DOI: 10.1007/s10535-014-0441-6 BIOLOGIA PLANTARUM 58 (4): 627-640, 2014 627 Natural genetic variation in MIR172 isolated from Brassica species S.M. SHIVARAJ, P. DHAKATE, P. MAYEE, M.S. NEGI, and A. SINGH* Department of Biotechnology, TERI University, 10 Institutional Area, Vasant Kunj, Delhi -110070, India Abstract The present study reports a natural variation in microRNA172 (MIR172) family members isolated from six species of genus Brassica. The analysis of nucleotide polymorphism across 44 Brassica MIR172 homologs revealed a higher conservation in the predicted precursors relative to flanking regions. Single nucleotide polymorphisms (SNPs) were detected in miRNA and miRNA*. The 21-nt miRNA sequence was conserved in all MIR172 members except MIR172a. However, the miRNA* sequence was conserved only in MIR172a compared to A. thaliana. Non-canonical Brassica variants of precursor miR172a were detected wherein SNP at 5’ terminal in mature miR172a resulted in a sequence identical to mature miR172e. SNPs and indels in precursors resulted in varied stem-loop structures of differing stabilities (ΔG) implying a differential efficiency of miRNA biogenesis. A sequence based phylogram revealed ortholog specific groupings of MIR172 irrespective of genetic background. A Northern analysis in Brassica juncea displayed the cumulative expression of miR172 isoforms in all tissues representing different developmental stages with levels gradually increasing from vegetative to reproductive stages. Detection of high content of miR172 in roots indicates the possibility of additional roles of Brassica miR172 in root development. Additional key words: Arabidopsis thaliana, evolution, flowering, orthologs, polyploidy, single nucleotide polymorphism. Introduction MicroRNAs (miRNAs) are a class of small non-coding RNAs (~21-nt) known to regulate target gene expression by directing transcript cleavage, translation repression (Rhoades et al. 2006, Rogers and Chen 2013), methylation (Chellappan et al. 2010, Wu et al. 2010), and histone modification (Chuang and Jones 2007, Singh and Campbell 2013). Although most miRNA loci are transcribed by RNA polymerase II (Lee et al. 2004, Xie et al. 2005), a few mammalian and viral miRNAs are transcribed by RNA polymerase III (Borchert et al. 2006, Diebel et al. 2010). In plants, the primary transcript forms a stem-loop structure which is processed in a step-wise manner by DICER-LIKE 1 (DCL1) to release a precursor of miRNA followed by a miRNA:miRNA* duplex (Reinhart et al. 2002, Chen 2005). The mechanistic aspects of miRNA biogenesis have been described in detail by Rogers and Chen (2013). Deep sequencing and expressed sequence tag (EST) based predictions have led to characterization of majority of plant miRNAs (Zhang et al. 2006, Fahlgren et al. 2007, Subramanian et al. 2008, Sunkar et al. 2008, Colaiacovo et al. 2010). Comparative genomics approaches have also been employed to isolate orthologs of miRNA families (Kusumanjali et al. 2011, Kumari et al. 2013). Characterization of miRNA gene sequences from complex plant genomes has provided insights into evolution of miRNA homologs. Whereas paralogs are considerably diverse except in the region mapping to mature miRNA (Rhoades et al. 2006), orthologs display lower sequence divergence even in the regions flanking mature miRNA (Zhang et al. 2006). Plant miRNAs are implicated in regulation of key developmental phases (Khraiwesh et al. 2012, Liu et al. 2013). The miR172 regulates processes, such as flowering, tuberization, and nodulation (Aukerman and Sakai 2003, Martin et al. 2009, Yan et al. 2013). In  Submitted 16 October 2013, last revision 11 February 2014, accepted 25 February 2014. Abbreviations: DAS - days after sowing; pre-miRNA - precursor microRNA; SNP - single nucleotide polymorphism. Acknowledgements: This work was supported by grants (BT/PR10071/AGR/36/31/2007 and BT/PR628/AGR/36/674/2011) received from the Department of Biotechnology, Govt. of India. The authors thank Dr. S.B. Tripathi and Dr. A. Agnihotri for offering valuable suggestions and sharing germplasm resources. Karuna Kusumanjali and Gunjan Kumari are acknowledged for technical assistance. The contribution of Mr. Hari Ram Gupta in field-related activities is duly recognized. Financial assistance as SRF to S.M. Shivaraj and Pratiksha Mayee from the Department of Biotechnology, Govt. of India, and to Priyanka Dhakate from the Council of Scientific and Industrial Research, Govt. of India is gratefully acknowledged. Infrastructural support from TERI and the TERI University are duly acknowledged. * Corresponding author: fax: (+91) 11 26122874, e-mail: asingh@teri.res.in