Comparative genomics and time-course transcriptomics uncover homoeologous exchange events reshaping glucosinolate metabolism in Brassica napus.

Improving glucosinolate (GSL) profiles in rapeseed (Brassica napus)-high in leaves for pathogen resistance but low in seeds for meal quality-is a key breeding goal, yet its genetic basis remains unclear. Here, we present a chromosome-level genome assembly for ZY821, an elite high-GSL variety, generated using long-read sequencing and Hi-C scaffolding. Comparative analysis with the low-GSL variety ZS11 identified three major homoeologous exchange (HE) events and extensive structural variation. Notably, an A09-C09 HE event replaced the low-expression BnaC09.MYB28 allele with the high-expression BnaA09.MYB28 allele, resulting in elevated MYB28s expression and thereby increased GSL accumulation in ZY821, whereas a deletion of BnaA09.MYB28 in ZS11 significantly reduced the expression of multiple putative downstream targets in the GSL biosynthesis pathway, leading to a reduction in GSL content. This mechanism was supported by population-level HE analysis and time-course transcriptomes across 116 RNA-Seq samples. Furthermore, joint differential expression and co-expression network analyses uncovered several novel candidate genes implicated in GSL metabolism. Collectively, our study provides new mechanistic insights into the genetic control of GSL accumulation, with significant implications for breeding optimized GSL profiles.