The SQUAMOSA promoter binding-like protein (SPL) is a specific transcription factor that affects plant growth and development. The SPL gene family has been explored in various plants, but information about these genes in alfalfa is limited. This study, based on the whole genome data of alfalfa SPL, the fundamental physicochemical properties, phylogenetic evolution, gene structure, cis-acting elements, and gene expression of members of the MsSPL gene family were analyzed by bioinformatics methods. We identified 82 SPL sequences in the alfalfa, which were annotated into 23 genes, including 7 (30.43%) genes with four alleles, 10 (43.47%) with three, 3 (13.04%) with two, 3 (13.04%) with one allele. These SPL genes were divided into six groups, that are constructed from A. thaliana, M. truncatula and alfalfa. Chromosomal localization of the identified SPL genes showed arbitary distribution. The subcellular localization predictions showed that all MsSPL proteins were located in the nucleus. A total of 71 pairs of duplicated genes were identified, and segmental duplication mainly contributed to the expansion of the MsSPL gene family. Analysis of the Ka/Ks ratios indicated that paralogs of the MsSPL gene family principally underwent purifying selection. Protein–protein interaction analysis of MsSPL proteins were performed to predict their roles in potential regulatory networks. Twelve cis-acting elements including phytohormone and stress elements were detected in the regions of MsSPL genes. We further analyzed that the MsSPLs had apparent responses to abiotic stresses such as drought and salt and the biotic stress of methyl jasmonate. These results provide comprehensive information on the MsSPL gene family in alfalfa and lay a solid foundation for elucidating the biological functions of MsSPLs. This study also provides valuable on the regulation mechanism and function of MsSPLs in response to biotic and abiotic stresses.

The WHY family is a group of plant-specific transcription factors, that can bind to single-stranded DNA molecules and play a variety of functions in plant nuclei and organelles, participating in the regulation of plant leaf senescence. It has been identified and analyzed in many species, however, the systematic identification and analysis of the WHY genes family have not yet been reported in alfalfa (Medicago sativa L.). Therefore, to explore the function of alfalfa the WHY genes, and 10 MsWHY genes were identified and further characterized their evolutionary relationship and expression patterns by analyzing the recently published genome of alfalfa. Comprehensive analysis of the chromosome location, physicochemical properties of the protein, evolutionary relationship, conserved motifs, and responses to abiotic stresses of the WHY gene family in alfalfa using bioinformatics methods. The results showed that 10 MsWHY genes were distributed on 10 chromosomes, and collinearity analysis showed that many MsWHYs might be derived from segmental duplications, and these genes are under purifying selection. Based on phylogenetic analyses, the WHY gene family of alfalfa can be divided into four subfamilies: I-IV subfamily, and approximately all the WHY genes within the same subfamily share similar gene structures. The 10 MsWHY gene family members contained 10 motifs, of which motif 2 and motif 4 are the conserved motifs shared by these genes. Furthermore, the analysis of cis-regulatory elements indicated that regulatory elements related to transcription, cell cycle, development, hormone, and stress response are abundant in the promoter sequence of the MsWHY genes. Real-time quantitative PCR demonstrated that MsWHYs gene expression is induced by drought, salt, and methyl jasmonate. The present study serves as a basic foundation for future functional studies on the alfalfa WHY family.