Broccoli (Brassica oleracea var. italica) is a widely cultivated cruciferous vegetable valued for its abundant bioactive compounds and nutraceutical properties. Among these, anthocyanins are not only important secondary metabolites contributing to nutritional and medicinal benefits, but also influence stress tolerance and the commercial quality of broccoli through purple pigmentation. However, the molecular mechanisms regulating anthocyanin biosynthesis in broccoli remain poorly understood, partly due to the incomplete genomic resources currently available. In this study, we constructed a telomere-to-telomere gap-free assembly of the broccoli genome using a combination of Oxford Nanopore Technology ultralong reads, PacBio high-fidelity reads, and Hi-C datasets. The resulting genome is 633.61 Mb in length, with an N50 of 60.36 Mb, and comprises gap-free assemblies of all 18 chromosomes, including complete telomere-to-telomere assemblies for nine chromosomes. Using this high-quality reference, we identified BoF3’H, a key gene regulating anthocyanin accumulation, which controlling the purple coloration of broccoli buds. To validate the function of the BoF3’H gene in anthocyanin biosynthesis, we used CRISPR-Cas9 gene editing to target and knock out the BoF3’H gene. The bof3’h mutant exhibited an 81.4% reduction in cyanidin and delphinidin derivative levels compared with those in the control. Metabolomic and transcriptomic profiling showed that the expression of 12 anthocyanin-related genes, including PAL, C4H, CL3, CHS, F3’H, and ANS, was downregulated. These findings elucidate the molecular basis of anthocyanin regulation in broccoli and provide a foundational genomic resource for evolutionary studies, gene discovery, and future breeding.

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