Mutant screening in rapeseed: New genetic variation for low sinapine content
The use of proteins from rapeseed meal after the oil extraction of the seeds is limited due to the presence of antinutritive substances (phenolics, sinapine) which do not allow an adequate commercialisation. Sinapine is the major compound of all phenolics in B. napus seeds. It contributes to the bitter taste and astringency of rapeseed products. As it forms complexes with proteins, rapeseed protein extracted from the meal is of poor quality and very limited use. Our aim is to reduce the sinapine content in the seeds and thus increase the protein quality.
We are trying to identify mutations in key genes of the phenylpropanoid pathway to prevent sinapine accumulation in seeds of B. napus. The major candidates of our approach were two genes involved in the last steps of the sinapine biosynthesis in rapeseed: BnREF1 and BnSGT coding for a sinapaldehyde/coniferylaldehyde dehydrogenase and an UDP-glucose:sinapate glucosyltransferase.
Chemical mutagenesis has been carried out by seed treatment with EMS (ethylmethanesulfonate) leading to point mutations. If these mutations show beneficial trait expression in crop species, they can be directly integrated in traditional breeding programs without involvement of transgenic methods.
Two EMS treated mutant M2 populations were created, one derived from a yellow seeded spring rapeseed line and another from a winter rapeseed cultivar. In preliminary experiments an EMS concentration of 1% was found suitable as monitored by survival rate and phenotypic variation.
Regions of the target gene sequences were subjected to the development of specific primers. Suitable fragments for TILLING were detected in BnaA.SGTa and BnaC.SGT.a two of four gene loci present in rapeseed and for the 9 exon regions of BnaA.REF1.a and BnaC.REF1.a. After screening of both EMS M2 populations, 135 missense and 13 nonsense mutations have been detected in the BnSGT genes, 162 missense, 3 nonsense, 8 splice site mutations in the BnREF1 genes. Measurements of sinapoylesters in seeds of the mutant M3-4 generation only showed minor, non-significant decreases as expected for the down-regulation of only one gene locus out of two. After combination of single nonsense mutations in both loci by crossing significant reductions in sinapine contents between 47 and 71 % were found, as well as reductions of the respective enzyme activities.
Zentrum für Molekulare Biowissenschaften (ZBM), University Kiel
Institut für Klinische Molekularbiologie (IKMB), University Kiel
Botanical Institut, University Kiel
Norddeutsche Pflanzenzucht Hans-Georg Lembke KG
H.-J. Harloff, S. Lemcke, J. Mittasch, A. Frolov, J. Wu, F. Dreyer, G. Leckband, C. Jung (2012): A mutation screening platform for rapeseed (Brassica napus L.) and the detection of sinapine biosynthesis mutants. Theoretical and Applied Genetics 124, 957-969
N. Emrani, H.-J. Harloff, O. Gudi, F. Kopisch-Obuch, C. Jung (2015): Reduction in sinapine content in rapeseed (Brassica napus L.) by induced mutations in sinapine biosynthesis genes, Molecular Breeding, 35 (2015) 1-11
The project was funded by BMBF.
Updated: 26.10.2016 Responsible for this webpage: Dr. H.-J. Harloff