| Summary: | <p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) posttranscriptionally down-regulate gene expression by binding target mRNAs. Analysis of the evolution of miRNA binding sites is helpful in understanding the co-evolution between miRNAs and their targets. To understand this process in plants a comparative analysis of miRNA-targeted duplicated gene pairs derived from a well-documented whole genome duplication (WGD) event in combination with a population genetics study of six experimentally validated miRNA binding sites in rice (<it>O. sativa</it>) was carried out.</p> <p>Results</p> <p>Of the 1,331 pairs of duplicate genes from the WGD, 41 genes (29 pairs) were computationally predicted to be miRNA targets. Sequence substitution analysis indicated that the synonymous substitution rate was significantly lower in the miRNA binding sites than their 5' and 3' flanking regions. Of the 29 duplicated gene pairs, 17 have only one paralog been targeted by a miRNA. This could be due to either gain of a miRNA binding site after the WGD or because one of the duplicated genes has escaped from being a miRNA target after the WGD (loss of miRNA binding site). These possibilities were distinguished by separating miRNAs conserved in both dicots and monocot plants from rice-specific miRNAs and by phylogenetic analysis of miRNA target gene families. The gain/loss rate of miRNA binding sites was estimated to be 3.0 × 10<sup>-9 </sup>gain/loss per year. Most (70.6%) of the gains/losses were due to nucleotide mutation. By analysis of cultivated (<it>O. sativa</it>; <it>n </it>= 30) and wild (<it>O. rufipogon</it>; <it>n </it>= 15) rice populations, no segregating site was observed in six miRNA binding sites whereas 0.12–0.20 SNPs per 21-nt or 1.53–1.80 × 10<sup>-3 </sup>of the average pairwise nucleotide diversity (π) were found in their flanking regions.</p> <p>Conclusion</p> <p>Both molecular evolution and population genetics support the hypothesis that conservation of miRNA binding sites is maintained by purifying selection through elimination of deleterious alleles. Nucleotide mutations play a major role in the gain/loss of miRNA binding sites during evolution.</p>
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