Global identification and analysis of microRNAs involved in salt stress responses in two alfalfa (Medicago sativa ‘Millennium’) lines

Alfalfa is an important economic crop; a mutant (M) strain was identified during planting and production. M plants consistently had better relative water content and relative electrical conductivity under higher salt conditions compared with the wild type (WT) plants, suggesting that M plants have higher tolerance for salt. To understand the microRNAs (miRNAs) involved in salt stress response in alfalfa, 128 miRNAs were identified from the WT and M alfalfa plants under normal and saline conditions. Of the 128 miRNAs, 29 and 23 differentially expressed miRNAs were identified in the M vs. WT control (M-CK vs. WT-CK) and salt-stressed M vs. WT (M-salt vs. WT-salt) comparison, respectively. These miRNAs responded to salt stress and showed different expression patterns after salt treatment. Their potential target genes were predicted and further analysed by GO classification and KEGG pathway analysis, where the majority of target genes were associated with plant growth and development, and exhibited significant changes in WT and M plants. In addition, compared with the WT plants, miR172-CNGC, miR319-CAX2, miR408-NHX and miR2590-CHX14/15 showed significant upregulation in M alfalfa plants, suggesting that M plants have higher ion transport levels. The differential expression profiles of miRNAs and putative target genes were further validated by quantitative real-time polymerase chain reaction. It is speculated that these miRNAs are involved in the increased salt tolerance of the M alfalfa plants.