Integrated dual-molecular marker analysis with SSR and UPLC-Q-TOF/MS fingerprints technology reveal the interrelation of the molecule-metabolite in Morus alba L. leaves

Morus alba L. (M. alba) leaves, valued for their medicinal and edible properties, possess substantial economic and social importance. Notably, diverse germplasm resources of M. alba exhibit considerable variation in both metabolite composition and pharmacological activity. However, the interrelationships among genetic diversity, metabolites profiles, and pharmacological activity across different M. alba varieties remain insufficiently elucidated. This study aimed to construct simple sequence repeat (SSR) and ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) fingerprints, together with antipyretic clustering diagrams, for 69 different varieties of M. alba leaves, and analyze the correlations among their genetic structures, metabolite profiles, and antipyretic activities. In this study, 69 M. alba leaf samples representing different varieties were collected, and their antipyretic effects were evaluated using a rat pharmacodynamic model. SSR fingerprints were generated with TP-M13 SSR molecular markers to screen core primer pairs for variety identification. Simultaneously, metabolite fingerprints were established using UPLC-Q-TOF/MS with common peaks calibrated and compounds qualitatively identified by mass spectrometry. Finally, multivariate chemometric approaches were applied to integrate SSR data and UPLC-Q-TOF/MS fingerprint profiles, and antipyretic activity results for clustering and correlation analysis. The dominant M. alba varieties, Yantai 792, Zhesang 3, 8036, Fengyuan 1, Laiwugusang, Xiajingusang, Gaoqing 792, Zhesang 1 and Heizhenzhu exhibited the strongest antipyretic activities. In contrast, Guiyou, Danbaisang, and Dashi showed weaker effects and were clustered into a single category in the molecule-metabolite-efficacy clustering diagram, thereby indicating the substantial influence of varieties differences on genetic diversity, metabolite composition, and pharmacological activity. Pearson correlation analysis further confirmed significant correlations among genetic components, metabolites, and pharmacological effects. In addition, a core marker set comprising 7 SSR loci was identified, together with 8 key chemical markers including cryptochlorogenic acid, neochlorogenic acid, isoquercitrin, rutin, nicotiflorin, astragalin, salicylic acid, and umbelliferone. These findings help identify the "molecule-metabolite" dual molecular markers with the bias of antipyretic effects for M. alba leaves varieties, and provide valuable insights for refining the medicinal value of M. alba leaves and promoting the utilization of high-value resources.