Background Alfalfa, the “queen of forage”, is the most extensively cultivated forage legume in the world. The development and yield of alfalfa are seriously limited by abiotic stress. MADS-box transcription factors are one of the largest gene families and play a pivotal role in plant development and abiotic stress. However, little is known regarding the MADS-box transcription factors in autotetraploid cultivated alfalfa. Results In the present study, we identified 120 MsMADS-box genes in the alfalfa genome. Phylogenetic analysis indicated that 75 type-I MsMADS-box genes were classified into the Mα, Mβ, and Mγ subgroups, and 45 type-II MsMADS-box genes were classified into 11 subgroups. The promoter region of MsMADS-box genes containing several hormone and stress related elements. Chromosomal location analysis revealed that 117 MsMADS-box genes were unevenly distributed on 32 chromosomes, and the remaining three genes were located on unmapped scaffolds. A total of nine pairs of segmental duplications and four groups of tandem duplications were found. Expression analysis showed that MsMADS-box genes were differentially expressed in various tissues and under abiotic stresses. qRT-PCR analysis revealed that the expression profiles of eight selected MsMADS-box genes were distinct under various stresses. Conclusions In this study, MsMADS-box genes were identified in the cultivated alfalfa genome based on autotetraploid level, and further confirmed by Gene Ontology (GO) analysis, phylogenetic analysis, sequence features and expression analysis. Taken together, these findings will provide clues for further study of MsMADS-box functions and alfalfa molecular breeding. Our study is the first to systematically identify and characterize the MADS-box transcription factors in autotetraploid cultivated alfalfa (Medicago sativa L.), and eight MsMADS-box genes were significantly involved in response to various stresses.

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