Genome-Wide Association Analysis Coupled With Transcriptome Analysis Reveals Candidate Genes Related to Salt Stress in Alfalfa (Medicago sativa L.)

Salt stress is the main abiotic factor affecting alfalfa yield and quality. However, knowledge of the genetic basis of the salt stress response in alfalfa is still limited. Here, a genome-wide association study (GWAS) involving 875,023 single-nucleotide polymorphisms (SNPs) was conducted on 220 alfalfa varieties under both normal and salt-stress conditions. Phenotypic analysis showed that breeding status and geographical origin play important roles in the alfalfa salt stress response. For germination ability under salt stress, a total of 15 significant SNPs explaining 9%–14% of the phenotypic variation were identified. For tolerance to salt stress in the seedling stage, a total of 18 significant SNPs explaining 12%–23% of the phenotypic variation were identified. Transcriptome analysis revealed 2,097 and 812 differentially expressed genes (DEGs) that were upregulated and 2,445 and 928 DEGs that were downregulated in the leaves and roots, respectively, under salt stress. Among these DEGs, many encoding transcription factors (TFs) were found, including MYB-, CBF-, NAC-, and bZIP-encoding genes. Combining the results of our GWAS analysis and transcriptome analysis, we identified a total of eight candidate genes (five candidate genes for tolerance to salt stress and three candidate genes for germination ability under salt stress). Two SNPs located within the upstream region of MsAUX28, which encodes an auxin response protein, were significantly associated with tolerance to salt stress. The two significant SNPs within the upstream region of MsAUX28 existed as three different haplotypes in this panel. Hap 1 (G/G, A/A) was under selection in the alfalfa domestication and improvement process.