1.5 mM; BSA 0.8mg/ ml; primers 1.5 ng/microliter each; glycerol 5%.

Polymerisation was done in a perkin Elmer 9600 : 1' at 95°C, followed by 35 cycles of (30" at 94°C, 30" at 60°C, 1'30" at 72°C) followed by 1'30" at 72°C.

The sequencing of 186 maize amplicon was then done with the primers used for DNA allele amplification. DNA sequences were edited and aligned. Sequence surronding polymorphism (see table I here-under were collected from these alignments .

LEGEND OF TABLE I (with references to the Bt2 gene for instance.)

Column 1 (Bt2) represents the name of the marker or gene .

Column 2 (Bt2-G2/G6-1) represents :

- the name of the maker (Bt2) If

- the genotype number (G2)

- the second genotype number (G6)

- and the number of the SNP (single nucleotide polymorphism) . So, in this case, it is a SNP found on a sequence nucleotide Bt2 between the genotypes (strains of maize) G2 and G6 and this SNP was numbered 1 (Sometimes there are several SNP between two genotypes for the same sequence)

Column 3 represents : similar to column 2, but with the codification of the marker/gene.

Column 4 represents sequence holding the SNP. Into brackets : [G/T] means that the sequence of G2 , at this position of Bt2 gene, is G, while for G6, it is T.

On the other hand, /G (CSU61-G1/G5-1A) means deletion of the base pair G in Gl compared to G5.

TABLE I csnpld

Bf2 Bt2-G2 G6-1 S71G2 G6-1 ATAATACTTGATATGCCATT[G/ TGTCCTCTTATTTTTAACAT

Ssu Ssu- G1/G5-1 S67G1/G5-1 ATGGCCTCGTCGGCCACTGC[AC]GTCGCTCCGTTCCATGGGCT

Ssu Ssu-G1/G3-1 S67G1/G3-1 GCCGCTCCTCCAGAAGCCTC[G/A]GCAACGTCAGCAACGGCGGA

Ssu SSU-G1/G3-2 S67G1/G3-2 GTGTTGCCCATCCCATCCCAIATJTTCCCAACCCCAAACGAACC

Ssu SSU-G1/G3-3 S67G1/G3-3 GTACCTGCCGCCGCTGTCGA(CG/AC]GGACGACCTGCTGAAGCAGG

Bt1 Bt1-G2 G3-1 S66G2/G3-1 AGTGAGCCCGCTTCTTATTC[ ηTAAGGTGATAGGTrrCTAAA

Bt1 BM-G2 G3-2 S66G1/G3-1 AATGTAATGGTACTCCGCGCπ^"/C]ATGGCTCTGGTACT AGGAA

Bt1 BU-G2/G3-3 S66G1/G2-1 AAATAGGCTCGGGCAATTAT[C 1CAGCTTAGGGACAGCAAGCG

Bre1 Bre1-G3 G6-1 S65G3/G6-1 TCCGCCCTGCCTCX;GGTTTT[A T]GCCCGACCTTCGAAACATTC

Bre1 Bre1-G3 G6-2 S65G3 G6-2 ACCACTGACGTAGCACCTCC[G T]ACTTCTCGTTGTAAAACCCC

Bre1 Bre1-G3/G5-1 S65G3G5-1 GGAGGTTCGCCTCATGTTATIC/ΗGTTGACGAGCCACATCCACT

Bre1 Bre1-G4 G6-1 S65G-J/G6-1 GCTCCGACTTCCAATCTTGA[A/C1CCTCCACCCTGCCTCCGGTT

ASG12 ASG12-G1/G3-1 S6 G1/G3-1 CTGGTRGAAATGTGTTGAAG[CA]TACTAGTGATGAACTGCTTG

ASG12 ASG12-G1/g3-2A S6 G1/g3-2A GCTGCTCCMGCGAGCCCGC[C G]CCGAAAAAGGAAAAAGGTGA

ASG12 ASG12-<31/g3-2B S64G1/g3-2B GCTGCTCCAAGCGAGCCCGC[C G]CCGAAAAAGGAAAAAGTTGA

ASG12 ASG12-G1/g3-3A S64G1/g3-3A CGCX:CCGAAAAAGGAAAAAG[GNITGAAGGTCCTTACTCACCGA

ASG12 ASG12-G1/g3-3B S6 G1/g3-3B CGCGCCGAAAAAGGAAAAAG[G ΗTGAAGGTCCTΤACTCACCGA

ASG12 ASG12-G1/gG3-4A S6 G1/gG3- A GAACCGGCX;ACAGTGCCTGAΠ^"/A]TTTGGCGGTGAGACCTCTTC

ASG12 ASG12-G1/g3-4A S64G1/g3- A GAACCGGCCACAGTGCCTGATT/A]TTTGGCX3GTGAGACTTCTTC

Sh2 Sh2-G5K36-1 S63G5 G6-1 CMTTGTTACCTGAGCMGA[T]TTTTGTGTACTTGACTTGTT

Sh2 Sh2-G4/G6-1 S63G4 G6-1 TACTGAGAGAATGCAACATC[ΑG]AGCATTCTGTGATTGGAGTC

Sh2 Sh2-G4G5-1A S63G4G5-1A TTTTAGTGTACΠGACTTGT[CMCTCCTCCACAGATGAAATAT

Sh2 SH2-G G5-1B S63G4 G5-1B TTTRTGTGTACTTGACTTGTTC/ΗCTCCTCCACAGATGAAATAT

Sh2 Sh2-G3 G6-1 S63G3G6-1 TCTGTGATTGGAGTCTGCTC[G/A]CGTGTCAGCTCTGGATGTGA

Sh1 Sh1-G5 G6-1 S57G5 G6-1 AACTACAAAAAGCATCTCXITIG IGGATTTGGCTATCTCCTTTT

Sh1 SM-G2/G5-1 S57G2 G5-1 TTAGCGCGAAAAAAAAACTC[ΗI 1 I I I I I I I GTCCTTTTACT

Sh1 Sh1-G2 G3-1 S57G2 G3-1 TCMTCCMTCMTTΓMTTΠ^{^}/CICTTCCTTTAAAAATATTATC

Sh1 S -G1/G2-1 S57G1 G2-1 TTACTACGAAAAACTCTTGA[G T]TCTAGGMTTTGAATTTGTG

Sh1 S -G1/G2-2A S57G1/G2-2A CTTCTTGGATTτTGCTATCTrr/C]CτTTTACTACGAAAAACTCT

Sh1 Sh1-G1/G2-2B S57G1/G2-2B CTCCTTGGATTTTGCTATCTrr/C]CTTTTACTACX3AAAAACTCT

Sh1 SM-G1/G2-3A S57G1/G2-3A rπTACTACGAAAAGCATCTrr/C]CTTGGArπTGCTATCπcT

Shi Sh1-G1/G2-3B S57G1 G2-3B TTTTACTACGAAAAGCATCT[T/C]CTTGGATTTTGCTATCTCCT

Shi S57G1 G2- S57G1/G2- GMGCCAAATCCTATTATΓΠJ/CJCTGCCTCTAGGGTCTGAATG

UAZ77 UAZ77-G4/G6-1 S56G4 G6-1 GTACACTGTTACMTCACAC[T/G]TAGTGAAGCGCAACACAGAT

UAZ77 UAZ77-G4 G6-2 S56G4G6-2 GCCTTATCATCX^TCTAGGTAN^'/AJTGGAGACGAGTGACCAGTCT

UAZ77 UAZ77-G G6-3 S56G G6-3 CTTTTCTTCAGACCCGAGCCFC ΗCCAATCGCGCCCTTCTGTGC

UAZ77 UAZ77-G G6-3 S56G /G6-3 CTTTTCTTC VGACXXX^GCC[C T< AATCGCGCCCTTΤTGTGC

UAZ77 UAZ77-G /G5-1A S56G4/G5-1A GAGCCCCCMTCGCGCC^rηC ηTGTGCCπGGCCTTGAGCTC

UAZ77 UAZ77-G4 G5-1A S56G4/G5-1A GAGCCTCCAATCGCGCCCTT[C/ηTGTGCCTTGGCCπGAGCTC

UAZ171 UAZ171G1/G3-1 S55G1Λ33-1 GAAGGAGCAGCAGCGCAAGG{AflACGTGTTCCAAGTCAACGTC

UMC17 UMC117-G2/G3-1 S54G2/G3-1 GTAGA G GCAAAAACXAn7]TTTTTTAGTGAAAAAACATA

UMC17 UMC117-G2Λ33-2 S54G2/G3-2 ATTGTGGCTAGAAACTTTGGpiJI I 1 1 1 1 lAAATTATGGTCAT

CSU109 CSU109-G5/G6-1 S53G5Λ36-1 GCAAAα^ACACCAATCTTC{GΛ AAATGAGCAAAGCAGAGACT

CSU109 CSU109-G5/G6-2 S53G5Λ36-2 CAGATCGGTTGTCCTCAGAGIAJAAGTCACCTACCTGCAAACX;

CSU109 CSU109-G5/G6-3 S53G5 G6-3 AAπCTACATAGGAGTCATG[CtηACAAGTACτTGTTrAAAGGA

CSU109 CSU109-G5 G6-4 S53G5G6-4 ACMGTACΠGTTTAAAGGA[CJCATGCCGGAATACACGCTGC

CSU109 CSU109-G5/G&SA S53G5/G6-5A GAGCGAGATCGATCCTGTTGN^{^}/CICATCCATCACTGCCATAGGA

CSU109 CSU109-G5G&5B S53GS/G&5B GAGCXΪΛGATCGATCCTGTTGN"A^CATCCATCACTGCCGTAGGA

CSU109 CSU109-G4/G6-1 S53G G6-1 TAGTCATAGO\A»\GCATGC{G/AITCGTGATGTAGCXSTTCACCC

CSU109 CSU109-G4Λ36-2 S53G4G6-2 CAARRGAAGAGGAAAAAAAAPT CTACATAGGAGTCATGTAC

CSU109 CSU10WS4/G5-1 S53G4/G5-1 CAGAGACTOW^AGGCGAALAAPIGGAGTCCACAATAGΠCGTC

CSU109 CSU109-G3KΪ5-1 S53G3Λ55-1 CCCACX3GCGGGAGATGGTGG[TRRAGAAGCGGAACCACCGAGC

CSU109 CSU10MS2/G6-1 S53G2/G6-1 ACTTGMAAAGGACATGCC{G#ΪGMTACACGCTGCCCAGGC

CSU109 CSU10WΪ2Λ33-1 S53G2G3-1 CCCAGGCCTTCCCV\CGGCGG)>VG]GATGGTGGTTAGAAGCGGAA

CSU109 CSU109-G1/G6-1 S53G1 G6-1 CAAAGCAGAGACTCCACY AG[AΛ3JC!GAACAGAGTCCGCAATAGT

CSU109 CSU10W31/G6-2 S53G1A36-2 GAACAΒAGTCCG<>VATAGRΗTΛ3ATCCTAATGCTACTTCGAGC

UMC130 UMC130-G3Λ36-1 S48G3Λ36-1 GATTCAGAAACAGTGGCGGC{A/GK3ATGTAGCATCAACACGCCC

CSU61 CSU61-GSG6-1 S45GSG6-1 ATGAGTATATTCAAGTCATAIΓCΠ'GTGAACTAGMTGTTATTT

CSU61 CSU61-G5Λ36-2A S45G5K36-2A CCTAGACGCTGACCGCCACA[G/AJAC«GCGGCGGCTGCCAAATC

CSU61 CSU61-G5A36-2B S 5G5Λ36-2B CCTAAACGCTGACCGCCACAIGAJACGGCGGCGGCTGCCAAATC

CSU61 CSU61-G5/G6-3 S 5G5/G6-3 TGAACAAAC ^TGCGCTACCJCTΗAGCTAGGTGTΤTTAAAGTAA

CSU61 CSU61-G4Λ36-1 S45G4/G6-1 TCCGCGGAAAC^AOVTCCGAIG/TΓΓTCTTGAGGATAACCGAGCT

CSU61 CSU61-G Λ35-1 S45G4/G5-1 GGGAGGGGAAAAAAAAAGAA[GAIAGCGTTGGTTGCGGTTCAGT

CSU61 CSU61-G G5-2 S45G4Λ35-2 GGCGGCTGCCAAATCCGCGG[AIAAACGACATCCGAGTTCTTG

CSU61 CSU61-G2 G4-1A S 5G2/G4-1A CTAGAATGTTATTTCΠCAC[CTA]GTΤGACCATGGAAAAAAACA

CSU61 CSU61-G2/G4-1B S 5G2/G4-1B CTAGMTGTTATTTCTTCACICTALGTTGACCATGGAAAGAAACA

CSU61 CSU61-G2Λ34-2A S45G2Λ54-2A TT< \CCGTTGACCATGGAAA[AΛ3Μ^AACAGTAATAAGTTCΠGT

CSU61 CSU61-G2Λ34-2B S45G2/G4-2B TTCVVCAGΠΒACCATGGAAATA ΘJAAACAGTAATAAGTTCTTGT

CSU61 CSU61-G1 G6-1 S45G1 G6-1 TTCTTC^CAGTTGACCATGGPAJAAAAAAACAGTAATAAGTTC

CSU61 CSU61-G1/G5-1A S45G1 G5-1A GAACCCACCGTGCCCTGGGAIGIGGGAAAAAAAAAGAAGAGCG

CSU61 CSU61-G1/G5-1B S45G1 G5-1B GAACCCACCGTGCCCTGGGA{RØ}GGGAAAAAAAAAGAAAAGCG

CSU61 CSU61-G1Λ35-2A S45G1Λ35-2A TGGGAGGGAAAAAAAAAGAALG/A)AGCGTTGGΠGCGGΠCAGT

CSU61 CSU61-G1 G5-3 S 5G1ΛS5J (MTACCAGCTAGGAATCGTAIAGJAAAAGCCTAGACGCTGACCG

UMC85 UMC95-G5 G6-1 S44G5/G6-1 GCTGCGTCAATCATCACTTCFT/ALCCCACAGGCGTCAAGTACAG

UMC95 UMC95-G3 G -1 S44G3/G4-1 GACAGATTCCAAAGTAGTCGTCYΗCGGCCAGGTCGAAAAAGAAT

U C95 U C95-G2/G6-1 S44G2/G6-1 GGCGCTGCGTCMTCATCACTAO CACCCACAGGCGTCAAGTA csnpld

UMC95 UMC95-G₂/G4-1A S4 G2/G4-1A TCGGTGTCACCACATGCATA[T/G]TCAGGACAGATTCCAAACTA

UMC95 U C95-G2/G4-1B S44G2/G4-1B TCGGTGTCACCACATGCATA T/G1TCAGGACAGATTCCAAAGTA

UMC95 U C95-G2/G4-2A S44G2/G4-2A GTCGCCGGCCAGGTCGAAAA[G/A]GMTACTCAGCAAAAGACCC

UMC95 UMC95-G2/G4-2B S44G2/G4-2B GTCGTCGGCCAGGTCGAAAA[G/A]GAATACTCAGCAAMGACCC

U C95 UMC95-G2/G3-1A S44G2/G3-1A TATTCAGGACAGATTCCAAA(C/G]TAGTCGCCGGCCAGGTCGAA

U C95 U C95-G2 G3-1B S44G2 G3-1B TAGTCAGGACAGATTCCAAAFC/GJTAGTCGCCGGCCAGGTCGAA

U C95 UMC95-G1/G6-1 S44G1/G6-1 GCGTCAAGTACAGATACGCA[A G]CACGCCTCAGCTTCGCCTTG

UMC95 U C95-G1/G2-1 S44G1/G2-1 CCTGGGACTCCGCAAATTGC[G/A]AGCACTCGGTGTCACCACAT

Wx1 Wx1-G2/G6-1 S43G2 G6-1 GCTGGTTCATTATCTGACCT[G/ΗGATTGCATTGCAGCTACAAG

Wx1 Wx1-G2 G6-2 S43G2/G6-2 CTGGATTGCATTGCAGCTAC[AGJAGAAGCCCGTGGAAGGCCGG

Wx1 Wx1-G2 G6-1B S43G2 G6-1B GCTGGTTCATTATCTGACCT[G/ΗGATTGCATTGCAGCTACGAG

Wx1 Wx1-G2/G6-2B S43G2/G6-2B CTTGATTGCATTGCAGCTAC[A G]AGAAGCCCGTGGAAGGCCGG

Wx1 Wx1-G2 G6-3 S43G2/G6-3 TCAGCCCCTACTACGCCGAA[G ]GAGCTCATCTCCGGCATCGC

Wx1 Wx1-G2 G5-1 S43G2/G5-1 TACCCGGAGCTGAACCTCCC[C G]GAGAGATTCAAGTCGTCCTT

Wx1 Wx1-G2 G4-1 S43G2 G4-1 TGCATGTGAACATTCATGAA[T/CJGGTAACCCACAACTGTTCGC

Wx1 Wx1-G6 G1-1 S43G6 G1-1 CTCCTACCAGGGCCGGTTCG[T]CCTTCTCCGACTACCCGGAG

Wx1 Wx1-G1/G6-1 S43G1/G6-1 TGAATGGTAACCCACAACTGFCNITCGCGTCCTGCTGGTTCATT

Wx1 Wx1-G1/G5-1 S43G1/G5-1 GCCGACAGGGTCCTCACCGT[G/C]AGCCCX;TACTACGCCGAAGA

Wx1 Wx1-G2/G6-1 S43G2/G6-1 GCTGGTTCATTATCTGACCTIGΠIGATTGCATTGCAGCTACAAG

Wx1 Wx1-G2 G6-1B S43G2/G6-1B GCTGGTTCATTATCTGACCT[G/ΗGATTGCATTGCAGCTACGAG

Wx1 Wx1-G2 G6-2 S43G2/G6-2 CTGGATTGCATTGCAGCTAC[A/G]AGAAGCCCGTGGAAGGCCGG

Wx1 Wx1-G2 G6-2B S43G2/G6-2B CTTGATTGCATTGCAGCTAC[A G]AGAAGCCCGTGGAAGGCCGG

Wx1 Wx1-G2/G6-5 S43G2/G6-5 TCAGCCCCTACTACGCCGAA[G/JGAGCTCATCTCCGGCATCGC

Wx1 Wx1-G2/G4-1 S43G2/G4-1 TGCATGTGAACATTCATGAA[T/C]GGTAACCCACAACTGTTCGC

Wx1 Wx1-G2 G3-1 S43G2/G3-1 CTGGTGGTGGTGCTTCTCTG[AAACJTGAAACTGAAACTGACTGCA

Wx1 Wx1-G2G3-3 S43G2/G3-3 GACCATCRRCACGTACTACC[TACXVLAGACCGCTTTCTGCATCCAC

Wx1 Wx1-G1/G6-1 S43G1/G6-1 CTGACCATCTTCACGTACTATCCTA/)CCAGACCGCTTTCTGCATCC

Wx1 WX1-G6/G1-1 S43G6K31-1 CTCCTACCAGGGCCGGTTCGΓT/ICCTTCTCCGACTACCCGGAG

Wx1 Wx1-G6Λ31-1 S43G6 G1-1 GAGATTCAAGTCGTCCTTCG[G/}ATTTCATCGACGGGTCTGTR

Wx1 WX1-G1/G6-2 S43G1/G6-2 TGAATGGTAACCCACAACTG[C T CGCGTCCTGCTGGTTCATT

Wx1 Wx1-G1/G6-3 S43G1/G6-3 GCCGACAGGGTCCTCACCGT[G/C]AGCX«CTACTACGCCGAAGA

Wx1 Wx1-G1/G5-1 S43G1/G5-1 TCTGACCATCTTCACGTACT[ACCT/]ACCAGACCGCTTTCTGCATC x1 Wx1-G1/G4-1 S43G1/G4-1 CTTGATTGCATTGCAGCTAC[G/A]AGAAGCCCGTGGAAGGCXX3G

Wx1 Wx1-G1/G4-1 S43G1/G4-1 CTGGATTGCATTGCAGCTAC[G/A]AGAAGCCCGTGGAAGGCCGG

Wx1 Wx1-G1/G3-1 S43G1/G3-1 GCTGGTTCATTATCTGAC TIT/GJGATTGCATTGCAGCTACGAG

Wx1 Wx1-G5/G6-1 S43G5 G6-1 AGAGATTCAAGTCGTCCTTqG/JGATTTCATCGACGGGTCTGT

UMC109 UMC109-G2/G6-1 S42G2Λ36-1 CTCCATGAAAAAGGTGCCGC{/G]TACTCTCTCAGTCAGCTACT

UMC109 UMC109-G2/G3-1A S42G2 G3-1A CTGCACTCCGATTGAGGGTC[C G]GAAGCAGGGCAGCGCGTGTG

UMC109 U C109-G2 G3-1B S42G2 G3-1B CTGCACTCCGATTGAGGGTC[C/GJGAAGCAGGGCAGCGCGTTGT

UMC109 U C109-G2 G3-1C S42G2 G3-1C CTGCACTCCGATTGAGGGTC[CG]GAAGCAGGGCAGCGCGTTTG

UMC109 UMC109-G2 G3-1D S42G2 G3-1D CTGCACTCCGATTGAGGGTqC/GJGAAGCAGGGCAGCGCGTTTT

UMC80 UMC80-G3/G5-1 S3 G3 G5-1 C^TGCCTCTGTTGATATTTTIG/CIGTGCACXITTTTGCTTGCAAC

U C80 UMC80-G3 G5-2 S34G3/G5-2 GATTTTGTAGGTTGATGCAT[CnTGTTrGATCTTTCTTATCTCC

UMCβO UMC60-G3 G5-3 S34G3 G5-3 TGCTTGCAACTAAATTAATqA G]TGCTCTATTTGACTAAGAGT

U C80 UMC80-G3/G4-1 S34G3 G4-1 ACATGTCXiW3GACGC^TGGTIC ]CCCAATATTGTTGTTGGAAG

UMC80 UMC80-G3 G4-2 S34G3/G4-2 TTGATCTTTCTTATCTCCTnyqCGAATTTGTTCTGTGTTATA

UMCβO UMC80-G3 G4-2B S34G3G4-2B TTGATCTTrCTTATCTCCTTpC]CGAATTTGTTCTGTGTATAC

UMC80 UMC80-G2/G5-1 S34G2Λ35-1 TGTAGGACTTGGAGAGCTTGfA/GITAATrTACACATGCCTCTGT

UMC80 UMC80-G2/G5-2 S34G2Λ35-2 CATGCCTCTGTTGATATTTTlGyC]GTGCACCTπTGCTTGCAAC

UMC80 UMC80-G2/G5-3 S34G2 G5-3 GATTTTGTAGGTTGATGCATIC/ηGTTTGATCTTTCTTATCTCC

UMC80 UMC80-G2Λ33-1 S34G2 G3-1 GAGACATπC«TACTCV^TA(αηAAττATTTGATGAAATTATT

UMC254 U C25 -G5/G6-1A S33G5/G6-1A AGTATCACAGACTAATCTGAPVG ATCTGGTTGCCACGAAAAC

UMC254 UMC254-G5 Gβ-1B S33G5Λ56-1B AGTATCACAGACTAATCTGAtA/GITATCTGGTTGCCACAAAAAC

UMC25 UMC254-G5Λ36-2 S33G5/G6-2 T<V^AAGTGGTGCAATCGCAA[rqCCACTTGGGCπ^GCCGTGGT

UMC25 UMC254-G5/G6-3 S33G5Λ36-3 (X^CTTGGGCTTGCCGTGGT ICGTATCGTACGCAGGTAGCA

U C254 UMC254-G5K36-4 S33G5Λ36-4 AGCAI 1 1 1 1 IGTTTTGI 1 1 HI/C]CCTTGGCAGACAACAGACAG

UMC25 UMC254-G5Λ36-5A S33G5K36-5A CAGTCCCGAGMT A TtC/JCAGAAAMGGTTrrGπτrT

UMC254 UMC254-G5A36-5B S33G5Λ56-5B CAGTCCCGAGMTCκκ^MTiαiCAGAAAAAGGTrrTGTTTTA

UMC254 U C254-G4R/G6-1A S33G4R G6-1A GGCAGACAACAGACAGATCV^G/CAJCATGCTTGCATTTACTCCCA

UMC2S4 UMC254-G4 /G6-1B S33G4R/G6-1B GGCAGACAACAGACAGATCA(AG«IA]CATGCTTGCATTTACTCTCA

U C254 UMC254-G3R/G6-1A S33G3R G6-1A GTGATCACAGACTAATCTGAPVGΠΆTCTGGTTGCCACGAAAAC

UMC254 UMC254-G3R/G6-1B S33G3R/G6-1B GTGATCACAGACTAATCTGAIAΛ3JTATCTGGTTGCCACAAAAAC

UMC254 UMC254-G3R G6-2A S33G3R G6-2A TCTG TATCTGGTTCKX VQG/AJAAAACCGGGACACAAGAGAG

UMC254 U C254-G3RG6-2B S33G3R G6-2B TCTGAGTATCTGGTTGCCAQG/ALAAAACCGGGACACAAGAGAG

UMC25 UMC254-G3/G6-3 S33G3 G6-3 TCAGTCAAACTCAGTCCCGA[A/GJAATCCCAAATCAGAAAAAGG

UMC254 U C254-G3 G5-1A S33G3Λ35-1A GGTTGCCACGAAAA XΪGGAICGJACAAGAGAGAAACTCAGAGT

UMC254 UMC254-G3 G5-1B S33G3/G5-1B GGTTGCΚ^CGAAAACCGGGA[C GJACAAGAGAGAAACTCAAAGT

UMC254 UMC254-G2R/G3-1A S33G2R/G3-1A ACX ^TGCTTGCATTTACTQCTNCAGTCAAACTCAGTCCCGM

UMC254 U C254-G2R/G3-1B S33G2R/G3-1B AC7\CATGCTTGCATTTACTqcyriCAGTCAAACTCAGTCCCGAA

U C254 UMC254-G1R/G2-1 S33G1R/G2-1 TATTATTCMTTTTGAATAAI/G1GAAGGAAATTTTAGCACCTC

ASG49 ASG49-G3/G5-1 S32G3 G5-1 ATTMTAMTGCATCCTCTG[C GJTAAAAAAACCCATTTTGAAT

ASG 9 ASG49-G3/G5-2 S32G3 G5-2 ATGAATTGAAGCTCTGAATA[(VnAGAATCCACCATTCTTCCGA

ASG 9 ASG49-G3/G5-3 S32G3 G5-3 GAATCCACCATTCTTCCGAAIA GICTGCTTCCTACAAAACTCGA

ASG49 ASG49-G3/G5-4 S32G3 G5-4 GAMGGATGTGTTTTTGATA[G A]CCTTCAGTCTTTCAGATGGA

ASG8 ASG8-G3/G5-1 S31G3 G5-1 CAATGTCTTGTTCGTTATCA(A G]CGAAAGTTTGAATCCCCACA

ASG8 ASG8-G3 G4-1 S31G3 G4-1 TGTATCGGCTAGTCTGGATG[G/AJTCGCACTGGCACTCAGTGCT csnpld

UMC132 UMC132-G4/G5-1 S29G4/G5-1 TCTATTCAGCAGTCTGAGAA[GCA C^"ΗAGGATGGTCGGCTTCTTCAG

UMC132 UMC13₂-G1/G5-1 S29G1/G5-1 CCTTACACTATTAACAGGCC[C ΗGTGATCTACCTGAATGCCTG

U C₂1 UMC21-G5/G6-1 S₂8G5/G6-1 CAAGAAGCCTCTTCAGTGTC[A C]GTCGTAGCTTCCTCAAGACC

UMC₂1 U C₂1-G5/G6-2 S₂8G5/G6-2 AGACCTTCCTGATGTGCGGA[T/C]GCTAATCCATGGAGCAGGGA

U C21 UMC21-G5 G6-2B S₂8G5/G6-2B MGACCTCCTGATGTGCGGA[T/QGCTAATCCATGGAGCAGGGA

UMC21 UMC21-G5/G6-3 S28G5/G6-3 CTAATCCATGGAGCAGGGAG[G A]AAGGGGCGAGGGGCAGCAAG

U C21 UMC21-G4 G5-1 S28G4Λ35-1 TCGTCGCGAATACAGCCGGG[G/QGAGGGGGTGGTCGCGACTGG

UMC21 UMC21-G3 G6-1 S28G3/G6-1 GTCGTAGCΠCCTCMGACC[T/]TCCTGATGTGCGGACGCTAA

U C21 UMC21-G3/G4-1 S28G3/G4-1 GAGTCGTCGCGAATACAGCC[A G]GGGGAGGGGGTGGTCGCGAC

UMC21 UMC21-G3/G4-2 S28G3/G4-2 AGGGGGTGGTCGCGACTGGA[T/G]CGCCCGAGCAGCGAGCAAGC

U C21 UMC21-G3/G4-3 S28G3 G4-3 MGCACATGTTTTMCCTTTΓT/GIATTCAAACTTTCCAGCCGTT

UMC21 UMC21-G3/G4-3B S28G3/G4-3B MGCACATGTTTTAACCTTTΠ^"/GIATTCAAACTTTCCAGCGTTA

UMC21 UMC21-G2/G6-1 S28G2 G6-1 GAATGTTGCTGTTATATTACFT/QCGTAGGTGACAAAGGGTTCA

UMC21 U C21-G2 G4-1 S28G2/G4-1 AGAAAAATTTACATAAAAAA[G/QCACACTCCATGATTGTTAAA

UMC21 UMC21-G2/G4-1B S28G2/G4-1B AGAAAAATTTACATAAAAAA[G/QCACACTCCATGATTGTTTAA

UMC21 UMC21-G2/G3-1 S28G2/G3-1 CTTTTATTCAAACTTTCCAG[/QCGTTAATTTGTTATCCGTTG

UMC21 U C21-G6/G1-1 S28G6/G1-1 TGTTGAACATGCTCTCAGGA[/CC]CCCCCTATTGTGACACAGCA

UMC21 UMC21-G1/G3-1 S28G1/G3-1 TACATCTTMC GC^CATGπ^"GπrηTAACX;TTTTATTCAAACTTT

UMC65 UMC65-G3/G6-1A S27G3/G6-1A AGTAATGTGTGACTGTGGGC[C G]CGTGTGACAGCTTTTACGTA

UMC65 UMC65-G3 G6-1B S27G3/G6-1B AGTAGTGTGTGACTGTGGGqC G]CGTGTGACAGCTTTTACGTA

UMC65 UMC65-G3 G6-2 S27G3 G6-2 TTCGCTTGGTAGCCGTAGCA[G/A]TATACTTTTACCGGCCACAG

U C65 UMC65-G3/G6-3 S27G3/G6-3 GGGCTTTGGGTTGTGAACTTICCA CJAAAAAAAAAAAAAATTTCCC

U C59 UMC59-G5/G6-1 UMC59-G5 G6-1 CCAAGAAAGArrAATGCTGG[ ηTAAAATATTGTTTCCAGTCT

U C59 UMC59-G5 G6-2 UMC59-G5/G6-2 AAAATCAGGACTGCGAAAAA[A C]CCAAGAAAGATTAATGCTGG

UMC59 UMC59-G5/G6-2B UMC59-G5 G6-2B AAAATCAGGACTGCGAAAAA[A C]CCAAGAAGATTAATGCTGGT

UMC59 UMC59-G5 G6-3 U C59-G5/G6-3 AAAGTGTGTGTTGTTGCCCAtG/AJATGArrCCATTCCACACAAG

UMC59 UMC59-G4/G5-1 UMC59-G4 G5-1 AGGACTGCGAAAAAACCAAG[/A1AAGATTAATGCTGGTAAAAT

UMC59 UMC59-G4 G5-2 UMC59-G4/G5-2 ATGCTGGTAAAATATTGTTTpqCAGTCTTTCACAAAGTGTGT

UMC59 UMC59-G3 G4-1 UMC59-G3/G4-1 CTACAAAAATCAGGACTGCG[/AJAAAAACCAAGAAGATTAATG

UMC59 UMC59-G3/G4-2 UMC59-G3/G4-2 TTGTTTCAGTCTTTCACAAA[/GηGTGTGTGTGCCAGATGATTC

UMC59 UMC59-G3/G4-3 U C59-G3/G4-3 TCACACACCGACCTGCX;TGG[ TlTATCAGGAACCATCCTCCTG

Ae1 Ae1-G4/G5-1 S23G4 G5-1 GGTGMTTGGTGATGCATGqT/GJGGGGGTGCTCGAGTTGGATG

Ae1 Ae1-G4 G5-2 S23G4 G5-2 TTCCAGTCGGATGAACTGGA[T/G1GTTCX3TCATCCACTCGTCAC

Ae1 Ae1-G3 G6-1 S23G3/G6-1 GGTGAATTGGTGATGCATGqA T]GGGGGTGCTCGAGTTGGATG

Ae1 Ae1-G5 G3-1 S23G5 G3-1 TTAAGTGAAGATGCCCAAAqCGIGTTAAACTTTCCATGGAACT

Ae1 Ae1-G5 G3-1B S23G5 G3-1B ATTAATGAAGATGCCCAAAqc GlGTTAAACTTTCCATGGAACT

Ae1 Ae1-G1/G6-1 S23G1/G6-1 TGATTCGGGTCTGTATGCGAtG T GTTGTGGTGGTGAACTGGT

Aβ1 Ae1-G1/G5-1 S23G1/G5-1 CGGGTCTGTATGCGAGTGTT[G A]TGGTGGTGAACTGGTGAATT

Ae1 Ae1-G1/G4-1 S23G1/G4-1 GTTCGCGGTTTCTGGGGCCG[G ηGGGCGGTGCTCGGTGGGGCC

UMC90 UMC90-G5Λ36-1 S22G5/G6-1 CAGAπGGTGTCGTπrACTAIA/GJAATTCAGTTCTGTCCATTTG

UMC90 U C90-G5 G6-2 S22G5 G6-2 AAGTAAGCATTCTTrATATGpTITACTTCCCATGATAAACTTT

UMC90 UMC90-G5/G6-3 S22G5/G6-3 CAMGGGCTTACTGTACTTTPCJCATCTTATTGGCAGGGCACC

UMC66 U C66-G5 G6-1 S19G5 G6-1 ACTTGGCCGGGGACGTCXSAqG/AJATCGTCGTAGCACTACTGGT

UMC66 UMC66-G5 G6-2 S19G5 G6-2 AGTACATGGCGAGCGTTGTA[G/C]CAGCTGCTTAGGTGATGTGG

Adh2 Adh2-G4/G6-1 S17G4 G6-1 CTATTTCCMGCTMCAACqC/GJCTCTTGGTCCCAACATCCTG

Adh2 Adh2-G3 G6-1 S17G3/G6-1 GGTTCTAAACATAGCTCGTqC/AJATTCATGATTCATCTCGAGC

UMC63 UMC63-G4/G6-1 S16G4/G6-1 TC^GCAAGCCTCXy AGGCTqCAJAATGGTCCAGTTACTTGGTT

UMC63 UMC63-G2/G6-1 S16G2/G6-1 GTGTGTAGCTTCATTCGCAArrG/ATTnTGAACAGCCTCTGCMGT

UMC63 UMC63-G2/G6-2A S16G2/G6-2A GTGCTTT(^TAAACCTAGAGn" C]TGACCAGCTGTGATTTCGGT

UMC63 UMC63-G2Λ36-2B S16G2 G6-2B GTGCrrTCGTAAACCTAGAG[rqTGACCAGCTGTGATπCGAT

UMC63 UMC63-G2Λ36-3A S16G2Λ36-3A GCTGACCAGCTGTGATTTCG[G/AN-GTATTCCAΑ3ΛCCACGAGT

U C63 UMC63-G1 G6-1 S16G1/G6-1 TGTGTAGCTTCATTCGCAAAIGΠTΠTGAACAGCCTCTGCAAGT

UMC63 UMC63-G1/G3-2A S16G1 G3-2A GTGCTTΑ^TAAACCTAGAGN7QTGACCAGCTGTGATTTCGAT

UMC63 U C63-G1/G3-2B S16G1Λ33-2B GTGCNCCGTAAACCTAGAGΠ"Λ^GACCAGCTGTGATRTCGGT

UMC63 UMC63-G1/G2-1 S16G1 G2-1 GTGTGTAGCTT(V0TCGCAA[A T1GTTTGAACAGCCTCTGCAAG

UMC102 UMC102-G5 G6-1 S14G5/G6-1 GCTC^GCTGCCGGAGTACGTIAΠIGGCTTGCTCTCCGGCCGGCC

UMC102 UMC102-G5/G6-1B S14G5/G6-1B ATAGCTCTGCCGGAGTACGTFANIGGCTTGCTCTCCGGCXXSGCC

ASG24 ASG24-G5 G6-1 S13G5/G6-1 TTTCACAACTCAACTGATTGIANTCTTGCTTTGATGTGGATTCT

ASG24 ASG24-G2/G6-1 S13G2/G6-1 TTGGTM7TTCAGAGCTAGAIOG1AACTTACTGTGGTACACGCX5

UMC49 UMC49-G4G6-1 S12G4G6-1 ACCTTTGCTGTGI 1 1 1 1 1 1 UI G]GTATTCGAATGGAGGGAGTA

UMC49 UMC49-G2/G5-1 S12G2/G5-1 AAAACAGCCAAGGTGGTGGTIC/G]AAAGGAAGGTGTCAGAAGGT

UMC49 UMC49-G2/G5-2 S12G2 G5-2 TCTGTTCGTT( VVTCTCTRTIA«]CAGTAAATATCCGTAATTAC

UMC49 U C49-G2 G5-3 S12G2/G5-3 CGTMTTACTTTGTTACTAQTA«IAGTAATTTTATATATATCCT

UMC49 UMC49-G2/G5-4 S12G2Λ35-4 TATATATATCCTCVVTTTCAAPVTTGAACAGTCAAAGTTAGTTTT

UMC49 U C49-G2/G5- B S12G2 G5-4B TATATATATCCTCATTTCAAJAΠTGAACAGTCAAAGTAGTTTTG

UMC49 UMC49-G2/G4-1 S12G2/G4-1 TATτTCTTATCCAGGATTGTπ'/qCTτTGGCCAAAGCATGGTAC

UMC49 U C49-G2/G4-2 S12G2/G4-2 CGTTCCATCTCTTTACAGTA{A/GlATATCCκ3TAATrACTTTGTT

UMC49 UMC49-G2/G4-3 S12G2/G4-3 ATCCGTAATTACTTTGTTAqTA ACJCTAAGTAATTTTATATATAT

UMC49 UMC49-G2/G3-4 S12G2/G3-4 GTAATTACTTTGTTACTACTIA/JAGTAATTTTATATATATCXJT

UMC49 UMC49-G1/G6-1 S12G1/G6-1 CTGTG I 1 1 1 1 1 1 1 1 GGTATT[G/qGAATGGAGGGAGTATTATTT

UMC49 UMC49-G1/G6-1B S12G1/G6-1B GCTGTG I I I I I I 1 lOGTATTtG/qGAATGGAGGGAGTATTATTT

UMC49 UMC49-G1/G5-1 S12G1 G5-1 ACTTAGATGATGAC< \GGTG[A/1AGAGTTTGGCACCTTTGCTG

U C49 UMC49-G1/G5-2 S12G1/G5-2 AGTTTGGCACCTTTGCTGTGrr/ll I I 1 1 I I IGGTATTGGAATG

UMC49 UMC49-G1/G5-3 S12G1/G5-3 CTTTACTGATτGGGTTACAAlA GμWSGTTATTTCTTATTCAGGC

UMC49 UMC49-G1/G5-4 S12G1/G5-4 AATTACTTTGTTACTACCAGrrJTAATTTTATATATATCCTCC

UMC131 UMC131-G4/G6-1 S10G4 G6-1 AGCGACAGGGATGTCGAGCA[G TTCTACGGAAGGCAATAATGAG csnpld

U C131 UMC131-G4G6-2 S1CX54/G&-2 AATTTGGGAAAATCAATGCA[GAACAC1ATCAGTGATTAATCCACATA

UMC131 UMC131-G3G6-1 S10G3G6-1 GCATGGCGGAGTGAGGGAGG(TG/]TGTGTGTGTGTGGCTCCACA

UMC131 U C131-G3/G6-2A S10G3G6-2A GGCCGCTACGCCATTTAGCG[G/A]ATTTGGGAAAATCAATGCAG

UMC131 UMC131-G3/G6-2B S10G3G6-2B GGCCGCTACGCCATTTAGCG[G/A1ATTTGGGAAAATCAATGCAC

UMC131 UMC131-G1/G6-1 S10G1/G6-1 CATCCCCGCCGGCAGAACAA[C/G]GTACGAGAAGGATGGAATGC

UMC53 UMC53-G5/G6-1 U C53-G5/G6-1 GTCCCAGATCAGGTCCACGTfT/qCGAGCTCGCTGTTCCCGCTT

U C53 UMC53-G5/G6-2 UMC53-G5G6-2 TGGTTCTTCACCACCACCGC[CG]CCGGGCGCGCCCAGCGCCTC

UMC53 UMC53-G4G6-1 UMC53-G4G6-1 GCAGCCTCAGGTACACGGGG[/A]AAGTCGGAGTGGTTCTTCAC

UMC53 U C53-G4/G6-2 UMC53-G4/G6-2 GCCGGGCGCGCCCAGCGCCT[/C]CGTCCCAGATCAGGTCCACG

U C53 UMC53-G3/G6-1 U C53-G3G6-1 GCACGTCGTTGGTGAAGAAG[ACCA]GCGGTACGGGTGCTTGTCGA

UMC53 U C53-G3/G5-1 UMC53-G3/G5-1 AGGTACACGGGGAAGTCGGA[GΛηTGGπCTTCACCACCACCGC

U C53 UMC53-G3G5-2 UMC53-G3/G5-2 CGACGGCGTCCAGCACCGAqG/JCCTCCGCCTTCACCCCGCGC

U C53 UMC53-G3G4-1 U C53-G3G4-1 GTCCACGTCGAGCTCGCTGT[CTJCCCGCTGCCCACGACGGCGT

U C53 U C53-G1/G4-1 UMC53-G1/G4-1 GCACGTCGTTGGTGAAGAAG[ACJAGCGGTACGGGTGCTTGTCG

UMC161 UMC161-G2G3-1 S06G2G3-1 NAACCAAACCCTGACTATTATT/CJAGGTAGATTAGACTAGACAC

UMC161 UMC161-G2G3-2 S06G2G3-2 ACGGTGAGGAGTGGCACATG[AC]GATGGAAAGTTCCTGTAGAC

UMC161 U C161-G2G3-2B S06G2/G3-2B ACGGTAAGGAGTGGCACATG[A/C]GATGGAAAGTTCCTGTAGAC

U C107 UMC107G2G4-1 S05G2G4-1 TATGCTTGGAAAGTGGGAAA[G/]GGGAACATACGATGGAGGAC

UMC67 UMC67-G5G6-1 S03G5G6-1 AMCMTMTTTTTACACAG[/ηTGCTAAGGTTTTACTGTTrr

UMC67 U C67-G2/G6-1 S03G2/G6-1 ATATCCATGTTGTCGCCTGC[TGJTGTGCGCTTGCTTGCCGCTA

U C76 UMC76-G4G6-1 S02G4G6-1 TTGCTGCTATGTTTACTGGG[/T]TGTAGAAAAAAAAATAATAT

UMC76 UMC76-G2/G6-1 S02G2G6-1 GCTCGGTAATAATTCTGGCTtCGJCGATGGCACCCATATTCCTC

U C76 U C76-G2/G6-1B S02G2G6-1B GCTCGGTAATAATTCTGGCTICGJCGATGGCACCCATATTCCTG

U C76 UMC76-G2/G5-1 S02G2G5-1 AAAACACGTGGTGTTTGTTA[G/A]GAAAGACCTAGTTTCTCGGC

UMC76 UMC76-G2/G5-1B S02G2/G5-1B AAATCACGTGGTGTTTGTTA[G/A]GAAAGACCTAGTTrCTCGGC

UMC76 UMC76-G2/G5-1 S02G2/G5-1 TAGTTTCTCGGCAATTGGCAlG/ηTGTGGAATGACCATCTCGTG

UMC76 UMC76-G2G5-1B S02G2G5-1B TAGTTTCTCGGCAATTGGCAfGηTGTGGAATGACCATCTCGTC

UMC76 UMC76-G2/G5-2 S02G2/G5-2 GTGTGGAATGACCATCTCGT[GCIGTGATGCCAGCATGCTGTTA

UMC76 UMC76-G2/G5-2B S02G2G5-2B GTGTGGAATGACCATCTCGT[G/C]GTGATGCCAGCATGCTACTA

UMC76 UMC76-G2/G5-3 S02G2/G5-3 ACCCTGTCAGGCTT∞ACAG[A/qTATMTATTTGTTGTGGTGT

U C76 UMC76-G2G5-3B S02G2G5-3B ACTCTGTCAGGCTTCCACAG[A/qTATAATATTTGTτGTGGTGT

UMC76 U C76-G2/G5-3C S02G2G5-3C ACTCTGTCAGGCTTCCACAGfACJTATAATATTTGTTGTGTGTG

U C76 UMC76-G2/G5-3D S02G2G5-3D ACCCTGTCAGGCTTCCACAG[AqTATAATATTTGTTGTGTGTG

Example 4 Analysis of Polymorphisms A. Preparation of Samples

Polymorphisms are detected in a target nucleic acid from a plant being analyzed. Target nucleic acids can be genomic or cDNA. 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 [or 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

Applica tions 1 , 17 (1991); PCR (eds. McPherson et al . , IRL

Press, Oxford); and U.S. Patent 4,683,202 (each of which is incorporated by reference for all purposes) .

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 . Na tl . 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 dependinq whether a polymorphism in question has already been characterized. The first type of analysis is sometimes referred to as de novo characterization. This analysis compares target sequences in different individual plants to identify points of variation, i.e., polymorphic sites. The de novo identification of the polymorphisms of the invention is described in the Examples section, The second type of analysis is determining which form(s) of a characterized polymorphism is (are) present in plants 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 member of a species but do not hybridize to the corresponding segment from another member due to the presence of different polymorphic forms in the respective segments from the two members . 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 example of which are described by Wo 95/11995 (incorporated by reference in its entirety for all purposes) . 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 forms 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 probe" exhibit complementarity to the second reference sequence. The inclusion of a second group (or further groups) can be particular useful for analysing 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 (i.e., 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 . 1 7 , 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 leading to a detectable product signifying 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 bo 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 Labora tory 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 differantiated 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 difference between alleles of target sequences.

Example 5 . Methods of Use After determining polymorphic form(s) present in a subject plant at one or more polymorphic sites, this information can be used in a number of methods .

A. Fingerprint Analysis

Analysis of which polymorphisms are present in a plant is useful in determining of which strain the plant is a member and in distinguishing one strain from another. A genetic fingerprint for an individual strain can be made by determining the nucleic acid sequence possessed by that individual strain that corresponds to a region of the genome known to contain polymorphisms. For a discussion of genetic fingerprinting in the animal kingdom, see, for example, Stokening et.al., Am. J. Hum . Genet . 48:370-382 (1991). The probability that one or more polymorphisms in an individual strain is the same as that in any other individual strain decreases as the number of polymorphic sites is increased.

The comparison of the nucleic acid sequences from two strains at one or multiple polymorphic sites can also demonstrate common or disparate ancestry. Since the polymorphic sites are within a large region in the genome, the probability of recombination between these polymorphic sites is low. That low probability means the haplotype (the set of all the disclosed polymorphic sites) set forth in this application should be inherited without change for at least several generations. Knowledge of plant strain or ancestry is useful, for example, in a plant breeding program or in tracing progeny of a proprietary plant. Fingerprints are also used to identify an individual strain and to distinguish or determine the relatedness of one individual strain to another. Genetic fingerprinting can also be useful in hybrid certification, the certification of seed lots, and the assertion of plant breeders rights under the laws of various countries .

B. Correlation of Polymorphisms with Phenotvpic Traits The polymorphisms of the invention may contribute to the phenotype of a plant 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. 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 a plant to a distinct mutation that is causally related to a certain phenotype . Phenotypic traits include characteristics such as growth rate, crop yield, crop quality, resistance to pathogens, herbicides, and other toxins, nutrient requirements, resistance to high temperature, freezing, drought, requirements for light and soil type, aesthetics, and height. Other phenotypic traits include susceptibility or resistance to diseases, such as plant cancers. Often polymorphisms occurring within the same gene correlate with the same phenotype.

Correlation is performed for a population of plants, which 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 plants, 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 K-squared test and statistically significant correlations between polymorphic form(s) and phenotypic characteristics are noted. 21

Correlations between characteristics and phenotype are useful for breeding for desired characteristics. By analogy, 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 :

Yijk n = μ + YSi + Pj; + X_k ~ 13ι + ... β₁₇ + PE„ + a_n +e_p where Yij_kpn is the milk, fat, fat percentage, SNF , SNF percentage, energy concentration, or lactation energy record; μ is an overall mean; YSi is the effect common to all cows calving in year-season; X^ is the effect common to cows in either the high or average selection line; βi to βπ 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.

One can test at least several hundreds of markers simultaneously in order to identify those linked to a gene or chromosomal region. For example, to identify markers linked to a gene conferring disease resistance, a DNA pool is constructed from plants of a segregating population that are resistant and another pool is constructed from plants that are sensitive to the disease. Those two DNA pools are identical except for the DNA sequences at the resistance gene locus and in the surrounding genomic area. Hybridization of such DNA pools to the DNA sequences listed in Table 1 allows the simultaneous testing of several hundreds of loci for polymorphisms . Allelic polymorphism-detecting sequences that show differences in hybridization patterns between such DNA pools will represent loci linked to the disease resistance gene .

The method just described can also be applied to rapidly identify rare alleles in large populations of plants . For example, nucleic acid pools are constructed from several individuals of a large population. The nucleic acid pools are hybridized to nucleic acids having the polymorphism-detecting sequences listed in Table I. The detection of a rare hybridization profile will indicate the presence of a rare allele in a specific nucleic acid pool. RNA pools are particularly suited to identify differences in gene expression.

C . Marker assisted back-cross

The markers are used to select, in back-cross populations, the plant that have the higher percentage of recurrent parent, while still remaining the genes given by the donor plant .

Example 6. Modified Polypeptides and Gene

Seguences

The invention further provides variant forms of nucleic acids and corresponding proteins. The nucleic acids comprise at least 10 contiguous amino acids of one of the sequences for example as described in Table I, in any of the allelic forms shown. Some nucleic acid encode full-length proteins .

Genes can be expressed in an expression vector in which a gene is operably linked to a native or other promoter. Usually, the promoter is an eukaryotic promoter for expression in a eukaryotic cell. The transcription regulation sequences typically include an heterologous promoter and U 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, and plant cells. 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 DNA fragments are introduced into cultured plant cells by standard methods including electroporation

(From et al . , Proc . Natl Acad . Sci , USA 82, 5824 (19853, infection by viral vectors such as cauliflower mosaic virus

(CaMV) (Hohn et al. , Molecular Biology of Plant Tumors, (Academic Press, New York, 1982) pp. 549-560; Howell, US 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al . , Na ture 327, 70-73 (1987) ), USQ of pollen as vector (WO 85/01856), or use of Agrobacterium tumefaciens transformed with a Ti plasmid in which DNA fragments are cloned. The Ti plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Horsch et al . , Science, 233, 496-498 (1984); Fraley et al . , Proc . Natl . Acad. Sci . USA 80, 4803 (1983)).

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 Enzymol ogy Volume 104, Academic Press, New York (1984); Sc:opeε, Protein Purification, Principles and Practice ' , 2nd Edition, Springer-Verlag, New York (1987); and Deutscher (ed) , Guide to Protein Purification ' Me thods 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 plants capable of expressing an exogenous variant gene and/or having one or both alleles of an endogenous variant gene inactivated. Plant regeneration from cultural protoplasts is described in Evans et al . , "Protoplasts Isolation and Culture," Handbook of Plant Cell Cul tures 1 , 124-176 (MacMillan Publishing Co., New York, 1983); Davey, "Recent Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts, (1983) - pp. 12-29, (Birkhauser, Basal 1983); Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," Protoplasts (1983) - pp. 31-41, (Birkhauser, Basel 1983); Binding, "Regeneration of Plants," Plant ProtopLasts , pp . 21 -12 , (CRC Press, Boca Raton, 1985). For example, a variant gene responsible for a disease-resistant phenotype can be introduced into the plant to simulate that phenotype. Expression of an exogenous variant gene is usually achieved by operably linking the qene to a promoter and optionally an enhancer. Inactivation of an exogenous variant genes can be achieved by forming a transgene in which a cloned variant genes is inactivated by insertion of a positive selection marker. See Capecchi, Science 244, 1288-1292 (1989) . Such transgenic plant are useful in a variety of screening assays. For example, the transgenic plant can then be treated with compounds of interest and the effect of those compounds on the disease resistance can be monitored. In another example, the transgenic plant can be exposed to a variety of environmental conditions to determine the effect of those conditions on the resistance to the disease.

In addition to substantially full-length polypeptides, 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 one allelic gene products but not to a second allelic gene product 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.

Example 7. 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 2? 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 Table I. Optional additional components of the kit include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates , means used to label ( or 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 .

Claims

1. A nucleic acid segment comprising at least 10 contiguous nucleotides from a vegetal sequence including a polymorphic site, notably a Single Nucleotide Polymorphism (SNP) or the complement of the segment.

2. A nucleic acid segment of claim 1, which is comprised in the sequence shown in Table I .

3. A nucleic acid segment of claim 1, less than 100 bases.

4. A nucleic acid segment of claim 1, that is DNA.

5. A nucleic acid segment of claim 1, that is RNA

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

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

8. An allele-specific oligonucleotide that hybridizes to a sequence of claim 1 or its complement.

9. An allele-specific oligonucleotide that hybridizes to a sequence of claim 8, sequence shown in Table 1.

10. The allele-specific oligonucleotide of claim 8, that is a probe

11. The allele-specific oligonucleotide of claim 10, wherein the central position of the probe aligns with the polymorphic site in the sequence.

12. The allele-specific oligonucleotide of claim 8, that is a primer.

13. The allele-specific oligonucleotide of claim 12, primer which comprises a sequence shown in Table I

14. The allele-specific oligonucleotide of claim 12, 3¹ end primer which comprises a sequence shown in Table I.

15. The method of analysing a nucleic acid, comprising : obtaining the nucleic acid from a subject; and determining a base occupying any one of the polymorphic sites shown in Table I .

16. The method of claim 15, wherein the determining comprises determining a set of bases occupying a set of the polymorphic sites shown in Table I.

17. The method of claim 16, wherein the nucleic acid is obtained from a plurality of subjects, and a base occupying one of the polymorphic positions is determined in each of the subjects, and the method further comprises testing each subject for the presence of a phenotype, and correlating the presence of the phenotype with the base.

18. Kit comprising at least one allele-specific oligonucleotide of claim 1 and optional additional composants (enzymes, buffers, instructions... )

19. Kit according to claim 18 comprising at least one allele-specific oligonucleotide of claim 2.

20 Use of the nucleic segments according to claims 1 to 17, to demonstrate common or disparate ancestry.

21. Use of the nucleic segments according to claims 1 to 17 in plant breeding.

22. Use of the nucleic acid segments according to claims 1 to 17 to trace progeny of a priority plant.

23. Use of the nucleic acid segments according to claims 1 to 17 in hybrid certification.

24. Use of the nucleic acid segments according to claims 1 to 17 to select in a back-cross population the plants that have the higher percentage of recurrent parent

(marker assisted back-cross) .

25. Use of the nucleic segments according to claim 1 to 17, wherein the polymorphisms, all of them or most of them, are linked to a group of genes involved in a given metabolic pathway.

26. Use according to 25, wherein the metabolic pathway is selected from the oil metabolic pathway, the starch metabolic pathway, the protein metabolic pathway, the aminoacids metabolic pathway, the lignin and the cell wall composition metabolic pathway and the pathogene resistance pathway