Identification of nitrogen starvation-responsive microRNAs in Chrysanthemum nankingense

MicroRNA (miRNA) is involved in many developmental processes and various abiotic stress responses in plants. As nitrogen is a limited element for plant growth, comparative analyses of miRNAs responding to low nitrogen stress is important for improving the nitrogen use efficiency (NUE). We used high-throughput sequencing to detect the response of miRNAs to low nitrogen stress in the roots and leaves of Chrysanthemum nankingense. Compared with the control, the differential expression was more than 2-fold in 81 miRNAs in roots and 101 miRNAs in leaves. The identified miRNAs showed overlapping or unique response to nitrate limitation in roots and leaves, including several members of known miRNA families with low nitrogen stress response, such as miR156, miR169, and miR393. The potential target genes of these miRNAs were also identified. The total amount of predicted target genes was 219, and the corresponding amount of matched miRNAs was 37 in roots and 44 in leaves. Moreover, we used 5' RLM-RACE to map the cleavage sites in four predicted target genes. The differential expression level of miRNAs and target genes was verified by quantitative real-time polymerase chain reaction (qRT-PCR). According to the functional characteristics of the predicted target genes, they were divided into three main categories: transcription factors, kinases, and metabolism.