High-throughput sequencing identification of novel and conserved miRNAs in the Brassica oleracea leaves
Plant microRNAs are short (~21 nt), non-coding molecules that regulate gene expression by targeting mRNA cleavage or protein translation inhibition. In this manner, they play many important roles in the cells of living organisms. One of the plant species in which the entire set of miRNAs has not been yet completely identified is Brassica oleracea var. capitata (cabbage). For this reason and for the economic and nutritional importance of this food crop, Illumina high-throughput small RNAs sequencing has been performed to discover novel and conserved miRNAs in the mature cabbage leaves. In this study, raw reads generated from three small RNA libraries were bioinformatically processed and further analyzed to select sequences homologous to known B. oleracea and other plant miRNAs. As a result of this step, 267 conserved miRNAs (belonging to 62 families) have been discovered. The remaining unannotated tags were used in the prediction and evaluation of novel miRNAs, which resulted in the 26 potential miRNAs proposal. The expression of 13 selected miRNAs was analyzed by northern blot hybridization. To determine the functions and processes that these molecules may potentially regulate in cabbage leaves, GO annotation, GO enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping of the predicted targets were performed. Taken together, for the first time, the large set of miRNAs was identified in the mature cabbage leaves. Potential target designation for these miRNAs may suggest their important role in the plant growth, development and other primary biological processes. The presented study not only supplements the knowledge about B. oleracea miRNAs, but also may be used in other researches concerning the improvement of the cabbage cultivation. Overall design: High-throughput sequencing of three small RNA libraries was performed to identified conserved and novel miRNAs in mature cabbage leaves, while the potential targets designation was carried out to suggest the functions and roles that identified miRNAs might play in this food crop.