Genome-wide Identification Of microRNAs Research Articles

Genome-Wide Identification of MicroRNAs that are Responsive to Virus/Viroid Infection in Nectarine Trees Through High-Throughput Sequencing

Genome-Wide Identification of MicroRNAs that are Responsive to Virus/Viroid Infection in Nectarine Trees Through High-Throughput Sequencing

Genome-wide identification of microRNAs and phased siRNAs in soybean roots under long-term salt stress.

Salinity stress, as the key limiting factor for agricultural productivity, can activate a series of molecular responses and alter gene expression in plants. Endogenous regulatory small RNAs, such as microRNAs (miRNAs) and phased siRNAs (phasiRNAs), play crucial roles during stress adaptation and prevent the injury from environmental circumstances. To identify long-term salt stress responsive miRNAs and phasiRNAs as well as their associated genes and pathways in soybean roots. Small RNA and degradome sequencing strategies were applied to genome widely investigate miRNAs and phasiRNAs in soybean roots under control and long-term salt stress conditions. In this study, stringent bioinformatic analysis led to the identification of 253 conserved and 38 novel miRNA candidates. Results of expression profiling, target and endogenous target mimics predictions provided valuable clues to their functional roles. Furthermore, 156 genes were identified to be capable of generating 21 nt and 24 nt phasiRNAs, in which 37 candidates were confirmed by degradome data for miRNA-directed cleavage. Approximately 90% of these phasiRNA loci were protein coding genes. And GO enrichment analysis pointed to "signal transduction" and "ADP binding" entries and reflected the functional roles of identified phasiRNA genes. Taken together, our findings extended the knowledge of salt responsive miRNAs and phasiRNAs in soybean roots, and provided valuable information for a better understanding of the regulatory events caused by small RNAs underlying plant adaptations to long-term salt stress.

Genome-Wide Identification of microRNAs in Response to Salt/Alkali Stress in Medicago truncatula through High-Throughput Sequencing.

Saline-alkaline stress is a universal abiotic stress that adversely affects plant growth and productivity. Saline-alkaline conditions results in plant abnormal transcriptome expression finally manifesting as defective phenotypes. Considerable research has revealed the active role of microRNA in various stress conditions. This study was aimed to identify novel miRNAs and the miRNA expression patterns in the leguminous model plant R108 (Medicago truncatula). The miRNA contained in the total RNA extracted from Medicago truncatula seedlings (72 h) that had been treated with solutions mimicking saline and alkaline soils was subjected to miRNA deep sequencing. The Illumina HiSeq sequencing platform was used to analyze nine small RNA libraries of three treatment groups: distilled water, 20 mM NaCl + Na2SO4 and 5 mM Na2CO3 + NaHCO3. Sequencing revealed that 876 miRNAs including 664 known miRNAs and 212 potential novel miRNAs were present in all the libraries. The miR159 family, miR156 family, miR2086-3p, miR396, miR166, miR319, miR167, miR5213-5p, miR1510 and miR2643 were among the most expressed miRNAs in all libraries. The results of miRNAs expression under treatments were validated by reverse-transcription quantitative PCR (RT-qPCR). Target gene prediction through computational analysis and pathway annotation analysis revealed that the primary pathways affected by stress were related to plant development, including metabolic processes, single-organism processes and response to the stimulus. Our results provide valuable information towards elucidating the molecular mechanisms of salt/alkali tolerance in Medicago truncatula and provide insight into the putative role of miRNAs in plant stress resistance.

Genome-wide identification of microRNAs in decidual natural killer cells from patients with unexplained recurrent spontaneous abortion.

This study revealed miRNA regulation and functional microarray-based profiles of miRNAs in the natural killer (NK) cells of the decidual tissue obtained from patients with unexplained recurrent spontaneous abortion (URSA). Patients with URSA were categorized based on the occurrence of at least two or more successive spontaneous abortions between 7th and 10th gestational week. Total RNA was isolated from the NK cells of the decidual tissue obtained from patients with induced abortion at about the 8th gestational week. The potential contribution of regulatory miRNAs to a genetic predisposition to URSA was characterized by comparison with healthy and fertile controls and bioinformatics analyses. Analysis of the miRNA expression profiles identified 50 miRNAs that were differentially expressed, including one down-regulated and 49 up-regulated miRNAs in the URSA group. MiRNA-Gene-Network, miRNA-GO-Network and miRNA-Gene-TF-Network were constructed. The key miRNAs, genes, GOs and core TFs in the network were determined. Our results suggest that a close relationship exists between the aberrant miRNAs and URSA. Furthermore, these findings support the notion that altered expression of miRNAs may contribute to the clinical diagnosis of URSA and the potential to develop novel strategies for therapeutic targets against URSA.

Genome-Wide Identification of MicroRNAs in Response to Cadmium Stress in Oilseed Rape (Brassica napus L.) Using High-Throughput Sequencing.

MicroRNAs (miRNAs) have important roles in regulating stress-response genes in plants. However, identification of miRNAs and the corresponding target genes that are induced in response to cadmium (Cd) stress in Brassica napus remains limited. In the current study, we sequenced three small-RNA libraries from B. napus after 0 days, 1 days, and 3 days of Cd treatment. In total, 44 known miRNAs (belonging to 27 families) and 103 novel miRNAs were identified. A comprehensive analysis of miRNA expression profiles found 39 differentially expressed miRNAs between control and Cd-treated plants; 13 differentially expressed miRNAs were confirmed by qRT-PCR. Characterization of the corresponding target genes indicated functions in processes including transcription factor regulation, biotic stress response, ion homeostasis, and secondary metabolism. Furthermore, we propose a hypothetical model of the Cd-response mechanism in B. napus. Combined with qRT-PCR confirmation, our data suggested that miRNAs were involved in the regulations of TFs, biotic stress defense, ion homeostasis and secondary metabolism synthesis to respond Cd stress in B. napus.

Genome-wide Identification of microRNAs that Respond to Drought Stress in Seedlings of Tertiary Relict Ammopiptanthus mongolicus

microRNAs (miRNAs) are important post-transcriptional regulators of gene expression in plant abiotic stress responses. Ammopiptanthus mongolicus is a typical evergreen woody xerophyte, which makes it an ideal model system for studying drought tolerance in woody plants. The response of miRNAs to drought stress is still unknown in this plant. In this research, we obtained 34 695 665 raw reads from two small RNA libraries constructed from control and drought-treated A. mongolicus seedlings by the Illumina deep sequencing technology. Length analysis revealed that reads of 20, 21, and 24 nucleotides accounted for the majority of the small RNAs in the two libraries. Sequence analyses identified 65 conserved miRNA sequences from 190 members of 31 miRNA families and 20 potential novel miRNAs from 17 families that were differentially expressed between the two libraries. The expression patterns of 25 of these miRNAs were significantly different in the two libraries, but only am-miR4a-c was downregulated during drought stress, the other 24 were upregulated. The expression trends determined by the HiSeq sequencing and by qRT-PCR were similar. Furthermore, 35 target genes for the conserved miRNA were markedly changed and 52 target genes for the potential novel miRNAs were predicted and their functions were annotated by computational analysis. Our results provide new molecular evidence for understanding the molecular mechanisms of drought response and tolerance in A. mongolicus.

Genome wide identification of microRNAs involved in fatty acid and lipid metabolism of Brassica napus by small RNA and degradome sequencing

Rapeseed (Brassica napus) is an important cash crop considered as the third largest oil crop worldwide. Rapeseed oil contains various saturation or unsaturation fatty acids, these fatty acids, whose could incorporation with TAG form into lipids stored in seeds play various roles in the metabolic activity. The different fatty acids in B. napus seeds determine oil quality, define if the oil is edible or must be used as industrial material. miRNAs are kind of non-coding sRNAs that could regulate gene expressions through post-transcriptional modification to their target transcripts playing important roles in plant metabolic activities. We employed high-throughput sequencing to identify the miRNAs and their target transcripts involved in fatty acids and lipids metabolism in different development of B. napus seeds. As a result, we identified 826 miRNA sequences, including 523 conserved and 303 newly miRNAs. From the degradome sequencing, we found 589 mRNA could be targeted by 236 miRNAs, it includes 49 novel miRNAs and 187 conserved miRNAs. The miRNA-target couple suggests that bna-5p-163957_18, bna-5p-396192_7, miR9563a-p3, miR9563b-p5, miR838-p3, miR156e-p3, miR159c and miR1134 could target PDP, LACS9, MFPA, ADSL1, ACO32, C0401, GDL73, PlCD6, OLEO3 and WSD1. These target transcripts are involving in acetyl-CoA generate and carbon chain desaturase, regulating the levels of very long chain fatty acids, β-oxidation and lipids transport and metabolism process. At the same, we employed the q-PCR to valid the expression of miRNAs and their target transcripts that involve in fatty acid and lipid metabolism, the result suggested that the miRNA and their transcript expression are negative correlation, which in accord with the expression of miRNA and its target transcript. The study findings suggest that the identified miRNA may play important role in the fatty acids and lipids metabolism in seeds of B. napus.

Genome-Wide Identification of MicroRNAs and Their Targets in the Leaves and Fruits of Eucommia ulmoides Using High-Throughput Sequencing.

MicroRNAs (miRNAs), a group of endogenous small non-coding RNAs, play important roles in plant growth, development, and stress response processes. Eucommia ulmoides Oliver (hardy rubber tree) is one of the few woody plants capable of producing trans-1, 4-polyisoprene (TPI), also known as Eu-rubber, which has been utilized as an industrial raw material and is extensively cultivated in China. However, the mechanism of TPI biosynthesis has not been identified in E. ulmoides. To characterize small RNAs and their targets with potential biological roles involved in the TPI biosynthesis in E. ulmoides, in the present study, eight small RNA libraries were constructed and sequenced from young and mature leaves and fruits of E. ulmoides. Further analysis identified 34 conserved miRNAs belonging to 20 families (two unclassified families), and 115 novel miRNAs seemed to be specific to E. ulmoides. Among these miRNAs, fourteen conserved miRNAs and 49 novel miRNAs were significantly differentially expressed and identified as Eu-rubber accumulation related miRNAs. Based on the E. ulmoides genomic data, 202 and 306 potential target genes were predicted for 33 conserved and 92 novel miRNAs, respectively; the predicted targets are mostly transcription factors and functional genes, which were enriched in metabolic pathways and biosynthesis of secondary metabolites. Noticeably, based on the expression patterns of miRNAs and their target genes in combination with the Eu-rubber accumulation, the negative correlation of expression of six miRNAs (Eu-miR14, Eu-miR91, miR162a, miR166a, miR172c, and miR396a) and their predicted targets serving as potential regulators in Eu-rubber accumulation. This study is the first to detect conserved and novel miRNAs and their potential targets in E. ulmoides and identify several candidate genes potentially controlling rubber accumulation, and thus provide molecular evidence for understanding the roles of miRNAs in regulating the TPI biosynthesis in E. ulmoides.

Genome-wide identification of microRNAs and their target genes in Cynoglossus semilaevis using computational approach

MicroRNAs (miRNAs) are endogenous non-protein-coding RNAs of approximately 22 nucleotides which regulate gene expression at the posttranscriptional level in plants and animals. Until recently, only limited numbers of miRNAs were identified in Cynoglossus semilaevis, which is an important commercial mariculture fish species in China. The recently sequenced genome of C. semilaevis provides an opportunity to identify some novel miRNAs in this species by computational approach. A total of 217 miRNAs belonging to 87 families were identified in C. semilaevis, including 96 known miRNAs and 121 putative novel miRNAs. To verify the predicted miRNAs, a qRT-PCR-based assay was performed, the results showed that randomly selected 6 miRNAs have different expression patterns in the liver, spleen, kidney and muscles tissues of C. semilaevis, and suggest that these miRNAs might be involved in tissue-specific expression. Following this, 20,614 potential target genes were predicted by searching the mRNA database, and these targets are involved in several key pathways. Finally, the analysis on GO enrichment and KEGG pathway showed that the predicted targets genes for these identified miRNAs participate in diverse physiological and metabolic processes. Taken together, the present study expands the collection of miRNAs and provides a foundation for further understanding the role of these miRNAs in the C. semilaevis.

Genome-wide identification of microRNAs in pomegranate (Punica granatum L.) by high-throughput sequencing.

BackgroundMicroRNAs (miRNAs), a class of small non-coding endogenous RNAs that regulate gene expression post-transcriptionally, play multiple key roles in plant growth and development and in biotic and abiotic stress response. Knowledge and roles of miRNAs in pomegranate fruit development have not been explored.ResultsPomegranate, which accumulates a large amount of anthocyanins in skin and arils, is valuable to human health, mainly because of its antioxidant properties. In this study, we developed a small RNA library from pooled RNA samples from young seedlings to mature fruits and identified both conserved and pomegranate-specific miRNA from 29,948,480 high-quality reads. For the pool of 15- to 30-nt small RNAs, ~50 % were 24 nt. The miR157 family was the most abundant, followed by miR156, miR166, and miR168, with variants within each family. The base bias at the first position from the 5’ end had a strong preference for U for most 18- to 26-nt sRNAs but a preference for A for 18-nt sRNAs. In addition, for all 24-nt sRNAs, the nucleotide U was preferred (97 %) in the first position. Stem-loop RT-qPCR was used to validate the expression of the predominant miRNAs and novel miRNAs in leaves, male and female flowers, and multiple fruit developmental stages; miR156, miR156a, miR159a, miR159b, and miR319b were upregulated during the later stages of fruit development. Higher expression of miR156 in later fruit developmental may positively regulate anthocyanin biosynthesis by reducing SPL transcription factor. Novel miRNAs showed variation in expression among different tissues. These novel miRNAs targeted different transcription factors and hormone related regulators. Gene ontology and KEGG pathway analyses revealed predominant metabolic processes and catalytic activities, important for fruit development. In addition, KEGG pathway analyses revealed the involvement of miRNAs in ascorbate and linolenic acid, starch and sucrose metabolism; RNA transport; plant hormone signaling pathways; and circadian clock.ConclusionOur first and preliminary report of miRNAs will provide information on the synthesis of biochemical compounds of pomegranate for future research. The functions of the targets of the novel miRNAs need further investigation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0807-3) contains supplementary material, which is available to authorized users.

Genome-Wide Identification of MicroRNAs in Leaves and the Developing Head of Four Durum Genotypes during Water Deficit Stress

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in plant development and abiotic stress responses. The miRNA transcriptome (miRNAome) under water deficit stress has been investigated in many plant species, but is poorly characterised in durum wheat (Triticum turgidum L. ssp. durum). Water stress during early reproductive stages can result in significant yield loss in durum wheat and this study describes genotypic differences in the miRNAome between water deficit tolerant and sensitive durum genotypes. Small RNA libraries (96 in total) were constructed from flag leaf and developing head tissues of four durum genotypes, with or without water stress to identify differentially abundant miRNAs. Illumina sequencing detected 110 conserved miRNAs and 159 novel candidate miRNA hairpins with 66 conserved miRNAs and five novel miRNA hairpins differentially abundant under water deficit stress. Ten miRNAs (seven conserved, three novel) were validated through qPCR. Several conserved and novel miRNAs showed unambiguous inverted regulatory profiles between the durum genotypes. Several miRNAs also showed differential abundance between two tissue types regardless of treatment. Predicted mRNA targets (130) of four novel durum miRNAs were characterised using Gene Ontology (GO) which revealed functions common to stress responses and plant development. Negative correlation was observed between several target genes and the corresponding miRNA under water stress. For the first time, we present a comprehensive study of the durum miRNAome under water deficit stress. The identification of differentially abundant miRNAs provides molecular evidence that miRNAs are potential determinants of water stress tolerance in durum wheat. GO analysis of predicted targets contributes to the understanding of genotypic physiological responses leading to stress tolerance capacity. Further functional analysis of specific stress responsive miRNAs and their interaction with targets is ongoing and will assist in developing future durum wheat varieties with enhanced water deficit stress tolerance.

Genome-wide identification of microRNAs and their targets in wild type and phyB mutant provides a key link between microRNAs and the phyB-mediated light signaling pathway in rice.

Phytochrome B (phyB), a member of the phytochrome family in rice, plays important roles in regulating a range of developmental processes and stress responses. However, little information about the mechanisms involved in the phyB-mediated light signaling pathway has been reported in rice. MicroRNAs (miRNAs) also perform important roles in plant development and stress responses. Thus, it is intriguing to explore the role of miRNAs in the phyB-mediated light signaling pathway in rice. In this study, comparative high-throughput sequencing and degradome analysis were used to identify candidate miRNAs and their targets that participate in the phyB-mediated light signaling pathway. A total of 720 known miRNAs, 704 novel miRNAs and 1957 target genes were identified from the fourth leaves of wild-type (WT) and phyB mutant rice at the five-leaf stage. Among them, 135 miRNAs showed differential expression, suggesting that the expression of these miRNAs is directly or indirectly under the control of phyB. In addition, 32 out of the 135 differentially expressed miRNAs were found to slice 70 genes in the rice genome. Analysis of these target genes showed that members of various transcription factor families constituted the largest proportion, indicating miRNAs are probably involved in the phyB-mediated light signaling pathway mainly by regulating the expression of transcription factors. Our results provide new clues for functional characterization of miRNAs in the phyB-mediated light signaling pathway, which should be helpful in comprehensively uncovering the molecular mechanisms of phytochrome-mediated photomorphogenesis and stress responses in plants.

Genome-Wide Identification of microRNAs and Their Targets in Cold-Stored Potato Tubers by Deep Sequencing and Degradome Analysis

Potato tubers, the economically important organ for food, processing raw materials, and seeds, are usually stored at different temperatures for various purposes. A general physiological process during storage is starch hydrolysis and accumulation of reducing sugars as breaking of tuber dormancy. The starch hydrolysis can be accelerated by low temperature. Valuable works have been reported on transcriptional and post-translational regulatory mechanisms that are involved in starch hydrolysis in stored potato tubers. However, little is known whether post-transcriptional events are also involved in this important process. In the present study, two small RNA libraries and two degradome libraries were constructed using 4- and 20 °C-stored potato tubers. Deep sequencing and whole genome-wide data analysis led to the identification of 53 known miRNAs and 60 novel miRNAs, largely increasing the number of miRNAs known in potato. Seventy genes were identified as miRNA targets by degradome analysis, indicating that miRNAs were active in gene expression regulation in the post-harvested tubers. The targets were potentially involved in various biological processes, such as stimulus response, signal transduction, transcriptional regulation, and metabolism. Further expression profiling revealed 11 miRNAs/miRNA*s and 34 targets that showed different patterns between two potato genotypes with distinct response to cold storage in terms of reducing sugar accumulation, implying a role of miRNAs in cold-induced sweetening. This study is the first genome-wide investigation of miRNAs and their targets in cold-stored potato tubers, thus broadening our perspectives on the roles of miRNAs in cold response, dormancy, and carbohydrate metabolism.

Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis

MicroRNAs (miRNAs) are emerging as essential regulators of biological processes. Somatic embryogenesis is one of the most important techniques for gymnosperm-breeding programs, but there is little understanding of its underlying mechanism. To investigate the roles of miRNAs during somatic embryogenesis in larch, we constructed a small RNA library from somatic embryos. High-throughput sequencing of the library identified 83 conserved miRNAs from 35 families, 16 novel miRNAs, and 14 plausible miRNA candidates, with a high proportion specific to larch or gymnosperms. qRT-PCR analysis demonstrated that both the conserved and novel or candidate miRNAs were expressed in larch. Several miRNA precursor sequences were obtained via RACE. We predicted 110 target genes using bioinformatics, and validated 9 of them by 5' RACE. 11 conserved miRNA families including 17 miRNAs with critical functions in plant development and six target mRNAs were detected by qRT-PCR in the larch SE. Stage-specific expression of miRNAs and their targets indicate their possible modulation on SE of larch: miR171a/b might exert function on PEMs, while miR171c acts in the induction process of larch SE; miR397 and miR398 mainly involved in modulation of PEM propagation and transition to single embryo; miR162 and miR168 exert their regulatory function during total SE process, especially during stages 5-8; miR156, miR159, miR160, miR166, miR167, and miR390 might play regulatory roles during cotyledonary embryo development. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving specific and common miRNAs operating post-transcriptionally during embryogenesis.

Genome-Wide Identification of MicroRNAs in Response to Low Nitrate Availability in Maize Leaves and Roots

BackgroundNitrate is the major source of nitrogen available for many crop plants and is often the limiting factor for plant growth and agricultural productivity especially for maize. Many studies have been done identifying the transcriptome changes under low nitrate conditions. However, the microRNAs (miRNAs) varied under nitrate limiting conditions in maize has not been reported. MiRNAs play important roles in abiotic stress responses and nutrient deprivation.Methodology/Principal FindingsIn this study, we used the SmartArray™ and GeneChip® microarray systems to perform a genome-wide search to detect miRNAs responding to the chronic and transient nitrate limiting conditions in maize. Nine miRNA families (miR164, miR169, miR172, miR397, miR398, miR399, miR408, miR528, and miR827) were identified in leaves, and nine miRNA families (miR160, miR167, miR168, miR169, miR319, miR395, miR399, miR408, and miR528) identified in roots. They were verified by real time stem loop RT-PCR, and some with additional time points of nitrate limitation. The miRNAs identified showed overlapping or unique responses to chronic and transient nitrate limitation, as well as tissue specificity. The potential target genes of these miRNAs in maize were identified. The expression of some of these was examined by qRT-PCR. The potential function of these miRNAs in responding to nitrate limitation is described.Conclusions/SignificanceGenome-wide miRNAs responding to nitrate limiting conditions in maize leaves and roots were identified. This provides an insight into the timing and tissue specificity of the transcriptional regulation to low nitrate availability in maize. The knowledge gained will help understand the important roles miRNAs play in maize responding to a nitrogen limiting environment and eventually develop strategies for the improvement of maize genetics.