Identification of apple miRNAs and their potential role in fire blight resistance

MicroRNAs (miRNAs) are key players in multiple biological processes; therefore, analysis and characterization of these small regulatory RNAs is a critical step toward a better understanding of animal and plant biology. In apple (Malus domestica), 200 microRNAs are known, which most probably represent only a fraction of miRNAome diversity. As a result, more effort is required to better annotate miRNAs and their functions in this economically important species. We performed deep sequencing of 12 small RNA libraries obtained for fire blight-resistant and fire blight-sensitive trees. In the sequencing results, we identified 116 novel microRNAs and confirmed a majority of previously reported apple miRNAs. We then experimentally verified selected candidates with RT-PCR and stem-loop quantitative PCR (qPCR) and performed differential expression analysis. Finally, we identified and characterized putative targets of all known apple miRNAs. The gene ontology (GO) enrichment analysis suggests prominent roles of miRNAs in response to stresses, including pathogen infection. In this study, we identified 116 new and confirmed the expression of 143 already known miRNAs. Moreover, our data suggests that apple microRNAs might be considered as regulators and markers of fire blight resistance. The analyses we performed allowed us to define four apple miRNAs potentially involved in fire blight resistance in apple trees: mdm-miR169a, mdm-miR160e, mdm-miR167b-g, and mdm-miR168a,b. These miRNAs are known to be involved in response to stresses across other plant species, usually by targeting stress response proteins. The relatively low number of candidates may result from the high variance of biological replicates and the fact that stress response miRNAs are usually induced by the stress factors and frequently expressed at a low level, or not expressed at all, in normal conditions. The results of our studies are freely available in an online database at http://lemur.amu.edu.pl/share/apple_miRNAs/ .