Identification of candidate genes associated with regreening stage in rapeseed (Brassica Rapa L.) by combined application of BSA-Seq and RNA-Seq

BACKGROUND: The regreening stage (RS) is a critical agronomic trait impacting yield potential in rapeseed (Brassica rapa L.), yet its underlying genetic mechanisms remain largely unexplored. Field observations indicate that early-regreening cultivars, like 'Hengyou 8', exhibit a longer grain-filling period and higher thousand-seed weight (TSW) compared to late-regreening cultivars like 'Hengyou 6'. This study aimed to systematically dissect the genetic basis of RS initiation to identify key regulatory genes and provide resources for molecular breeding. RESULTS: We constructed an F₂ population from a cross between 'Hengyou 8' and 'Hengyou 6' and employed Bulk Segregant Analysis sequencing (BSA-seq), which identified 11 quantitative trait loci (QTLs) associated with RS on chromosomes A01, A02, A04, A05, A06, A08, A09, and A10. Concurrently, transcriptome sequencing (RNA-seq) of shoot apical meristems and roots across the RS process revealed 17,242 differentially expressed genes (DEGs). Integrated analysis of BSA-seq and RNA-seq data pinpointed 15 high-confidence candidate genes within the QTL regions. These include NAC016, NAC017, MYC2, and DDE2, which are primarily involved in jasmonic acid (JA) metabolism, phytohormone signaling, cell development, and stress responses. Expression profiling showed distinct spatiotemporal patterns for these genes between the parental lines, suggesting their roles in modulating the timing of regreening. CONCLUSIONS: Our findings provide the first comprehensive genetic map of the regreening process in rapeseed, revealing a dynamic regulatory network centered on JA signaling and stress response pathways. The identified candidate genes and associated molecular markers establish a valuable resource and a solid theoretical foundation for future functional studies. More importantly, we have identified direct targets for marker-assisted selection (MAS). This provides a foundation for breeding novel rapeseed cultivars with optimized regreening timing and enhanced yield potential.