Detection of Low-Sinapine Mutants in Rapeseed by Application of TILLING

Rapeseed (Brassica napus L.) is one of the most important oilseed crops worldwide. The seed oil content ranges between 40 and 50% of dry matter, and the meal remaining after oil extraction contains about 40% protein which makes it one of the most widely used protein sources in animal feeds. However, the presence of antinutritive compounds like sinapine, phenolic acid and tannins affects the quality of rapeseed meal. Hence, the modification of the meal quality has been one of the main objectives of rapeseed breeding. Sinapine is a predominant phenolic compound that contributes to the bitter taste and astringency of rapeseed products. To reduce the sinapic acid ester content in the seeds and hence increase the protein quality, we have applied TILLING (Targeting Induced Local Lesions IN Genomes) to detect loss-of-function mutants in an EMS winter rapeseed population within major genes of the sinapin biosynthesis pathway. Two key genes of the sinapine biosynthesis in rapeseed, the sinapaldehyde/coniferylaldehyde dehydrogenase [BnaX.REF1], and the UDP-glucose:sinapate glucosyltransferase [BnaX.SGT] have been the major targets for our approach. Screening of the Express 617 EMS population (3488 M₂ plants) resulted in the identification of 135 mutations within BnaX.SGT and 341 within BnaX.REF1. Consequently, mutation frequencies of 1/22 and 1/12 kb were obtained. As mutant plants homozygous for single stop codon mutations did not show a phenotypical effect, i.e. reduced sinapine content, homozygous stop codon and splice site M₃ mutant plants were crossed to combine the most promising mutation events and the sinapine content and enzyme activity were measured in the subsequent F₂ progenies. The phenotyping of the F₂ plants showed up to 42 % reduction in sinapoylcholine (sinapine) content in plants homozygous for stop codon mutations in the BnaA.SGT.a and BnaC.SGT.a genes as compared to the wild type. The UDP-glucose:sinapate glucosyltransferase enzyme activity measurements in three developmental stages in seeds also showed a significant reduction due to nonsense mutations in single target genes. The result of our study shows the aptness of TILLING as a cost-effective, successful tool for the detection of the promising mutants with low sinapine content in rapeseed.