Identification And Functional Analysis Of MicroRNAs And Their Targets In Foxtail Millet (Setaria Italica)

microRNAs (miRNAs) are a class of～21 nt non-coding, endogenous RNAs that play important roles in various aspects such as organ development, cell differentiation and stress response. Foxtail millet is an important food and energy crops. It has excellent drought tolerance and water-use efficiency. In this study, we aimed to genome-wide characterize miRNAs in foxtail millet, and identify miRNA targets through the degradome sequencing and reveale miRNA-precursor processing modes. We also performed a global analysis of mRNA degradation under drought stress and uncovered diverse drought-response mechanisms in foxtail millet.Based on our sequenced small RNAs for shoots (14-day-old) and three small RNA datasets (leaf, flower and root) downloaded from a previously reported small RNA-seq study. We identified 43 known miRNAs,172 novel miRNAs and 2 mirtron precursor candidates in foxtail millet and studied their expression profiles in four different tissues. The result shown that, the different miRNA families showed distinct expression patterns and some members of the same miRNA family had different tissue-specific expression patterns. The stem-loop qRT-PCR was conducted to confirm the tissue-specific expression of 10 miRNAs. Potential targets of the foxtail millet miRNAs were predicted based on our strict criteria. In total,447 candidate target genes for 166 miRNAs were identified. GO analysis indicated that the targets of the miRNAs were involved in a wide range of regulatory functions and some specific biological processes. Four targets were confirmed by 5’RACE. A total of 69 pairs of syntenic miRNA precursors that were conserved between foxtail millet and sorghum were found.In order to identify miRNA targets and uncovered drought-response mechanisms of uncapped mRNA in foxtail millet, we profiled uncapped transcripts using degradome sequencing in foxtail millet seedlings. Degradome libraries were prepared from seedlings subjected to polyethylene glycol (PEG)-simulated drought and control conditions. The 238 predicted miRNA—target interactions were identified in the degradome sequencing data. The degradome sequencing analysis also suggested that two miRNA hairpin processing mechanisms—"loop-last" and "loop-first" processing—operate in foxtail millet.When both gene degradation (degradome-sequencing) and gene transcription (RNA-sequencing) data were considered, we classified drought-responsive genes into four types according to their characteristic changes. Type-I genes were characterized by transcript and uncapped transcript abundances changing in the same direction after drought treatment. The GO enrichment analysis indicated that these genes were enriched in various oxidationrelated enzymes, suggesting that these types of genes may be important regulators of reactive oxygen species removal to maintain redox balance under drought stress conditions. Genes in the type-Ⅱ category, comprising genes showing significant changes in uncapped transcript abundance after drought treatment but no changes in transcript abundance, could not be detected by analysis of RNA-seq data alone. Detailed functional annotation revealed that many of the type-Ⅱ genes belonged to diverse families of transcription factors such as WRKYs, MYBs and NACs. In contrast, the amounts of degraded type-IV gene mRNAs remained unchanged after drought treatment, whereas synthesized mRNA quantities showed obvious alterations. In the GO analysis, catalysis-related genes, such as hydrolases, isomerases and peroxidases were obviously over represented among type-Ⅳ genes, implying that these type-IV genes may serve as an intrinsic defense tool to resist drought stress. Finally we found 11 C4 photosynthesis-related enzymes encoded by drought-responsive genes.Furthermore, the function of sit-miR396, which accumulates preferentially in the root, were analyzed. Heterologous expression of sit-miR396 caused loss of smaller leaf area, more leaf number, shorter root and plant height in Arabidposis. Over-expression of sit-miR396 in foxtail millet, resulted in shorter plant height and root. The 10 potential targets of sit-miR396 were predicted. Functional annotation revealed that 10 targets are all growth regulator factors (GRFs), and 7 targets were identified in the degradome sequencing data. The qRT-PCR assay showed that the transcriptional levels of six GRFs decreased in sit-miR396 transgenic foxtail millet. Further studies about the role of sit-miR396 in root development are needed.