CA2274317A1 - Vegetal sequences including a polymorphic site and their uses - Google Patents
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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 alI or<~anisms undergo spontaneous mutation in the course of th~=ir continuing evolution generating variant forms of prog~snitor sequences (Gusella, d Ann, Rev. Biochem. 55, 831-854 (1~386)). 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 memf>ers of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant forms) 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-Kelley, 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 ~~resence 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 nurnber 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., LnlO 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 subject;.
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 loe DNA or RNA, and can be double- or single-stranded. Som.=_ segments are 10-20 or 10-50 bases long. Preferred segments include a diallelic polymorphic site. In a preferr~=d embodiment, the invention concerns nucleic acid segments from a fragment shown in Table I (corn).
The Invention further provides allele-specific oligonucieotides that hybridizes to a segment of a vegetal fragment, for example fracrment in Table I. These oligonucleotides can be probes or primers. Also provided are isolated nucleic acid" compris:~ng 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 furi~her 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 basE=s occupying a set of the n 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, 0ligonucleotide 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 5 the sequence to be amplified.
Linkage describes the tendency of genes, alleles, loci or genetic marker: to be inherited together as a result of their location on vhe 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 j=requency of greater than 1%, and more preferably greater than 10°s or 20°s 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, minisatellites, dinucleotide repeats, trinucleot:ide 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 sometime: 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).
SITE AND THEIR USES
The genomes of alI or<~anisms undergo spontaneous mutation in the course of th~=ir continuing evolution generating variant forms of prog~snitor sequences (Gusella, d Ann, Rev. Biochem. 55, 831-854 (1~386)). 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 memf>ers of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant forms) 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-Kelley, 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 ~~resence 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 nurnber 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., LnlO 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 subject;.
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 loe DNA or RNA, and can be double- or single-stranded. Som.=_ segments are 10-20 or 10-50 bases long. Preferred segments include a diallelic polymorphic site. In a preferr~=d embodiment, the invention concerns nucleic acid segments from a fragment shown in Table I (corn).
The Invention further provides allele-specific oligonucieotides that hybridizes to a segment of a vegetal fragment, for example fracrment in Table I. These oligonucleotides can be probes or primers. Also provided are isolated nucleic acid" compris:~ng 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 furi~her 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 basE=s occupying a set of the n 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, 0ligonucleotide 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 5 the sequence to be amplified.
Linkage describes the tendency of genes, alleles, loci or genetic marker: to be inherited together as a result of their location on vhe 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 j=requency of greater than 1%, and more preferably greater than 10°s or 20°s 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, minisatellites, dinucleotide repeats, trinucleot:ide 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 sometime: 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 arisefrom 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 INVED~~fION
I. Novel PolvmorlJhi~ms 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 ex rac ion 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 vuas 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 G1, 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 rind Nei-li distance) is 0.69.
This value is close to the maximum distance between two cultivated maize lines.
Among the 15 genetics distance between couple of these 6 lines . 8 are superior t:o 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.
Genotvoes G1=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 2 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 2d 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 SO1F is the forward primer for probe 1 SOlR 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 S01 1.01-1.02 SO1F CGCACGCACATTAGCTTTCG 610822 UMC76 S02 1.02-1.03 S02F ATTATTCGGCGTCCAGCCCC 610865 lO S02R TTACCAGCGGTGAGAGCTGC 610866 UMC67 S03 1.05-1.06 S03F CGTTCGTGTGGCATCAATCG 610864 UMC161 S06 1.10-1.11 S06F CAGACCTTGGTTGGAGGCAAC610824 UMC53 S08 2.01-20.2 S08F CGGACGTGATGCAAGTTTCG 610851 UMC131 S10 2.04-2.05 S10F2 TCCTTGGCACTCACGCTACC 610816 UMC49 S12 2.08-2.09 S12F CAGAGAGCCGTCTCGAATCG 610845 UMC102 S14 3.04-3.05 S14F TGCTGTGCTGTCACATGGCG 610801 UMC63 S16 3.08-3.09 S16F2 ACGCCCTGACAGAACCATCG 610857 Adh2 S17 4.03 S17F2 TGCCTGCTGCATCTCTAGCC X02915 UMC66 S19 4.06-4.07 S19F TGGAGTGTCCAAAGACCGACC610862 UMC90 S22 5.01 S22F GCAGGTGAACAATGCTGCCC 610870 Ael S23 5.05 S23F TCGCTGGGGTTTTAGCATTG L08065 UMC59 S26 6.01-6.02 S26F TCCAAAGCGAAAGCCTGATG 610853 UMC65 S27 6.03-6.04 S27F TTCCAGCTTTCCTCGGCACC 610860 UMC21 S28 6.04-6.05 S28F TGCAGATGTGCCTTTCCTGTG610830 UMC132 S29 6.06-6.07 S29F CGCACAGAGGCAGATGCAGC 610824 UMC254 S33 7.03-7.04 S33F CCGGGCGCAAAAGAATGTG 610832 UMC80 S34 7.04 S34F TCGCCTTTATCGGTGCAATG 610867 BNL9-11 S38 8.01-8.02 S38F2 CGAGGGAATGTCATCAACCC 610778 UMC109 S42 9.00-9.01 S42F GCACCGTCGTTTACCTCAAGC613177 Wxl S43 9.02-9.03 S43F CGTGCTACCTCAAGAGCAAC X03935 UMC95 S44 9.04-9.05 S44F CACTCGGAAGTCGGAATCGC 610872 CSU61 S45 9.05-9.06 S45F TCTCCACGAATCCCACCGTC T12691 UMC130 S48 10.02-10.03 S48F AAGGGGGAAGAAGGTCATC GI0824 CSU109 S53 2.09 S53F GCTTTCGGTTCCGGATAGCG T22721 UAZ77 S56 5.04 S56F2 GCAACCAACTGCAACATCGC T18762 Sh1 S57 9.01 S57F TGCTGTTATTGCGTGCCGTG X02382 Sh2 S63 3.09 S63F TTCTTCACTGCACCCCGATG M816D3 5 Bre1 565 6 S65F AGCAGCAGATCAGGCACACC U17897 Bt1 S66 5 S66F GGCAAGGATCGGAGTTGCTC M79333 Ssu S67 S67F GCAAGCAAGCAAGCAGCGAG D00170 1~ S67R GACCCGAAGCAAAACCGAAC D00170 Bt2 S71 4 S71F TGCCGAAAAAGGTGGCATTC Seq (Bae et al 1990) 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 10x . 3 microliter; dNTP's . 0.2 mM each; MgCl2 . 1.5 mM; BSA 0.8mg/
ml; primers 1.5 ng/microliter each; glycerol 50.
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) the genotype number (G2) - the second genotype number (G6) - and the number of the SNP (single nucleotide polymorphism). So, in this cage, 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 marl~:er/gene.
Column 4 represent~~ 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-Gl/G5-1A) means deletion of the base pair G in G7_ compared to G5.
iz TABLE I
csnpld Ssu 8t2-G2/G6-1 S71G2IG6-tATAATACTTGATATGCCATT[G/1~TGTCCTCTTATTTTTAA
S
CAT
Ssu su- G1JG5-1 S67G1/GS-1ATGGCCTCGTCGGCCACTGC[A/C]GTCGCTCCGTTCCATG
S
GGCT
Ssu su-G11G3-1 S67G1/G3-1GCCGCTCCTCCAGAAGCCTC[G/A]GCAACGTCAGCAACG
GCGGA
Ssu Ssu-G1/G3-2 S67G11G3-2GTGTTGCCCATCCCATCCCA[A!Z]TTCCCAACCCCAA
ACGAACC
Bti Ssu-G1/G3-3 S67GiIG33 GTACCTGCCGCCGCTGTCGA[CG/AC]GGACGACCTGCTG
B
AAGCAGG
Bti tt-G2/G3-1 S66G2/G3-1AGTGAGCCCGCTTCTTATTC[/T]TAAGGTGATAGGTTTCTA
B
AA
Bt1 ti-G2/G3-2 S66Gi/G3-iAATGTAATGGTACTCCGCGC[T/C]ATGGCTCTGGTACTTA
GGAA
Bre1 Bt1-G2/G3-3 S66G1/G2-1AAATAGGCTCGGGCAATTAT[C/jCAGCTTAGGGACAGCAAGC
B
G
Bret rei-G3/G6-1 S65G3/Gtr1TCCGCCCTGCCTCCGGTTTT[M']GCCCGACCTTCGAAACATT
B
C
Br ret-G3/Gti-2S65G3IG6-2ACCACTGACGTAGCACCTCC[G/T]ACTTCTCGTTGTAA
AACCCC
e Bre1-G3/G5-1S65G3IG5-1GGAGGTTCGCCTCATGTTAT[C!f]GTTGACGAGCCA
B
i CATCCACT
re Bre1-GMG6-1 S65G41G6-1GCTCCGACTTCCAATCTTGA[A/C]CCTCCACCCTGC
CTCCGGTT
~Gi2 ASG12-G1/G3-1S64G1/G3-1CTGGTTGAAATGTGTTGAAG[C/A]TACTAGTGATGAA
CTGCTTG
ASG1 ASGi2-Gl/g3-2AS64G1/g3-2AGCTGCTCCAAGCGAGCCCGC[C!G]CCGAAAAAGG
AAAAAGGTGA
2 ASG12-GlIg3-28S64G1/g3-2BGCTGCTCCAAGCGAGCCCGC[C/G]CCGAAAAAGG
AS ~
AAAAA( G12 ASG12-GlIg3-3AS64G11g3-3A,TTGA
ASGi2 CGCCCCGAAAAAGGAAAAAG[G/T]TGAAGGTCCTTAC
TCACCGA
ASGi ASGi2'Gi/g3~BS64G1I93-3BCGCGCCGAAAAAGGAAAAAG[G/T]TGAAGGTCCTTA
CTCACCGA
2 ASG12-Gl/gG3-4AS64G1/gG3-4AGAACCGGCCACAGTGCCTGA[TIA)T'TTGGCGGTGAG
ACCTCTTC
Sh2 ASG12-Gl/g3-4AS64GtJg3-4AGAACCGGCCACAGTGCCTGA[T/A]TTTGGCGGTGAGAC
TTCTTC
S~ Sh2-G5/G6-1 S63G5/G6-1CAATTGTTACCTGAGCAAGA(f/]TTTTGTGTACTTGACTT
GTT
Sh2 Sh2~4/G6-1 S6:iG4/Gti-1TACTGAGAGAATGCAACATC[C/G]AGCATTCTGTGATTGGAGTC
5h2-G4IG5-1 S63GMG5-i TTTTAGTGTACTTGACTTGT[C/T]CTCCTCCACAGATGAAATAT
Sh2 A A
Sh2-G41G5-1 St~G4lGS-1TTTTTGTGTACTTGACTTGT[G7]CTCCTCCACAGATGAAATAT
Sh2 B B
Sh2-G3/G6-1 S63G3/G6-iTCTGTGATTGGAGTCTGCTC[G1A]CGTGTCAGCTCTGGATGTGA
Sh1 Sh1-G5/G6-1 S57G5JG6-1AACTACJ1AAAAGCATCTCCT[GIT]GGATTTGGCTATCTCCTTTT
Shi Sh1-G21G5-i S57G2/G5-1TTAGCGCCAAAAAAAApCTC[/T)TTTTTTTTTGTCCTTTTACT
Shi Sh1-G21G3-1 S57G2/G3-1TCAATCCAATCAATTTAATT[TIC]CTTCCTTTAAAAATATTATC
Sh1 Shl~l/G2-1 S57G11G2-iTTACTACGAAAAACTCTTGA[GIT]TCTAGGAATTTGAATTTGTG
Sh1 Sh1-G1/G2-2AS57G1/G2-2ACTTCTTGGATTTTGCTATCT[T/C]CTTTTACTACGAAAAACTCT
Sht Sh1-G1IG2-2BS57G1IG2-2BCTCCTTGGATTTTGCTATCT[T/C]CTTTTACTACGAAAMCTCT
Sh1 Sh1-G1/G2~A S57G11G2~ATTTTACTACGAAAAGCATCT[TIC]CTTGGATTTTGCTATCTTCT
Sh1 Sh1-G1/G238 S57G1/G2~BTTTTACTACGAAAAGCATCT[TIC]CTTGGATTTTGCTATCTCCT
Sh1 S57G1/G2-4 S57G1JG2-dGAAGCCAAATCCTATTATTT[T/C]CTGCCTCTAGGGTCTGAATG
UAZ77-GNG6-iS56G4lG6-1GTACACTGTTACAATCACAC[fIG]TAGTGAAGCGCAACACAGAT
UAZ77-G4/G6-2S56GNGti-2GCCTTATCATCCTCTAGGTA[f/A]TGGAGACGAGTGACCAGTCT
UAZ77-G4IG63S56GMGti-3CTTTTCTTCAGACCCGAGCC[CITjCCAATCGCGCCCTTCTGTGC
U
AZ77 UAZ77.G4lG63SS6G4IG63 CTTTTCTTCAGACCCGAGCC[CJT)CCAATCGCGCCCTTTTGTGC
UAZ77~41G5-1AS56G4/G5-1AGAGCCCCCAATCGCGCCCTT[GT]TGTGCCTTGGCCTTGAGCTC
UAZT7.G41G5-1AS56G4IG5-1AGAGCCTCCAATCGCGCCCTT[GT]TGTGCCTTGGCCTTGAGCTC
UAZ171G1IG3-1S55G1/G3-1GAAGGAGCAGCAGCGCAAGG(NJACGTGTTCCAAGTCAACGTC
UMC17 UMC117-G2IG3-1S54G2/G3-1GTAGAAAGTTAGCAAAAACA[T~7T-~TTAGTGAAAAAACATA
UMC17 UMC117~2Xa3-2S54G21G3-2ATTGTGGCTAGAAACTTTGG[I~TTITTITApp~TTATGGTCAT
CSU109CSU70S~SfG6-1S53G5lG&1 GCAAACCMCACCAp~TCTTC[G/C]AAATGAGCAAAGCAGAGACT
CSU109CSU1l79.G51G6-2S53G5/G6-2CAGATCGGTTGTCCTCAGAG[AJJAAGTCACCTACCTGCAAACC
CSU109CSU1 3 S53G5/Gf,-3AATTCTAG4TAGGAGTCATG[G1]ACAAGTACTTGTTTAAAGGA
CSU109CSUIt~5IG6-4S53GSIGfi-4ACAAGTACTTGTTTAAAGGA(GjCATGCCGGAATACACGCTGC
~~~A GAGCGAGATCGATCCTGTTG[T/C]CATCCATCACTGCCATAGGA
CSUt09CSU109-GSIG6.SBS53GSIGSSBGAGCGAGATCGATCCTGTTG[TI~CATCCATCA
CTGCCGTAGGA
CtaU109CSU1t79.G4/Gfr.1S53G4IG6-1~
TAGTCATAGCHA~f'.AGCATGaG//~JTCG~,ATGTAGCGTTCACCC
CSU109CSUtOA.GMG6 S53G4/G6-2CAATTGAAGAGG4~1AAAAppA(/~CT~pTTCATGTAC
CSU109.G4AG5-1S53G4/GS-1~G~GAGTCCACAATAOTTCGTC
CSU109.C311p5-1S53G3K35.1CCCACCGCGG~TGGTGG[T/JTAGAAGG;GAACCACCGAGC
CSUIt~CSU109.G?hGG-1S53G2K~6-1ACTTGTTTMI~GGA~CATGCC(<i~GGApTq~CTGCCCAGGC
CSU109CSU1~IG3-1 S53G2Aa.1 OCCAGGCCTTCOCACGGCGG[AK33]GATGGTGGTTAGAAGCGGAA
TCCGCAATAGT
CSU109CSU109.G1~G6-2S53G1~Gfs.2GJ1ACI4C',AGTCCGCMTAGTT[T/~CaATCCTAATGCTi4CTTOGAGC
UMC130UMC130~31G6.1S48G3IG6-1GATTCJ1GAAACA~GTGGCGGC[AK~GATGTAGCATCAACACGCCC
CSUtiI.GS/G6-1S45ta51G6-1ATGAGTATATTC~AAGTCATA[TICjTGTGAACTAGAATGTTATTT
CSU61 CSU61-G5Ki6-2AS45GSlGfr2ACCTAGJ40GCTGACCGOC~AC~A[G//ypCGGCGG~CTGCG1AATC
CSU6I.f~G6 S45GSIG6-2BCCTAAACGCTGI1CC(~
2B CCqC
, CSUB1 CSUB1-G5b6.3S~SGSIG6-3/~G/Np~G~CTGCCAMTC
TGAAA'.~MACCATGCGCTACC(GTjAGCTACiGTGTTTTMAC' TM
CSU61 CSU61-G~i6-1&45G~AG6..1s TCOGCGGJ1AACAACATCCGA(G!1'[TTCTTGAGOATAACCCAGCT
CSUB1 CSU61.G4/G5.1S45G4K35..1GGGAGGGGAMAqA~pGp~GJTTGGTTGCGGTTCAGT
CSU61 CSU61-G4fG5-2S45G4/GS2 GGCGGCTGCCAAAT~GCaG[!A)AppCGA~TtxGAGTTCTTG
CSU6i CSU61-G2IG4-1AS45G2IG4-1ACTAGMTGTTATTTCTTCAC[GA]GTTGACCATGGMAAAAACA
CSU61 CSU61-G2IG4-18S45G2/G4-1BCTAGAATGTTATTTCTTCAC[GI~GTTGACCATGGAAAGAAACA
CSU61 CSU61-G?IG4-2AS45G21G4-2ATTCACCCTTGACCATGGMA[AIIpTp,ATAAGTTCTTGT
CSU61 CSU61-G2K34-2BS45G21G4-28TTC~ACJIGTTGACCATGGAAA(NC~)I4AACAGTMTMGTTCTTGT
CSU61 CSU61-Gt/G6-1S45G1IG6-1TTCTTG~CA,GTTGACCATGG[IA~AAAAAAAG4GTMTAAGTTC
CSU61 CSU61.G1IG5.1AS45G1/G5.1AGAACCCACCOTGCCCTGOGA[/G)<3G
CSUB1 CSU61~1IG5-1BS45G11G5_iBGAACCCACCGTGCCCT
CSU61 CSU61-G11G5-2AS15G1Ki5-2ATGGGAGGGNJIAAAqAAA~C'sAA[LiI~AGCt;',TTGGTTGCGGTTCAGT
CSU6i CSU61-G1/G53S45G1K's5~3CGTACC~1ICCTqGGMTCGTA(AK;~UIApGCCTAGACGCTGACCG
lJMC85 UMC95GSJG6-iS44G5/G6-iGCTGCGTCMTCI1TG1C1TC[T/A]CCCACI1GGCGTCAAGTACAG
U~AC85 UMC95~G3IG4-1S44G3K's4-1GACAGATTCCAAAGTAGTCG[GT)CGGCCACGTCGAAAAAGAAT
UMC9rG2/G6-1S44G2/G6-1GGCGCTGCGTCAATCATCAC[MjTCACCCACAGGCGTCMGTA
csnpld UMC95 UMC95-G2IG4-tAS44G21G4-1ATCGGTGTCACCnCATGCATA,[TlG]TCAGGACAGATTCCAAACTA
UMC95 UMC95-G21G4-iBS44G2/G4-1BTCGGTGTCACCACATGCATA[TlG[TCAGGACAGATTCCAAAGTA
UMC95 UMC95-G?JG4-2AS44G2IG4-2AGTCGCCGGCCAGGTCGMAA(G/A]GAATACTCAGCAAAAGACCC
UMC95 UMC9SG2IG4-2BS44G2/G4-2BGTCGTCGGCCA~GGTCGAAAA[G/A]GAATACTCAGCAAAAGACCC
UMC95 UMC95-G21G3-1AS44G2/G3-1ATATTCAGGACAGATTCCAAA[GG]TAGTCGCCGGCCAGGTCGAA
UMC95 UMC95-G21G3-1S44G2/G3-1TAGTCAGGACAGATTCCAAA(GG]TAGTCGCCGGCCAGGTCGAA
B B
UMC95 UMC95-GIIG(i-1S44G1/Gti-1GCGTCAAGTACAGATACGCA[AIG]CACGCCTCAGCTTCGCCTTG
UMC95 UMC95-GllG2-1S44G1/G2-1CCTGGGACTCG:~CAAATTGC[GlA)A1GCACTCGGTGTCACCACAT
Wx1 Wx1-G?JGtr1 S43G2/G&1GCTGGTTCATTA,TCTGACC1'[GlT]GATTGCATTGCAGCTACAAG
Wx1 Wxl~?JG6-2 S43G21G6-2CTGGATTGCATTGCAGCTAC(A/G]AGMGCCCGTGGMGGCCGG
Wx1 Wx1-G2/Gt~IB S43G21G&1BGCTGGTTCA1TA,TCTGACCT'[GlT]GATTGCATTGCAGCTACGAG
Wxi Wx1-G2lGti-2BS43G2/Gtr28CTTGA1TGCATTGCAGCTAC[A/G]AGAAGCCCGTGGAAGGCCGG
Wxt Wx1-G2/Gti-3 S43GTJGti-3TCAGCCCCTACI'ACGCCGAA[G/JGAGCTCATCTCCGGCATCGC
Wxi Wx1-G2/G5-1 S43G2/G5-1TACCCGGAGCTGAACCTCCC[GG]GAGAGATTCAAGTCGTCCTT
Wx1 Wx1-G21G4-1 S43G21G4-1TGCATGTGAACATTCATGAA(f/CjGGTMCCCACAACTGTTCGC
Wx1 Wx1-G6IG1-1 S43G6/G1-1CTCCTACCAGG(~CCGGTTCG[T/]CCTTCTCCGACTACCCGGAG
Wx1 Wx1-GI/Gfr1 S43G11Gti-1TGAATGGTMCCCACAACTG[GTJTCGCGTCCTGCTGGTTCATT
Wx1 Wx1 ~11G5-1 S43G11G5-1GCCGACAGGGTCCTCACCGT[GIC]AGCCCCTACTACGCCGAAGA
Wx1 Wxi ~2lGfi-1 S43G2/G6-1GCTGGTTCATTATCTGACCT(G/T[GATTGCATTGCAGCTACAAG
Wx1 Wx1-G2/G6-1 S43G21G6-1GCTGGTTCATTATCTGACCT(G/T]GATTGCATTGCAGCTACGAG
B B
Wx1 Wx1-G2/G6-2 S43G2iGti-2CTGGATTGCATTGCAGCTAC(AJG]AGAAGCCCGTGGAAGGCCGG
Wx1 Wx1-G21G6-2B S43G21Gfr28CTTGATTGCATTGCAGCTAC[AIG]AGMGCCCGTGGAAGGCCGG
Wx1 Wx1-G2/G65 S43G21G6-5TCAGCCCCTACT'ACGCCGAA[G~GAGCTCATCTCCGGCATCGC
Wxt Wx1-G2IG4-1 S43G2JG4-1TGCATGTGMCATTCATGAA[T/C]GGTAACCCACAACTGTTCGC
Wx1 Wx1-G2IG3-1 S43G21G3-1CTGGTGGTGGTGCTTCTCTG[AAAG]TGAAACTGAAACTGACTGCA
Wx1 Wx1-G2/G3-3 S43G2IG3~3GACCATCTTCACGTACTACC(fACG JAGACCGCTTTCTGCATCCAC
Wx1 Wx1-Gl/Gti-1 S43G1/Gti-1CTGACCATCTTCACGTACTA,[CCTAI]CCAGACCGCTTTCTGCATCC
Wx1 Wx1-GtiIG1-1 S43Gti/G1-1CTCCTACCAGGGCCGGTTCG[T>jCCTTCTCCGACTACCCGGAG
Wx1 Wx1-GtilG1-1 S43G6IG1-1GAGATTCMGTCGTCCTTCG(G/jATTTCATCGACGGGTCTGTT
Wx1 Wxi-G1/G6-2 S43G1/G6-2TGAATGGTAACCCACAACTG(GT]TCGCGTCCTGCTGGTTCATT
Wx1 Wxt-G1IG6-3 S43G1/Gti3GCCGACAGGGTf:CTCACCGT[GlC]AGCCCCTACTACGCCGAAGA
Wx1 Wxl.G1/GS-1 S43G1IG5-1TCTGACCATCTT~CACGTACT[ACCTIjACCAGACCGCTTTCTGCATC
Wxi Wx1-G11G4-1 S43G1/G4-tCTTGATTGCATT~~.~CAGCTAC(GlA]AGMGCCCGTGGAAGGCCGG
Wxi Wxt-G11G4-1 S43G1IG4-1CTGGATTGCATTGCAGCTAC[G/A]AGAAGCCCGTGGAAGGCCGG
Wx1 Wxt-G1/G3-1 S43G11G3-1GCTGGTTCATTATCTGACCT(T/G]GATTGCATTGCAGCTACGAG
Wxt Wx1-GSIG6-1 S43GS1G6-1AGAGATTCMGTCGTCCTTC[G~GATTTCATCGACGGGTCTGT
UMC109 UMC109-G2IGti-1S42G2/G6-1CTCG~1TGAAAAAGGTGCCGC[/G]TACTCTCTCAGTCAGCTACT
UMCIt><J UMC109-G2/G3-1AS42G2IG3-1ACTGCACTCCGATTGAGGGTC[C/G]GMGCAGGGCAGCGCGTGTG
UMC109 UMC109-G2IG3-1S42G2IG3-1CTGCACTCCGA'iTGAGGGTC[GGjGMGCAGGGCAGCGCGTTGT
B B
UMCIt><J UMC109-G2/G3-1CS42G21G3-1CCTGCACTCCGATTGAGGGTC[GG]GAAGCAGGGCAGCGCGTTTG
UMC1t79 UMCIf~-G21G3-1DS42G2IG3-1DCTGCACTCCGATTGAGGGTC[CJG]GMGCAGGGCAGCGCGTTTT
UM(~.80 UMC80.G3IG5-1S34G3NG5.1CATGCCTCTGTTGATATTTT[G/C]GTGCACCTTTTGCTTGCMC
UMCt30 UMCBf>G3/G5-2S34G3/G5-2GATTTTGTAGGTTGATGCAT[Cl~jGTTTGATCTTTCTTATCTCC
UMC80 UMC80-G3/GS3S34G31G5~3TGCTTGCAAiCTAAATTMTC[AIG]TGCTCTATTTGACTMGAGT
UMC80 UMC80-G31G4-1S34G31G4-1ACATGTC.C~4GGACGCATGGIjCIJCCCAATAT'TGTTGTTGGMG
UMC80 UMC80-G31G4-2S34G31G4-2TTGATCTTTCTTATCTCCTT[!C]CGMTTTGTTCTGTGTTATA
UMCtiO UMC80-G31G4-2BS34G31G4-2BTTGATCTT'TCTTATCTCCTT[/C]CGMTTTGTTCTGTGTATAC
UMC80 UMC80.G2/G5-1S34G?aGS_1TGTAGGACTTGGAGAGCTTG(AIG]TMTTTACACATGCCTCTGT
UMC80 UMC80.G2lGS-2S34GZb5-2CATGCCTCTGTTGATATiTT(GJCjGTGCACCTTTTGCTTGCMC
UMC80 UMC8~Gr3 S34G2Ki53GATTTTGTAGGTTGATGCAT[C>nGTTTGATCTTTCTTATCTCC
UMC80 UMC804~G3-1S34G21G3-1GAGACATTTCCTACTCMT
A jCn]Ai4TTATTTGATGAMTTATT
UMC254 UMC254.G51G6-tAS33GSfG&1AAGTATCACMiACTMTCTGA[NGjTATCTGGTTGCf:AOGAAMC
UMC254 UMC254~G6.1BS33G5lGrrlBAGTATCACAQJ1CTMTCTGA[AK3]TATCTGGTTGOCACAAAAAC
UMC254 UMC254-G5fG6-2S33G51G6-2TCAAAGTGGTGCAATCGCAA[TICjCGA~CTTGGGCTTGCC(iTGGT
UMC254 UMC254-G51G6.3S33GSrGS~CCI1CTTGGGCTTGCCGTGGT(CIJCGTATCGTACGCAGGTAGCA
UMC254 UMC254-G51G6-4S33GSK36-4AGCATTTTTfGTTTTGTTTT[TJC]CCTTGGCAGACMCI1GACAG
tl~AC254 UMC254-G';~G65AS33GS/G65ACAGTCCCOAGANTCCCAAAT[GjCAQAAAAAGGTTTTGTTTTT
tlAAC254 UMC254-G51G8.58S33GSIG6~58CAGTCCCGAGMTCCCAAAT(GJCAGIUW1I1GOTTTTGTTTTA
UMC254 UMC254.G4RIG6-1AS33G4R/GfrlAGGC;AuC~AC~1A~CA~C'~CA~CATCA[AG1CNCATGCTTGCATTTACTCCCA
UMC254 UMC254-G4RIG6-1S33G4RKi6-1GGCAGACAACAGiACAraATCA[AG/C~CATGCTTGCATTTACTCTCA
B B
UMC254 UMC254-G3RIG6-1AS33G3RI08-1AGTGATCACAGACTMTCTGA[NGjTATCTGGTTGCCACGMMC
UMC254 UMC254G3RIG6-1S33G3RIG8-1GTGATCAf:AGACTMTCTGA(AIGjTATCTGGTTGCCACAAAAAC
B B
UMC254 UMC2S4~G3RIG6-2AS33G3WG6-2ATGTGMTATCTGGTTGCCAC[G/AjAAAAiCCOGGACACMCiAGAG
UMC254 UMC254~i3R/Gtr2BS33G3R/G8-2BTCTGAGTATCTGGTTGCCAC[G/AjAAAACCGGGACACMGAGAG
UMC254 UMC254.G3/G6-3S33G3~ TCAGTCAAACTCAGTCCCGA[AK3]AATCCCAAATCAGAAAMGG
UMC254 UMC254~3X35-1AS33G3IG5-1AGGTTGCCACOIV~1ACCOGGA(CIG TCAGAGT
UMCZ54 UMC254~G31G5-1S33G3JG5-1GGTTGCCACGAiIAA~CCGGGA[GG)ACAAf"aAGAGAAACTCAAAGT
B B
UMC254 UMC254-G2R/G3-tAS33G2R1G3-1AACGCATGCTTGCATTTACTC[CJTJCAGTCAAACTCAGTCCCGAA
UMC254 UMC254-G2R/G3-1S33G2R1G3-1ACACATGCTTGCATTTACTC[GT)CAGTCAAACTCAGTCCCGAA
B B
UMC254 UMC254-G1R/G2-1S33G1R/G2-1TATTATTCAATTTTGAATM[IGjGAAGOAAATTTTAGG1,CCTC
ASG49 ASG49-G3IG5-1S32G3IG~r1ATTMTAMTGCATCCTCTG[GGjTAAAAAAACCCATTTTGMT
ASG49 ASG49-G3/G5-2S32G31G~r2ATGMTTGMGCTCTGMTA[GITjAGMTCCACCATTCTTCCGA
ASG48 ASG43-G31G5-3S32G31G5-3GMTCG4Cf:A1TC1TCCGAA(A!G]CTGCTTCCTACAAAACTCGA
ASG49 ASG49-G3IG5-4S32G3/GS-4GAAAGGATGTGZTTTTGATA[G!A)CCTTCAGTCTTTCAGATGGA
ASGB ASGS-G3lGS-1S31G31Gr1CAATGTCTTGTTCGTTATCA(A/G)CGAAAGTTTGMTCCCCACA
ASG8 ASG8~G31G4-1S31G3IG4-1TGTATCGGCTAGTCTGGATG[GIAjTCGCACTGGCACTCAGTGCT
csnpid UMC132UMC132-G4/GS-1S29G4lGS-1 TCTATTCAGCAGTCTGAGAA[GCAICT]AGGATGGTCGGCTTCTTCAG
UMC132UMC132-G11G5-1S29G1/G5-1 CCTTACACTATTAACAGGCC[GT]GTGATCTACCTGAATGCCTG
UMC21UMC21-G5/G6-1S28GS/G&1 CAAGAAGCCTCTTCAGTGTC[A/C]GTCGTAGCTTCCTCAAGACC
UMC21UMC21-G51G6-2S28G5IG6-2 AGACCTTCCTGATGTGCGGA[T!C]GCTAATCCATGGAGCAGGGA
UMC21UMC21-GSIG6-28S28GS/G6-2BAAGACCTCCTGATGTGCGGA[f/C]GCTAATCCATGGAGCAGGGA
UMC21UMC21-G5/G6-3S28G51G6-3 CTAATCCATGGAGCAGGGAG[G/A]AAGGGGCGAGGGGCAGCAAG
UMC21UMC21-GMGS-1S28G4IG5-1 TCGTCGCGAATACAGCCGGG(G/C]GAGGGGGTGGTCGCGACTGG
UMC21UMC21-G3/G6-1528G3/G6-1 GTCGTAGCTTCCTCAAGACC[TIJTCCTGATGTGCGGACGCTAA
UMC21UMC21-G3IG4-1S28G3/G4-1 GAGTCGTCGCGAATACAGCC[A/G]GGGGAGGGGGTGGTCGCGAC
UMC21UMC21-G31G4-2S28G3IG4-2 AGGGGGTGGTCGCGACTGGA[T1G]CGCCCGAGCAGCGAGCAAGC
UMC21UMC21-G3/G4-3528G3IG4-3 AAGCACATGTTTTAACCTTT[T/G]ATTCAAACTTTCCAGCCGTT
UMC21UMC21-G31G4-3BS28G3/G4-3BAAGCACATGTTTTAACCTTT[T/G]ATTCAAACTTTCCAGCGTTA
UMC21UMC21-G2/G6-1S28G2IG6-1 GAATGTTGCTGTTATATTAC[TIC]CGTAGGTGACAAAGGGTTCA
UMC21UMC21-G21G4-1S28G21G4-1 AGAAAAATTTACATAAAAAA[G/C]CACACTCCATGATTGTTAAA
UMC21UMC21-G2/G4-1S28G2/G4-1 AGAAAAATTTACATAAAAAA[GIC)CACACTCCATGATTGTTTAA
B B
UMC21UMC21-G2IG3-1S28G21G3-1 CTTTTATTCAAHCTTTCCAG[/C]CGTTAATTTGTTATCCGTTG
UMC21UMC21-G6IG1-1S28G6/G1-1 TGTTGAACATGCTCTCAGGA[/CC]CCCCCTATTGTGACACAGCA
UMC21UMC21-G1IG3-1S28G1IG3-1 TACATCTTAACAAGCACATG[TGIT'11 jTMCCTTTTATTCAAACTTT
UMC65UMC65-G3IGfi-1AS27G3/G&1A AGTAATGTGTGACTGTGGGC[GG]CGTGTGACAGCTTTTACGTA
UMC65UMC65-G3/G6-1S27G3IG6-1 AGTAGTGTGTGACTGTGGGC[GG]CGTGTGACAGCTTTTACGTA
B B
UMC65UMC65-G3/G6-2S27G3/G6-2 TTCGCTTGGTAGCCGTAGCA[G/A]TATACTTTTACCGGCCACAG
UMC65UMC65-G3/GB-3S27G3/G6-3 GGGCTTTGGGTTGTGAACTT[CCAIC TTTCCC
UMC59UMC59-GSIG6-1UMC59-GSIGfr1CCAAGAAAGATTAATGCTGG[/T]TAAAATATTGTTTCCAGTCT
UMC59UMC59-G51G6-2UMC59-G5/G6-2AAAATCAGGACTGCGAAAAA(A/C]CCAAGAAAGATTAATGCTGG
UMC59UMC59~5/G6-2BUMC59-GS/G6-2BAAAATG~GGACTGCGAAAAA[A!C]CCAAGMGATTAATGCTGGT
UMC59UMC59-G5/G6-3UMC59-G51G&3AAAGTGTGTGTTGTTGCCCA[GIA]ATGATTCCATTCCACACAAG
UMC59UMC59-G4IG5-1UMC59-G4/G5-1AGGACTGCGAAAAAACCAAG[lA]AAGATTAATGCTGGTAAAAT
UMC59UMC59-G4/G5-2UMG59-G4IG5-2ATGCTGGTAAAATATTGTTT[/C]CAGTCTTTCACAAAGTGTGT
UMC59UMC59-G3IG4-1UMC59-G3/G41CTACAAAAATCAGGACTGCG[lA]AAAMCCMGAAGATTAATG
UMC59UMC59-G31G4-2UMC59-G3/G4-2TTGTTTCAGTCTTTCACAAA[/GT]GTGTGTGTGCCAGATGATTC
UMC59UMC59-G3/G4-3UMC59~31G4-3TCACACACCGACCTGCCTGG[/T]TATCAGGAACCATCCTCCTG
Ae1 Ael~4/G5-1 S23G4JG5-1 GGTGMTTGGTGATGCATGC[TIG]GGGGGTGCTCGAGTTGGATG
Ae1 Ae1-G4IG5-2 S23G4lGS-2 TTCCAGTCGGATGAACTGGA[TlG)GTTCGTCATCCACTCGTG~C
Ae1 Ae1-G3JG6-1 S23G3IG6-1 GGTGAATTGGTGATGCATGC[AIT]GGGGGTGCTCGAGTTGGATG
Ae1 Aei-G51G3-t S23G51G3-1 TTAAGTGAACATGCCCAAAC[GG]GTTAAACTTTCCATGGAACT
Ae1 Ae1-G5IG3-1 S23G5/G3-18ATTAATGAAGATGCCCAAAC(GG]GTTAAACTTTCCATGGAACT
B
Ae1 Aet-G1JG6-1 S23G11G6-1 TGATTCGGGTCTGTATGCGA[G/nTGTTGTGGTGGTGAACTGGT
Ae1 Ae1-G1/G5-1 S23G1/G5-1 CGGGTCTGTATGCGAGTGTT[G/A]TGGTGGTGAACTGGTGAATT
Aet Ae1-G1/G4-1 S23G1/G4-1 GTTCGCGGTTTCTGGGGCCG[GIT]GGGCGGTGCTCGGTGGGGCC
UMC90UMC90-GS/G6-1S22G5/G6-1 CAGATTGGTGTCGTTTACTA[A!G]AATTCAGTTCTGTCCATTTG
UMC90UMC90.G5lG6-2S22G~2 AAGTAAGCATTCTTTATATG[ITjTACTTCCCATGATAAACTTT
UMC90UMC90~5IG6-3S22GSIG63 CAAAGGGCTTACTGTACTTT[IC]CATCTTATTGGCAGGGCACC
UMC66UMC66-GS/G6-1S19G~1 ACTTGGCCGGGGACGTCGAC[GIA]ATCGTCGTAGCACTACTGGT
UMC66UMC66GSIG62 S19GSIG6-2 AGTACATGGCGAGCGTTGTA[G/C]CAGCTGCTTAGGTGATGTGG
Adh2 Adh2-G4IG6-1S17G4IG6-1 CTATTTCG1AGCTAACAACC[C!G]CTCTTGGTCCCAACATCCTG
Adh2 Adh2-G3IG6-1S17G31G6-1 GGTTCTAAACATAGCTCGTC[GI1JATTCATGATTCATCTCGAGC
UMC63UMC63.G4IG6-1S16G4/G6-1 TCAGCAAGCCTCCAAGGCTC[GA)AATGGTCCAGTTACTTGGTT
UMC63UMC63G2/G&1 S16G21G6-1 GTGTGTAGCTTCATTCGCAA[TGIAT]TTTGAA~CAGCCTCTGCAAGT
UMC63UMC63-G21G6-2AS16G21G6-2AGTGCTTTCGTAMCCTAGAG[TIC]TGAiCCAGCTGTGATTTCGGT
UMC63UMC63-G2IG6-2BS16G2IG6-2BGTGCTTTCGTAAA~CCTAGAG[TKxTGA~OCA,GCTGTGATTTCGAT
UMC63UMC63~21Grr3AS16G2~G63A GCTGACCAGCTGTGATTTCG(GJI~]1'GTATTCCAOGACGA~CGAGT
UMC63UMC63-G1IG6-1S16G11G6-1 TGTGTAGCTTCATTCGCA~AjGtTJTTTGJ1A~CAGCCTCTGC.AAGT
UMC63UMC63.G1IG3-2AS16G1JG3-2AGTGCTTCCGTAAACCTAGAO[TfC]TGACCAGCTGTGATTTCGAT
UMC63UMC63-G1/G3-2BS16G11G3-2BGTGCTTCCGTAMCCTAGAG[T1C]TGA~7CAGCTGTGATTTCGGT
UMC63UMC63-G1IG2-1S16G1IG2-1 GTGTGTAGCTTCATTaGCAA[[NT]GTnG~C;CCTCTGCMG
UMC102UMC102-G5IG6-1S14G51G6-1 GCTCAGCTGCCGGAGTA~CG
T(NT]GGCTTGCTCTCCGGCCGGCC
T(AITJGGCTTGCTCTCCGGCCrGCC
ASG24ASG24-GS1G6-1Sl3GSlG6-1 TTTCACAACTCAAiCTGATTG(M]CTTGCTTTGATGTQOATTCT
ASG24ASG24-GZ/G6.1S13G2IG6-1 TTGGTMTTTCAGA,CCTAGA[CI~GjMCTTACTGTGGTACAOGCC
UMC49UMC49~,41G6-1S12G4lG6-1 ACCTTTGCTGTGTTT1T11'T[TlrajGTATTOGMTGGA~GGGAGTA
UMC49UMC49-G2/Gr1S12G2JG5-1 AAAACAGCCAAGOTGGTGOT[CK3]AA~UCsGAAGGTGTCAQ~AAGGT
UMC49UMC49-G21G5-2S12G2IGr2 TCTGTTCGTTCCATCTCTTT[AIGJCI1GTAMTATCCGTMTTAC
UMC49UMC49-G2lGS-3S12G2IG53 CGTMTTACTTTGTTACTAC(TAfC]I4GTMTTTTATATATATCCT
UMC49UMC49-G21G5-4S12G2IG5.4 TATATATATCCTCATTTCAA[AfnGr4A~CAGTCAAAGTTAGTTTT
UMC49UMC49-G2/G5-4BS12G21G~r48TATATATATCCTCATTTCAPv(NT]GAA~CAGTCAAAGTAGTTTTG
UMC49UMC49-G2/G4-1S12G2IG4-1 TATTTCTTATCCAGGATTGT[TIC)CT1TGGCCAAAGCATGGTAC
UMC49UMC49-G2IG4-2S12G2JG4-2 CGTTCCATCTCTTTACAGTA[ING]ATATCCOTMTTACTTTGTT
UMC49UMC49-G21G4-3S12G2/G43 ATCCGTMTTACTTTGTTA~TA/ACjCTMGTMTTTTATATATAT
UMC49UMC49-G2IG3-4S12G21G3-4 GTAATTACT'iTGTfACTACT[AIJAOTMTTTTATATATATCCT
UMC49UMC49-GllG6-1S12G1IG6-1 CTGTGTTTTTTT1TGGTATT[G>C)GMTOGJ4CiGGAGTATTATTT
UMC49UMC49-G1/G6-1BS12G1/G6-1BGCTGTG1ZT11nTGGTATT[OIC]OMTGGAGGGAGTATTATTT
[N/AGAGTTTGGCACCITTGCTG
UMC49UMC49-G1/G5-2S12G1/G5-2 AGTTTGGCACCTTTGCTGTG(TQTTTTTTTTGGTATTGGAATG
UMC49UMC49-G11G5-3Sl2GtIG5-3 CTTTACTGATTGGGTTACAA(AIG]AGGTTATTTCTTATTCAGGC
UMC49UMC49-G1IG5-4S12G11G5-4 AATTACTTTGTTACTACCAG[Tl]TMTTTTATATATATCCTCC
UMC131UMC131-G41G6-iS10G4JG6-1 AGCGACAGGGATGTCGAGCA[GfT]CTACGGAAGGCAATAATGAG
csnpld 2AATTTGGGAAAATCAATGCA[GAA/CAC]ATCAGTGATTAATCCACATA
UMC131 UMC131-G3/G6-1 S10G31G6-1GCATGGCGGAGTGAGGGAGG[TGQTGTGTGTGTGTGGCTCCACA
2AGGCCGCTAGGCCATTTAGCG[G!A]ATTTGGGAAAATCAATGCAG
2BGGCCGCTACGCCATTTAGCG[G/A]ATTTGGGAAAATCAATGCAC
UMC131 UMC131-G1IG6-1 S10G1/G6-1CATCCCCGCCGGCAGAACAA[GG]GTACGAGAAGGATGGAATGC
UMC53 UMC53~5JG6-1 UMC53-G5IG6-1GTCCCAGATCAGGTCCACGT[1/C]CGAGCTCGCTGTTCCCGCTT
UMC53 UMC53-GSIG6-2 UMC53~SIG6-2TGGTTCTTCACCfvCCACCGC[GG]CCGGGCGCGCCCAGCGCCTC
UMC53 UMC53-GMG6-1 UMC53-GMG6-1GCAGCCTCAGGT~4CACGGGG[/A]AAGTCGGAGTGGTTCT1CAC
UMC53 UMC53-G4JG6-2 UMC53-G4IG6-2GCCGGGCGCGCC;CAGCGCCT[/C]GGTCCCAGATCAGGTCCACG
UMC53 UMC53-G3/G6-1 UMC53-G3/G6-tGCACGTCGTTGG'f GAAGAAG[AGCA]GCGGTACGGGTGCTTGTCGA
UMC53 UMC53.G3lG5-1 UMC53-G31G5-1AGGTACACGGGG~4AGTCGGA[G/T]TGGTTCTTCACCACCACCGC
UMC53 UMC53-G3/G5-2 UMC53-G3/G5-2CGACGGCGTCCAGCAC;CGAC[G/JCCTCCGCCTTCACCCCGCGC
UMC:53 UMC53-G3/G4-1 UMC53-G31G4-1GTCCACGTCGAGf:TCGCTGT[G1]CCC;GCTGCCCACGACGGCGT
UMC53 UMC53-G1/G4-1 UMC53-G1/G4-1GCACGTCGTTGG'fGAAGMG[A/CJAGCGGTACGGGTGCTTGTCG
1NAACCAAACCCTG'~qCTATTA[TIC]AGGTAGATTAGACTAGACAC
UMC161 UMCt61-G21G3-2 SO6G2/G3-2ACGGTGAGGAGT<aGCACATG[A/C]GATGGAAAGTTCCTGTAGAC
28ACGGTAAGGAGTC~GCACATG[A/C]GATGGAAAGTTCCTGTAGAC
UMC107 UMC107G21G4-1 S05G21G4-1TATGCTTGGAAAGTGGGAAA[G/JGGGAACATACGATGGAGGAC
UMC67 UMC67-GS/G6-1 S03GSIG6-1AAACAATAATTTTTACACAG[ITJTGCTMGGTTTTACTGTTTT
UMC67 UMC67-G21G6-1 S03G21G6-1ATATCCATGTTGTI:GCCTGC[/TG]TGTGCGCTTGCTTGCCGCTA
UMC76 UMC76~4/G6-1 S02G4lG6-1TTGCTGCTATGTTTACTGGG[ITjTGTAGAAAAAAAAATAATAT
UMC76 UMC76-G21G6-1 S02G2/G6-1GCTCGGTAATAATTCTGGCT[GG]CGATGGCACCCATATTCCTC
UMC76 UMC76-G?JG6-1 B S02G2/G6-1GCTCGGTAATAATTCTGGCTjGG]CGATGGCACCCATATTCCTG
B
UMC76 UMC76-G2/G5-1 S02G21G5-1AAAACACGTGGTGTTTGTTA[GIA]GAAAGACCTAGTTTCTCGGC
UMC76 UMC76-G2/GS-1 B S02G2IG5-1AAATCACGTGGTGTTTGTTA[G/A]GAAAGACCTAGTTTCTC;GGC
B
UMC76 UMC76-G2/G5.1 S02G21G5-1TAGTTTCTCGGCAATTGGCA[G/T]TGTGGAATGACGATCTCGTG
UMC76 UMC76-G2/G5-1 B S02G21G5-1TAGTTTCTCGGCAATTGGCA[GIT]TGTGGAATGACCATCTCGTC
B
UMC76 UMC76~2/G5-2 S02G2/G5-2GTGTGGAATGAC:CATCTCGT[G/C]GTGATGCCAGCATGCTGTTA
UMC76 UMC76~2/GS-2B S02G2IG5-28GTGTGGAATGACCATCTC:GT[GlC]GTGATGCCAGCATGCTACTA
UMC76 UMC76-G2lGS-3 S02G21G5-3ACCCTGTCAGGCTTCCACAG[AIC]TATAATAT1TGTTGTGGTGT
UMC76 UMC76-G2/G5-3B S02G2IGS-3BACTCTGTCAGGCTTCCACAG[A/C]TATAATATTTGTTGTGGTGT
UMC76 UMC76-G2/G5-3C S02G2/G53CACTCTGTCAGGCTTCCACAG[A/C]TATAATATTTGTTGTGTGTG
UMC76 UMC76-G2/GS3D S02G21G5-3DACCCTGTCAGGCTTCCACAG[A/C]TATMTATTTGTTGTGTGTG
WO 9$/30717 PCT/EP97/07134 Example 4 Analysis of Polvmor hi ms 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 Applications 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. 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 Polvmort~hisms in Taraet 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 WO 98/30717 PCT/EP97l07134 1 '~
analysis is determining which forms) 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 Pro es 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 t~~ the corresponding segment from another member due to the presence of different polymorphic forms in the respe~~tive 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 .L6 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 Arravs The polymorphisms can also be identified by hybridization to nucleic acid arrays, some example of which are described by Wo 95/11995 (i.ncorporated by reference in 1~
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 Acld 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 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.
1g 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., C;SHP, New York 1989); Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).
5. Denaturincr Gradient Gel Electrot~horesis 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 5~echnology, Principles and Applications for DNA Amplification, (W. H. Freeman and Co, New York, 1992), Chapter 7.
6. Finale-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 sinc3le stranded PCR products, as described in Orita et al . , ~~roc, Nat . Acad. Sci . 8 6 , 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 forms) present in a subject plant at one or more polymorphic sites, this information can be used in a number of methods.
A. Fingerprint Analvsis 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 10 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 15 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 20 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 Polvmorphisms with Phenotv~ic Tr i The polymorphism;s 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 _~nclude 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 ~~f 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 statistica7.ly significant correlations between polymorphic forms) and phenotypic characteristics are noted.
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 O 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:
Yijkpn = ~ + YSi + Pj; + X~; ~ 131 + ... 1317 + PE11 + a" +ep where Yijgpnis the milk, fat, fat percentage, SNF, SNF
percentage, energy concentration, or lactation energy record;
~ 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; (31 to 131 are the binomial regressions of production record on mtDNA D-loop sequence polymorphisms; PEn is permanent environmental effect common to all records of cow n; an 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 ep 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 z3 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 hack-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 Polvaeptides and Gene ~eauences 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 expre:~sed in an expression vector in which a gene is operably 7. inked 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 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. Nat1 Acad. Sci, USA 82, 5824 (19853, infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et a1. , 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., Nature 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 (2984);
Fraley et al., Proc. Natl. Acad. Sci. USA 80, 4803 (1983)).
The protein may be isolated by conventional 5 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, ~~rinciples and Practice', 2nd 10 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 15 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 20 described in Evans et al., "Protoplasts Isolation and Culture," Handbook of Plant Cell Cultures .2 , 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, 25 Basal 1983); Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrar.~t Crops," Protoplasts (1983) - pp. 31-41, (Birkhauser, Basel :1983); Binding, "Regeneration of Plants," Plant ProtopLasts, pp. 22-73, (CRC Press, Boca Baton, 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 blologically 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, Pr~incip.Ies 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 z~
allele-specific oligonucleotide~s 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 ca.n 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 (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and t:he appropriate-buffers for reverse transcription, PCR, or hybridization reactions.
Usually, the kit also contains instructions for carrying out the methods.
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 INVED~~fION
I. Novel PolvmorlJhi~ms 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 ex rac ion 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 vuas 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 G1, 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 rind Nei-li distance) is 0.69.
This value is close to the maximum distance between two cultivated maize lines.
Among the 15 genetics distance between couple of these 6 lines . 8 are superior t:o 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.
Genotvoes G1=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 2 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 2d 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 SO1F is the forward primer for probe 1 SOlR 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 S01 1.01-1.02 SO1F CGCACGCACATTAGCTTTCG 610822 UMC76 S02 1.02-1.03 S02F ATTATTCGGCGTCCAGCCCC 610865 lO S02R TTACCAGCGGTGAGAGCTGC 610866 UMC67 S03 1.05-1.06 S03F CGTTCGTGTGGCATCAATCG 610864 UMC161 S06 1.10-1.11 S06F CAGACCTTGGTTGGAGGCAAC610824 UMC53 S08 2.01-20.2 S08F CGGACGTGATGCAAGTTTCG 610851 UMC131 S10 2.04-2.05 S10F2 TCCTTGGCACTCACGCTACC 610816 UMC49 S12 2.08-2.09 S12F CAGAGAGCCGTCTCGAATCG 610845 UMC102 S14 3.04-3.05 S14F TGCTGTGCTGTCACATGGCG 610801 UMC63 S16 3.08-3.09 S16F2 ACGCCCTGACAGAACCATCG 610857 Adh2 S17 4.03 S17F2 TGCCTGCTGCATCTCTAGCC X02915 UMC66 S19 4.06-4.07 S19F TGGAGTGTCCAAAGACCGACC610862 UMC90 S22 5.01 S22F GCAGGTGAACAATGCTGCCC 610870 Ael S23 5.05 S23F TCGCTGGGGTTTTAGCATTG L08065 UMC59 S26 6.01-6.02 S26F TCCAAAGCGAAAGCCTGATG 610853 UMC65 S27 6.03-6.04 S27F TTCCAGCTTTCCTCGGCACC 610860 UMC21 S28 6.04-6.05 S28F TGCAGATGTGCCTTTCCTGTG610830 UMC132 S29 6.06-6.07 S29F CGCACAGAGGCAGATGCAGC 610824 UMC254 S33 7.03-7.04 S33F CCGGGCGCAAAAGAATGTG 610832 UMC80 S34 7.04 S34F TCGCCTTTATCGGTGCAATG 610867 BNL9-11 S38 8.01-8.02 S38F2 CGAGGGAATGTCATCAACCC 610778 UMC109 S42 9.00-9.01 S42F GCACCGTCGTTTACCTCAAGC613177 Wxl S43 9.02-9.03 S43F CGTGCTACCTCAAGAGCAAC X03935 UMC95 S44 9.04-9.05 S44F CACTCGGAAGTCGGAATCGC 610872 CSU61 S45 9.05-9.06 S45F TCTCCACGAATCCCACCGTC T12691 UMC130 S48 10.02-10.03 S48F AAGGGGGAAGAAGGTCATC GI0824 CSU109 S53 2.09 S53F GCTTTCGGTTCCGGATAGCG T22721 UAZ77 S56 5.04 S56F2 GCAACCAACTGCAACATCGC T18762 Sh1 S57 9.01 S57F TGCTGTTATTGCGTGCCGTG X02382 Sh2 S63 3.09 S63F TTCTTCACTGCACCCCGATG M816D3 5 Bre1 565 6 S65F AGCAGCAGATCAGGCACACC U17897 Bt1 S66 5 S66F GGCAAGGATCGGAGTTGCTC M79333 Ssu S67 S67F GCAAGCAAGCAAGCAGCGAG D00170 1~ S67R GACCCGAAGCAAAACCGAAC D00170 Bt2 S71 4 S71F TGCCGAAAAAGGTGGCATTC Seq (Bae et al 1990) 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 10x . 3 microliter; dNTP's . 0.2 mM each; MgCl2 . 1.5 mM; BSA 0.8mg/
ml; primers 1.5 ng/microliter each; glycerol 50.
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) the genotype number (G2) - the second genotype number (G6) - and the number of the SNP (single nucleotide polymorphism). So, in this cage, 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 marl~:er/gene.
Column 4 represent~~ 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-Gl/G5-1A) means deletion of the base pair G in G7_ compared to G5.
iz TABLE I
csnpld Ssu 8t2-G2/G6-1 S71G2IG6-tATAATACTTGATATGCCATT[G/1~TGTCCTCTTATTTTTAA
S
CAT
Ssu su- G1JG5-1 S67G1/GS-1ATGGCCTCGTCGGCCACTGC[A/C]GTCGCTCCGTTCCATG
S
GGCT
Ssu su-G11G3-1 S67G1/G3-1GCCGCTCCTCCAGAAGCCTC[G/A]GCAACGTCAGCAACG
GCGGA
Ssu Ssu-G1/G3-2 S67G11G3-2GTGTTGCCCATCCCATCCCA[A!Z]TTCCCAACCCCAA
ACGAACC
Bti Ssu-G1/G3-3 S67GiIG33 GTACCTGCCGCCGCTGTCGA[CG/AC]GGACGACCTGCTG
B
AAGCAGG
Bti tt-G2/G3-1 S66G2/G3-1AGTGAGCCCGCTTCTTATTC[/T]TAAGGTGATAGGTTTCTA
B
AA
Bt1 ti-G2/G3-2 S66Gi/G3-iAATGTAATGGTACTCCGCGC[T/C]ATGGCTCTGGTACTTA
GGAA
Bre1 Bt1-G2/G3-3 S66G1/G2-1AAATAGGCTCGGGCAATTAT[C/jCAGCTTAGGGACAGCAAGC
B
G
Bret rei-G3/G6-1 S65G3/Gtr1TCCGCCCTGCCTCCGGTTTT[M']GCCCGACCTTCGAAACATT
B
C
Br ret-G3/Gti-2S65G3IG6-2ACCACTGACGTAGCACCTCC[G/T]ACTTCTCGTTGTAA
AACCCC
e Bre1-G3/G5-1S65G3IG5-1GGAGGTTCGCCTCATGTTAT[C!f]GTTGACGAGCCA
B
i CATCCACT
re Bre1-GMG6-1 S65G41G6-1GCTCCGACTTCCAATCTTGA[A/C]CCTCCACCCTGC
CTCCGGTT
~Gi2 ASG12-G1/G3-1S64G1/G3-1CTGGTTGAAATGTGTTGAAG[C/A]TACTAGTGATGAA
CTGCTTG
ASG1 ASGi2-Gl/g3-2AS64G1/g3-2AGCTGCTCCAAGCGAGCCCGC[C!G]CCGAAAAAGG
AAAAAGGTGA
2 ASG12-GlIg3-28S64G1/g3-2BGCTGCTCCAAGCGAGCCCGC[C/G]CCGAAAAAGG
AS ~
AAAAA( G12 ASG12-GlIg3-3AS64G11g3-3A,TTGA
ASGi2 CGCCCCGAAAAAGGAAAAAG[G/T]TGAAGGTCCTTAC
TCACCGA
ASGi ASGi2'Gi/g3~BS64G1I93-3BCGCGCCGAAAAAGGAAAAAG[G/T]TGAAGGTCCTTA
CTCACCGA
2 ASG12-Gl/gG3-4AS64G1/gG3-4AGAACCGGCCACAGTGCCTGA[TIA)T'TTGGCGGTGAG
ACCTCTTC
Sh2 ASG12-Gl/g3-4AS64GtJg3-4AGAACCGGCCACAGTGCCTGA[T/A]TTTGGCGGTGAGAC
TTCTTC
S~ Sh2-G5/G6-1 S63G5/G6-1CAATTGTTACCTGAGCAAGA(f/]TTTTGTGTACTTGACTT
GTT
Sh2 Sh2~4/G6-1 S6:iG4/Gti-1TACTGAGAGAATGCAACATC[C/G]AGCATTCTGTGATTGGAGTC
5h2-G4IG5-1 S63GMG5-i TTTTAGTGTACTTGACTTGT[C/T]CTCCTCCACAGATGAAATAT
Sh2 A A
Sh2-G41G5-1 St~G4lGS-1TTTTTGTGTACTTGACTTGT[G7]CTCCTCCACAGATGAAATAT
Sh2 B B
Sh2-G3/G6-1 S63G3/G6-iTCTGTGATTGGAGTCTGCTC[G1A]CGTGTCAGCTCTGGATGTGA
Sh1 Sh1-G5/G6-1 S57G5JG6-1AACTACJ1AAAAGCATCTCCT[GIT]GGATTTGGCTATCTCCTTTT
Shi Sh1-G21G5-i S57G2/G5-1TTAGCGCCAAAAAAAApCTC[/T)TTTTTTTTTGTCCTTTTACT
Shi Sh1-G21G3-1 S57G2/G3-1TCAATCCAATCAATTTAATT[TIC]CTTCCTTTAAAAATATTATC
Sh1 Shl~l/G2-1 S57G11G2-iTTACTACGAAAAACTCTTGA[GIT]TCTAGGAATTTGAATTTGTG
Sh1 Sh1-G1/G2-2AS57G1/G2-2ACTTCTTGGATTTTGCTATCT[T/C]CTTTTACTACGAAAAACTCT
Sht Sh1-G1IG2-2BS57G1IG2-2BCTCCTTGGATTTTGCTATCT[T/C]CTTTTACTACGAAAMCTCT
Sh1 Sh1-G1/G2~A S57G11G2~ATTTTACTACGAAAAGCATCT[TIC]CTTGGATTTTGCTATCTTCT
Sh1 Sh1-G1/G238 S57G1/G2~BTTTTACTACGAAAAGCATCT[TIC]CTTGGATTTTGCTATCTCCT
Sh1 S57G1/G2-4 S57G1JG2-dGAAGCCAAATCCTATTATTT[T/C]CTGCCTCTAGGGTCTGAATG
UAZ77-GNG6-iS56G4lG6-1GTACACTGTTACAATCACAC[fIG]TAGTGAAGCGCAACACAGAT
UAZ77-G4/G6-2S56GNGti-2GCCTTATCATCCTCTAGGTA[f/A]TGGAGACGAGTGACCAGTCT
UAZ77-G4IG63S56GMGti-3CTTTTCTTCAGACCCGAGCC[CITjCCAATCGCGCCCTTCTGTGC
U
AZ77 UAZ77.G4lG63SS6G4IG63 CTTTTCTTCAGACCCGAGCC[CJT)CCAATCGCGCCCTTTTGTGC
UAZ77~41G5-1AS56G4/G5-1AGAGCCCCCAATCGCGCCCTT[GT]TGTGCCTTGGCCTTGAGCTC
UAZT7.G41G5-1AS56G4IG5-1AGAGCCTCCAATCGCGCCCTT[GT]TGTGCCTTGGCCTTGAGCTC
UAZ171G1IG3-1S55G1/G3-1GAAGGAGCAGCAGCGCAAGG(NJACGTGTTCCAAGTCAACGTC
UMC17 UMC117-G2IG3-1S54G2/G3-1GTAGAAAGTTAGCAAAAACA[T~7T-~TTAGTGAAAAAACATA
UMC17 UMC117~2Xa3-2S54G21G3-2ATTGTGGCTAGAAACTTTGG[I~TTITTITApp~TTATGGTCAT
CSU109CSU70S~SfG6-1S53G5lG&1 GCAAACCMCACCAp~TCTTC[G/C]AAATGAGCAAAGCAGAGACT
CSU109CSU1l79.G51G6-2S53G5/G6-2CAGATCGGTTGTCCTCAGAG[AJJAAGTCACCTACCTGCAAACC
CSU109CSU1 3 S53G5/Gf,-3AATTCTAG4TAGGAGTCATG[G1]ACAAGTACTTGTTTAAAGGA
CSU109CSUIt~5IG6-4S53GSIGfi-4ACAAGTACTTGTTTAAAGGA(GjCATGCCGGAATACACGCTGC
~~~A GAGCGAGATCGATCCTGTTG[T/C]CATCCATCACTGCCATAGGA
CSUt09CSU109-GSIG6.SBS53GSIGSSBGAGCGAGATCGATCCTGTTG[TI~CATCCATCA
CTGCCGTAGGA
CtaU109CSU1t79.G4/Gfr.1S53G4IG6-1~
TAGTCATAGCHA~f'.AGCATGaG//~JTCG~,ATGTAGCGTTCACCC
CSU109CSUtOA.GMG6 S53G4/G6-2CAATTGAAGAGG4~1AAAAppA(/~CT~pTTCATGTAC
CSU109.G4AG5-1S53G4/GS-1~G~GAGTCCACAATAOTTCGTC
CSU109.C311p5-1S53G3K35.1CCCACCGCGG~TGGTGG[T/JTAGAAGG;GAACCACCGAGC
CSUIt~CSU109.G?hGG-1S53G2K~6-1ACTTGTTTMI~GGA~CATGCC(<i~GGApTq~CTGCCCAGGC
CSU109CSU1~IG3-1 S53G2Aa.1 OCCAGGCCTTCOCACGGCGG[AK33]GATGGTGGTTAGAAGCGGAA
TCCGCAATAGT
CSU109CSU109.G1~G6-2S53G1~Gfs.2GJ1ACI4C',AGTCCGCMTAGTT[T/~CaATCCTAATGCTi4CTTOGAGC
UMC130UMC130~31G6.1S48G3IG6-1GATTCJ1GAAACA~GTGGCGGC[AK~GATGTAGCATCAACACGCCC
CSUtiI.GS/G6-1S45ta51G6-1ATGAGTATATTC~AAGTCATA[TICjTGTGAACTAGAATGTTATTT
CSU61 CSU61-G5Ki6-2AS45GSlGfr2ACCTAGJ40GCTGACCGOC~AC~A[G//ypCGGCGG~CTGCG1AATC
CSU6I.f~G6 S45GSIG6-2BCCTAAACGCTGI1CC(~
2B CCqC
, CSUB1 CSUB1-G5b6.3S~SGSIG6-3/~G/Np~G~CTGCCAMTC
TGAAA'.~MACCATGCGCTACC(GTjAGCTACiGTGTTTTMAC' TM
CSU61 CSU61-G~i6-1&45G~AG6..1s TCOGCGGJ1AACAACATCCGA(G!1'[TTCTTGAGOATAACCCAGCT
CSUB1 CSU61.G4/G5.1S45G4K35..1GGGAGGGGAMAqA~pGp~GJTTGGTTGCGGTTCAGT
CSU61 CSU61-G4fG5-2S45G4/GS2 GGCGGCTGCCAAAT~GCaG[!A)AppCGA~TtxGAGTTCTTG
CSU6i CSU61-G2IG4-1AS45G2IG4-1ACTAGMTGTTATTTCTTCAC[GA]GTTGACCATGGMAAAAACA
CSU61 CSU61-G2IG4-18S45G2/G4-1BCTAGAATGTTATTTCTTCAC[GI~GTTGACCATGGAAAGAAACA
CSU61 CSU61-G?IG4-2AS45G21G4-2ATTCACCCTTGACCATGGMA[AIIpTp,ATAAGTTCTTGT
CSU61 CSU61-G2K34-2BS45G21G4-28TTC~ACJIGTTGACCATGGAAA(NC~)I4AACAGTMTMGTTCTTGT
CSU61 CSU61-Gt/G6-1S45G1IG6-1TTCTTG~CA,GTTGACCATGG[IA~AAAAAAAG4GTMTAAGTTC
CSU61 CSU61.G1IG5.1AS45G1/G5.1AGAACCCACCOTGCCCTGOGA[/G)<3G
CSUB1 CSU61~1IG5-1BS45G11G5_iBGAACCCACCGTGCCCT
CSU61 CSU61-G11G5-2AS15G1Ki5-2ATGGGAGGGNJIAAAqAAA~C'sAA[LiI~AGCt;',TTGGTTGCGGTTCAGT
CSU6i CSU61-G1/G53S45G1K's5~3CGTACC~1ICCTqGGMTCGTA(AK;~UIApGCCTAGACGCTGACCG
lJMC85 UMC95GSJG6-iS44G5/G6-iGCTGCGTCMTCI1TG1C1TC[T/A]CCCACI1GGCGTCAAGTACAG
U~AC85 UMC95~G3IG4-1S44G3K's4-1GACAGATTCCAAAGTAGTCG[GT)CGGCCACGTCGAAAAAGAAT
UMC9rG2/G6-1S44G2/G6-1GGCGCTGCGTCAATCATCAC[MjTCACCCACAGGCGTCMGTA
csnpld UMC95 UMC95-G2IG4-tAS44G21G4-1ATCGGTGTCACCnCATGCATA,[TlG]TCAGGACAGATTCCAAACTA
UMC95 UMC95-G21G4-iBS44G2/G4-1BTCGGTGTCACCACATGCATA[TlG[TCAGGACAGATTCCAAAGTA
UMC95 UMC95-G?JG4-2AS44G2IG4-2AGTCGCCGGCCAGGTCGMAA(G/A]GAATACTCAGCAAAAGACCC
UMC95 UMC9SG2IG4-2BS44G2/G4-2BGTCGTCGGCCA~GGTCGAAAA[G/A]GAATACTCAGCAAAAGACCC
UMC95 UMC95-G21G3-1AS44G2/G3-1ATATTCAGGACAGATTCCAAA[GG]TAGTCGCCGGCCAGGTCGAA
UMC95 UMC95-G21G3-1S44G2/G3-1TAGTCAGGACAGATTCCAAA(GG]TAGTCGCCGGCCAGGTCGAA
B B
UMC95 UMC95-GIIG(i-1S44G1/Gti-1GCGTCAAGTACAGATACGCA[AIG]CACGCCTCAGCTTCGCCTTG
UMC95 UMC95-GllG2-1S44G1/G2-1CCTGGGACTCG:~CAAATTGC[GlA)A1GCACTCGGTGTCACCACAT
Wx1 Wx1-G?JGtr1 S43G2/G&1GCTGGTTCATTA,TCTGACC1'[GlT]GATTGCATTGCAGCTACAAG
Wx1 Wxl~?JG6-2 S43G21G6-2CTGGATTGCATTGCAGCTAC(A/G]AGMGCCCGTGGMGGCCGG
Wx1 Wx1-G2/Gt~IB S43G21G&1BGCTGGTTCA1TA,TCTGACCT'[GlT]GATTGCATTGCAGCTACGAG
Wxi Wx1-G2lGti-2BS43G2/Gtr28CTTGA1TGCATTGCAGCTAC[A/G]AGAAGCCCGTGGAAGGCCGG
Wxt Wx1-G2/Gti-3 S43GTJGti-3TCAGCCCCTACI'ACGCCGAA[G/JGAGCTCATCTCCGGCATCGC
Wxi Wx1-G2/G5-1 S43G2/G5-1TACCCGGAGCTGAACCTCCC[GG]GAGAGATTCAAGTCGTCCTT
Wx1 Wx1-G21G4-1 S43G21G4-1TGCATGTGAACATTCATGAA(f/CjGGTMCCCACAACTGTTCGC
Wx1 Wx1-G6IG1-1 S43G6/G1-1CTCCTACCAGG(~CCGGTTCG[T/]CCTTCTCCGACTACCCGGAG
Wx1 Wx1-GI/Gfr1 S43G11Gti-1TGAATGGTMCCCACAACTG[GTJTCGCGTCCTGCTGGTTCATT
Wx1 Wx1 ~11G5-1 S43G11G5-1GCCGACAGGGTCCTCACCGT[GIC]AGCCCCTACTACGCCGAAGA
Wx1 Wxi ~2lGfi-1 S43G2/G6-1GCTGGTTCATTATCTGACCT(G/T[GATTGCATTGCAGCTACAAG
Wx1 Wx1-G2/G6-1 S43G21G6-1GCTGGTTCATTATCTGACCT(G/T]GATTGCATTGCAGCTACGAG
B B
Wx1 Wx1-G2/G6-2 S43G2iGti-2CTGGATTGCATTGCAGCTAC(AJG]AGAAGCCCGTGGAAGGCCGG
Wx1 Wx1-G21G6-2B S43G21Gfr28CTTGATTGCATTGCAGCTAC[AIG]AGMGCCCGTGGAAGGCCGG
Wx1 Wx1-G2/G65 S43G21G6-5TCAGCCCCTACT'ACGCCGAA[G~GAGCTCATCTCCGGCATCGC
Wxt Wx1-G2IG4-1 S43G2JG4-1TGCATGTGMCATTCATGAA[T/C]GGTAACCCACAACTGTTCGC
Wx1 Wx1-G2IG3-1 S43G21G3-1CTGGTGGTGGTGCTTCTCTG[AAAG]TGAAACTGAAACTGACTGCA
Wx1 Wx1-G2/G3-3 S43G2IG3~3GACCATCTTCACGTACTACC(fACG JAGACCGCTTTCTGCATCCAC
Wx1 Wx1-Gl/Gti-1 S43G1/Gti-1CTGACCATCTTCACGTACTA,[CCTAI]CCAGACCGCTTTCTGCATCC
Wx1 Wx1-GtiIG1-1 S43Gti/G1-1CTCCTACCAGGGCCGGTTCG[T>jCCTTCTCCGACTACCCGGAG
Wx1 Wx1-GtilG1-1 S43G6IG1-1GAGATTCMGTCGTCCTTCG(G/jATTTCATCGACGGGTCTGTT
Wx1 Wxi-G1/G6-2 S43G1/G6-2TGAATGGTAACCCACAACTG(GT]TCGCGTCCTGCTGGTTCATT
Wx1 Wxt-G1IG6-3 S43G1/Gti3GCCGACAGGGTf:CTCACCGT[GlC]AGCCCCTACTACGCCGAAGA
Wx1 Wxl.G1/GS-1 S43G1IG5-1TCTGACCATCTT~CACGTACT[ACCTIjACCAGACCGCTTTCTGCATC
Wxi Wx1-G11G4-1 S43G1/G4-tCTTGATTGCATT~~.~CAGCTAC(GlA]AGMGCCCGTGGAAGGCCGG
Wxi Wxt-G11G4-1 S43G1IG4-1CTGGATTGCATTGCAGCTAC[G/A]AGAAGCCCGTGGAAGGCCGG
Wx1 Wxt-G1/G3-1 S43G11G3-1GCTGGTTCATTATCTGACCT(T/G]GATTGCATTGCAGCTACGAG
Wxt Wx1-GSIG6-1 S43GS1G6-1AGAGATTCMGTCGTCCTTC[G~GATTTCATCGACGGGTCTGT
UMC109 UMC109-G2IGti-1S42G2/G6-1CTCG~1TGAAAAAGGTGCCGC[/G]TACTCTCTCAGTCAGCTACT
UMCIt><J UMC109-G2/G3-1AS42G2IG3-1ACTGCACTCCGATTGAGGGTC[C/G]GMGCAGGGCAGCGCGTGTG
UMC109 UMC109-G2IG3-1S42G2IG3-1CTGCACTCCGA'iTGAGGGTC[GGjGMGCAGGGCAGCGCGTTGT
B B
UMCIt><J UMC109-G2/G3-1CS42G21G3-1CCTGCACTCCGATTGAGGGTC[GG]GAAGCAGGGCAGCGCGTTTG
UMC1t79 UMCIf~-G21G3-1DS42G2IG3-1DCTGCACTCCGATTGAGGGTC[CJG]GMGCAGGGCAGCGCGTTTT
UM(~.80 UMC80.G3IG5-1S34G3NG5.1CATGCCTCTGTTGATATTTT[G/C]GTGCACCTTTTGCTTGCMC
UMCt30 UMCBf>G3/G5-2S34G3/G5-2GATTTTGTAGGTTGATGCAT[Cl~jGTTTGATCTTTCTTATCTCC
UMC80 UMC80-G3/GS3S34G31G5~3TGCTTGCAAiCTAAATTMTC[AIG]TGCTCTATTTGACTMGAGT
UMC80 UMC80-G31G4-1S34G31G4-1ACATGTC.C~4GGACGCATGGIjCIJCCCAATAT'TGTTGTTGGMG
UMC80 UMC80-G31G4-2S34G31G4-2TTGATCTTTCTTATCTCCTT[!C]CGMTTTGTTCTGTGTTATA
UMCtiO UMC80-G31G4-2BS34G31G4-2BTTGATCTT'TCTTATCTCCTT[/C]CGMTTTGTTCTGTGTATAC
UMC80 UMC80.G2/G5-1S34G?aGS_1TGTAGGACTTGGAGAGCTTG(AIG]TMTTTACACATGCCTCTGT
UMC80 UMC80.G2lGS-2S34GZb5-2CATGCCTCTGTTGATATiTT(GJCjGTGCACCTTTTGCTTGCMC
UMC80 UMC8~Gr3 S34G2Ki53GATTTTGTAGGTTGATGCAT[C>nGTTTGATCTTTCTTATCTCC
UMC80 UMC804~G3-1S34G21G3-1GAGACATTTCCTACTCMT
A jCn]Ai4TTATTTGATGAMTTATT
UMC254 UMC254.G51G6-tAS33GSfG&1AAGTATCACMiACTMTCTGA[NGjTATCTGGTTGCf:AOGAAMC
UMC254 UMC254~G6.1BS33G5lGrrlBAGTATCACAQJ1CTMTCTGA[AK3]TATCTGGTTGOCACAAAAAC
UMC254 UMC254-G5fG6-2S33G51G6-2TCAAAGTGGTGCAATCGCAA[TICjCGA~CTTGGGCTTGCC(iTGGT
UMC254 UMC254-G51G6.3S33GSrGS~CCI1CTTGGGCTTGCCGTGGT(CIJCGTATCGTACGCAGGTAGCA
UMC254 UMC254-G51G6-4S33GSK36-4AGCATTTTTfGTTTTGTTTT[TJC]CCTTGGCAGACMCI1GACAG
tl~AC254 UMC254-G';~G65AS33GS/G65ACAGTCCCOAGANTCCCAAAT[GjCAQAAAAAGGTTTTGTTTTT
tlAAC254 UMC254-G51G8.58S33GSIG6~58CAGTCCCGAGMTCCCAAAT(GJCAGIUW1I1GOTTTTGTTTTA
UMC254 UMC254.G4RIG6-1AS33G4R/GfrlAGGC;AuC~AC~1A~CA~C'~CA~CATCA[AG1CNCATGCTTGCATTTACTCCCA
UMC254 UMC254-G4RIG6-1S33G4RKi6-1GGCAGACAACAGiACAraATCA[AG/C~CATGCTTGCATTTACTCTCA
B B
UMC254 UMC254-G3RIG6-1AS33G3RI08-1AGTGATCACAGACTMTCTGA[NGjTATCTGGTTGCCACGMMC
UMC254 UMC254G3RIG6-1S33G3RIG8-1GTGATCAf:AGACTMTCTGA(AIGjTATCTGGTTGCCACAAAAAC
B B
UMC254 UMC2S4~G3RIG6-2AS33G3WG6-2ATGTGMTATCTGGTTGCCAC[G/AjAAAAiCCOGGACACMCiAGAG
UMC254 UMC254~i3R/Gtr2BS33G3R/G8-2BTCTGAGTATCTGGTTGCCAC[G/AjAAAACCGGGACACMGAGAG
UMC254 UMC254.G3/G6-3S33G3~ TCAGTCAAACTCAGTCCCGA[AK3]AATCCCAAATCAGAAAMGG
UMC254 UMC254~3X35-1AS33G3IG5-1AGGTTGCCACOIV~1ACCOGGA(CIG TCAGAGT
UMCZ54 UMC254~G31G5-1S33G3JG5-1GGTTGCCACGAiIAA~CCGGGA[GG)ACAAf"aAGAGAAACTCAAAGT
B B
UMC254 UMC254-G2R/G3-tAS33G2R1G3-1AACGCATGCTTGCATTTACTC[CJTJCAGTCAAACTCAGTCCCGAA
UMC254 UMC254-G2R/G3-1S33G2R1G3-1ACACATGCTTGCATTTACTC[GT)CAGTCAAACTCAGTCCCGAA
B B
UMC254 UMC254-G1R/G2-1S33G1R/G2-1TATTATTCAATTTTGAATM[IGjGAAGOAAATTTTAGG1,CCTC
ASG49 ASG49-G3IG5-1S32G3IG~r1ATTMTAMTGCATCCTCTG[GGjTAAAAAAACCCATTTTGMT
ASG49 ASG49-G3/G5-2S32G31G~r2ATGMTTGMGCTCTGMTA[GITjAGMTCCACCATTCTTCCGA
ASG48 ASG43-G31G5-3S32G31G5-3GMTCG4Cf:A1TC1TCCGAA(A!G]CTGCTTCCTACAAAACTCGA
ASG49 ASG49-G3IG5-4S32G3/GS-4GAAAGGATGTGZTTTTGATA[G!A)CCTTCAGTCTTTCAGATGGA
ASGB ASGS-G3lGS-1S31G31Gr1CAATGTCTTGTTCGTTATCA(A/G)CGAAAGTTTGMTCCCCACA
ASG8 ASG8~G31G4-1S31G3IG4-1TGTATCGGCTAGTCTGGATG[GIAjTCGCACTGGCACTCAGTGCT
csnpid UMC132UMC132-G4/GS-1S29G4lGS-1 TCTATTCAGCAGTCTGAGAA[GCAICT]AGGATGGTCGGCTTCTTCAG
UMC132UMC132-G11G5-1S29G1/G5-1 CCTTACACTATTAACAGGCC[GT]GTGATCTACCTGAATGCCTG
UMC21UMC21-G5/G6-1S28GS/G&1 CAAGAAGCCTCTTCAGTGTC[A/C]GTCGTAGCTTCCTCAAGACC
UMC21UMC21-G51G6-2S28G5IG6-2 AGACCTTCCTGATGTGCGGA[T!C]GCTAATCCATGGAGCAGGGA
UMC21UMC21-GSIG6-28S28GS/G6-2BAAGACCTCCTGATGTGCGGA[f/C]GCTAATCCATGGAGCAGGGA
UMC21UMC21-G5/G6-3S28G51G6-3 CTAATCCATGGAGCAGGGAG[G/A]AAGGGGCGAGGGGCAGCAAG
UMC21UMC21-GMGS-1S28G4IG5-1 TCGTCGCGAATACAGCCGGG(G/C]GAGGGGGTGGTCGCGACTGG
UMC21UMC21-G3/G6-1528G3/G6-1 GTCGTAGCTTCCTCAAGACC[TIJTCCTGATGTGCGGACGCTAA
UMC21UMC21-G3IG4-1S28G3/G4-1 GAGTCGTCGCGAATACAGCC[A/G]GGGGAGGGGGTGGTCGCGAC
UMC21UMC21-G31G4-2S28G3IG4-2 AGGGGGTGGTCGCGACTGGA[T1G]CGCCCGAGCAGCGAGCAAGC
UMC21UMC21-G3/G4-3528G3IG4-3 AAGCACATGTTTTAACCTTT[T/G]ATTCAAACTTTCCAGCCGTT
UMC21UMC21-G31G4-3BS28G3/G4-3BAAGCACATGTTTTAACCTTT[T/G]ATTCAAACTTTCCAGCGTTA
UMC21UMC21-G2/G6-1S28G2IG6-1 GAATGTTGCTGTTATATTAC[TIC]CGTAGGTGACAAAGGGTTCA
UMC21UMC21-G21G4-1S28G21G4-1 AGAAAAATTTACATAAAAAA[G/C]CACACTCCATGATTGTTAAA
UMC21UMC21-G2/G4-1S28G2/G4-1 AGAAAAATTTACATAAAAAA[GIC)CACACTCCATGATTGTTTAA
B B
UMC21UMC21-G2IG3-1S28G21G3-1 CTTTTATTCAAHCTTTCCAG[/C]CGTTAATTTGTTATCCGTTG
UMC21UMC21-G6IG1-1S28G6/G1-1 TGTTGAACATGCTCTCAGGA[/CC]CCCCCTATTGTGACACAGCA
UMC21UMC21-G1IG3-1S28G1IG3-1 TACATCTTAACAAGCACATG[TGIT'11 jTMCCTTTTATTCAAACTTT
UMC65UMC65-G3IGfi-1AS27G3/G&1A AGTAATGTGTGACTGTGGGC[GG]CGTGTGACAGCTTTTACGTA
UMC65UMC65-G3/G6-1S27G3IG6-1 AGTAGTGTGTGACTGTGGGC[GG]CGTGTGACAGCTTTTACGTA
B B
UMC65UMC65-G3/G6-2S27G3/G6-2 TTCGCTTGGTAGCCGTAGCA[G/A]TATACTTTTACCGGCCACAG
UMC65UMC65-G3/GB-3S27G3/G6-3 GGGCTTTGGGTTGTGAACTT[CCAIC TTTCCC
UMC59UMC59-GSIG6-1UMC59-GSIGfr1CCAAGAAAGATTAATGCTGG[/T]TAAAATATTGTTTCCAGTCT
UMC59UMC59-G51G6-2UMC59-G5/G6-2AAAATCAGGACTGCGAAAAA(A/C]CCAAGAAAGATTAATGCTGG
UMC59UMC59~5/G6-2BUMC59-GS/G6-2BAAAATG~GGACTGCGAAAAA[A!C]CCAAGMGATTAATGCTGGT
UMC59UMC59-G5/G6-3UMC59-G51G&3AAAGTGTGTGTTGTTGCCCA[GIA]ATGATTCCATTCCACACAAG
UMC59UMC59-G4IG5-1UMC59-G4/G5-1AGGACTGCGAAAAAACCAAG[lA]AAGATTAATGCTGGTAAAAT
UMC59UMC59-G4/G5-2UMG59-G4IG5-2ATGCTGGTAAAATATTGTTT[/C]CAGTCTTTCACAAAGTGTGT
UMC59UMC59-G3IG4-1UMC59-G3/G41CTACAAAAATCAGGACTGCG[lA]AAAMCCMGAAGATTAATG
UMC59UMC59-G31G4-2UMC59-G3/G4-2TTGTTTCAGTCTTTCACAAA[/GT]GTGTGTGTGCCAGATGATTC
UMC59UMC59-G3/G4-3UMC59~31G4-3TCACACACCGACCTGCCTGG[/T]TATCAGGAACCATCCTCCTG
Ae1 Ael~4/G5-1 S23G4JG5-1 GGTGMTTGGTGATGCATGC[TIG]GGGGGTGCTCGAGTTGGATG
Ae1 Ae1-G4IG5-2 S23G4lGS-2 TTCCAGTCGGATGAACTGGA[TlG)GTTCGTCATCCACTCGTG~C
Ae1 Ae1-G3JG6-1 S23G3IG6-1 GGTGAATTGGTGATGCATGC[AIT]GGGGGTGCTCGAGTTGGATG
Ae1 Aei-G51G3-t S23G51G3-1 TTAAGTGAACATGCCCAAAC[GG]GTTAAACTTTCCATGGAACT
Ae1 Ae1-G5IG3-1 S23G5/G3-18ATTAATGAAGATGCCCAAAC(GG]GTTAAACTTTCCATGGAACT
B
Ae1 Aet-G1JG6-1 S23G11G6-1 TGATTCGGGTCTGTATGCGA[G/nTGTTGTGGTGGTGAACTGGT
Ae1 Ae1-G1/G5-1 S23G1/G5-1 CGGGTCTGTATGCGAGTGTT[G/A]TGGTGGTGAACTGGTGAATT
Aet Ae1-G1/G4-1 S23G1/G4-1 GTTCGCGGTTTCTGGGGCCG[GIT]GGGCGGTGCTCGGTGGGGCC
UMC90UMC90-GS/G6-1S22G5/G6-1 CAGATTGGTGTCGTTTACTA[A!G]AATTCAGTTCTGTCCATTTG
UMC90UMC90.G5lG6-2S22G~2 AAGTAAGCATTCTTTATATG[ITjTACTTCCCATGATAAACTTT
UMC90UMC90~5IG6-3S22GSIG63 CAAAGGGCTTACTGTACTTT[IC]CATCTTATTGGCAGGGCACC
UMC66UMC66-GS/G6-1S19G~1 ACTTGGCCGGGGACGTCGAC[GIA]ATCGTCGTAGCACTACTGGT
UMC66UMC66GSIG62 S19GSIG6-2 AGTACATGGCGAGCGTTGTA[G/C]CAGCTGCTTAGGTGATGTGG
Adh2 Adh2-G4IG6-1S17G4IG6-1 CTATTTCG1AGCTAACAACC[C!G]CTCTTGGTCCCAACATCCTG
Adh2 Adh2-G3IG6-1S17G31G6-1 GGTTCTAAACATAGCTCGTC[GI1JATTCATGATTCATCTCGAGC
UMC63UMC63.G4IG6-1S16G4/G6-1 TCAGCAAGCCTCCAAGGCTC[GA)AATGGTCCAGTTACTTGGTT
UMC63UMC63G2/G&1 S16G21G6-1 GTGTGTAGCTTCATTCGCAA[TGIAT]TTTGAA~CAGCCTCTGCAAGT
UMC63UMC63-G21G6-2AS16G21G6-2AGTGCTTTCGTAMCCTAGAG[TIC]TGAiCCAGCTGTGATTTCGGT
UMC63UMC63-G2IG6-2BS16G2IG6-2BGTGCTTTCGTAAA~CCTAGAG[TKxTGA~OCA,GCTGTGATTTCGAT
UMC63UMC63~21Grr3AS16G2~G63A GCTGACCAGCTGTGATTTCG(GJI~]1'GTATTCCAOGACGA~CGAGT
UMC63UMC63-G1IG6-1S16G11G6-1 TGTGTAGCTTCATTCGCA~AjGtTJTTTGJ1A~CAGCCTCTGC.AAGT
UMC63UMC63.G1IG3-2AS16G1JG3-2AGTGCTTCCGTAAACCTAGAO[TfC]TGACCAGCTGTGATTTCGAT
UMC63UMC63-G1/G3-2BS16G11G3-2BGTGCTTCCGTAMCCTAGAG[T1C]TGA~7CAGCTGTGATTTCGGT
UMC63UMC63-G1IG2-1S16G1IG2-1 GTGTGTAGCTTCATTaGCAA[[NT]GTnG~C;CCTCTGCMG
UMC102UMC102-G5IG6-1S14G51G6-1 GCTCAGCTGCCGGAGTA~CG
T(NT]GGCTTGCTCTCCGGCCGGCC
T(AITJGGCTTGCTCTCCGGCCrGCC
ASG24ASG24-GS1G6-1Sl3GSlG6-1 TTTCACAACTCAAiCTGATTG(M]CTTGCTTTGATGTQOATTCT
ASG24ASG24-GZ/G6.1S13G2IG6-1 TTGGTMTTTCAGA,CCTAGA[CI~GjMCTTACTGTGGTACAOGCC
UMC49UMC49~,41G6-1S12G4lG6-1 ACCTTTGCTGTGTTT1T11'T[TlrajGTATTOGMTGGA~GGGAGTA
UMC49UMC49-G2/Gr1S12G2JG5-1 AAAACAGCCAAGOTGGTGOT[CK3]AA~UCsGAAGGTGTCAQ~AAGGT
UMC49UMC49-G21G5-2S12G2IGr2 TCTGTTCGTTCCATCTCTTT[AIGJCI1GTAMTATCCGTMTTAC
UMC49UMC49-G2lGS-3S12G2IG53 CGTMTTACTTTGTTACTAC(TAfC]I4GTMTTTTATATATATCCT
UMC49UMC49-G21G5-4S12G2IG5.4 TATATATATCCTCATTTCAA[AfnGr4A~CAGTCAAAGTTAGTTTT
UMC49UMC49-G2/G5-4BS12G21G~r48TATATATATCCTCATTTCAPv(NT]GAA~CAGTCAAAGTAGTTTTG
UMC49UMC49-G2/G4-1S12G2IG4-1 TATTTCTTATCCAGGATTGT[TIC)CT1TGGCCAAAGCATGGTAC
UMC49UMC49-G2IG4-2S12G2JG4-2 CGTTCCATCTCTTTACAGTA[ING]ATATCCOTMTTACTTTGTT
UMC49UMC49-G21G4-3S12G2/G43 ATCCGTMTTACTTTGTTA~TA/ACjCTMGTMTTTTATATATAT
UMC49UMC49-G2IG3-4S12G21G3-4 GTAATTACT'iTGTfACTACT[AIJAOTMTTTTATATATATCCT
UMC49UMC49-GllG6-1S12G1IG6-1 CTGTGTTTTTTT1TGGTATT[G>C)GMTOGJ4CiGGAGTATTATTT
UMC49UMC49-G1/G6-1BS12G1/G6-1BGCTGTG1ZT11nTGGTATT[OIC]OMTGGAGGGAGTATTATTT
[N/AGAGTTTGGCACCITTGCTG
UMC49UMC49-G1/G5-2S12G1/G5-2 AGTTTGGCACCTTTGCTGTG(TQTTTTTTTTGGTATTGGAATG
UMC49UMC49-G11G5-3Sl2GtIG5-3 CTTTACTGATTGGGTTACAA(AIG]AGGTTATTTCTTATTCAGGC
UMC49UMC49-G1IG5-4S12G11G5-4 AATTACTTTGTTACTACCAG[Tl]TMTTTTATATATATCCTCC
UMC131UMC131-G41G6-iS10G4JG6-1 AGCGACAGGGATGTCGAGCA[GfT]CTACGGAAGGCAATAATGAG
csnpld 2AATTTGGGAAAATCAATGCA[GAA/CAC]ATCAGTGATTAATCCACATA
UMC131 UMC131-G3/G6-1 S10G31G6-1GCATGGCGGAGTGAGGGAGG[TGQTGTGTGTGTGTGGCTCCACA
2AGGCCGCTAGGCCATTTAGCG[G!A]ATTTGGGAAAATCAATGCAG
2BGGCCGCTACGCCATTTAGCG[G/A]ATTTGGGAAAATCAATGCAC
UMC131 UMC131-G1IG6-1 S10G1/G6-1CATCCCCGCCGGCAGAACAA[GG]GTACGAGAAGGATGGAATGC
UMC53 UMC53~5JG6-1 UMC53-G5IG6-1GTCCCAGATCAGGTCCACGT[1/C]CGAGCTCGCTGTTCCCGCTT
UMC53 UMC53-GSIG6-2 UMC53~SIG6-2TGGTTCTTCACCfvCCACCGC[GG]CCGGGCGCGCCCAGCGCCTC
UMC53 UMC53-GMG6-1 UMC53-GMG6-1GCAGCCTCAGGT~4CACGGGG[/A]AAGTCGGAGTGGTTCT1CAC
UMC53 UMC53-G4JG6-2 UMC53-G4IG6-2GCCGGGCGCGCC;CAGCGCCT[/C]GGTCCCAGATCAGGTCCACG
UMC53 UMC53-G3/G6-1 UMC53-G3/G6-tGCACGTCGTTGG'f GAAGAAG[AGCA]GCGGTACGGGTGCTTGTCGA
UMC53 UMC53.G3lG5-1 UMC53-G31G5-1AGGTACACGGGG~4AGTCGGA[G/T]TGGTTCTTCACCACCACCGC
UMC53 UMC53-G3/G5-2 UMC53-G3/G5-2CGACGGCGTCCAGCAC;CGAC[G/JCCTCCGCCTTCACCCCGCGC
UMC:53 UMC53-G3/G4-1 UMC53-G31G4-1GTCCACGTCGAGf:TCGCTGT[G1]CCC;GCTGCCCACGACGGCGT
UMC53 UMC53-G1/G4-1 UMC53-G1/G4-1GCACGTCGTTGG'fGAAGMG[A/CJAGCGGTACGGGTGCTTGTCG
1NAACCAAACCCTG'~qCTATTA[TIC]AGGTAGATTAGACTAGACAC
UMC161 UMCt61-G21G3-2 SO6G2/G3-2ACGGTGAGGAGT<aGCACATG[A/C]GATGGAAAGTTCCTGTAGAC
28ACGGTAAGGAGTC~GCACATG[A/C]GATGGAAAGTTCCTGTAGAC
UMC107 UMC107G21G4-1 S05G21G4-1TATGCTTGGAAAGTGGGAAA[G/JGGGAACATACGATGGAGGAC
UMC67 UMC67-GS/G6-1 S03GSIG6-1AAACAATAATTTTTACACAG[ITJTGCTMGGTTTTACTGTTTT
UMC67 UMC67-G21G6-1 S03G21G6-1ATATCCATGTTGTI:GCCTGC[/TG]TGTGCGCTTGCTTGCCGCTA
UMC76 UMC76~4/G6-1 S02G4lG6-1TTGCTGCTATGTTTACTGGG[ITjTGTAGAAAAAAAAATAATAT
UMC76 UMC76-G21G6-1 S02G2/G6-1GCTCGGTAATAATTCTGGCT[GG]CGATGGCACCCATATTCCTC
UMC76 UMC76-G?JG6-1 B S02G2/G6-1GCTCGGTAATAATTCTGGCTjGG]CGATGGCACCCATATTCCTG
B
UMC76 UMC76-G2/G5-1 S02G21G5-1AAAACACGTGGTGTTTGTTA[GIA]GAAAGACCTAGTTTCTCGGC
UMC76 UMC76-G2/GS-1 B S02G2IG5-1AAATCACGTGGTGTTTGTTA[G/A]GAAAGACCTAGTTTCTC;GGC
B
UMC76 UMC76-G2/G5.1 S02G21G5-1TAGTTTCTCGGCAATTGGCA[G/T]TGTGGAATGACGATCTCGTG
UMC76 UMC76-G2/G5-1 B S02G21G5-1TAGTTTCTCGGCAATTGGCA[GIT]TGTGGAATGACCATCTCGTC
B
UMC76 UMC76~2/G5-2 S02G2/G5-2GTGTGGAATGAC:CATCTCGT[G/C]GTGATGCCAGCATGCTGTTA
UMC76 UMC76~2/GS-2B S02G2IG5-28GTGTGGAATGACCATCTC:GT[GlC]GTGATGCCAGCATGCTACTA
UMC76 UMC76-G2lGS-3 S02G21G5-3ACCCTGTCAGGCTTCCACAG[AIC]TATAATAT1TGTTGTGGTGT
UMC76 UMC76-G2/G5-3B S02G2IGS-3BACTCTGTCAGGCTTCCACAG[A/C]TATAATATTTGTTGTGGTGT
UMC76 UMC76-G2/G5-3C S02G2/G53CACTCTGTCAGGCTTCCACAG[A/C]TATAATATTTGTTGTGTGTG
UMC76 UMC76-G2/GS3D S02G21G5-3DACCCTGTCAGGCTTCCACAG[A/C]TATMTATTTGTTGTGTGTG
WO 9$/30717 PCT/EP97/07134 Example 4 Analysis of Polvmor hi ms 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 Applications 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. 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 Polvmort~hisms in Taraet 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 WO 98/30717 PCT/EP97l07134 1 '~
analysis is determining which forms) 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 Pro es 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 t~~ the corresponding segment from another member due to the presence of different polymorphic forms in the respe~~tive 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 .L6 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 Arravs The polymorphisms can also be identified by hybridization to nucleic acid arrays, some example of which are described by Wo 95/11995 (i.ncorporated by reference in 1~
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 Acld 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 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.
1g 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., C;SHP, New York 1989); Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).
5. Denaturincr Gradient Gel Electrot~horesis 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 5~echnology, Principles and Applications for DNA Amplification, (W. H. Freeman and Co, New York, 1992), Chapter 7.
6. Finale-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 sinc3le stranded PCR products, as described in Orita et al . , ~~roc, Nat . Acad. Sci . 8 6 , 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 forms) present in a subject plant at one or more polymorphic sites, this information can be used in a number of methods.
A. Fingerprint Analvsis 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 10 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 15 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 20 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 Polvmorphisms with Phenotv~ic Tr i The polymorphism;s 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 _~nclude 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 ~~f 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 statistica7.ly significant correlations between polymorphic forms) and phenotypic characteristics are noted.
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 O 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:
Yijkpn = ~ + YSi + Pj; + X~; ~ 131 + ... 1317 + PE11 + a" +ep where Yijgpnis the milk, fat, fat percentage, SNF, SNF
percentage, energy concentration, or lactation energy record;
~ 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; (31 to 131 are the binomial regressions of production record on mtDNA D-loop sequence polymorphisms; PEn is permanent environmental effect common to all records of cow n; an 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 ep 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 z3 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 hack-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 Polvaeptides and Gene ~eauences 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 expre:~sed in an expression vector in which a gene is operably 7. inked 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 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. Nat1 Acad. Sci, USA 82, 5824 (19853, infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et a1. , 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., Nature 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 (2984);
Fraley et al., Proc. Natl. Acad. Sci. USA 80, 4803 (1983)).
The protein may be isolated by conventional 5 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, ~~rinciples and Practice', 2nd 10 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 15 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 20 described in Evans et al., "Protoplasts Isolation and Culture," Handbook of Plant Cell Cultures .2 , 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, 25 Basal 1983); Dale, "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrar.~t Crops," Protoplasts (1983) - pp. 31-41, (Birkhauser, Basel :1983); Binding, "Regeneration of Plants," Plant ProtopLasts, pp. 22-73, (CRC Press, Boca Baton, 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 blologically 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, Pr~incip.Ies 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 z~
allele-specific oligonucleotide~s 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 ca.n 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 (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and t:he appropriate-buffers for reverse transcription, PCR, or hybridization reactions.
Usually, the kit also contains instructions for carrying out the methods.
Claims (26)
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, 3' 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
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| US3206996P | 1996-12-02 | 1996-12-02 | |
| US60/032,069 | 1996-12-02 | ||
| PCT/EP1997/007134 WO1998030717A2 (en) | 1996-12-02 | 1997-12-02 | Vegetal sequences including a polymorphic site and their uses |
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| CA2274317A1 true CA2274317A1 (en) | 1998-07-16 |
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| CA002274317A Abandoned CA2274317A1 (en) | 1996-12-02 | 1997-12-02 | Vegetal sequences including a polymorphic site and their uses |
Country Status (4)
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| EP (1) | EP0944741A2 (en) |
| AU (1) | AU7206698A (en) |
| CA (1) | CA2274317A1 (en) |
| WO (1) | WO1998030717A2 (en) |
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| EP0944741A2 (en) * | 1996-12-02 | 1999-09-29 | Biocem S.A. | Vegetal sequences including a polymorphic site and their uses |
| US6207387B1 (en) * | 1998-10-06 | 2001-03-27 | Emory University | Molecular diagnostics for galactosemia |
| US6670464B1 (en) | 1998-11-17 | 2003-12-30 | Curagen Corporation | Nucleic acids containing single nucleotide polymorphisms and methods of use thereof |
| AU1928801A (en) * | 1999-11-24 | 2001-06-04 | Curagen Corporation | Nucleic acids containing single nucleotide polymorphisms and methods of use thereof |
| JP2004508003A (en) * | 1999-11-30 | 2004-03-18 | キュラジェン コーポレイション | Nucleic acids containing single nucleotide polymorphisms and methods of using the same |
| AU2739401A (en) * | 1999-12-27 | 2001-07-09 | Curagen Corporation | Nucleic acids containing single nucleotide polymorphisms and methods of use thereof |
| EP1250456A2 (en) * | 2000-01-07 | 2002-10-23 | Curagen Corporation | Nucleic acids containing single nucleotide polymorphisms and methods of use thereof |
| WO2008143993A2 (en) * | 2007-05-17 | 2008-11-27 | Monsanto Technology Llc | Corn polymorphisms and methods of genotyping |
| US20130244936A1 (en) | 2010-06-04 | 2013-09-19 | Vincent Goffin | Constitutively active prolactin receptor variants as prognostic markers and therapeutic targets to prevent progression of hormone-dependent cancers towards hormone-independence |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0317239A3 (en) * | 1987-11-13 | 1990-01-17 | Native Plants Incorporated | Method and device for improved restriction fragment length polymorphism analysis |
| EP0402401A4 (en) * | 1988-02-22 | 1991-09-11 | Pioneer Hi-Bred International | Genetic linkages between agronomically important genes and restriction fragment length polymorphisms |
| JPH05505311A (en) * | 1990-11-06 | 1993-08-12 | ザ ルブリゾル コーポレーション | Compositions and methods for analyzing genomic variation |
| US5437697A (en) * | 1992-07-07 | 1995-08-01 | E. I. Du Pont De Nemours And Company | Method to identify genetic markers that are linked to agronomically important genes |
| US5332408A (en) * | 1992-08-13 | 1994-07-26 | Lakeside Biotechnology, Inc. | Methods and reagents for backcross breeding of plants |
| DK0785281T3 (en) * | 1995-07-28 | 2004-09-06 | Sapporo Breweries | Method for genetic differentiation of hop species |
| EP0944741A2 (en) * | 1996-12-02 | 1999-09-29 | Biocem S.A. | Vegetal sequences including a polymorphic site and their uses |
| US6114116A (en) * | 1996-12-02 | 2000-09-05 | Lemieux; Bertrand | Brassica polymorphisms |
| CA2286864A1 (en) * | 1997-01-10 | 1998-07-16 | Pioneer Hi-Bred International, Inc. | Hybridization-based genetic amplification and analysis |
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1997
- 1997-12-02 EP EP97955036A patent/EP0944741A2/en not_active Withdrawn
- 1997-12-02 WO PCT/EP1997/007134 patent/WO1998030717A2/en not_active Application Discontinuation
- 1997-12-02 CA CA002274317A patent/CA2274317A1/en not_active Abandoned
- 1997-12-02 AU AU72066/98A patent/AU7206698A/en not_active Abandoned
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| EP0944741A2 (en) | 1999-09-29 |
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| WO1998030717A3 (en) | 1999-04-29 |
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