A mutant Brassica napus (canola) population for the identification of new genetic diversity via TILLING and next generation sequencing

PLoS One. 2013 Dec 20;8(12):e84303. doi: 10.1371/journal.pone.0084303. eCollection 2013.

Abstract

We have generated a Brassica napus (canola) population of 3,158 EMS-mutagenised lines and used TILLING to demonstrate that the population has a high enough mutation density that it will be useful for identification of mutations in genes of interest in this important crop species. TILLING is a reverse genetics technique that has been successfully used in many plant and animal species. Classical TILLING involves the generation of a mutagenised population, followed by screening of DNA samples using a mismatch-specific endonuclease that cleaves only those PCR products that carry a mutation. Polyacrylamide gel detection is then used to visualise the mutations in any gene of interest. We have used this TILLING technique to identify 432 unique mutations in 26 different genes in B. napus (canola cv. DH12075). This reflects a mutation density ranging from 1/56 kb to 1/308 kb (depending on the locus) with an average of 1/109 kb. We have also successfully verified the utility of next generation sequencing technology as a powerful approach for the identification of rare mutations in a population of plants, even in polyploid species such as B. napus. Most of the mutants we have identified are publically available.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Brassica napus / genetics*
  • Brassica napus / growth & development
  • DNA Mutational Analysis / methods*
  • DNA Primers / genetics
  • Electrophoresis, Polyacrylamide Gel / methods
  • Genetic Variation / genetics*
  • Molecular Sequence Data
  • Mutagenesis / genetics*
  • Mutation Rate

Substances

  • DNA Primers

Grants and funding

Funding for this study was provided by the following grants: the National Research Council Genomics Health Initiative Biorenewable Oil for Food and Fuel program (to A.G.S.); Genome Canada/Genome Alberta Designing Oilseeds for Tomorrow's Markets (to G.W.H.); AVAC Ltd. Bioactive Oils Programme (to G.W.H.); and Canadian Foundation for Innovation project #19834 (to G.W.H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.