Comparative genomics reveals origin of MIR159A-MIR159B paralogy, and complexities of PTGS interaction between miR159 and target GA-MYBs in Brassicaceae.

Whole-genome and segmental duplications coupled with sequence and functional diversification are responsible for gene family expansion, and morphological and adaptive diversity. Although broad contours of such processes are understood, detailed investigations on regulatory elements, such as miRNA-transcription factor modules, especially in non-model crop plants with complex genomes, are few. The present study was performed to understand evolutionary history of MIR159 family, and changes in the miRNA-binding site (MBS) of the targets MYB33, MYB65, and MYB101 that may affect post-transcriptional gene silencing. We established orthology and paralogy between members of MIR159 family by reconstructing the phylogeny based on 240 precursor sequences sampled across green plants. An unambiguous paralogous relationship between MIR159A and MIR159B was observed only in Brassicaceae which prompted us to analyze the origin of this paralogy. Comparative micro-synteny of ca. 100 kb genomic segments surrounding MIR159A, MIR159B, and MIR159C loci across 15 genomes of Brassicaceae revealed segmental duplication that occurred in the common ancestor of Brassicaceae to be responsible for origin of MIR159A-MIR159B paralogy; extensive gene loss and rearrangements were also encountered. The impact of polyploidy was revealed when the three sub-genomes-least fractionated (LF), moderately fractionated (MF1), and most fractionated (MF2) sub-genomes of Brassica and Camelina sativa-were analyzed. Extensive gene loss was observed among sub-genomes of Brassica, whereas those in Camelina were largely conserved. Analysis of the target MYBs revealed the complete loss of MYB33 homologs in a Brassica lineage-specific manner. Our findings suggest that mature miR159a/b /c are capable of targeting MYB65 across Brassicaceae, MYB33 in all species except Brassica, and MYB101 only in Arabidopsis thaliana. Comparative analysis of the mature miRNA sequence and the miRNA-binding site (MBS) in MYB33, MYB65, and MYB101 showed the complexity of regulatory network that is dependent on strict sequence complementarity potentially leading to regulatory diversity.