Identification Of Micrornas And Selection Of Suitable Inner Reference Genes For Normalization Of Micrornas Expression In Peach

MicroRNAs are a class of small, endogenous, non-coding RNA molecules that play a crucial role in various biological processes, including growth and development, tissues or organ differentiation and stress responses. Peach is an economically important group of cultivated fruits and is considered as a model species in the Rosaceae. However the regulatory network of miRNA and their target genes has not been reported previously. Thus, identification of miRNA and their targets can provide more information about the regulatory network of miRNAs and advances our understanding of miRNA functions during growth and development in peach. First of all, we predicted miRNAs and their targets using peach genome by bioinformatics method. Some of the predicted miRNAs and their targets were validated by RT-qPCR and RLM-RACE (RNA ligase-mediated5’rapid amplification of cDNA ends) method, respectively. In addition, conserved miRNAs and peach-specific miRNAs were identified by high-throughput sequencing. The targets of conserved miRNAs and species-specific miRNAs were found by degradome analysis. Finally, suitable inner reference genes for normalization of microRNAs expression were selected in peach during abiotic stresses. The main results are as follows:1. We predicted peach miRNAs and their targets using peach genome and miRBase database by bioinformatics method.110miRNA, belonging to37miRNA families, and43miRNA target genes were predicted based on the miRNA high conservatism in plant and the stem-loop structure of miRNA precursor. Subsequently,25conserved miRNA was validated and qualified in different tissues by RT-qPCR. It was shown that miRNAs were differentialy expressed in different tissues. Furthermore, two target genes were experimentally verified by detection of the miRNA-mediated mRNA cleavage sites in peach using RLM-RACE method. It was shown that the cleavage sites were located in the miRNA and its target gene pairing region between10nt and11nt.2.721conserved miRNA, belonging to46miRNA families, and341potential peach-specific miRNA were identified by high-throughput sequencing.30conserved miRNA and15peach-specific miRNA candidates were qualified in different tissues by RT-qPCR.3. Three degradome libraries were constructed from peach leaves, stems and flowers for deep sequencing. After analysis,655and85targets of conserved miRNAs and peach-specific miRNAs were detected.4. Two common used housekeeping genes and15ppe-miRNAs (Prunus persica miRNAs) were selected as reference candidates, and their expression stability was investigated in different peach tissues as well as after three abiotic stress treatments. After analysis by GeNorm and NormFinder, it was found that the pair of miR5059and miR5072was the best reference genes in peach during abiotic stresses...