Genome-Wide Comprehensive Analysis of the CDPK Gene Family in Three Alfalfa Germplasms Revealed That MsCDPK9.Zm1 Positively Regulates the Physiological Salt Tolerance of Alfalfa

Alfalfa (Medicago sativa L.) is a widely cultivated perennial forage legume, yet its productivity is severely constrained by soil salinization. Calcium-dependent protein kinases (CDPKs) play critical roles in plant salt-stress signaling; however, systematic characterization of the CDPK gene family in alfalfa and functional validation of its members remain limited. In this study, we performed genome-wide identification and functional characterization of the CDPK gene family across three alfalfa germplasms. We identified 18, 90, and 26 MsCDPK members in M. sativa ‘Zhongmu No. 1’, M. sativa ‘Zhongmu No. 4’, and M. sativa subsp. caerulea, respectively, which were classified into four subfamilies based on phylogenetic analysis. Collinearity analysis revealed that segmental duplication was the primary driver of MsCDPK gene family expansion, whereas tandem duplication was not detected. Notably, expression pattern analysis identified MsCDPK9.Zm1 as a salt-responsive gene. Overexpression of MsCDPK9.Zm1 in alfalfa significantly enhanced physiological salt tolerance, as evidenced by increased antioxidant enzyme activities and reduced oxidative damage under salt stress. Luciferase complementation imaging assays further supported the physical association between MsCDPK9.Zm1 and RBOHB. These findings further our understanding of the CDPK gene family in alfalfa and identify MsCDPK9.Zm1 as a candidate gene for molecular breeding of salt-tolerant alfalfa.