Integrated whole-genome resequencing and mRNA-miRNA analysis reveals selection signals and regulatory networks of antipyretic effect variation in Morus alba var. multicaulis

Lusang (Morus alba var. multicaulis) represents both the inception and paradigm of artificial cultivation and breeding of mulberry (Morus spp.) in China, marking the onset of mulberry domestication. However, the genetic structure underlying this domestication process remains poorly understood, and no reports to date have described the selection signals and their candidate genes, or mRNA–miRNA regulatory networks associated with the pharmacological traits of Lusang. In this study, we performed whole-genome resequencing and integrated mRNA–miRNA analyses to investigate the population structure, genetic diversity, and selection signals of Lusang. Principal component analysis, phylogenetic reconstruction, and population structure analysis yielded congruent results, consistently grouping into three distinct clades. Notably, this classification aligned with previous grouping based on antipyretic activity and metabolite clustering, jointly supporting the notion that the medicinal varieties of mulberry leaves (the leaves of Morus alba) can be categorized according to genetic divergence. Integrated correlation analysis revealed overall correlations among genomic, metabolomic, and efficacy datasets. Furthermore, we identified several miRNA–gene regulatory axes (miR167c‑ACT, miR159c‑F3H/FLS, and miR828a‑UGT76B1) that are potentially involved in regulating the differential accumulation of key antipyretic compounds such as cryptochlorogenic acid, neochlorogenic acid, rutin, isoquercitrin, and astragalin. These regulatory relationships may contribute to the observed variation in antipyretic efficacy among cultivars. Collectively, our findings suggested that divergence in the accumulation of key antipyretic compounds was likely driven by genetic variation at the regulatory level, including selection signals in non-coding regions and their consequent effects on gene expression and miRNA-mediated networks. This study provides molecular insights into the pharmacological variation of Lusang from an integrated genotype–metabolite–pharmacological effect perspective.