Pan-genome analysis of the SPL gene family and its function exploration in seed weight and yield in Brassica napus L

Improving rapeseed yield is a key goal for breeders, with seed weight (SW) being a critical factor in enhancing the yield of rapeseed varieties. Increasing research on SW has focused on the SQUAMOSA promoter binding protein-like (SPL) family, making the SPL genes a primary target in our study. We conducted a whole-genome analysis of eight rapeseed varieties, identifying 72 BnaSPL genes, which include 53 core genes, 14 dispensable genes, and five specific genes. Based on evolutionary relationships, these BnaSPL genes were classified into six subgroups. An evaluation of Ka/Ks values revealed that seven BnaSPL genes are under positive selection, while 44 are subject to purifying selection. Expression profiling across different tissues and qRT-PCR analysis confirmed that 26 BnaSPL genes are highly expressed during seed development. Further analysis indicated that eight genes exhibit variations in structure and motifs across at least two rapeseed varieties, although all retain conserved SBP domains. The diversity in cis-acting elements suggests potential functional variability among these genes. Haplotype analysis of 2311 germplasm resources identified four key genes—BnaSPL2–3, BnaSPL3–3, BnaSPL7–4, and BnaSPL16–4—whose haplotypes were associated with differences in thousand-seed weight (TSW) and yield across rapeseed varieties. Notably, these yield increases were stable and unaffected by external environmental factors, such as low or high salinity stress. Our findings underscore the potential of BnaSPL2–3, BnaSPL3–3, BnaSPL7–4, and BnaSPL16–4 in breeding rapeseed varieties with higher TSW and yield.