Understanding the intricate regulatory mechanisms underlying the anthocyanin content (AC) in fruits and vegetables is crucial for advanced biotechnological customization. In this study, we generated high-quality haplotype-resolved genome assemblies for two mulberry cultivars: the high-AC ‘Zhongsang5801’ (ZS5801) and the low-AC ‘Zhenzhubai’ (ZZB). Additionally, we conducted a comprehensive analysis of genes associated with AC production. Through genome-wide association studies (GWAS) on 112 mulberry fruits, we identified MaVHAG3, which encodes a vacuolar-type H+-ATPase G3 subunit, as a key gene linked to purple pigmentation. To gain deeper insights into the genetic and molecular processes underlying high AC, we compared the genomes of ZS5801 and ZZB, along with fruit transcriptome data across five developmental stages, and quantified the accumulation of metabolic substances. Compared to ZZB, ZS5801 exhibited significantly more differentially expressed genes (DEGs) related to anthocyanin metabolism and higher levels of anthocyanins and flavonoids. Comparative analyses revealed expansions and contractions in the flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) genes, resulting in altered carbon flow. Co-expression analysis demonstrated that ZS5801 displayed more significant alterations in genes involved in late-stage AC regulation compared to ZZB, particularly during the phase stage. In summary, our findings provide valuable insights into the regulation of mulberry fruit AC, offering genetic resources to enhance cultivars with higher AC traits.

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