Genetic markers for myb28

Abstract

The present invention relates to a method for determining the genotype of a Cruciferous vegetable plant for a plant with an increased glucosinolate level, comprising obtaining a sample of nucleic acids from said plant or a portion thereof and detecting in said nucleic acids a polymorphism at the Myb28 locus that is genetically linked to an increased glucosinolate level. The polymorphism may comprises at least one of: a) a single nucleotide polymorphism (SNP) at a position corresponding to nucleotide 83, 136, 226, 563, 610, 830, 995, 1116, 1513, 1577, 1606, 1620, 1825, 1863, 1877 or 2026 of SEQ ID NO: 1, or b) a polymorphism in the number of nucleotides present between nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1, or c) a polymorphism in the number of nucleotides present between nucleotides 836 and 837, between nucleotides 867 and 868, or between nucleotides 943 and 944 of SEQ ID NO: 1.

1 . A method for determining the genotype of a Cruciferous vegetable plant with an increased glucosinolate level, comprising obtaining a sample of nucleic acids from said plant or a portion thereof and detecting in said nucleic acids a polymorphism at the Myb28 locus that is genetically linked to an increased glucosinolate level. 
     
     
         2 . The method according to  claim 1 , wherein:
 (a) the step of detecting comprises PCR or DNA hybridization;   (b) determining the genotype comprises a co-dominant assay;   (c) the polymorphism is detected by a screening method comprising use of at least a first sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23;   (d) the method further comprises assaying the phenotype of a Cruciferous vegetable plant for an increased level of glucosinolate;   (e) the glucosinolate is 4-methylsulphinylbutyl glucosinolate (MSB), 3-methylsulphinylpropyl glucosinolate (MSP) or combinations thereof;   (f) the Cruciferous vegetable plant comprises at least one glucosinolate in amount of at least 10 micromol/g dry weight;   (g) the Cruciferous vegetable plant comprises 4-methylsulphinylbutyl glucosinolate (MSB), 3-methylsulphinylpropyl glucosinolate (MSP) or combinations thereof in an amount of at least 10 micromol/g dry weight;   (h) the polymorphism comprises at least one of:   i. a single nucleotide polymorphism (SNP) at a position corresponding to nucleotide 83, 136, 226, 563, 610, 830, 995, 1513, 1577, 1606, 1620, 1825, 1863, 1877 or 2026 of SEQ ID NO: 1, or   ii. a polymorphism in the number of nucleotides present between nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1, or   iii. a polymorphism in the number of nucleotides present between nucleotides 836 and 837, between nucleotides 867 and 868, or between nucleotides 943 and 944 of SEQ ID NO: 1; or   (i) the Cruciferous vegetable plant is broccoli;   
     
     
         3 .- 5 . (canceled) 
     
     
         6 . The method according to  claim 1 , wherein the polymorphism comprises (a) at least one of a single nucleotide polymorphism (SNP) at a position corresponding to nucleotide 83, 136, 226, 563, 610, 830, 995, 1116, 1513, 1577, 1606, 1620, 1825, 1863, 1877 or 2026 of SEQ ID NO: 1 or combinations thereof; (b) a deletion of one or more of the nucleotides present between nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1; (c) a deletion of all of the nucleotides present between nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1; (d) a deletion of at least one nucleotide at a position corresponding to nucleotide 324, 325, 326, 327, 328, 329, 330, 331, 522, 523, 784, 785, 910, 911, 912, 913, 1366, 1367, 1368, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, or 2055 of SEQ ID NO: 1; (e) a deletion of the nucleotides at the following positions: 324-331, 522-523, 784-785, 910-913, 1366-1368, 1812-1820 or 2047-2055 of SEQ ID NO: 1 or combinations thereof; or (f) an insertion of one or more nucleotides between the nucleotides 836 and 837, 867 and 868, or 943 and 944 of SEQ ID NO: 1. 
     
     
         7 . The method according to  claim 6  wherein (a) the insertion between the nucleotides 836 and 837 is of two nucleotides; (b) the insertion between the nucleotides 836 and 837 is of TT, (c) the insertion between the nucleotides 867 and 868 is of one nucleotide, (d) the insertion between the nucleotides 867 and 868 is A, (e) the insertion between the nucleotides 943 and 944 is 13 nucleotides or up to 13 nucleotides, or (f) the insertion between the nucleotides 943 and 944 is TATTAAAAAAGTA. 
     
     
         8 .- 13 . (canceled) 
     
     
         14 . A method of producing a Cruciferous vegetable plant having Myb28-mediated increased glucosinolate levels, which method comprises selecting first progeny plants comprising a polymorphism at the Myb28 locus that is genetically linked to increased glucosinolate levels. 
     
     
         15 . The method according to  claim 14 , which method comprises the steps of: (a) crossing a Cruciferous vegetable plant having an increased glucosinolate level with a second Cruciferous vegetable; and (b) selecting at least a first progeny Cruciferous vegetable plant comprising a polymorphism at the Myb28 locus that is genetically linked to increasing glucosinolate levels. 
     
     
         16 . The method according to  claim 14 , wherein:
 (a) the step of selecting comprises PCR or DNA hybridization;   (b) the polymorphism is detected by a screening method comprising use of an oligonucleotide comprising a sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23;   (c) the selecting comprises detecting a co-dominant genetic marker;   (d) the plant is transformed with a myb28 gene comprising SEQ ID NO: 1 except for at least one polymorphism selected from the group consisting of:
 i) a single nucleotide polymorphism (SNP) at a position corresponding to nucleotide 83, 136, 226, 563, 610, 830, 995, 1116, 1513, 1577, 1606, 1620, 1825, 1863, 1877 or 2026 of SEQ ID NO: 1, or 
 ii) a polymorphism in the number of nucleotides present between nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1, or 
 iii) a polymorphism in the number of nucleotides present between nucleotides 836 and 837, between nucleotides 867 and 868, or between nucleotides 943 and 944 of SEQ ID NO: 1; 
   (e) the glucosinolate is 4-methylsulphinylbutyl glucosinolate (MSB), 3-methylsulphinylpropyl glucosinolate (MSP) or combinations thereof;   (f) the Cruciferous vegetable plant comprises at least one glucosinolate in amount of at least 10 micromol/g dry weight;   (g) the Cruciferous vegetable plant comprises 4-methylsulphinylbutyl glucosinolate (MSB), 3-methylsulphinylpropyl glucosinolate (MSP) or combinations thereof in an amount of at least 10 micromol/g dry weight;   (h) the polymorphism comprises at least one of:
 i) a single nucleotide polymorphism (SNP) at a position corresponding to nucleotide 83, 136, 226, 563, 610, 830, 995, 1116, 1513, 1577, 1606, 1620, 1825, 1863, 1877 or 2026 of SEQ ID NO: 1, or 
 ii) a polymorphism in the number of nucleotides present between nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1, or 
 iii) a polymorphism in the number of nucleotides present between nucleotides 836 and 837, between nucleotides 867 and 868, or between nucleotides 943 and 944 of SEQ ID NO: 1; or 
   (i) the Cruciferous vegetable plant is broccoli.   
     
     
         17 .- 19 . (canceled) 
     
     
         20 . The method according to  claim 15 , wherein selecting the first progeny further comprises selecting the progeny based on the presence of one or more genetic markers from the second Cruciferous vegetable plant genetically linked to at least a first additional trait. 
     
     
         21 . The method of  claim 20 , wherein the additional trait is selected from the group consisting of: yield, disease resistance, emergence vigor, vegetative vigor, stress tolerance, plant height, inflorescence quality, inflorescence diameter, inflorescence weight, inflorescence size, inflorescence shape, inflorescence colour, and number of days to flowering. 
     
     
         22 . (canceled) 
     
     
         23 . The method according to  claim 14 , wherein the polymorphism (a) comprises at least one of a single nucleotide polymorphism (SNP) at a position corresponding to nucleotide 83, 136, 226, 563, 610, 830, 995, 1116, 1513, 1577, 1606, 1620, 1825, 1863, 1877 or 2026 of SEQ ID NO: 1 or combinations thereof; (b) comprises a deletion of one or more of the nucleotides present between nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1; (c) comprises a deletion of all of the nucleotides present between nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1; (d) comprises a deletion of at least one nucleotide at a position corresponding to nucleotide 324, 325, 326, 327, 328, 329, 330, 331, 522, 523, 784, 785, 910, 911, 912, 913, 1366, 1367, 1368, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1820, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, or 2055 of SEQ ID NO: 1; (e) comprises a deletion of the nucleotides at the following positions: 324-331, 522-523, 784-785, 910-913, 1366-1368, 1812-1820 or 2047-2055 of SEQ ID NO: 1 or combinations thereof; or (f) comprises an insertion of one or more nucleotides between the nucleotides 836 and 837, 867 and 868, or 943 and 944 of SEQ ID NO: 1. 
     
     
         24 . The method according to  claim 23 , wherein (a) the insertion between the nucleotides 836 and 837 is of two nucleotides; (b) the insertion between the nucleotides 836 and 837 is of TT, (c) the insertion between the nucleotides 867 and 868 is of one nucleotide, (d) the insertion between the nucleotides 867 and 868 is A, (e) the insertion between the nucleotides 943 and 944 is 13 nucleotides or up to 13 nucleotides, or (f) the insertion between the nucleotides 943 and 944 is TATTAAAAAAGTA. 
     
     
         25 . The method according to  claim 14 , the method further comprising the step of: (c) crossing the progeny plant with itself or a third plant to produce a progeny plant of a subsequent generation. 
     
     
         26 . The method according to  claim 25 , wherein the method further comprising the steps of: (d) crossing the progeny plant of a subsequent generation with itself or a second plant; and (e) repeating steps (c) and (d) for an additional 3-10 generations to produce an inbred Cruciferous vegetable plant comprising an increased level of glucosinolate, wherein the progeny plant of at least one subsequent generation is screened for the presence of a polymorphism at the Myb28 locus genetically linked to glucosinolate production. 
     
     
         27 . The method according to  claim 26 , wherein:
 (a) said progeny plant of a subsequent generation is selected for crossing based on the presence of glucosinolates and a desired trait;   (b) the progeny plant of a subsequent generation is selected at each generation for crossing based on the presence of an increased glucosinolate level and the desired trait; or   (c) step (e) is repeated with sufficient inbreeding to obtain an inbred Cruciferous vegetable plant that comprises an increased glucosinolate trait and otherwise comprises the agronomic traits of the second Cruciferous vegetable plant.   
     
     
         28 .- 33 . (canceled) 
     
     
         34 . The method according to  claim 23 , wherein the polymorphism is one which is within 5 cM of said polymorphism. 
     
     
         35 . (canceled) 
     
     
         36 . The method according to  claim 24  wherein the plant is transformed with a myb28 gene comprising SEQ ID NO: 24 or a sequence which has a least 97% (such as at least 98% or at least 99%) identity with SEQ ID NO: 24. 
     
     
         37 . A plant or a part thereof produced by the method of  claim 14 . 
     
     
         38 . A seed that produces the plant of  claim 37 . 
     
     
         39 . The method of  claim 1 , which method further comprises the step of storing the result of the step of detecting the polymorphism on a computer readable medium. 
     
     
         40 . A computer readable medium produced by the method of  claim 39 . 
     
     
         41 . A method of producing the edible portion of a Cruciferous vegetable plant (e.g. broccoli plant) comprising: (a) obtaining the plant of  claim 37 ; and (b) collecting the edible portion of the Cruciferous vegetable plant (e.g. the inflorescence of a broccoli plant) produced by the plant. 
     
     
         42 . A recombinant nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:
 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23.   
     
     
         43 . A primer or probe that amplifies and/or hybridizes to at least one polymorphism at a position corresponding to nucleotide 83, 136, 226, 563, 610, 830, 995, 1116, 1513, 1577, 1606, 1620, 1825, 1863, 1877 or 2026 of SEQ ID NO:1; or that amplifies and/or hybridizes to a polymorphism present between positions corresponding to nucleotides 323 and 332, between nucleotides 521 and 524, between nucleotides 783 and 786, between nucleotides and 909 and 914, between nucleotides 1365 and 1369, between 1811 and 1821, or between nucleotides 2046 and 2056 of SEQ ID NO: 1; or that amplifies and/or hybridizes to a polymorphism present between nucleotides 836 and 837, between nucleotides 867 and 868, or between nucleotides 943 and 944 of SEQ ID NO: 1. 
     
     
         44 . The primer or probe according to  claim 43  that amplified and/or hybridizes a polymorphism comprising an insertion of one or more nucleotides between the nucleotides 836 and 837, 867 and 868, or 943 and 944 of SEQ ID NO: 1. 
     
     
         45 . The primer or probe according to  claim 44  wherein (a) the insertion between the nucleotides 836 and 837 is of two nucleotides; (b) the insertion between the nucleotides 836 and 837 is TT; (c) the insertion between the nucleotides 867 and 868 is of one nucleotide; (d) the insertion between the nucleotides 867 and 868 is A; (e) the insertion between the nucleotides 943 and 944 is 13 nucleotides or up to 13 nucleotides; (f) the insertion between the nucleotides 943 and 944 is TATTAAAAAAGTA. 
     
     
         46 . An isolated nucleic acid comprising a sequence of at least 18 contiguous nucleotides that are conserved between SEQ ID NO: 1 and SEQ ID NO: 24 when aligned. 
     
     
         47 . (canceled)