Genome-wide identification and in silico characterisation of microRNAs, their targets and processing pathway genes in Phaseolus vulgaris L.

Common bean (Phaseolus vulgaris L., Fabaceae) is a globally important staple crop, which is an important source of calories, protein and essential micronutrients. At the genomic level little is known regarding the small non-coding RNAs within the common bean genome. One of the most important classes of such small non-coding RNAs is microRNAs (miRNAs), which control mRNA and protein expression levels in many eukaryotes. Computational methods have been applied to identify putative miRNAs in the genomes of different organisms. In this study, our objective was to comprehensively identify and characterise miRNAs from the genome and transcriptome of P. vulgaris, including both mature and precursor miRNA forms. We also sought to identify the putative proteins involved in miRNA processing and the likely target genes of common bean miRNAs. We identified 221 mature miRNAs and 136 precursor miRNAs distributed across 52 different miRNA families in the P. vulgaris genome. Amongst these, we distinguished 129 novel mature miRNAs and 123 miRNA precursors belonging to 24 different miRNA families. We also identified 31 proteins predicted to participate in the miRNA-processing pathway in P. vulgaris. Finally, we also identified 483 predicted miRNA targets, including many which corroborate results from other species, suggesting that miRNA regulatory systems are evolutionarily conserved and important for plant development. Our results expand the study of miRNAs and their target genes in common bean, and provide new opportunities to understand their roles in the biology of this important staple crop.

New Insights on Coffea miRNAs: Features and Evolutionary Conservation.

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.