Ho/ll canola with resistance to clubroot disease

Abstract

This disclosure concerns a plant of the genus, Brassica, or parts thereof, which comprise one or more traits selected from the group consisting of high oleic acid content, low linolenic acid content, increased herbicide resistance, restorer of cytoplasmic male sterility, and increased clubroot disease ( Plasmodiophora brassicae ) resistance, compared to a wild-type plant of the same species. This disclosure further relates to wild-type and mutant alleles of genes involved in these traits, molecular markers linked thereto, and methods of their use.

What may be claimed is: 
     
         1 . A method for identifying a plant comprising a mutation in a fad2 gene contributing to a high oleic acid phenotype in  Brassica napus,  the method comprising:
 isolating genomic DNA from a plant; and   screening the isolated genomic DNA for a nucleic acid molecular marker in  B. napus  linkage group N5 or N1, wherein presence of the nucleic acid molecular marker is indicative of a mutation in a fad2 gene contributing to a high oleic acid phenotype in  Brassica napus.      
     
     
         2 . The method according to  claim 1 , wherein the mutation is a single nucleotide polymorphism that results in a truncatedfad2 expression product. 
     
     
         3 . The method according to  claim 1 , wherein the molecular marker is a single nucleotide polymorphism at the position corresponding to position 411 of the  B. napus  fad2 gene. 
     
     
         4 . The method according to  claim 3 , wherein the molecular marker is a thymidine nucleotide at the position corresponding to position 411 of the  B. napus  fad2 gene. 
     
     
         5 . The method according to  claim 4 , wherein screening the isolated genomic DNA for the nucleic acid molecular marker comprises determining the presence in the isolated genomic DNA for a nucleic acid molecule that is specifically hybridizable with the complement of SEQ ID NO:7. 
     
     
         6 . A plant identified by the method according to  claim 1 . 
     
     
         7 . A method for identifying a plant comprising a mutation in a fad3 gene contributing to a low linolenic acid phenotype in  Brassica napus,  the method comprising:
 isolating genomic DNA from a plant; and   screening the isolated genomic DNA for a nucleic acid molecular marker in  B. napus  linkage group N4 or N14, wherein presence of the nucleic acid molecular marker is indicative of a mutation in a fad3 gene contributing to a low linolenic acid phenotype in  Brassica napus.      
     
     
         8 . The method according to  claim 7 , wherein the mutation is a single nucleotide polymorphism that results in abnormal splicing of afad3 expression product. 
     
     
         9 . The method according to  claim 7 , wherein the molecular marker is a single nucleotide polymorphism at the position corresponding to the first base of a 5′ splice site of the third intron of the  B. napus  fad32 gene. 
     
     
         10 . The method according to  claim 9 , wherein the molecular marker is an adenine nucleotide at the position corresponding to the first base of a 5′ splice site of the third intron of the  B. napus  fad32 gene. 
     
     
         11 . The method according to  claim 10 , wherein screening the isolated genomic DNA for the nucleic acid molecular marker comprises determining the presence in the isolated genomic DNA for a nucleic acid molecule that is specifically hybridizable with the complement of SEQ ID NO:12. 
     
     
         12 . A plant identified by the method according to  claim 7 . 
     
     
         13 . A method for introducing a high oleic acid phenotype in a  Brassica  plant, the method comprising:
 crossing a first  Brassica  plant comprising a single nucleotide mutation in a fad2 gene that results in a truncated fad2 expression product, with a second Brassica plant, to generate F 1    Brassica  plants;   using marker-assisted selection to identify an F 1    Brassica  plant comprising a nucleic acid molecular marker in  B. napus  linkage group N5 or N1, wherein presence of the nucleic acid molecular marker is indicative of a mutation in a fad2 gene contributing to a high oleic acid phenotype in  Brassica napus;  and   propagating the identified F 1    Brassica  plant, thereby introducing a high oleic acid phenotype in a  Brassica  plant.   
     
     
         14 . The method according to  claim 13 , wherein identifying the F 1    Brassica  plant comprises determining the presence in the F 1    Brassica  plant of a nucleic acid molecule that is specifically hybridizable with the complement of SEQ ID NO:7, wherein the nucleic acid molecule is not specifically hybridizable with the complement of SEQ ID NO:9. 
     
     
         15 . A method for introducing a high oleic acid phenotype in a  Brassica  plant, the method comprising introducing into the  Brassica  plant a mutant fad2 gene encoding a polypeptide comprising SEQ ID NO:8. 
     
     
         16 . The method according to  claim 15 , wherein the mutant fad2 gene comprises SEQ ID NO:7. 
     
     
         17 . The method according to  claim 15 , wherein the mutant fad2 gene is introduced into the  Brassica  plant by crossing with a  Brassica  plant comprising the mutant fad2 gene. 
     
     
         18 . A method for introducing a low linolenic acid phenotype in a  Brassica  plant, the method comprising:
 crossing a first  Brassica  plant comprising a single nucleotide mutation in a fad3 gene that results in abnormal splicing of a fad3 expression product, with a second Brassica plant, to generate F 1    Brassica  plants;   using marker-assisted selection to identify an F 1    Brassica  plant comprising a nucleic acid molecular marker in  B. napus  linkage group N4 or N14, wherein presence of the nucleic acid molecular marker is indicative of a mutation in a fad3 gene contributing to a low linolenic acid phenotype in  Brassica napus;  and   propagating the identified F 1    Brassica  plant, thereby introducing a low linolenic acid phenotype in a  Brassica  plant.   
     
     
         19 . The method according to  claim 18 , wherein identifying the F 1    Brassica  plant comprises determining the presence in the F 1    Brassica  plant of a nucleic acid molecule that is specifically hybridizable with the complement of SEQ ID NO:12, wherein the nucleic acid molecule is not specifically hybridizable with the complement of SEQ ID NO:13. 
     
     
         20 . A method for introducing a high oleic acid phenotype in a  Brassica  plant, the method comprising introducing into the  Brassica  plant a mutant fad3 gene comprising SEQ ID NO:12. 
     
     
         21 . The method according to  claim 19 , wherein the mutant fad3 gene is introduced into the  Brassica  plant by crossing with a  Brassica  plant comprising the mutant fad3 gene. 
     
     
         22 . A method for introducing a high oleic acid phenotype in a  Brassica  plant, the method comprising:
 crossing a first  Brassica  plant with a second  Brassica  plant comprising means for introducing a high oleic acid phenotype into a plant of the genus,  Brassica,  to generate F 1    Brassica  plants;   identifying an F 1    Brassica  plant comprising means for introducing a high oleic acid phenotype into a plant of the genus,  Brassica;  and   propagating the identified F 1    Brassica  plant, thereby introducing a high oleic acid phenotype in a  Brassica  plant.   
     
     
         23 . A method for identifying a  Brassica  plant comprising a high oleic acid phenotype, the method comprising:
 isolating nucleic acid molecules from a  Brassica  plant; and   contacting the isolated nucleic acid molecules with means for identifying a plant carrying a gene contributing to a high oleic acid phenotype in a plant of the genus,  Brassica,  to produce a detectable signal that is indicative of the presence of a high oleic acid phenotype in the  Brassica  plant.   
     
     
         24 . A method for introducing a low linolenic acid phenotype in a  Brassica  plant, the method comprising:
 crossing a first  Brassica  plant with a second Brassica plant comprising means for introducing a low linolenic acid phenotype into a plant of the genus,  Brassica,  to generate F 1    Brassica  plants;   identifying an F 1    Brassica  plant comprising means for introducing a low linolenic acid phenotype into a plant of the genus,  Brassica;  and   propagating the identified F 1    Brassica  plant, thereby introducing a low linolenic acid phenotype in a  Brassica  plant.   
     
     
         25 . A method for identifying a  Brassica  plant comprising a low linolenic acid phenotype, the method comprising:
 isolating nucleic acid molecules from a  Brassica  plant; and   contacting the isolated nucleic acid molecules with means for identifying a plant carrying a gene contributing to a low linolenic acid phenotype in a plant of the genus,  Brassica,  to produce a detectable signal that is indicative of the presence of a low linolenic acid phenotype in the  Brassica  plant.