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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Abbreviations
- BAC:
-
Bacterial artificial chromosome
- CODDLE:
-
Codons optimized to discover deleterious lesions
- EMS:
-
Ethylmethane sulfonate
- IRD label:
-
Infrared detection label
- PCR:
-
Polymerase chain reaction
- REF1:
-
Reduction of epidermal fluorescence1
- SGT:
-
UDP-glucose:sinapic acid glucosyltransferase
- TILLING:
-
Targeting-induced local lesions in genomes
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Acknowledgments
We thank Gislind Bräcker, Meike Pfeiler, Anja Henning, Ingrid Otschik for technical assistance and Monika Bruisch for excellent support in the greenhouse. This project was funded by the seed companies Norddeutsche Pflanzenzucht Hans-Georg Lembke KG (Hohenlieth, Germany), Deutsche Saatveredelung AG (Lippstadt, Germany) and KWS SAAT AG (Einbeck, Germany), and the German Federal Ministry for Education, Research and Technology BMBF (GABI Future grant no. 0315052C). We especially thank Dr. G. Rakow (AAFC, Canada) for providing the spring rapeseed genotype YN01-429, Dr. Frank Breuer (KWS SAAT AG) and Dr. Zeljko Micic (Deutsche Saatveredelung AG) for Express 617 M2 and M3 seed propagation and M2 leaf sampling. We thank the Zentrum für Molekulare Biowissenschaften (ZBM), University of Kiel for providing the facilities for DNA isolation and normalization and especially Prof. Dr. Axel Scheidig for CEL1 preparation. We thank Prof. Dr. Wolfgang Bilger and Jens Hermann from the Institute of Botany, University Kiel for performing additional HPLC analyses. We are also grateful to the Institute for Clinical Molecular Biology, University Kiel for considerable Sanger sequencing and especially Prof. Dr. Philip Rosenstiel for the critical reading of the manuscript.
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Communicated by R. Visser.
Hans-Joachim Harloff and Susanne Lemcke have equally contributed.
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Harloff, HJ., Lemcke, S., Mittasch, J. et al. A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theor Appl Genet 124, 957–969 (2012). https://doi.org/10.1007/s00122-011-1760-z
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DOI: https://doi.org/10.1007/s00122-011-1760-z