Computational identification of microRNAs in the strawberry (Fragaria × ananassa) genome sequence and validation of their precise sequences by miR-RACE Jian Han a,1 , Aying Li a,1 , Hong Liu b , Xicheng Wen a , Mizhen Zhao b , Nadira Bilkish Korir a , Nicholas Kibet Korir c , Chen Wang a , Jinggui Fang a, ⁎ a College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China b Department of Horticulture, Nanjing Jinling Institute of Technology, Nanjing 210038, China c Department of Agricultural Technology, Kenyatta University, P.O. Box, 43844-00100 Nairobi, Kenya abstract article info Article history: Accepted 12 November 2013 Available online 11 December 2013 Keyword: Strawberry Bioinformatics MicroRNAs miRNA RACE Genome database In plants, microRNAs (miRNAs) play significant roles in post-transcriptional gene regulation and have been found to control many genes involved in different biological and metabolic processes. Extensive studies were carried out to discover miRNAs and analyze their functions in model plant species, such as in Arabidopsis and rice that have been reported. In this research, we used bioinformatics to predict microRNAs in an important strawberry rootstock cultivar to discover and validate precise sequences of microRNAs in strawberry. By adopting a range of filtering criteria, we obtained 59 potential miRNAs belonging to 40 miRNA families from the Fragaria vesca genome. Using two specific5′ and 3′ miRNA RACE PCR reactions and a sequence-directed cloning method, we accurately determined 34 precise sequences of candidate miRNAs, while six other sequences exhibited some minor divergence in their termini nucleotides, and 19 miRNAs that could not be cloned owing to expression abundance may be too low or these mirRNAs predicted could not be existing in strawberry. Potential target genes were further predicted for the miRNAs above. The expression of the 16 miRNAs unreported and having exact sequences and their targets by experiment could be detected in different tissues of strawberry ranging from roots, stems, leaves, flowers and fruits by qRT-PCR and some of them showed differential expression in var- ious tissues. The functional analysis of 16 miRNAs and their targets was carried out. Finally, we conclude that there are 34 mirRNAs in strawberry and their targets play vital roles not only in growth and development, but also in diverse physiological processes. These results show that regulatory miRNAs exist in agronomically impor- tant strawberry and might have an important function in strawberry growth and development. © 2013 Elsevier B.V. All rights reserved. 1. Introduction MicroRNAs are a series of non-coding RNAs that regulate gene expres- sion at the post-transcriptional level, either by endonucleolytic cleavage or by translational inhibition, and they also play important roles in gene silencing (Chen, 2004; Lauter et al., 2005; Llave et al., 2002). In plants, miRNAs are transcribed by RNA polymerase II into long primary tran- scripts (pri-miRNAs) (B. Zhang et al., 2006; Chen et al., 2005), and they are cut into miRNA precursors (pre-miRNAs) with typical hairpin struc- ture(s). Mature miRNAs are generated from the stem portion of single stranded stem–loop precursors by complexes containing the nuclear RNase III enzyme and the ribonuclease III-like enzyme Dicer (DCL1) (Kurihara and Watanabe, 2004), then the mature miRNAs are incorporat- ed into the RNA-induced silencing complex (RISC) and guide RISC to com- plementary mRNA targets. Finally, the RISC inhibits translation elongation or triggers the degradation of target mRNAs (Lin et al., 2005). In recent years, the discovery of many small RNAs has aroused a great deal of inter- est in post-transcriptional regulation of gene expression during develop- ment and other biological processes. Increasing evidence indicates that miRNAs play major roles in key aspects of plant development and their responses to environmental stresses (Allen et al., 2005; Berezikov et al., 2006; Carthew and Sontheimer, 2009; Chen et al., 2005). The fact that a large number of known miRNAs in the plant kingdom ranging from mosses and ferns to higher flowering plants are vastly evolutionarily con- served has been used as a practical indicator for prediction of miRNAs by homology searches in other species (B. Zhang et al., 2006; Sunkar et al., 2008) and provides powerful evidence supporting prediction and valida- tion of conserved miRNAs and their target genes from all plant species by bioinformatics and experimental methods (Axtell and Bartel, 2005; Fahlgren et al., 2007). Gene 536 (2014) 151–162 Abbreviations: miRNAs, microRNAs; qRT-PCR, quantitative real-time PCR; DCL1, the ri- bonuclease III-like enzyme Dicer; RISC, the RNA-induced silencing complex; UPE, unpaired energy; LMW, low molecular weight; EST, expressed sequence tag; Fve-mirRNA, Fragaria vesca microRNA; CDS, coding sequence. ⁎ Corresponding author. E-mail address: fanggg@njau.edu.cn (J. Fang). 1 These authors contributed equally to this study. 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.11.023 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene