Genome wide identification,characterization and validation of novel miRNA-based SSR markers in pomegranate ( Punica granatum L.)

A total of 17,439 mature miRNAs (~ 21 nt) earlier generated through RNA seq in the pomegranate were used for in silico analysis. After complexity reduction, a total of 1922 representative mature miRNAs were selected and used as query sequences against pomegranate genome to retrieve 2540 homologous contigs with flanking regions (~ 800). By using pre-miRNA prediction web server, a total of 1028 true contigs harbouring pri-miRNAs encoding 1162 pre-miRNAs were identified. Survey of these sequences for SSRs yielded a total of 1358 and 238 SSRs specific to pri-miRNA and pre-miRNAs, respectively. Of these, primer pairs were designed for 897 pri-miRNA and 168 pre-miRNA SSRs. In pri-miRNA sequences, hexa-nucleotides repeats were found to be most abundant (44.18%) followed by mono- (18.41%) and di-nucleotide (17.01%), which is also observed in pre-miRNA sequences. Further, a set of 51 randomly selected pre-miRNA-SSRs was examined for marker polymorphism. The experimental validation of these markers on eight pomegranate genotypes demonstrated 92.15% polymorphism. Utility of these functional markers was confirmed via examination of genetic diversity of 18 pomegranate genotypes using 15 miRNA-SSRs. Further, potential application of miRNA-SSRs for discovery of trait specific candidate genes was showed by validating 51 mature miRNA against publically available 2047 EST sequences of pomegranate by target and network analysis. In summary, the current study offers novel functional molecular markers for pomegranate genetic improvement.     

A bioinformatics-based update on microRNAs and their targets in rainbow trout (Oncorhynchus mykiss)

MicroRNAs (miRNAs) participate in various vitally biological processes via controlling target genes activity and thousands of miRNAs have been identified in many species to date, including 18,698 known animal miRNA in miRBase. However, there are only limited studies reported in rainbow trout (Oncorhynchus mykiss) especially via the computational-based approaches. In present study, we systematically investigated the miRNAs in rainbow trout using a well-developed comparative genome-based homologue search. A total of 196 potential miRNAs, belonging to 124 miRNA families, were identified, most of which were firstly reported in rainbow trout. The length of miRNAs ranged from 17 to 24 nt with an average of 20 nt while the length of their precursors varied from 47 to 152 nt with an average of 85 nt. The identified miRNAs were not evenly distributed in each miRNA family, with only one member per family for a majority, and multiple members were also identified for several families. Nucleotide U was dominant in the pre-miRNAs with a percentage of 30.04%. The rainbow trout pre-miRNAs had relatively high negative minimal folding free energy (MFE) and adjusted MFE (AMFE). Not only the mature miRNAs but their precursor sequences are conserved among the living organisms. About 2466 O. mykiss genes were predicted as potential targets for 189 miRNAs. Gene Ontology (GO) analysis showed that nearly 2093, 2107, and 2081 target genes are involved in cellular component, molecular function, and biological processes respectively. KEGG pathway enrichment analysis illuminated that these miRNAs targets might regulate 105 metabolic pathways, including those of purine metabolism, nitrogen metabolism, and oxidative phosphorylation. This study has provided an update on rainbow trout miRNAs and their targets, which represents a foundation for future studies.     

Discovery of novel microRNAs in rat kidney using next generation sequencing and microarray validation

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. The latest version of the miRBase database (Release 18) includes 1,157 mouse and 680 rat mature miRNAs. Only one new rat mature miRNA was added to the rat miRNA database from version 16 to version 18 of miRBase, suggesting that many rat miRNAs remain to be discovered. Given the importance of rat as a model organism, discovery of the completed set of rat miRNAs is necessary for understanding rat miRNA regulation. In this study, next generation sequencing (NGS), microarray analysis and bioinformatics technologies were applied to discover novel miRNAs in rat kidneys. MiRanalyzer was utilized to analyze the sequences of the small RNAs generated from NGS analysis of rat kidney samples. Hundreds of novel miRNA candidates were examined according to the mappings of their reads to the rat genome, presence of sequences that can form a miRNA hairpin structure around the mapped locations, Dicer cleavage patterns, and the levels of their expression determined by both NGS and microarray analyses. Nine novel rat hairpin precursor miRNAs (pre-miRNA) were discovered with high confidence. Five of the novel pre-miRNAs are also reported in other species while four of them are rat specific. In summary, 9 novel pre-miRNAs (14 novel mature miRNAs) were identified via combination of NGS, microarray and bioinformatics high-throughput technologies.     

Investigation on the Conserved MicroRNA Genes in Higher Plants

Analysis of evolving microRNA repertoires within the plant domain can further corroborate our understanding of genome evolution and plasticity. An extensive collection of relatively unbiased miRBase-registered plant miRNAs and predicted unlisted MIRs from 23 plant ESTs were examined. As a result, 4324 pre-miRNAs were predicted and classified in 656 miRNA gene families with mostly being transposons (57.81%). From 216 newly identified pre-miRNAs, 103 distinct types belonged to reduced complexity/repeated regions. Collinearity between the numbers of miRNAs in each species with the relevant sizes of genomes was absent. Duplications of MIRs were evident, with higher MIR paralogs in Liliopsida compared with dicots. Due to the lack of an apparent pattern of phylogeny, Dollo maximum parsimony was used that established the acceleration of gains and potential losses of miRNA gene families within Mesangiospermae during the last 200 million years ago. Phylogenetic analysis of Liliopsida in contrast to Eudicotyledons agreed with the reconstructed tree based on the possible expansion of distinguished MIR families. In marked contrast to dicots, the degrees of resemblance in Liliopsida were higher than their direct predecessors. Analyses of recent monophyletic lineages were illustrative of miRNA horizontal genes transfer.

     

Characterization of microRNAs from sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>) using computational and experimental analyses

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA* and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA* had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3′UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA* in sheep.     

Identification and characterization of microRNAs in Asiatic cotton (Gossypium arboreum L.)

To date, no miRNAs have been identified in the important diploid cotton species although there are several reports on miRNAs in upland cotton. In this study, we identified 73 miRNAs, belonging to 49 families, from Asiatic cotton using a well-developed comparative genome-based homologue search. Several of the predicted miRNAs were validated using quantitative real time PCR (qRT-PCR). The length of miRNAs varied from 18 to 22 nt with an average of 20 nt. The length of miRNA precursors also varied from 46 to 684 nt with an average of 138 ±120 nt. For a majority of Asiatic cotton miRNAs, there is only one member per family; however, multiple members were identified for miRNA 156, 414, 837, 838, 1044, 1533, 2902, 2868, 5021 and 5142 families. Nucleotides A and U were dominant, accounted for 62.95%, in the Asiatic cotton pre-miRNAs. The Asiatic cotton pre-miRNAs had high negative minimal folding free energy (MFE) and adjusted MFE (AMFE) and high MFE index (MFEI). Many miRNAs identified in Asiatic cotton suggest that miRNAs also play a similar regulatory mechanism in diploid cotton.     

月季‘绿萼’花器官发育相关microRNA的鉴定及分析

利用高通量测序技术,构建了中国古老月季‘绿萼’(Rosa chinensis ‘Viridiflora’)和‘月月粉’(R.chinensis‘Old Blush’)花蕾期的microRNA(miRNA)文库,并对其进行了测序和序列分析。结果显示,在‘绿萼’文库中,鉴定到已知的miRNA成熟体39个,miRNA前体42个;预测到新的miRNA成熟体56个,前体57个。在‘月月粉’文库中,鉴定到已知RNA成熟体39个,已知miRNA前体40个;预测到新的miRNA成熟体53个,前体57个。与‘月月粉’相比,‘绿萼’中差异表达的miRNA有31个,其中17个上调、14个下调。荧光定量PCR实验结果表明,miR156、miR398和miR535在2种月季的花蕾期表达上调,而miR167、miR172和miR396表达下调。进一步检测miR172和miR156在2种月季不同花器官中的表达差异,发现miR172在‘绿萼’的花瓣、雌、雄蕊中表达显著下调,提示miR172可能通过负调控其靶基因RcAP2的表达,在‘绿萼’花器官发育过程中起重要作用。     

Interrogation of rabbit miRNAs and their isomiRs

Rabbit (Oryctolagus cuniculus) is the only lagomorph animal of which the genome has been sequenced. Establishing a rabbit miRNA resource will benefit subsequent functional genomic studies in mammals. We have generated small RNA sequence reads with SOLiD and Solexa platforms to identify rabbit miRNAs, where we identified 464 pre-miRNAs and 886 mature miRNAs. The brain and heart miRNA libraries were used for further in-depth analysis of isomiR distributions. There are several intriguing findings. First, several rabbit pre-miRNAs form highly conserved clusters. Second, there is a preference in selecting one strand as mature miRNA, resulting in an arm selection preference. Third, we analyzed the isomiR expression and validated the expression of isomiR types in different rabbit tissues. Moreover, we further performed additional small RNA libraries and defined miRNAs differentially expressed between brain and heart. We conclude also that isomiR distribution profiles could vary between brain and heart tissues.