In their study, a total of 23 MIR1444s were identified. Populus and Idesia species were found to contain two MIR1444 genes, while Salix included only one. Populus and Idesia MIR1444b genes and Salix MIR1444s were phylogenetically separated from Populus and Idesia MIR1444a genes. Ptr-miR1444a and ptr-miR1444b showed sequence divergence. Compared with ptr-miR1444b, ptr-miR1444a started 2 nt upstream of precursor, resulting in differential regulation of PPO targets. Sequence alignment showed the MIR1444 genes exhibited extensive similarity to their PPO targets and suggest characteristics of MIRs originated from targets through an inverted gene duplication event. A genome sequence comparison showed that MIR1444 genes in Populus and Idesia were expanded through the Salicoid genome duplication event, while a copy of the MIR1444 gene was lost in Salix through DNA segment deletion during chromosome rearrangement.
These results provide significant information for the origin of plant miRNAs and the mechanism of Salicaceae gene evolution and divergence.
Proposed model for MIR1444 origin and evolution – MIR1444 genes were originated from PPOs through an inverted gene duplication event. It resulted in tail-to-tail orientations of complete or partial PPO gene sequences, which were diversified through sequence mutation to shorten and gain of bulges in the foldback structure. Continuous mutation generates MIR1444 genes with sequences and mismatch patterns in and surrounding the miR1444:miR1444* region. MIR1444 genes were expanded through the Salicoid genome duplication event and then further diversified through sequence mutation. A copy of the duplicated MIR1444 genes was lost through DNA segment deletion during chromosome rearrangement in Salix. Pink arrows indicate transcription direction of PPO genes.