ORIGINAL ARTICLE Sequence and functional characterization of MIRNA164 promoters from Brassica shows copy number dependent regulatory diversification among homeologs Aditi Jain 1,2 & Saurabh Anand 1 & Neer K Singh 1 & Sandip Das 1 Received: 1 December 2017 /Revised: 12 February 2018 /Accepted: 26 February 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract The impact of polyploidy on functional diversification of cis-regulatory elements is poorly understood. This is primarily on account of lack of well-defined structure of cis-elements and a universal regulatory code. To the best of our knowledge, this is the first report on characterization of sequence and functional diversification of paralogous and homeologous promoter elements associated with MIR164 from Brassica. The availability of whole genome sequence allowed us to identify and isolate a total of 42 homologous copies of MIR164 from diploid species—Brassica rapa (A-genome), Brassica nigra (B-genome), Brassica oleracea (C-genome), and allopolyploids-Brassica juncea (AB-genome), Brassica carinata (BC-genome) and Brassica napus (AC-genome). Additionally, we retrieved homologous sequences based on comparative genomics from Arabidopsis lyrata, Capsella rubella, and Thellungiella halophila, spanning ca. 45 million years of evolutionary history of Brassicaceae. Sequence comparison across Brassicaceae revealed lineage-, karyotype, species-, and sub-genome specific changes providing a snapshot of evolutionary dynamics of miRNA promoters in polyploids. Tree topology of cis-elements associated with MIR164 was found to re-capitulate the species and family evolutionary history. Phylogenetic shadowing identified transcription factor binding sites (TFBS) conserved across Brassicaceae, of which, some are already known as regulators of MIR164 expression. Some of the TFBS were found to be distributed in a sub-genome specific (e.g., SOX specific to promoter of MIR164c from MF2 sub-genome), lineage-specific (YABBY binding motif, specific to C. rubella in MIR164b), or species-specific (e.g., VOZ in A. thaliana MIR164a) manner which might contribute towards genetic and adaptive variation. Reporter activity driven by promoters associated with MIR164 paralogs and homeologs was majorly in agreement with known role of miR164 in leaf shaping, regulation of lateral root development and senescence, and one previously un-described novel role in trichome. The impact of polyploidy was most profound when reporter activity across three MIR164c homeologs were compared that revealed negligible overlap, whereas reporter activity among two homeologs of MIR164a displays significant overlap. A copy number dependent cis-regulatory divergence thus exists in MIR164 genes in Brassica juncea. The full extent of regulatory diversification towards adaptive strategies will only be known when future endeavors analyze the promoter function under duress of stress and hormonal regimes. Keywords Brassicaceae . miRNA . Polyploid . Paralogs . miR164 . Promoter . Homeolog . Phylogenetic shadowing Introduction Polyploidization is a major evolutionary force providing op- portunities for organism to diversify and innovate in form and function (Adams and Wendel 2005; Soltis et al. 2009; Madlung and Wendel 2013). Study of paralogous genes resulting from duplication event is central to understanding how polyploidy can act as a driver for phenotypic and adap- tive novelties (Kellogg 2004; Stastny et al. 2005; te Beest et al. 2012). Diversification of a gene function can either occur through sequence level changes causing altered protein Saurabh Anand and Neer K Singh contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10142-018-0598-8) contains supplementary material, which is available to authorized users. * Sandip Das sdas@botany.du.ac.in; sandipdas04@gmail.com 1 Department of Botany, University of Delhi, New Delhi 110007, India 2 Present address: Department of Biotechnology, TERI University, Plot No. 10, Institutional Area, Vasant Kunj, New Delhi 110070, India Functional & Integrative Genomics https://doi.org/10.1007/s10142-018-0598-8