A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants.

Harloff, H. J., Lemcke, S., Mittasch, J., Frolov, A., Wu, J. G., Dreyer, F., Leckband, G. and Jung, Christian (2012) A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theoretical and Applied Genetics, 124 (5). pp. 957-969. DOI 10.1007/s00122-011-1760-z.

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Supplementary data:

Abstract

We developed two mutant populations of oilseed rape (Brassica napus L.) using EMS (ethylmethanesulfonate) as a mutagen. The populations were derived from the spring type line YN01-429 and the winter type cultivar Express 617 encompassing 5,361 and 3,488 M-2 plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8x pooling strategy. Genes of the sinapine biosynthesis pathway were chosen for determining the mutation frequencies and for creating novel genetic variation for rapeseed breeding. The extraction meal of oilseed rape is a rich protein source containing about 40% protein. Its use as an animal feed or human food, however, is limited by anti-nutritive compounds like sinapine. The targeting induced local lesions in genomes (TILLING) strategy was applied to identify mutations of major genes of the sinapine biosynthesis pathway. We constructed locus-specific primers for several TILLING amplicons of two sinapine synthesis genes, BnaX.SGT and BnaX.REF1, covering 80-90% of the coding sequences. Screening of both populations revealed 229 and 341 mutations within the BnaX.SGT sequences (135missense and 13 nonsense mutations) and the BnaX.REF1 sequences (162 missense, 3 nonsense, 8 splice site mutations), respectively. These mutants provide a new resource for breeding low-sinapine oilseed rape. The frequencies of missense and nonsense mutations corresponded to the frequencies of the target codons. Mutation frequencies ranged from 1/12 to 1/22 kb for the Express 617 population and from 1/27 to 1/60 kb for the YN01-429 population. Our TILLING resource is publicly available. Due to the high mutation frequencies in combination with an 89 pooling strategy, mutants can be routinely identified in a cost-efficient manner. However, primers have to be carefully designed to amplify single sequences from the polyploid rapeseed genome.

Document Type: Article
Additional Information: Univ Kiel, Plant Breeding Inst, D-24098 Kiel, Germany. Leibniz Inst Plant Biochem, Dept Secondary Metab, D-06120 Halle, Saale, Germany. Norddeutsch Pflanzenzucht Hans Georg Lembke KG, Hohenlieth, Germany. Jung, C (reprint author), Univ Kiel, Plant Breeding Inst, Olshausenstr 40, D-24098 Kiel, Germany. h.harloff@plantbreeding.uni-kiel.de; s.lemcke@plantbreeding.uni-kiel.de; juliane.mittasch@googlemail.com; andrej.frolov@bbz.uni-leipzig.de; jgwu@zju.edu.cn; f.dreyer@npz.de; g.leckband@npz.de; c.jung@plantbreeding.uni-kiel.de
Keywords: sinapate ester content chemically-induced mutations arabidopsis-thaliana point mutations site mutation genes expression discovery canola identification
Research affiliation: Kiel University > Kiel Marine Science
OceanRep > The Future Ocean - Cluster of Excellence
Kiel University
Refereed: Yes
Open Access Journal?: No
DOI etc.: 10.1007/s00122-011-1760-z
ISSN: 0040-5752
Projects: Future Ocean
Date Deposited: 14 May 2014 10:03
Last Modified: 31 Jul 2019 06:29
URI: http://oceanrep.geomar.de/id/eprint/24011

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