A mutation screening platform for rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) and the detection of sinapine biosynthesis mutants |
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Authors: | Hans-Joachim?Harloff Susanne?Lemcke Juliane?Mittasch Andrej?Frolov Jian?Guo?Wu Felix?Dreyer Gunhild?Leckband Email author" target="_blank">Christian?JungEmail author |
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Institution: | (1) Plant Breeding Institute, Christian-Albrechts-University of Kiel, Olshausenstr. 40, 24098 Kiel, Germany;(2) Department of Secondary Metabolism, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany;(3) Present address: College of Agriculture and Biotechnology, University of Zhejiang, Hangzhou, China;(4) Norddeutsche Pflanzenzucht Hans-Georg Lembke KG, Hohenlieth, Germany |
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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 M2 plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8× 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 antinutritive 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 (135 missense 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 8× 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. |
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