Polynucleotide markers

Abstract

The invention relates to polynucleotides that are closely linked to the bolting gene or B gene within the sugar beet genome and can be used for the development of molecular markers. The invention further relates to molecular markers and kits comprising said markers that can be used for mapping, identification and isolation of the bolting gene or S gene in the sugar beet genome and to discriminate between the annual and bienniai genotype or between different haplotypes within plant groupings of sugar beet plants exhibiting a biennial genotype. The invention also relates to assays and methods of breeding sugar beet plants involving said markers.

1 - 63 . (canceled) 
     
     
         64 . An isolated nucleic acid molecule having at least 95%, 90%, 85%, 80% or 75% sequence identity to SEQ ID NO: 52, wherein the nucleic acid encodes a polypeptide associated with bolting in sugar beet. 
     
     
         65 . The isolated nucleic acid molecule of  claim 64 , wherein the nucleic acid comprises SEQ ID NO: 52. 
     
     
         66 . An isolated nucleic acid molecule encoding a polypeptide having at least 95%, 90%, 85%, 80% or 75% sequence identity to SEQ ID NO: 6, wherein the polypeptide is associated with bolting in sugar beet. 
     
     
         67 . The isolated nucleic acid molecule of  claim 66 , wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6. 
     
     
         68 . An expression vector comprising a nucleic acid molecule comprising the nucleic acid sequence of  claim 64 . 
     
     
         69 . An amplicon or informative fragment thereof comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 51, SEQ ID NO: 52 or any allelic variations thereof, wherein the amplicon or informative fragment can be used to identify the absence or presence of an allele associated with annuality in a sugar beet plant. 
     
     
         70 . A polynucleotide including an informative fragment thereof, comprising a nucleotide sequence that has the nucleotide sequence depicted in SEQ ID NO: 5 or variants thereof, wherein said variants comprise a nucleotide sequence selected from the group of nucleotide sequences consisting of:
 a) a sequence having a G at position 3695, a C at position 3827, a T at position 3954, a T at position 5284, a G at position 5714, a G at position 10954, a T at position 11043, a C at position 11143, a C at position 11150, an A at position 11220, a C at position 11238, an A at position 11299, an A at position 11391, a G at position 12053, a G at position 12086, a T at position 12127, an A at position 12193, a G at position 12337, and a G at position 12837, representing annual allele 1; or   b) a sequence having a G at position 3695, an A at position 3827, an A at position 3954, a C at position 5284, a T at position 5714, an A at position 10954, a G at position 11043, a C at position 11143, a C at position 11150, a C at position 11220, a C at position 11238, a T at position 11299, a G at position 11391, an A at position 12053, a G at position 12086, a C at position 12127, a G at position 12193, a G at position 12337, and an A at position 12837, representing biennial allele 7.   
     
     
         71 . A method of identifying the absence or presence of an allele associated with annuality in a sugar beet plant, the method comprising:
 a) obtaining a genomic sample from a sugar beet plant;   b) contacting the genomic sample comprising DNA with a pair of primers that, when used in a nucleic-acid amplification reaction with genomic DNA from sugar beet; produces an amplicon that can be used to identify the absence or presence of an allele associated with annuality.   c) amplifying a fragment from said genomic sample using the primer pair of b) wherein the primer pair is complementary and binds to the nucleotide sequence of b)   d) detecting the amplicon that can be used to identify the absence or presence of an allele associated with annuality in a sugar beet plant.   
     
     
         72 . The method of  claim 71 , wherein the genomic region from which the fragment is amplified comprises SEQ ID NO: 5 or any allelic variations thereof. 
     
     
         73 . The method of  claim 71 , wherein said amplicon comprises a polymorphism that is diagnostic for the B allele at the B locus and allows to discriminate between the annual and biennial genotype or between different haplotypes within plant groupings of sugar beet plants exhibiting a biennial or annual genotype. 
     
     
         74 . The method of  claim 73 , wherein the polymorphism is located at position 3827, 3954, 5284, 5714, 10954, 11220, 11391, 12053, 12127, or 12837 in the B locus of the sugar beet genome. 
     
     
         75 . The method of  claim 73 , wherein the primer pair amplifies an informative fragment from a coding region of the BVPRR7 gene comprising a single nucleotide polymorphism comprising a A/C. SNP at position 3827, a A/T SNP at position 3954, a T/G SNP at position 5714, a C/A SNP at position 11220, a G/A SNP at position 11391, a A/G SNP at position 12053, or a C/T SNP at position 12127 
     
     
         76 . The method of  claim 71 , wherein the amplified fragment from said genomic sample comprises the nucleic acid sequence or informative fragments selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 51, SEQ ID NO: 52. 
     
     
         77 . The method of  claim 71 , wherein the primer pair is complementary and binds to a nucleotide sequence present in the promoter region of the BvPRRR7 gene as depicted in SEQ ID NO: 51. 
     
     
         78 . The method of  claim 71 , wherein the method is used to identify annual seed contaminations in commercial biennial sugar beet seed. 
     
     
         79 . The method of  claim 71 , wherein the primer pair comprises the nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 27, or SEQ ID NO: 28. 
     
     
         80 . A method of identifying the absence or presence of an allele associated with annuality in a sugar beet plant the method comprising:
 a) obtaining a genomic sample from a sugar beet plant;   e) contacting the genomic sample comprising DNA with a probe, wherein the probe hybridizes under high stringency conditions with genomic DNA from sugar beet and can identify the absence or presence of an allele associated with annuality.   b) Subjecting the sample and probe to high stringency hybridization conditions; and   c) Detecting the hybridization of the probe to the DNA wherein the presence or absence of hybridization can be used to identify the absence or presence of an allele associated with annuality in a sugar beet plant.   
     
     
         81 . The method of  claim 80 , wherein the genomic region for which the probe hybridizes under high stringency conditions comprises SEQ ID NO: 5 or allelic variations thereof. 
     
     
         82 . An allelic discrimination assay for detecting a polymorphism in a genomic region of the sugar beet genome co-segregating with the annuality phenotype, wherein the polymorphism is diagnostic for the B allele at the B locus and allows to discriminate between the annual and biennial genotype comprising a molecular marker, the assay comprising:
 a) obtaining a genomic sample from a sugar beet plant;   b) contacting the genomic sample comprising DNA with a pair of primers that, when used in a nucleic-acid amplification reaction with genomic DNA from sugar beet; produces an amplicon that can be used to identify the absence or presence of an allele associated with annuality.   c) amplifying a fragment from said genomic sample using the primer pair of b) wherein the primer pair is complementary and binds to the nucleotide sequence of b)   d) detecting the amplicon that can be used to identify the absence or presence of an allele associated with annuality in a sugar beet plant.   
     
     
         83 . The assay of  claim 82 , wherein the primer pair comprises the nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 27, or SEQ ID NO: 28. 
     
     
         84 . A pair of primers which anneals to a nucleotide sequence within the coding region of the BvPRR7 gene as depicted in SEQ ID NO: 5 and amplifies an informative fragment from said coding sequence comprising a polymorphism, wherein the polymorphism comprises at least one of the SNPs selected from the group consisting of a A/C SNP at position #3827, an A/T SNP at position #3954, a T/G SNP at position #5714, a C/A SNP at position #11220, a G/A SNP at position #11391, a A/G SNP at position #12053, or a C/T SNP at position #12127. 
     
     
         85 . The pair of primers of  claim 84  comprising a forward primer F3806 as depicted in SEQ ID NO 27 and a reverse primer R3807 as depicted in SEQ ID NO 28 for amplifying a fragment comprising SNP #3827. 
     
     
         86 . The pair of primers of  claim 84  comprising a forward primer PRR7-F as depicted in SEQ ID NO: 7 and a reverse primer PRR7-R as depicted in SEQ ID NO: 8 for amplifying a fragment comprising SNP #160.