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

## TL;DR

This study identifies and characterizes key RNA silencing genes in alfalfa and their roles in stress responses, offering insights for improving stress resilience in crops.

## Contribution

The first genome-wide analysis of DCL, AGO, and RDR gene families in autotetraploid alfalfa and their abiotic stress responses.

## Key findings

- 31 MsDCL, 82 MsAGO, and 52 MsRDR genes were identified and classified into evolutionary clades.
- Gene duplication, especially segmental duplication, drove the expansion of these gene families.
- Six core stress-responsive genes were identified and validated under salt and drought stress.

## Abstract

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 (Medicago sativa L.), identifying 31 MsDCL, 82 MsAGO, and 52 MsRDR 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 cis-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—MsDCL11, MsAGO54, MsAGO60, MsAGO73, MsAGO76, and MsRDR37—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.