Abstract
Background: MicroRNAs (miRNAs) are small, endogenously transcribed, non-protein-coding RNAs that play important roles in regulation of gene expression in animals and plants. Here, selective constraints on the novel precursor microRNA159 (pre-miR159) gene were investigated in 42 Phalaenopsis species (Orchidaceae). Methods/results: A novel precursor microRNA159 gene was isolated from 42 Phalaenopsis species using a new microRNA-PCR (miR-PCR) approach. Sequencing of pre-miR159 genes revealed differences from the canonical pre-miR159 gene in Phalaenopsis species and other plants. Results demonstrated that the 5' and 3' fold-back arms and the terminal loop of the novel pre-miR159 gene have undergone purifying selection and selective constraint for stabilizing the secondary hairpin structure. Two conserved motifs within the 5' fold-back arm had the highest purifying selective pressure within the novel pre-miR159 gene. Evidence of sequence co-evolution between the 5' and 3' fold-back regions was observed. Conclusions: Functional selective pressure might arise from the constraint of forming a hairpin structure and demonstrate co-evolution of sequences between the 5' and 3' fold-back regions of the novel pre-miR159 gene in Phalaenopsis species.