Journal of Plant Physiology 168 (2011) 824–830 Contents lists available at ScienceDirect Journal of Plant Physiology journal homepage: www.elsevier.de/jplph Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons Mingxiong Pang a , Chaozhu Xing a,d , Nick Adams a , Laura Rodriguez-Uribe a , S.E. Hughs c , Stephen F. Hanson b , Jinfa Zhang a,∗ a Department of Plant and Environmental Sciences, New Mexico State University, MSC 3Q, Las Cruces, NM 88003, USA b Department of Entomology, Plant Pathology and Weed Science, New Mexico State University, Las Cruces, NM 88003, USA c Southwest Cotton Ginning Research Laboratory, Mesilla Park, NM 88047, USA d China Cotton Research Institute, Anyang, Henan 455000, China article info Article history: Received 5 May 2010 Received in revised form 19 October 2010 Accepted 30 October 2010 Keywords: Boll development Cotton Days post anthesis (DPA) MicroRNA (miRNA) miRNA-AFLP Quantitative RT-PCR abstract MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and character- ization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expres- sion comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression lev- els in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two culti- vated cotton cultivars, Pima Phy 76 (Gossypium barbadense) and Acala 1517-99 (Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA- AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirma- tion using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies. © 2010 Elsevier GmbH. All rights reserved. Introduction MicroRNAs (miRNAs) represent a class of non-coding small RNAs that are generally ∼21 nucleotides (nt) in length and function as post-transcriptional negative regulators through base pairing to nearly complementary sequences in the target mRNAs (Reinhart et al., 2002; Carrington and Ambros, 2003; Bartel, 2004). miRNAs regulate their targets through mRNA cleavage and translational inhibition (Bartel and Bartel, 2003; Carrington and Ambros, 2003; Yu et al., 2005; Bushati and Cohen, 2007) and are involved in plant development, hormone signaling, and responses to various environmental stresses (Jones-Rhoades and Bartel, 2004; Sunkar Abbreviations: AFLP, amplified fragment length polymorphism; DPA, days post anthesis; EST, expressed sequenced tag; PSA, pre-selective amplification; qRT-PCR, quantitative reverse transcription-polymerase chain reaction. ∗ Corresponding author. Tel.: +1 575 646 3438; fax: +1 575 646 6041. E-mail address: jinzhang@nmsu.edu (J.F. Zhang). and Zhu, 2004; Eckardt, 2005; Mallory et al., 2005; Zhang et al., 2006). miRNAs are processed from single-stranded, hairpin RNA precursors (Bartel, 2004). Although some miRNAs are transcribed by RNA polymerase III in both animals and plants, the major- ity of miRNA genes exist as independent transcriptional units, transcribed by RNA polymerase II into long primary transcripts (termed pri-miRNAs) which are then processed to mature miR- NAs (Bartel, 2004; Kurihara and Watanabe, 2004; Parizotto et al., 2004). miRNAs have been identified by both cloning and com- putational approaches based on the key features of all currently known miRNAs, which include a folded hairpin RNA precursor that is evolutionarily conserved (Ambros et al., 2003). High throughput sequencing has now become the method of choice in confirming existing miRNAs and identifying new ones in various plant species including cotton (Kwak et al., 2009; Pang et al., 2009; Ruan et al., 2009). The identification and characterization of the entire reper- toire of miRNAs and their target genes from model organisms is of fundamental importance to understanding regulatory networks that they modulate. 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.10.006