Citrus reticulata ‘Chachi’ (CRC) has long been recognized for its nutritional benefits, health-promoting properties, and pharmacological potential. Despite its importance, the bioactive components of CRC and their biosynthetic pathways have remained largely unexplored. In this study, we introduce a gap-free genome assembly for CRC, which has a size of 312.97 Mb and a contig N50 size of 32.18 Mb. We identified key structural genes, transcription factors, and metabolites crucial to flavonoid biosynthesis through genomic, transcriptomic, and metabolomic analyses. Our analyses reveal that 409 flavonoid metabolites, accounting for 83.30% of the total identified, are highly concentrated in the early stage of fruit development. This concentration decreases as the fruit develops, with a notable decline in compounds such as hesperetin, naringin, and most polymethoxyflavones observed in later fruit development stages. Additionally, we have examined the expression of 21 structural genes within the flavonoid biosynthetic pathway, and found a significant reduction in the expression levels of key genes including 4CL, CHS, CHI, FLS, F3H, and 4′OMT during fruit development, aligning with the trend of flavonoid metabolite accumulation. In conclusion, this study offers deep insights into the genomic evolution, biosynthesis processes, and the nutritional and medicinal properties of CRC, which lay a solid foundation for further gene function studies and germplasm improvement in citrus.

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