Prediction of potential novel microRNAs in soybean when in symbiosis.

MicroRNAs (miRNAs) are small molecules, noncoding proteins that are involved in many biological processes, especially in plants; among these processes is nodulation in the legume. Biological nitrogen fixation is a key process, with critical importance to the soybean crop. This study aimed to identify the potential of novel miRNAs to act during the root nodulation process. We utilized a set of transcripts that were differentially expressed in soybean roots 10 days after inoculation with Bradyrhizobium japonicum, which were obtained in a previous study, and performed a set of computational analyses that led us to select new miRNAs potentially involved in nodulation. Among these analyses, the set of transcripts were submitted to an in silico annotation of noncoding RNAs, including a search of similarity against miRNA public databases, ab initio tools for miRNA identification, structural search against miRNA families, prediction of the secondary structure of miRNA precursors, and prediction of the sequences of mature miRNAs. Subsequently, we applied filter procedures based on miRNA selections described in the literature (e.g., free energy value). In the next step, a manual curation inspection of the annotation was performed and the top candidates were selected and used for prediction of potential target genes, which were later checked manually in the database of the soybean genome. This prediction led us to the identification of 9 potential new miRNAs; among these, 4 were conserved in other plants. Moreover, we predicted their target genes might play important roles in the regulation of nodulation

Identification of novel soybean microRNAs involved in abiotic and biotic stresses

<p>Abstract</p> <p>Background</p> <p>Small RNAs (19-24 nt) are key regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in eukaryotes. Current studies have demonstrated that microRNAs (miRNAs) act in several plant pathways associated with tissue proliferation, differentiation, and development and in response to abiotic and biotic stresses. In order to identify new miRNAs in soybean and to verify those that are possibly water deficit and rust-stress regulated, eight libraries of small RNAs were constructed and submitted to Solexa sequencing.</p> <p>Results</p> <p>The libraries were developed from drought-sensitive and tolerant seedlings and rust-susceptible and resistant soybeans with or without stressors. Sequencing the library and subsequent analyses detected 256 miRNAs. From this total, we identified 24 families of novel miRNAs that had not been reported before, six families of conserved miRNAs that exist in other plants species, and 22 families previously reported in soybean. We also observed the presence of several isomiRNAs during our analyses. To validate novel miRNAs, we performed RT-qPCR across the eight different libraries. Among the 11 miRNAs analyzed, all showed different expression profiles during biotic and abiotic stresses to soybean. The majority of miRNAs were up-regulated during water deficit stress in the sensitive plants. However, for the tolerant genotype, most of the miRNAs were down regulated. The pattern of miRNAs expression was also different for the distinct genotypes submitted to the pathogen stress. Most miRNAs were down regulated during the fungus infection in the susceptible genotype; however, in the resistant genotype, most miRNAs did not vary during rust attack. A prediction of the putative targets was carried out for conserved and novel miRNAs families.</p> <p>Conclusions</p> <p>Validation of our results with quantitative RT-qPCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. The identification of differentially expressed plant miRNAs provides molecular evidence for the possible involvement of miRNAs in the process of water deficit- and rust-stress responses.</p

Comparison of Small RNA Profiles of Glycine max and Glycine soja at Early Developmental Stages

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MicroRNA and cDNA-Microarray as Potential Targets against Abiotic Stress Response in Plants: Advances and Prospects

Abiotic stresses, such as temperature (heat and cold), salinity, and drought negatively affect plant productivity; hence, the molecular responses of abiotic stresses need to be investigated. Numerous molecular and genetic engineering studies have made substantial contributions and revealed that abiotic stresses are the key factors associated with production losses in plants. In response to abiotic stresses, altered expression patterns of miRNAs have been reported, and, as a result, cDNA-microarray and microRNA (miRNA) have been used to identify genes and their expression patterns against environmental adversities in plants. MicroRNA plays a significant role in environmental stresses, plant growth and development, and regulation of various biological and metabolic activities. MicroRNAs have been studied for over a decade to identify those susceptible to environmental stimuli, characterize expression patterns, and recognize their involvement in stress responses and tolerance. Recent findings have been reported that plants assign miRNAs as critical post-transcriptional regulators of gene expression in a sequence-specific manner to adapt to multiple abiotic stresses during their growth and developmental cycle. In this study, we reviewed the current status and described the application of cDNA-microarray and miRNA to understand the abiotic stress responses and different approaches used in plants to survive against different stresses. Despite the accessibility to suitable miRNAs, there is a lack of simple ways to identify miRNA and the application of cDNA-microarray. The elucidation of miRNA responses to abiotic stresses may lead to developing technologies for the early detection of plant environmental stressors. The miRNAs and cDNA-microarrays are powerful tools to enhance abiotic stress tolerance in plants through multiple advanced sequencing and bioinformatics techniques, including miRNA-regulated network, miRNA target prediction, miRNA identification, expression profile, features (disease or stress, biomarkers) association, tools based on machine learning algorithms, NGS, and tools specific for plants. Such technologies were established to identify miRNA and their target gene network prediction, emphasizing current achievements, impediments, and future perspectives. Furthermore, there is also a need to identify and classify new functional genes that may play a role in stress resistance, since many plant genes constitute an unexplained fraction

Identification of miRNAs and their targets by high-throughput sequencing and degradome analysis in cytoplasmic male-sterile line NJCMS1A and its maintainer NJCMS1B of soybean

Table S1. Summary of small RNA annotations from NJCMS1A and NJCMS1B. Table S2. Known miRNAs identified in NJCMS1A and NJCMS1B. Table S3. Family member distribution in conserved miRNA families. Table S4. Summary of miRNA families found in NJCMS1A and NJCMS1B. Table S5. Novel miRNAs on the other arm of known pre-miRNAs. Table S6. Novel miRNAs identified in NJCMS1A and NJCMS1B. Table S7-1. High-confidence known miRNAs identified in NJCMS1A and NJCMS1B. Table S7-2. High-confidence novel miRNAs identified in NJCMS1A and NJCMS1B. Table S8-1. The up-regulated miRNAs identified in NJCMS1A and NJCMS1B. Table S8-2. The down-regulated miRNAs identified in NJCMS1A and NJCMS1B. Table S9. The targets of miRNAs identified in NJCMS1A and NJCMS1B. Table S10. Targets of novel miRNAs in NJCMS1A and NJCMS1B. Table S11. Primers used in this study. (ZIP 637 kb

Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume <it>Phaseolus vulgaris </it>(common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of <it>Phaseolus vulgaris</it>.</p> <p>Results</p> <p>Small RNA libraries were generated from roots, flowers, leaves, and seedlings of <it>P. vulgaris</it>. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies.</p> <p>Conclusions</p> <p>This work represents the first massive-scale RNA sequencing study performed in <it>Phaseolus vulgaris </it>to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of <it>P. vulgaris </it>miRNAs in relation to those of other legumes.</p

Transcriptional and Post-Transcriptional Regulation of Nodule-Specific Gene Expression in Soybean

Lateral roots and nodules are two important nutrient acquisition organs in soybean. The evolutionary origin of nodules from lateral roots has been highly hypothesized based on morphological similarities and genetic studies, but gene expression profiles during the formation of these organs have not been compared. In addition, the role of post-transcriptional gene regulation during nodule development has not been thoroughly explored. Bridging these knowledge gaps is crucial to develop genetic/biotechnological strategies to optimize nutrient acquisition and sustainable production of crops. To answer some of the outstanding questions about regulation of gene expression during nodule development, (i) global transcriptome analyses of lateral root and nodule were compared to identify organ-specific enrichment patterns of transcription factors and hormone signaling elements; (ii) small RNA and degradome/Parallel Analysis of RNA Ends (PARE) libraries were generated to identify miRNAs and their cleavage products respectively in nodule tissues; (iii) miRNA qPCR quantification methods were optimized; and (iv) the effect of misexpression of selected miRNAs on nodule development were evaluated. Analysis of transcriptome data showed very little overlap in transcription factor expression profiles between emerging nodules and emerging lateral roots. The expression profiles of certain key hormone biosynthesis and signaling genes were distinct between nodules and lateral roots. Interestingly, members of gene families associated with shoot axillary meristem formation were enriched in nodules, but not in lateral roots. Analysis of small RNA and PARE libraries resulted in the identification of 497 previously unknown miRNA precursors and validated 353 miRNA-target pairs. These and additional results suggested that inverse expression of miRNA and target is likely to be one of the mechanisms that direct nodule-specific gene expression. In addition, methods for miRNA quantification by qPCR were optimized, and a potential role for miR169 in regulating hormone homeostasis during nodule development was identified through functional assays. In summary, nodules might have adopted not only the developmental pathways of lateral roots, but also shoot axillary meristems. Furthermore, the inverse expression of miRNAs and their targets between nodules and adjacent root tissues might be a mechanism that spatially limits target gene expression to the nodule and/or root tissues