Abstract
Winter damage, especially in northern climates, is a major limitation of the utilization of perennial forages such as alfalfa. Therefore, improving freezing tolerance is imperative in alfalfa genetic breeding. However, freezing tolerance is a complex trait that is determined by many genes. To understand the complex regulation mechanisms of freezing tolerance in alfalfa, we performed small RNA sequencing analysis under cold (4°) and freezing (-8°) stress. The sequencing results revealed that 173 known, and 24 novel miRNAs were expressed, and that the expression of 35 miRNAs was affected by cold and/or freezing stress. Meanwhile, 105 target genes cleaved by these miRNAs were characterized by degradome sequencing. These targets were associated with biological regulation, cellular processes, metabolic processes, and response to stress. Interestingly, most of them were characterized as transcription factors (TFs), including auxin response factors, SBP, NAC, AP2/ERF, and GRF, which play important roles in plant abiotic responses. In addition, important miRNAs and mRNAs involved in nodulation were also identified, for example, the relationship between miR169 and the TF CCAAT (also named as NF-YA/HAP2), which suggested that nodulation has an important function in freezing tolerance in alfalfa. Our results provide valuable information to help determine the molecular mechanisms of freezing tolerance in alfalfa, which will aid the application of these miRNAs and their targets in the improvement of freezing tolerance in alfalfa and related plants.