Bioinformatics prediction of miRNAs in the Prunus persica genome with validation of their precise sequences by miR-RACE

We predicted 262 potential MicroRNAs (miRNAs) belonging to 70 miRNA families from the peach (Prunus persica) genome and two specific 5′ and 3′ miRNA rapid amplification of cDNA ends (miR-RACE) PCR reactions and sequence-directed cloning were employed to accurately validate 61 unique P. persica miRNAs (Ppe-miRNAs) sequences belonging to 61 families comprising 97 Ppe-miRNAs. Validation of the termini nucleotides in particular can define the real sequences of the Ppe-miRNAs on peach genome. Comparison between predicted and validated Ppe-miRNAs through alignment revealed that 43 unique orthologous sequences were identical, while the remaining 18 exhibited some divergences at their termini nucleotides. Quantitative real-time polymerase chain reaction (qRT-PCR) was further employed to analyze the expression of all the 61 miRNAs and 10 putative targets of 8 randomly selected Ppe-miRNAs in peach leaves, flowers and fruits at different stages of development, where both the miRNAs and the putative target genes showed tissue-specific expression.