Comprehensive high-throughput sequencing, evolutionary and functional analyses reveal the conservation and diversification of miR166s in regulating pleiotropic traits between rapeseed and Arabidopsis

Rapeseed yield is quantitatively controlled by polygenes and multiple environmental factors, it is difficult to regulate pleiotropic characteristics via a single gene. MicroRNAs (miRNAs) are considered fine-tuners owning broad impacts on complex traits. However, numerous studies have focused only on screening miRNAs that regulate a single trait, and no miRNAs have been cloned and proven to be key regulators controlling pleiotropic characteristics in rapeseed. Here, high-throughput sequencing analysis both revealed three differentially expressed miRNAs among extreme materials regards to silique length (SL) and harvest index (HI). Among them, bna-miR166f was the main candidate miRNAs expressed differentially among multiple-yield cultivars. Then, transgenic experience with heterologous expression (overexpression and knockdown) confirmed that bna-miR166f regulates pleiotropic traits, including flowering time, silique development and branch numbers by targeting to all the five HD-Zip IIIs in Arabidopsis. However, based on the transgenic validation of bna-miR166f in Arabidopsis and plentiful miRNA-Seq data among yield-related cultivars in rapeseed, it was found that partially opposite phenotypic traits (especially SL) are exhibited between Arabidopsis and rapeseed. Joint miRNA/mRNA-Seq analysis revealed no negative expression profiles between bna-miR166f and the 17 putative BnaHD-Zip IIIs. Phylogenetic analysis revealed high evolutionary conservation of miR166, while the HD-Zip family experienced severe genome triplication in rapeseed, and the partial functional diversity between Arabidopsis and rapeseed may be caused by evolutionary divergence of the targets. Taken together, these results revealed the pivotal role of bna-miR166f in the genetic control of pleiotropic characteristics, thereby providing a theoretical basis for molecular breeding to improve rapeseed yield in the future. •The yield of based material restricted development of rape production and biodiesel.•bna-miR166f was identified among multiple differently yielding cultivars.•bna-miR166f play pleiotropic roles in mediating yield related traits in Arabidopsis.•bna-miR166f targets to all the five HD-Zip III members in homologous Arabidopsis.•Conservation but function diversity of miR166s between rapeseed and Arabidopsis.