Genome-Wide Identification and Functional Divergence of the Chloride Channel (CLC) Gene Family in Autotetraploid Alfalfa (Medicago sativa L.)

Chloride channel proteins (CLCs) are essential anion transporters involved in plant growth, osmotic regulation, and ion homeostasis. However, their genome-wide characterization in tetraploid alfalfa (<i>Medicago sativa</i> L.) remains unexplored. In this study, a total of 35 CLC family members were identified and underwent comprehensive bioinformatic analyses. Phylogenetic and structural analyses divided them into six subfamilies and two subclasses based on conserved residues such as GxGIPE. Members within the same subclass shared conserved domains and similar motif patterns. Analysis of duplication events indicated that 48 segmental duplications were the primary driving force behind the expansion of this gene family. Promoter analysis revealed abundant light, hormone, and stress-responsive cis-elements, suggesting multiple regulatory functions. Gene expression profiling demonstrated that salt, drought stress, and ABA treatment significantly induced the expression levels of some genes. Among them, <i>MsCLC2</i> and <i>MsCLC18</i> from Group c exhibited more than fivefold upregulation under both salt and drought stress, significantly higher than other members. Subcellular localization confirmed <i>MsCLC18</i> on the plasma membrane, potentially regulating Cl^- efflux through a Cl^-/H⁺ antiporter mechanism to alleviate Cl^- toxicity. These findings provide a theoretical foundation for the function study of <i>CLC</i> genes in alfalfa and offer new insights into the molecular evolution of polyploid plants under abiotic stress.