Recent advances in plant genomics have characterized transposable elements (TEs) as key contributors to genome structure and gene regulation. This study focuses on the remarkably high abundance of short interspersed nuclear elements (SINEs) in the genus Trifolium. Using the allotetraploid horticultural plant white clover (Trifolium repens L.) as the study organism, we systematically investigate lineage-specific SINE amplification, genomic distribution, insertional preferences, and their regulatory effects on gene expression. Our analyses reveal that SINEs are significantly more abundant in Trifolium than in other angiosperms. Comparative genomic analyses further indicate that SINE accumulation is closely associated with polyploidization and domestication. Gene Ontology (GO) enrichment analyses demonstrate that SINEs are preferentially enriched in stress responsive genes. Expression analyses further showed that, within duplicated gene pairs, genes with SINE insertions in their upstream promoter regions exhibit significantly higher transcript levels compared with genes without such insertions. Under drought, cold, and cadmium stress, these SINE-associated genes exhibit upregulation, and our data analysis shows a strong correlation between the presence of SINE insertions and stress-induced upregulation of gene expression. This study demonstrates that SINE insertions in upstream promoter regions modulate transcriptional regulatory networks involved in stress responses, contributing to broad ecological adaptation in white clover.

Soil salinization is a source of major abiotic stress that severely limits the production of alfalfa (Medicago sativa L.), a globally critical forage legume for sustainable livestock production. Its complex autotetraploid genome and self-incompatibility greatly hinder salt tolerance genetic improvement, while the post-transcriptional regulatory mechanism of alfalfa salt stress response remains largely uncharacterized. This study aimed to narrow the gap between genome-wide genetic signals and causal regulatory mechanisms and identify core post-transcriptional regulators of alfalfa salt tolerance via a multi-layered integrative analysis pipeline. We performed a genome-wide association study (GWAS) using 220 globally collected alfalfa accessions, combined with public transcriptome integration, co-expression network analysis, 3′ untranslated region (3′UTR) motif discovery, and AlphaFold2-based protein-RNA docking simulation. We identified 20 significant salt tolerance-associated loci and prioritized the CCCH-type zinc-finger RNA-binding protein (RBP) MsCCCH20 as the core candidate regulator. We further screened 35 high-confidence target genes of MsCCCH20, detected conserved AU/AG-rich 3′UTR motifs, and provided structural predictions consistent with potential sequence-specific interactions (ipTM 0.70–0.79). Our findings establish a robust framework linking genetic association signals to post-transcriptional regulatory networks and provide high-confidence candidate genes and functional markers for the molecular breeding of salt-tolerant alfalfa.