Abstract
BACKGROUND: Alfalfa (Medicago sativa L.) is an important high-quality forage crop. Low temperature is an abiotic stress factor that affects the distribution and productivity of alfalfa. To further understand the molecular response to low temperature, and to identify additional genes and metabolic pathways associated with cold tolerance in alfalfa, in this study we conducted transcriptome sequencing, weighted gene co-expression network analysis, KEGG pathway enrichment analysis, and quantitative real-time PCR validation in alfalfa cultivars subjected to low-temperature treatment. RESULTS: Weighted gene co-expression network analysis revealed that three gene modules were significantly negatively correlated with the semi-lethal temperature for alfalfa. Genes in the three modules were used to construct gene co-expression networks, from which MS.gene46105, MS.gene044087, MS.gene76894, MS.gene44620, MS.gene22005, MS.gene045060, MS.gene31405, and MS.gene74761 were selected as important genes associated with cold tolerance. Quantitative real-time PCR analysis of these eight genes validated the reliability of the transcriptome sequencing data. In addition, further analysis of the genes within the three modules revealed that several transcription factors (AP2/ERF, bZIP, C3H, NAC, and others) and metabolic pathways (N-glycan biosynthesis, citrate cycle, glycolysis/gluconeogenesis, and carbon metabolism, and others) responded well to the low temperature. CONCLUSIONS: Three gene modules, eight genes, several transcription factors and multiple metabolic pathways associated with cold tolerance were screened. This results will provide a valuable reference for further clarification of the cold tolerance mechanism and breeding for cold tolerance in alfalfa.