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.     

Prediction and validation of conservative microRNAs of <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Potato (Solanum tuberosum) is an important crop around the world, and accounts for a significant amount of the food consumed by humans. However, little information is available about potato miRNAs which play important regulatory roles in plant growth and development. In the present study, computational prediction of potential miRNAs from potato revealed 71 miRNAs belonging to 48 families. Amongst these 71 mRNAs, 65 were predicted for the first time. Most potato miRNA families have one to three members, and sequence analysis showed that the candidate pre-miRNA sequences varied from 48 to 224 bp in length. To verify the predicted miRNAs, specific stem-loop RT primers were designed and real-time PCR assays were used to profile the expression levels of seven miRNAs from different tissues of potato. The results showed that all the selected miRNAs were successfully amplified. Most of them had their highest expression levels in leaves, and the lowest levels in the stem, while miR159 and miR164 presented a different expression pattern. The specific expression levels of each miRNAs in the tested tissues may be related to their particular functions in regulating potato vegetative growth and organ development.     

Conserved and novel miRNAs in the legume <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> in response to stress

MicroRNAs (miRNAs) are small RNA molecules recognized as important regulators of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data. We are interested in the identification of miRNAs in Phaseolus vulgaris (common bean) to uncover different plant strategies to cope with adverse conditions and because of its relevance as a crop in developing countries. Here we present the identification of conserved and candidate novel miRNAs in P. vulgaris present in different organs and growth conditions, including drought, abscisic acid treatment, and Rhizobium infection. We also identified cDNA sequences in public databases that represent the corresponding miRNA precursors. In addition, we predicted and validated target mRNAs amongst reported EST and cDNAs for P. vulgaris. We propose that the novel miRNAs present in common bean and other legumes, are involved in regulation of legume-specific processes including adaptation to diverse external cues. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bioinformatic identification and expression analysis of new microRNAs from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

As an essential regulatory component in plants, microRNAs (miRNAs) have been intensively studied over the past decade. Although hundreds of miRNAs have been identified and analyzed in many important crops and model plants, very little is known about the function of common wheat (Triticum aestivum L.) miRNAs. In this study, we performed computational prediction of novel wheat miRNAs based on BLAST searches of the expressed sequence tag database. The expression profiles of all miRNAs were performed for both vegetative and reproductive tissues to identify developmentally regulated miRNAs. A total of 19 new miRNAs belonging to 12 MIR families were identified using stringent criteria for miRNA annotation. For all of the miRNAs, the secondary structures of their precursor sequences were predicted. Two pairs of distinct miRNAs were found to be located on the same precursor. The predicted miRNAs were experimentally verified by a stem-loop qRT-PCR-based assay. The expression profiles were performed in both vegetative and reproductive tissues to find the potential correlations between the developmental phase and miRNA activity. Thirteen out of 19 miRNAs were upregulated at certain phases of plant development, and three of them (miR319, miR395, and miR171) showed the greatest expression in young spikes during microsporogenesis. Our results provide useful information for future studies of miRNA-mediated regulation of flower and grain development in wheat.     

Genome-wide profiling of novel and conserved <Emphasis Type="Italic">Populus</Emphasis> microRNAs involved in pathogen stress response by deep sequencing

MicroRNAs (miRNAs) are small RNAs, generally of 20–23 nt, that down-regulate target gene expression during development, differentiation, growth, and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity, and mechanical stresses have been performed; however, there are few reports profiling the miRNA expression patterns during pathogen stress. We obtained almost 38 million raw reads through Solexa sequencing of two libraries from Populus inoculated and uninoculated with canker disease pathogen. Sequence analyses identified 74 conserved miRNA sequences belonging to 37 miRNA families from 154 loci in the Populus genome and 27 novel miRNA sequences from 35 loci, including their complementary miRNA* strands. Intriguingly, the miRNA* of three conserved miRNAs were more abundant than their corresponding miRNAs. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of 33 miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and 63 target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of plant defense responses during pathogen infection.     

Identification and comparative analysis of microRNAs in <Emphasis Type="Italic">Pinus massoniana</Emphasis> infected by <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>

Pine wilt disease seriously endangers pine tree growth and can lead to wilting and death. In this study, Masson pine (Pinus massoniana) was used as the donor plant to study miRNA expression by high-throughput sequencing in needle leaves of trees during the first 3 days after infection by Bursaphelenchus xylophilus. The results showed that the number of differentially expressed miRNAs first increased and then decreased in samples within 3 days of infection, with the highest expression in samples collected on day 2. Analysis of miRNA target genes in needles on different days after infection revealed enrichment in two pathways: plant hormone signal transduction and RNA transport. Further comparison of the samples indicated differential expression of 10 miRNAs on different days after infection, and the corresponding target genes, such as those participating in plant hormone signal transduction, were also significantly enriched. In addition, the concentrations of zeatin and indole acetic acid in needle tissues were significantly lower after infection than in the control sample. The above results indicated that B. xylophilus infection influenced the expression of miRNAs related to plant hormone signal transduction in P. massoniana, decreasing plant hormone synthesis and ultimately affecting the growth of Masson pine.     

Identification and comparative analysis of the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> hemocytes microRNAs in response to <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> infection

MicroRNAs (miRNAs) are a class of noncoding RNA molecules that function as negative regulators of gene expression and play important roles in a wide spectrum of biological processes, including in immune response. However, the physiological regulation function of Pinctada fucata miRNAs, specially their immunomodulation has not been explored yet. Here, two small RNA libraries from hemocytes of P. fucata with or without Vibrio alginolyticus infection were constructed and sequenced using the high-throughput Illumina deep sequencing technology. In total, 11,939,992 and 11,083,327 raw reads, corresponding to 10,993,546 and 9,988,179 clean reads, were respectively obtained in the control and infected libraries. A total of 276 miRNAs, including 225 known miRNAs and 51 putative novel miRNAs, were identified by bioinformatic analysis. By using pairwise comparison between two libraries, 93 miRNAs were found to be significantly differentially expressed, with 42 and 51 miRNAs exhibiting up-regulation and down-regulation, respectively. Thereinto, some known miRNAs were considered to be immune-related. Real-time PCR were implemented for 6 miRNAs co-expressed in the control and infected samples, and agreement was confirmed between the high-throughput sequencing and real-time PCR data. After miRNA targets were predicted, GO and KEGG pathway enrichment analysis were performed, and the results indicated that ten of the differentially expressed miRNAs were involved in immune-related pathways, and might participate in the host immune response to V. alginolyticus. These results of identification and comparative analysis of miRNAs might deepen our understanding of host-pathogen interactions and immune defense mechanisms in P. fucata.     

Deep sequencing discovery and profiling of conserved and novel miRNAs in the ovule of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Key message

High-throughput sequencing and subsequent analysis identified multiple miRNAs closely related to ovule, indicating that miRNAs are important in Ginkgo biloba ovule.

Abstract

MicroRNAs (miRNAs) are small, noncoding, regulatory RNAs that play crucial regulatory roles in the process of plant growth and development. However, limited information regarding their functions in gymnosperm reproduction is available. Here, we used high-throughput sequencing combined with computational analysis to identify and characterize miRNAs from ovules of G. biloba, and identified 34 conserved miRNA families and 99 novel miRNAs. The precursor sequences of several of the conserved and novel miRNAs were further validated by RT-PCR and sequencing. Furthermore, we found that some target genes, e.g. MYB, homeodomain-leucine zipper (HD-ZIPIII) and auxin response factor (ARF), may be involved in ovule development, and that the significantly enriched pathways of some miRNA targets were related to plant–pathogen interactions and the biosynthesis of secondary metabolites. Twenty-six conserved miRNA families were found to be expressed in both leaves and ovules, while miRNA156, miRNA164, miRNA167, miRNA169, miRNA172 and miRNA390 were up-regulated in ovules. Thus, multiple miRNAs closely related to G. biloba ovule development were identified, resulting in a greater understanding of the important regulatory functions of miRNAs in plant ovules.

     

Identification of novel small RNAs in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

To date, the majority of plant small RNAs (sRNA) have been identified in rice, poplar and Arabidopsis. To identify novel tomato sRNAs potentially involved in tomato specific processes such as fruit development and/or ripening, we cloned 4,018 sRNAs from tomato fruit tissue at the mature green stage. From this pool of sRNAs, we detected tomato homologues of nine known miRNAs, including miR482; a poplar miRNA not conserved in Arabidopsis or rice. We identified three novel putative miRNAs with flanking sequence that could be folded into a stem-loop precursor structure and which accumulated as 19-24nt RNA. One of these putative miRNAs (Put-miRNA3) exhibited significantly higher expression in fruit compared with leaf tissues, indicating a specific role in fruit development processes. We also identified nine sRNAs that accumulated as 19–24nt RNA species in tomato but genome sequence was not available for these loci. None of the nine sRNAs or three putative miRNAs possessed a homologue in Arabidopsis that had a precursor with a predicted stem-loop structure or that accumulated as a sRNA species, suggesting that the 12 sRNAs we have identified in tomato may have a species specific role in this model fruit species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.