Abstract
Background: Morus alba L., a perennial woody plant with significant economic and ecological value, contains abundant phytochemicals, including flavonoids, which play crucial roles in plant defense and human health. Flavonoids are a diverse group of secondary metabolites with antioxidant, anti-inflammatory, and antimicrobial properties, and are widely found in plant tissues and contribute to both plant fitness and human nutrition. Results: Through comparative transcriptome analysis between Black Mulberry (HS) and Medicinal Mulberry (YS), we identified numerous differentially expressed genes (DEGs) associated with flavonoid biosynthesis. Key structural genes, including CHS1, CHI, F3H, F3’H, and ANS, as well as transcription factors (TFs) such as MYB306, BHLH106, and BHLH153, exhibited cultivar-specific expression patterns that strongly correlated with flavonoid accumulation during fruit development. Notably, the flavonoid content was consistently higher in YS than in HS, especially at later developmental stages. Furthermore, weighted gene co-expression network analysis (WGCNA) revealed a key module enriched with genes involved in flavonoid synthesis and related regulatory pathways, providing new insights into the transcriptional regulation of flavonoid accumulation in M. alba L. Conclusions: The study extends the understanding of flavonoid biosynthesis in plants by highlighting the cultivar-specific regulation of key biosynthetic genes and transcription factors. The results offer a theoretical basis for improving M. alba L. varieties and enhancing their nutritional and medicinal values. By integrating multi-omics approaches, future research can further unravel the complex regulatory networks governing flavonoid accumulation and explore their applications in agriculture, food, and medicine. Supplementary Information: The online version contains supplementary material available at 10.1186/s12870-025-08070-9.