ORIGINAL ARTICLE microRNA profiling of root tissues and root forming explant cultures in Medicago truncatula Rodney P. Eyles • Philip H. Williams • Stephen J. Ohms • Georg F. Weiller • Huw A. Ogilvie • Michael A. Djordjevic • Nijat Imin Received: 19 February 2013 / Accepted: 8 March 2013 / Published online: 10 April 2013 Ó Springer-Verlag Berlin Heidelberg 2013 Abstract Plant root architecture is regulated by the ini- tiation and modulation of cell division in regions contain- ing pluripotent stem cells known as meristems. In roots, meristems are formed early in embryogenesis, in the case of the root apical meristem (RAM), and during organo- genesis at the site of lateral root or, in legumes, nodule formation. Root meristems can also be generated in vitro from leaf explants cultures supplemented with auxin. mi- croRNAs (miRNAs) have emerged as regulators of many key biological functions in plants including root develop- ment. To identify key miRNAs involved in root meristem formation in Medicago truncatula, we used deep sequencing to compare miRNA populations. Comparisons were made between: (1) the root tip (RT), containing the RAM and the elongation zone (EZ) tissue and (2) root forming callus (RFC) and non-root forming callus (NRFC). We identified 83 previously reported miRNAs, 24 new to M. truncatula, in 44 families. For the first time in M. truncatula, members of conserved miRNA families miR165, miR181 and miR397 were found. Bioinformatic analysis identified 38 potential novel miRNAs. Selected miRNAs and targets were validated using Taqman miRNA assays and 5 0 RACE. Many miRNAs were differentially expressed between tissues, particularly RFC and NRFC. Target prediction revealed a number of miRNAs to target genes previously shown to be differentially expressed between RT and EZ or RFC and NRFC and important in root development. Additionally, we predict the miRNA/ target relationships for miR397 and miR160 to be con- served in M. truncatula. Amongst the predictions, were AUXIN RESPONSE FACTOR 10, targeted by miR160 and a LACCASE-like gene, targeted by miR397, both are miRNA/target pairings conserved in other species. Keywords Auxin Á Cytokinin Á Deep sequencing Á Elongation zone Á Root tip Abbreviations AGO Argonaute AP2 Apetala-2 ARF Auxin response factor CIP Cow intestinal phosphotase DCL Dicer-like ENDO1 Endonuclease-1 EZ Elongation zone HB1 Homeobox-1 HD-ZIP1 Homeodomain-zip1 LOB Lateral organ boundary NRFC Non-root forming callus QC Quiescent centre qRT-PCR Quantitative real-time PCR Electronic supplementary material The online version of this article (doi:10.1007/s00425-013-1871-7) contains supplementary material, which is available to authorized users. R. P. Eyles Á H. A. Ogilvie Á M. A. Djordjevic (&) Á N. Imin Plant Science Division, Research School of Biology, College of Medicine, Biology and Environment, Australian National University, Canberra, ACT 0200, Australia e-mail: michael.djordjevic@anu.edu.au P. H. Williams Á G. F. Weiller Bioinformatics Laboratory, Research School of Biology, Division of Plant Sciences, College of Medicine, Biology and Environment, Australian National University, Canberra, ACT 0200, Australia S. J. Ohms ACRF Biomolecular Resource Facility, John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia 123 Planta (2013) 238:91–105 DOI 10.1007/s00425-013-1871-7