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Keywords:

  • Brassica napus;
  • EMS mutagensis;
  • FAE1;
  • mutant population;
  • point mutations;
  • seed erucic acid content;
  • TILLING

Summary

  • • 
    Two ethylmethanesulfonate (EMS) mutant populations of the semi-winter rapeseed cv. Ningyou7 were constructed with high mutant load, to provide a TILLING platform for functional genomics in Brassica napus, and for introduction of novel allelic variation in rapeseed breeding.
  • • 
    Forward genetic screening of mutants from the M2 populations resulted in identification of a large number of novel phenotypes. Reverse genetic screening focused on the potentially multi-paralogous gene FAE1 (fatty acid elongase1), which controls seed erucic acid synthesis in rapeseed. A B. napus BAC library was screened, and loci in a reference mapping population (TNDH) were mapped to conclude that there are two paralogous copies of FAE1, one on each of the B. napus A and C genomes.
  • • 
    A new procedure is demonstrated to identify novel mutations in situations where two or more very similar paralogous gene copies exist in a genome. The procedure involves TILLING of single plants, using existing SNPs as a positive control, and is able to distinguish novel mutations based on primer pairs designed to amplify both FAE1 paralogues simultaneously.
  • • 
    The procedure was applied to 1344 M2 plants, with 19 mutations identified, of which three were functionally compromised with reduced seed erucic acid content.