Functional analysis and mutagenesis of glucosinolate synthesis genes for breeding oilseed rape (Brassica napus) with lower glucosinolate content

Rapeseed is a major oil crop in the temperate and sub-tropical regions of the world. For utilization as animal feed, it is crucial to reduce the content of anti-nutritive compounds like glucosinolates (GSLs) in the rapeseed meal which can have severe metabolic effects on poultry, fish and livestock. Reducing the GSL content to <18 µmol/g dry weight in the seeds is a major breeding target. While genes involved in GSL biosynthesis have been described in rapeseed, comprehensive functional analyses are missing. By knocking out the aliphatic GSL biosynthesis genes BnMYB28 and BnCYP79F1 and the GSL transporter BnGTR2, we aimed to reduce the GSL content in seeds of rapeseed. After analyzing the gene expression of single paralogs, we used an ethyl methanesulfonate (EMS) treated population of the inbred winter rapeseed Express617 to detect functional mutations in the two gene families. We demonstrate that independent knock-out mutants of the two genes possessed significantly reduced total and aliphatic GSLs, primarily progoitrin, in the seeds. Compared to the wildtype Express617 control plants, progoitrin levels were decreased by 55.3% and 32.4% in double mutants of BnMYB28 and BnCYP79F1, respectively. Our study provides a strong basis for breeding rapeseed with improved meal quality in the future. 

We have developed a TILLING by whole-genome sequencing (TbyWGS) platform containing >78 million high confidence EMS-induced C→T and G→A mutations from whole-genome sequencing data from 1,988 M2 plants. On average, each plant possessed ~39,000 mutations with a frequency of 1/23.6 kb on the genome level. ~82% of the mutations were located in 5 kb upstream or downstream of gene coding regions or in intergenic regions. 18% were located within gene coding regions. The web-based resource enables the user-friendly detection of EMS mutations in any genomic region of interest.


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