Conditionally Significant eQTL and TWAS Analyses Identify MsRD26 as a Major Candidate Positive Regulator of Salt Tolerance in Medicago sativa L

Natural variation in gene expression bridges genetic polymorphisms and phenotypic divergence, yet the regulatory architecture underlying salt stress responses remains largely unexplored in alfalfa. Here, we generated 528 RNA-seq libraries from 176 alfalfa accessions under well-watered and salt stress conditions and identified 12,901 differentially expressed genes. Through integration of population transcriptomics, eQTL mapping, TWAS, and Mendelian randomization, we constructed a genome-wide regulatory landscape comprising 62,423 to 67,405 eQTLs, 346 distant eQTL hotspots, and a predictive TF-eGene interaction network. We prioritized 1,401 genes whose expression variations are associated with salt tolerance. Among these, we functionally validated MsRD26, a NAC transcription factor, as a major candidate positive regulator of salt tolerance. This study provides a comprehensive resource of regulatory variants and candidate genes for salt tolerance in alfalfa, with implications for genomics-assisted breeding in this polyploid forage crop.