A telomere-to-telomere Brassica napus genome identifies BnaWRKY44 regulation of seed coat color and oil.
Brassica napus yellow-seeded genotypes have higher seed oil content and quality than black-seeded types, but the mechanisms controlling seed coat color (SCC) remain unclear, partly due to limited gap-free references. We generated the telomere-to-telomere genome assembly of the black-seeded cultivar Zhongyou 821 using PacBio HiFi, ultralong ONT, and Hi-C sequencing. Using this reference, genome-wide association study (GWAS) of 504 accessions identified BnaWRKY44 as a major SCC candidate gene. CRISPR-Cas9 knockout of BnaWRKY44 lightened SCC, increased oil content and unsaturated fatty acids, and improved oil quality. Haplotype analysis defined the elite BnaWRKY44Hap4 allele and enabled development of a diagnostic CAPS marker for breeding. Transcriptomic, Y1H and dual-LUC assays showed that BnaWRKY44 directly binds and regulates the BnaVPT1 promoter. BnaVPT1 knockout confirmed its roles in SCC lightening and flavonoid biosynthesis. This work provides a T2T rapeseed resource and a BnaWRKY44-BnaVPT1 module for breeding high-oil, yellow-seeded rapeseed.