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.