Computational identification of microRNAs in peach expressed sequence tags and validation of their precise sequences by miR-RACE

Twenty-two potential miRNAs from seven miRNA families were first predicted from more than 80,857 EST sequences of peach (Prunus persica). Using two specific 50 and 30 miRNA RACE (miR-RACE) PCR reactions and sequence-directed cloning, we accurately determined the precise sequences, especially both ends, of eight candidate miRNAs. The sequencing results demonstrated that the ppe-miRNAs were conserved to those that were predicted computationally except ppe-miR171b. We validated the existence of two members (ppe-miR171a and miR171b) of the miR171 family in peach that belonged to different precursors. qRT-PCR was further employed in analyzing expression of the eight miRNAs in peach leaves, flowers, and fruits at different developing stages, where some of the miRNAs showed tissue-specific expression.