Highly preserved roles of Brassica MIR172 in polyploid Brassicas: ectopic expression of variants of Brassica MIR172 accelerates floral transition.

Functional characterization of regulatory genes governing flowering time is a research priority for breeding earliness in crop Brassicas. Highly polyploid genomes of Brassicas pose challenges in unraveling homeolog gene function. In Arabidopsis, five MIR172 paralogs control flowering time and floral organ identity by down-regulating AP2 and AP2-like genes. The impact of homeolog diversification on MIR172 loci, however, needs to be examined in morphologically diverse Brassicas. Herein, we analyze fractionation status and phylogeny of MIR172 and target AP2 from Brassicas and compare functionality of MIR172 variants representing distinct sub-genomes and progenitor genomes. Copy number analysis revealed higher retention of MIR172 loci relative to AP2 in diploid and amphi-diploid Brassica species. Dendrogram of 87 MIR172 sequences from Brassicaceae showed five major clusters corresponding to MIR172a-MIR172e which further separated into sub-genome and progenitor genome specific clades. Similar groupings were observed in the phylogeny of 11 Brassica AP2 and AP2-like genes. Over-expression of a pair of natural variants for each of MIR172b, MIR172d and MIR172e representing sub-genomes, progenitor genomes and species of Brassicas displayed floral acceleration in all transgenic lines indicating a strong selection pressure on MIR172. All gain-of-function lines, except 35S::MIR172e and 35S::MIR172e' displayed floral organ defects implying altered target spectrum of MIR172e relative to MIR172b and MIR172d. Expression of MIR172e caused marginal earliness in flowering time in B. juncea. In conclusion, this study demonstrates tightly preserved role of homeologs and natural variants of MIR172 family in mediating flowering in Brassicas and suggests their deployment for introgression of early flowering trait.