Genome-wide identification and characterization of DCL, AGO, and RDR gene families and their abiotic stress responses in alfalfa (Medicago sativa L.)

Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) proteins are core components of the plant RNA silencing pathway and play critical roles in plant growth, development, and stress adaptation. In this study, we performed the first genome-wide analysis of these gene families in autotetraploid alfalfa (<i>Medicago sativa</i> L.), identifying 31 <i>MsDCL</i>, 82 <i>MsAGO</i>, and 52 <i>MsRDR</i> genes, followed by systematic characterization. Phylogenetic analysis classified these genes into distinct evolutionary clades, and gene duplication events-particularly segmental duplications-were identified as the primary drivers of family expansion. Promoter analysis revealed an abundance of hormone- and stress-responsive <i>cis</i>-elements in their regulatory regions. Expression profiling across six tissues indicated both constitutive and tissue-specific expression patterns. Transcriptome sequencing under salt and drought stress identified six core stress-responsive candidate genes-<i>MsDCL11</i>, <i>MsAGO54</i>, <i>MsAGO60</i>, <i>MsAGO73</i>, <i>MsAGO76</i>, and <i>MsRDR37</i>-which were further validated by RT-qPCR. These findings provide a theoretical foundation for future functional studies of RNA silencing pathway genes and offer valuable genetic resources for the molecular breeding of stress-resilient alfalfa.