Natural variation and artificial selection at the BnaC2.MYB28 locus modulate Brassica napus seed glucosinolate

The degradation products of glucosinolates (GSLs) greatly lower the nutritional value of rapeseed (Brassica napus) meal; thus, reduction of seed GSL content (SGC) has become an important objective of rapeseed breeding. In our previous study, we finely mapped a major QTL (qGSL-C2) for SGC to a 49-kb collinear region on B. rapa chromosome A2. Here, we experimentally validated that BnaC2.MYB28, encoding an R2R3-MYB transcription factor, is the causal gene of qGSL-C2. BnaC2.MYB28 is a nucleus-localized protein mainly expressed in vegetative tissues. Knockout of BnaC2.MYB28 in the high-SGC parent G120 reduced SGC to a value lower than that in the low-SGC parent ZY50, while overexpression of BnaC2.MYB28 in both parental lines (G120 and ZY50) led to extremely high SGC, indicating that BnaC2.MYB28 acts as a positive regulator of SGC in both parents. Molecular characterization revealed that BnaC2.MYB28 forms a homodimer and specifically interacts with BnaMYC3. Moreover, BnaC2.MYB28 can directly activate the expression of GSL biosynthesis genes. Differential expression abundance resulting from the polymorphic promoter sequences, in combination with the different capability in activating downstream genes involved in aliphatic GSL biosynthesis, caused the functional divergence of BnaC2.MYB28 in SGC regulation between the parents. Natural variation of BnaC2.MYB28 was highly associated with SGC in natural germplasm and has undergone artificial selection in modern low-GSL breeding. This study provides important insights into the core function of BnaC2.MYB28 in regulating SGC and a promising strategy for manipulating SGC in rapeseed.