Dual genetic loci and flavonoid metabolism orchestrate fruiting body coloration in Flammulina filiformis: a multi-omic roadmap for fungal pigmentation

BACKGROUND: The fruiting bodies of macrofungi exhibit diverse coloration, traditionally attributed to melanin and carotenoid biosynthesis. This study is the first to reveal that flavonoids, rather than these classical pigments, are the predominant contributors to yellow pigmentation in the Flammulina filiformis. OBJECTIVE: To uncover the genetic basis and key regulatory genes involved in pigment formation in F. filiformis fruiting bodies, and to establish a model framework for studying color genetics in macrofungi. METHODS: Metabolomic profiling was conducted on yellow and white F. filiformis fruiting bodies to identify key pigment components. A segregating population was constructed, followed by integrated multi-omics analyses-including bulk segregant analysis (BSA), genome-wide association study (GWAS), and transcriptomics-to map regulatory loci and candidate genes. Functional roles were validated via genetic transformation and protein structural modeling. RESULTS: Flavonoid accumulation was identified as the biochemical hallmark of pigmented fruiting bodies. Genetic analysis revealed a dual regulatory mechanism: a qualitative locus governing pigmentation presence and a quantitative trait determining color intensity. Combined BSA and GWAS pinpointed a major locus, Ffcrs, within a recombination-suppressed region. Transcriptomic analysis identified two key regulators, Ffakr (a transcriptional activator) and Ffpal (encoding phenylalanine ammonia-lyase). Functional verification via transformation, structural modeling, and metabolite profiling in transgenic lines confirmed their essential roles in flavonoid biosynthesis and pigmentation. CONCLUSION: This study uncovers a flavonoid-based pigmentation mechanism in F. filiformis and elucidates a complex genetic architecture shaped by both qualitative and quantitative loci, providing a new paradigm for understanding pigment formation in macrofungi. The identified regulatory factors establish a molecular foundation for the precise manipulation of economically important pigmentation traits in edible mushroom.