Heat shock transcription factors (HSFs) are important regulatory factors in plant stress responses to various biotic and abiotic stresses and play important roles in growth and development. The HSF gene family has been systematically identified and analyzed in many plants but it is not in the tetraploid alfalfa genome. We detected 104 HSF genes (MsHSFs) in the tetraploid alfalfa genome (“Xinjiangdaye” reference genome) and classified them into three subgroups: 68 in HSFA, 35 in HSFB and 1 in HSFC subgroups. Basic bioinformatics analysis, including genome location, protein sequence length, protein molecular weight and conserved motif identification, was conducted. Gene expression analysis revealed tissue-specific expression for 13 MsHSFs and tissue-wide expression for 28 MsHSFs. Based on transcriptomic data analysis, 21, 11 and 27 MsHSFs responded to drought stress, cold stress and salt stress, respectively, with seven responding to all three. According to RT–PCR, MsHSF27/33 expression gradually increased with cold, salt and drought stress condition duration; MsHSF6 expression increased over time under salt and drought stress conditions but decreased under cold stress. Our results provide key information for further functional analysis of MsHSFs and for genetic improvement of stress resistance in alfalfa.

Alfalfa (Medicago sativa L.) is an economically important forage legume. As a result of the extensive and intensive cultivation of alfalfa planting and the gradual expansion of planting areas, pest occurrence has increased in frequency. Aphids are one of the main pests that threaten the growth and productivity of alfalfa. After an aphid outbreak, alfalfa yield and quality are commonly greatly reduced. At present, there are few studies on alfalfa plants infested with aphids, so it is imperative to study the regulatory mechanisms of aphid infestation tolerance in alfalfa. In this study, alfalfa plants from the variety “Wudi” were investigated, and pea aphids were selected for inoculation. The transcriptome and metabolome data were analyzed at three time points (0 d, 1 d, and 4 d), revealing 3458 differentially expressed genes and 358 differential metabolites. Trend analysis of DEGs and DAMs revealed that the former were significantly enriched in three distinct trends, whereas the latter were significantly enriched in only one. Moreover, 117 important hub genes associated with alfalfa response to aphid infestation were screened by WGCNA. By mapping DEGs and DAMs to KEGG pathways, it was found that the “phenylpropanoid biosynthesis”, “flavonoid biosynthesis”, and “isoflavonoid biosynthesis” pathways play an important role in alfalfa responses to aphid infestation. These results further elucidate the regulatory mechanism of alfalfa in response to aphid infestation and provide valuable information for breeding new aphid-resistant plant varieties.

Soil salinization severely restricts the growth and forage yield of Medicago sativa (alfalfa), a globally critical leguminous forage crop, yet the regulato