Medicago sativa L. is the most important legume plant, but its drought resistance mechanism is still unclear. This study analyzed the physiological and transcriptome responses of three Medicago sativa L. varieties with different drought resistance after 0h, 6h, 12h, and 24h of drought stress. Through transcriptome sequencing, we identified 22,386 DEGs in G3 variety, 18,083 DEGs in G8 variety, and 24,587 DEGs in LD variety at 6h of drought stress; we identified 24,469 DEGs in G3 variety, 28,824 DEGs in G8 variety, and 22,497 DEGs in LD variety at 12h of drought stress; we identified 23,279 DEGs in G3 variety, 20,263 DEGs in G8 variety, and 21,541 DEGs in LD variety at 24h of drought stress. The functional enrichment analysis, temporal expression pattern analysis, and weighted gene co-expression network analysis (WGCNA) of the differentially expressed genes (DEGs) showed phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism, glycolysis/gluconeogenesis, glutathione metabolism, and biosynthesis of amino acid responses to drought stress in alfalfa. The differential expression of genes during phenylpropanoid biosynthesis, starch and sucrose metabolism, and the glutathione metabolism pathway was further studied, and it was speculated that PAL, COMT, 4CL, CCR, CAD, HXK, INV, SUS, WAXY, AGP, GST, and APX1 played important roles in the alfalfa drought stress response.
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