Integrated transcriptomic and WGCNA approaches identify a regulatory hub gene for flavonol glycoside biosynthesis in extreme chemotypes of Epimedium

Epimedium , a traditional Chinese medicinal herb belonging to the perennial Epimedium genus of the Berberidaceae family, has a documented medicinal history spanning over 2,000 years. However, reports on the regulatory mechanisms underlying the biosynthesis of its key flavonol glycosides (epimedin A, B, C, and icariin) remain scarce. This study employed High Performance Liquid Chromatography (HPLC) and RNA Sequencing (RNA-seq) strategies to elucidate the regulatory network governing flavonol glycoside synthesis in Epimedium . By conducting HPLC based quantification of flavonol glycosides in the leaves of 22 Epimedium species grown under identical environmental conditions, five species exhibiting extreme flavonol glycoside accumulation were identified. Based on their content profiles, the Epimedium accessions were classified into extremely high, inter-mediate, and extremely low groups. Interestingly, we found that an endangered Epimedium species ( Epimedium truncatum ) has a much higher content of epimedin C than other pharmacopoeial species, which may be the reason for its gradual disappearance. RNA-seq analysis of these five selected species, combined with Weighted Gene Co-expression Network Analysis (WGCNA), revealed five hub genes central to the regulation of flavonol glycoside synthesis, along with their associated interaction networks. Among these, the expression levels of three MYB-type transcription factors were negatively correlated with epimedin C synthesis. In contrast, the expression of a previously unreported 2”-O-glycosyltransferase showed a positive correlation with the accumulation of epimedin C, while the expression of a rhamnosyltransferase was positively correlated with the synthesis of epimedin A, B and icariin. We further validated the five hub genes in Epimedium using qPCR and quantified their expression levels across different medicinal parts. These findings suggest that these five genes can serve as marker genes indicative of flavonol glycoside content in Epimedium . In summary, this study integrates transcriptomic analysis of five Epimedium species with extreme chemotypes to unravel the intrinsic gene regulatory network underlying the biosynthesis of Epimedium flavonol glycosides, providing a foundation for chemotype-oriented breeding of Epimedium .