Flavonoid biomarkers and co-expression gene networks characterized in ten Hemerocallis citrina accessions via multi-omics.

Hemerocallis citrina Baroni (H. citrina), a traditional medicinal and edible plant, is rich in bioactive flavonoids. This study investigated flavonoid metabolites in H. citrina flowers from ten accessions using UPLC-MS/MS-based metabolomics. A total of 642 flavonoids and 14 tannins were identified in the flowers. Among these, 375 flavonoids were classified as flavonols and flavone, representing for 39.33% and 17.53%, respectively. The identification of 309 differential flavonoid metabolites was achieved through analyses of nine pairwise comparison groups. These differential flavonoids in flowers of H. citrina could be distinctly categorized into two groups, clearly separating the 7 edible accessions from the 3 non-edible accessions. Significant variations in 14 flavonoid markers were observed among 10 H. citrina accessions originating from different regions, as determined by hierarchical clustering and ROC analyses. Transcriptomic analyses revealed that the majority of differentially expressed genes were enriched in the flavonoid metabolism pathway among these accessions. Integrated transcriptomic and metabolic analyses identified 14 differential flavonoid metabolites (DFMs) and 47 differentially expressed genes (DEGs) associated with flavonoid biosynthesis through Pearson's correlation analysis and WGCNA analyses. qRT-PCR validation of 13 DEGs confirmed the consistency of transcriptomic data. A flavonoid-gene correlation network indicated that the 14 DFMs might be directly regulated by 17 DEGs, comprising 13 flavnoid metabolism-related genes and 4 transcript factors. These findings provide biological and chemical insights into flavonoid metabolic difference across H. citrina origins, offering a theoretical basis for food and medical applications, and enabling clear differentiation between edible and non-edible H. citrina flowers in the market.