Degradome Analysis Research Articles

Drought stress-induced changes of microRNAs in diploid and autotetraploid Paulownia tomentosa

Drought stress adversely affects plant productivity. Growth and timber production of Paulownia trees are limited under drought stress. Changes in gene expression patterns and miRNA in different ploidy of Paulownia tomentosa have been investigated. However, the responses of P. tomentosa to drought stress at the microRNA (miRNA) level have not been reported so far. To identify miRNA candidates and their target genes involved in the drought stress response in diploid and tetraploid P. tomentosa, four small RNA and four degradome libraries from diploid and autotetraploid P. tomentosa under normal and drought stress conditions were constructed and sequenced. A total of 41 conserved and 90 novel miRNAs were identified. Among these miRNAs, 67 (26 conserved and 41 novel) and 53 (six conserved and 47 novel) were significantly differentially expressed in response to drought stress in diploid and autotetraploid P. tomentosa, respectively. Degradome analysis identified 356 candidate miRNA target genes that encoded proteins with functions that included plant defense, transcriptional regulation, and hormone metabolism. In particular, miR4 and miR156 were identified only in autotetraploid P. tomentosa under drought stress. These results will help us build a foundation for future studies of the biological functions of miRNA-mediated gene regulation in P. tomentosa.

Identification and Functional Analysis of microRNAs Involved in the Anther Development in Cotton Genic Male Sterile Line Yu98-8A.

Hybrid vigor contributes in a large way to the yield and quality of cotton (Gossypium hirsutum) fiber. Although microRNAs play essential regulatory roles in flower induction and development, it is still unclear if microRNAs are involved in male sterility, as the regulatory molecular mechanisms of male sterility in cotton need to be better defined. In this study, two independent small RNA libraries were constructed and sequenced from the young buds collected from the sporogenous cell formation to the meiosis stage of the male sterile line Yu98-8A and the near-isogenic line. Sequencing revealed 1588 and 1536 known microRNAs and 347 and 351 novel miRNAs from male sterile and male fertile libraries, respectively. MicroRNA expression profiles revealed that 49 conserved and 51 novel miRNAs were differentially expressed. Bioinformatic and degradome analysis indicated the regulatory complexity of microRNAs during flower induction and development. Further RT-qPCR and physiological analysis indicated that, among the different Kyoto Encyclopedia Gene and Genomes pathways, indole-3-acetic acid and gibberellic acid signaling transduction pathways may play pivotal regulatory functions in male sterility.

The legume miR1514a modulates a NAC transcription factor transcript to trigger phasiRNA formation in response to drought.

Recent studies have identified microRNAs as post-transcriptional regulators involved in stress responses. miR1514a is a legume microRNA that is induced in response to drought stress in Phaseolus vulgaris (common bean) and shows differential accumulation levels in roots during water deficit in two cultivars with different drought tolerance phenotypes. A recent degradome analysis revealed that miR1514a targets the transcripts of two NAC transcription factors (TFs), Phvul.010g121000 and Phvul.010g120700. Furthermore, expression studies and small RNA-seq data indicate that only Phvul.010g120700 generates phasiRNAs, which also accumulate under water deficit conditions. To confirm these results, we over-expressed miR1514a in transgenic hairy roots, and observed a reduced accumulation of Phvul.010g120700 and an increase in NAC-derived phasiRNAs; inhibition of miR1514a activity resulted in the opposite effect. Moreover, we determined that a NAC-derived phasiRNA associates with ARGONAUTE 1 (AGO1), suggesting that it is functional. In addition, a transcriptome analysis of transgenic hairy roots with reduced miR1514a levels revealed several differentially expressed transcripts, mainly involved in metabolism and stress responses, suggesting they are regulated by the NAC TF and/or by phasiRNAs. This work therefore demonstrates the participation of miR1514 in the regulation of a NAC transcription factor transcript through phasiRNA production during the plant response to water deficit.

Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30% of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust resistance breeding programs.

Genome-wide mining for microRNAs and their targets in Betula luminifera using high-throughput sequencing and degradome analyses

microRNAs (miRNAs) play vital roles in plants regulating a panoply of biological processes, such as development, hormone signaling, and stress response, by inhibiting target genes at the post-transcriptional level. However, the roles of miRNAs in Betula luminifera remain elusive. To mine for B. luminifera miRNAs and targets, we used a deep sequencing approach to analyze the sRNAs and degradome sequencing of mixed samples, including roots, stems, and leaves. A total of 114 known miRNAs or miRNA*s from 44 families, and 24 novel miRNAs and 17 miRNA*s plus 15 plausible miRNA candidates were identified, of which 36 known miRNAs, 29 miRNA*s, and all novel miRNAs had precursor sequences. Additionally, 49 targets for 19 known miRNA families and seven miRNA*s, and seven targets for novel miRNAs were identified using a high-throughput degradome-sequencing approach. The conserved miRNA targets were mainly transcription factors, whereas the miRNA* targets were mainly protein-coding genes, with preferential propensity to functional enzymes. A Gene Ontology analysis showed that the predicted targets were classified into 62 biological processes, 20 cellular components, and 28 molecular functions, respectively. We found two different targets for miR396a* and miR396c*, and the target changed when the miR156d precursor cleavage site was shifted toward the 5′-end by two nucleotides, indicating the diverse regulatory roles of MIRNA genes. Furthermore, three targets identified by degradome sequencing were validated further through 5′ rapid amplification of cDNA ends. The expression patterns of the randomly selected miRNAs varied among different tissues. miR164 expression was induced under nitrogen starvation, with tissue-specific expression patterns, and was negatively correlated with the NAC1 target gene in roots and leaves, but not in stems. This study is a transcriptome-based analysis of miRNAs and the degradome, providing useful information to explore the functions of miRNAs and their target genes in B. luminifera.

Identification of miRNAs and their targets in wild tomato at moderately and acutely elevated temperatures by high-throughput sequencing and degradome analysis.

MicroRNAs (miRNAs) are 19–24 nucleotide (nt) noncoding RNAs that play important roles in abiotic stress responses in plants. High temperatures have been the subject of considerable attention due to their negative effects on plant growth and development. Heat-responsive miRNAs have been identified in some plants. However, there have been no reports on the global identification of miRNAs and their targets in tomato at high temperatures, especially at different elevated temperatures. Here, three small-RNA libraries and three degradome libraries were constructed from the leaves of the heat-tolerant tomato at normal, moderately and acutely elevated temperatures (26/18 °C, 33/33 °C and 40/40 °C, respectively). Following high-throughput sequencing, 662 conserved and 97 novel miRNAs were identified in total with 469 conserved and 91 novel miRNAs shared in the three small-RNA libraries. Of these miRNAs, 96 and 150 miRNAs were responsive to the moderately and acutely elevated temperature, respectively. Following degradome sequencing, 349 sequences were identified as targets of 138 conserved miRNAs, and 13 sequences were identified as targets of eight novel miRNAs. The expression levels of seven miRNAs and six target genes obtained by quantitative real-time PCR (qRT-PCR) were largely consistent with the sequencing results. This study enriches the number of heat-responsive miRNAs and lays a foundation for the elucidation of the miRNA-mediated regulatory mechanism in tomatoes at elevated temperatures.

High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis).

BackgroundG1 + HBP is a male sterile cybrid line with nuclear genome from Hirado Buntan pummelo (C. grandis Osbeck) (HBP) and mitochondrial genome from “Guoqing No.1” (G1, Satsuma mandarin), which provides a good opportunity to study male sterility and nuclear-cytoplasmic cross talk in citrus. High-throughput sRNA and degradome sequencing were applied to identify miRNAs and their targets in G1 + HBP and its fertile type HBP during reproductive development.ResultsA total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2) and transport inhibitor response 1 (TIR1). Eight target genes were confirmed to be sliced by corresponding miRNAs using 5’ RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between sterile line G1 + HBP and fertile line HBP were identified. Differential expression of miRNAs and their target genes between two lines was validated by quantitative RT-PCR, and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulatory mechanism of miR167a was investigated by yeast one-hybrid and dual-luciferase assays that one dehydrate responsive element binding (DREB) transcription factor binds to miR167a promoter and transcriptionally repress miR167 expression.ConclusionOur study reveals the altered expression of miRNAs and their target genes in a male sterile line of pummelo and highlights that miRNA regulatory network may be involved in floral bud development and cytoplasmic male sterility in citrus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2882-0) contains supplementary material, which is available to authorized users.

Differential expression of microRNAs and potential targets under drought stress in barley

Drought is a crucial environmental constraint limiting crop production in many parts of the world. microRNA (miRNA) based gene regulation has been shown to act in several pathways, including crop response to drought stress. Sequence based profiling and computational analysis have revealed hundreds of miRNAs and their potential targets in different plant species under various stress conditions, but few have been biologically verified. In this study, 11 candidate miRNAs were tested for their expression profiles in barley. Differences in accumulation of only four miRNAs (Ath-miR169b, Osa-miR1432, Hv-miRx5 and Hv-miR166b/c) were observed between drought-treated and well-watered barley in four genotypes. miRNA targets were predicted using degradome analysis of two, different genotypes, and genotype-specific target cleavage was observed. Inverse correlation of mature miRNA accumulation with miRNA target transcripts was also genotype dependent under drought treatment. Drought-responsive miRNAs accumulated predominantly in mesophyll tissues. Our results demonstrate genotype-specific miRNA regulation under drought stress and evidence for their role in mediating expression of target genes for abiotic stress response in barley.

Comparison of miRNAs and Their Targets in Seed Development between Two Maize Inbred Lines by High-Throughput Sequencing and Degradome Analysis.

MicroRNAs (miRNAs) play an important role in plant growth, development, and response to environment. For identifying and comparing miRNAs and their targets in seed development between two maize inbred lines (i.e. PH6WC and PH4CV), two sRNAs and two degradome libraries were constructed. Through high-throughput sequencing and miRNA identification, 55 conserved and 24 novel unique miRNA sequences were identified in two sRNA libraries; moreover, through degradome sequencing and analysis, 137 target transcripts corresponding to 38 unique miRNA sequences were identified in two degradome libraries. Subsequently, 16 significantly differentially expressed miRNA sequences were verified by qRT-PCR, in which 9 verified sequences obviously target 30 transcripts mainly involved with regulation in flowering and development in embryo. Therefore, the results suggested that some miRNAs (e.g. miR156, miR171, miR396 and miR444) related reproductive development might differentially express in seed development between the PH6WC and PH4CV maize inbred lines in this present study.

Genome-wide Investigation of microRNAs and Their Targets in Brassica rapa ssp. pekinensis Root with Plasmodiophora brassicae Infection

Increasing evidence has revealed that microRNAs play a pivotal role in the post transcriptional regulation of gene expression in response to pathogens in plants. However, there is little information available about the expression patterns of miRNAs and their targets in Chinese cabbage (Brassica rapa ssp. pekinensis) under Plasmodiophora brassicae stress. In the present study, using deep sequencing and degradome analysis, a genome-wide identification of miRNAs and their targets during P. brassicae stress was performed. A total of 221 known and 93 potentially novel miRNAs were successfully identified from two root libraries of one control (635-10CK) and P. brassicae-treated Chinese cabbage samples (635-10T). Of these, 14 known and 10 potentially novel miRNAs were found to be differentially expressed after P. brassicae treatment. Degradome analysis revealed that the 223 target genes of the 75 miRNAs could be potentially cleaved. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that the putative target genes of the miRNAs were predominately involved in selenocompound metabolism and plant hormone signal transduction. Then the expression of 12 miRNAs was validated by quantitative real-time PCR (qRT-PCR). These results provide insights into the miRNA-mediated regulatory networks underlying the stress response to the plant pathogen P. brassicae.

Identification of microRNAs and their targets in Paulownia fortunei plants free from phytoplasma pathogen after methyl methane sulfonate treatment.

MicroRNAs (miRNAs) play major roles in plant responses to various biotic and abiotic stresses by regulating gene expression at the transcriptional and post-transcriptional levels. Paulownia witches' broom (PaWB) disease caused by phytoplasmas reduces Paulownia production worldwide. In this study, we investigated the miRNA-mediated plant response to PaWB phytoplasma by Illumina sequencing and degradome analysis of Paulownia fortunei small RNAs (sRNAs). The sRNA and degradome libraries were constructed from healthy and diseased P.fortunei plants and the plants free from phytoplasma pathogen after 60mgL(-1) methyl methane sulfonate treatment. A total of 96 P.fortunei-conserved miRNAs and 83 putative novel miRNAs were identified. Among them, 37 miRNAs (17 conserved, 20 novel) were found to be differentially expressed in response to PaWB phytoplasma infection. In addition, 114 target genes for 18 of the conserved miRNA families and 33 target genes for 15 of the novel miRNAs in P.fortunei were detected. The expression patterns of 14 of the PaWB phytoplasma-responsive miRNAs and 12 target genes were determined by quantitative real-time polymerase chain reaction (qPCR) experiments. A functional analysis of the miRNA targets indicated that these targeted genes may regulate transcription, stress response, nitrogen metabolism, and various other activities. Our results will help identify the potential roles of miRNAs involved in protecting P.fortunei from diseases.

Deep sequencing leads to the identification of eukaryotic translation initiation factor 5A as a key element in Rsv1-mediated lethal systemic hypersensitive response to Soybean mosaic virus infection in soybean.

Rsv1, a single dominant resistance locus in soybean, confers extreme resistance to the majority of Soybean mosaic virus (SMV) strains, but is susceptible to the G7 strain. In Rsv1-genotype soybean, G7 infection provokes a lethal systemic hypersensitive response (LSHR), a delayed host defence response. The Rsv1-mediated LSHR signalling pathway remains largely unknown. In this study, we employed a genome-wide investigation to gain an insight into the molecular interplay between SMV G7 and Rsv1-genotype soybean. Small RNA (sRNA), degradome and transcriptome sequencing analyses were used to identify differentially expressed genes (DEGs) and microRNAs (DEMs) in response to G7 infection. A number of DEGs, DEMs and microRNA targets, and the interaction network of DEMs and their target mRNAs responsive to G7 infection, were identified. Knock-down of one of the identified DEGs, the eukaryotic translation initiation factor 5A (eIF5A), diminished the LSHR and enhanced viral accumulation, suggesting the essential role of eIF5A in the G7-induced, Rsv1-mediated LSHR signalling pathway. This work provides an in-depth genome-wide analysis of high-throughput sequencing data, and identifies multiple genes and microRNA signatures that are associated with the Rsv1-mediated LSHR.

Regulation of Small RNAs and Corresponding Targets in Nod Factor-Induced Phaseolus vulgaris Root Hair Cells.

A genome-wide analysis identified the set of small RNAs (sRNAs) from the agronomical important legume Phaseolus vulgaris (common bean), including novel P. vulgaris-specific microRNAs (miRNAs) potentially important for the regulation of the rhizobia-symbiotic process. Generally, novel miRNAs are difficult to identify and study because they are very lowly expressed in a tissue- or cell-specific manner. In this work, we aimed to analyze sRNAs from common bean root hairs (RH), a single-cell model, induced with pure Rhizobium etli nodulation factors (NF), a unique type of signal molecule. The sequence analysis of samples from NF-induced and control libraries led to the identity of 132 mature miRNAs, including 63 novel miRNAs and 1984 phasiRNAs. From these, six miRNAs were significantly differentially expressed during NF induction, including one novel miRNA: miR-RH82. A parallel degradome analysis of the same samples revealed 29 targets potentially cleaved by novel miRNAs specifically in NF-induced RH samples; however, these novel miRNAs were not differentially accumulated in this tissue. This study reveals Phaseolus vulgaris-specific novel miRNA candidates and their corresponding targets that meet all criteria to be involved in the regulation of the early nodulation events, thus setting the basis for exploring miRNA-mediated improvement of the common bean–rhizobia symbiosis.

Small RNA sequencing and degradome analysis of developing fibers of short fiber mutants Ligon-lintles-1 (Li 1 ) and -2 (Li 2 ) revealed a role for miRNAs and their targets in cotton fiber elongation.

BackgroundThe length of cotton fiber is an important agronomic trait that directly affects the quality of yarn and fabric. Understanding the molecular basis of fiber elongation would provide a means for improvement of fiber length. Ligon-lintless-1 (Li1) and −2 (Li2) are monogenic and dominant mutations that result in an extreme reduction in the length of lint fiber on mature seeds. In a near-isogenic state with wild type cotton these two short fiber mutants provide an effective model system to study the mechanisms of fiber elongation. Plant miRNAs regulate many aspects of growth and development. However, the mechanism underlying the miRNA-mediated regulation of fiber development is largely unknown.ResultsSmall RNA libraries constructed from developing fiber cells of the short fiber mutants Li1 and Li2 and their near-isogenic wild type lines were sequenced. We identified 24 conservative and 147 novel miRNA families with targets that were detected through degradome sequencing. The distribution of the target genes into functional categories revealed the largest set of genes were transcription factors. Expression profiles of 20 miRNAs were examined across a fiber developmental time course in wild type and short fiber mutations. We conducted correlation analysis between miRNA transcript abundance and the length of fiber for 11 diverse Upland cotton lines. The expression patterns of 4 miRNAs revealed significant negative correlation with fiber lengths of 11 cotton lines.ConclusionsOur results suggested that the mutations have changed the regulation of miRNAs expression during fiber development. Further investigations of differentially expressed miRNAs in the Li1 and Li2 mutants will contribute to better understanding of the regulatory mechanisms of cotton fiber development. Four miRNAs negatively correlated with fiber length are good candidates for further investigations of miRNA regulation of important genotype dependent fiber traits. Thus, our results will contribute to further studies on the role of miRNAs in cotton fiber development and will provide a tool for fiber improvement through molecular breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2715-1) contains supplementary material, which is available to authorized users.

Discovery and profiling of microRNAs and their targets in Paulownia ‘Yuza 1’ plants via high-throughput sequencing and degradome analysis

Polyploid breeding is an effective method that has been used widely for the development of new varieties of plants. MicroRNAs (miRNAs) are a class of endogenous small RNAs that regulate gene expression at the post-transcriptional level. To understand the functions of miRNAs in autotetraploid and diploid Paulownia ‘Yuza 1’ plants, two small RNA libraries and two degradome sequencing libraries of the autotetraploid and the corresponding diploid plants were constructed and analyzed. A total of 49 conserved miRNAs that were grouped into 15 families, and 25 novel miRNAs were obtained from two sRNA libraries. Among these miRNAs, 28 were differentially expressed at a significant level in the autotetraploid relative to the diploid. Moreover, the miRNA target genes were identified by degradome sequencing and some of the differently expressed miRNAs and their target genes were validated by quantitative RT-PCR analysis. This study has provided valuable information on P. ‘Yuza 1’ miRNAs and their targets, which will enrich the Paulownia miRNA information resources and will contribute to our understanding of miRNA-mediated regulation in autotetraploid Paulownia plantlets.

Identification of miRNAs and their targets through high-throughput sequencing and degradome analysis in male and female Asparagus officinalis.

BackgroundMicroRNAs (miRNAs), a class of non-coding small RNAs (sRNAs), regulate various biological processes. Although miRNAs have been identified and characterized in several plant species, miRNAs in Asparagus officinalis have not been reported. As a dioecious plant with homomorphic sex chromosomes, asparagus is regarded as an important model system for studying mechanisms of plant sex determination.ResultsTwo independent sRNA libraries from male and female asparagus plants were sequenced with Illumina sequencing, thereby generating 4.13 and 5.88 million final clean reads, respectively. Both libraries predominantly contained 24-nt sRNAs, followed by 21-nt sRNAs. Further analysis identified 154 conserved miRNAs, which belong to 26 families, and 39 novel miRNA candidates seemed to be specific to asparagus. Comparative profiling revealed that 63 miRNAs exhibited significant differential expression between male and female plants, which was confirmed by real-time quantitative PCR analysis. Among them, 37 miRNAs were significantly up-regulated in the female library, whereas the others were preferentially expressed in the male library. Furthermore, 40 target mRNAs representing 44 conserved and seven novel miRNAs were identified in asparagus through high-throughput degradome sequencing. Functional annotation showed that these target mRNAs were involved in a wide range of developmental and metabolic processes.ConclusionsWe identified a large set of conserved and specific miRNAs and compared their expression levels between male and female asparagus plants. Several asparagus miRNAs, which belong to the miR159, miR167, and miR172 families involved in reproductive organ development, were differentially expressed between male and female plants, as well as during flower development. Consistently, several predicted targets of asparagus miRNAs were associated with floral organ development. These findings suggest the potential roles of miRNAs in sex determination and reproductive developmental processes in asparagus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0770-z) contains supplementary material, which is available to authorized users.

Identification of miRNAs and Their Targets in the Liverwort Marchantia polymorpha by Integrating RNA-Seq and Degradome Analyses.

Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem–loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in embryophytes.

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.

BackgroundCytoplasmic male sterility (CMS) provides crucial breeding materials that facilitate hybrid seed production in various crops, and thus plays an important role in the study of hybrid vigor (heterosis), in plants. However, the CMS regulatory network in soybean remains unclear. MicroRNAs (miRNAs) play crucial roles in flower and pollen development by targeting genes that regulate their expression in plants. To identify the miRNAs and their targets that exist in the soybean CMS line NJCMS1A and its maintainer NJCMS1B, high-throughput sequencing and degradome analysis were conducted in this study.ResultsTwo small RNA libraries were constructed from the flower buds of the soybean CMS line NJCMS1A and its maintainer NJCMS1B. A total of 105 new miRNAs present on the other arm of known pre-miRNAs, 23 new miRNA members, 158 novel miRNAs and 160 high-confidence soybean miRNAs were identified using high-throughput sequencing. Among the identified miRNAs, 101 differentially expressed miRNAs with greater than two-fold changes between NJCMS1A and NJCMS1B were discovered. The different expression levels of selected miRNAs were confirmed by stem-loop quantitative real-time PCR. A degradome analysis showed that 856 targets were predicted to be targeted by 296 miRNAs, including a squamosa promoter-binding protein-like transcription factor family protein, a pentatricopeptide repeat-containing protein, and an auxin response factor, which were previously shown to be involved in floral organ or anther development in plants. Additionally, some targets, including a MADS-box transcription factor, NADP-dependent isocitrate dehydrogenase and NADH-ubiquinone oxidoreductase 24 kDa subunit, were identified, and they may have some relationship with the programmed cell death, reactive oxygen species accumulation and energy deficiencies, which might lead to soybean male sterility.ConclusionsThe present study is the first to use deep sequencing technology to identify miRNAs and their targets in the flower buds of the soybean CMS line NJCMS1A and its maintainer NJCMS1B. The results revealed that the miRNAs might participate in flower and pollen development, which could facilitate our understanding of the molecular mechanisms behind CMS in soybean.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2352-0) contains supplementary material, which is available to authorized users.

A highly specific microRNA-mediated mechanism silences LTR retrotransposons of strawberry.

Small RNAs are involved in a plethora of functions in plant genomes. In general, transcriptional gene silencing is mediated by 24-nucleotide siRNAs and is required for maintaining transposable elements in a silenced state. However, microRNAs are not commonly associated with transposon silencing. In this study, we performed small RNA transcriptome and degradome analyses of the Rosaceae model plant Fragaria vesca (the woodland strawberry) at the genome-wide level, and identified miRNA families and their targets. We report a highly specific mechanism of LTR retrotransposon silencing mediated by an abundant, ubiquitously expressed miRNA (fve-miR1511) generated from a single locus. This miRNA specifically targets LTR retroelements, silencing them post-transcriptionally by perfectly pairing to the highly conserved primer binding site for methionyl initiator tRNA that is essential for reverse transcription. We investigated the possible origins of this miRNA, and present evidence that the pre-miR1511 hairpin structure probably derived from a locus coding for tRNA(iM) (et) through a single microinversion event. Our study shows that this miRNA targets retrotransposons specifically and constitutively, and contributes to features such as genome stability, size and architecture in a far more direct way than previously thought.

Genome-Wide Dissection of the MicroRNA Expression Profile in Rice Embryo during Early Stages of Seed Germination.

The first 24 hours after imbibition (HAI) is pivotal for rice seed germination, during which embryo cells switch from a quiescent state to a metabolically active state rapidly. MicroRNAs (miRNAs) have increasingly been shown to play important roles in rice development. Nevertheless, limited knowledge about miRNA regulation has been obtained in the early stages of rice seed germination. In this study, the small RNAs (sRNAs) from embryos of 0, 12, and 24 HAI rice seeds were sequenced to investigate the composition and expression patterns of miRNAs. The bioinformatics analysis identified 289 miRNA loci, including 59 known and 230 novel miRNAs, and 35 selected miRNAs were confirmed by stem-loop real-time RT-PCR. Expression analysis revealed that the dry and imbibed seeds have unique miRNA expression patterns compared with other tissues, particularly for the dry seeds. Using three methods, Mireap, psRNATarget and degradome analyses, 1197 potential target genes of identified miRNAs involved in various molecular functions were predicted. Among these target genes, 39 had significantly negative correlations with their corresponding miRNAs as inferred from published transcriptome data, and 6 inversely expressed miRNA-target pairs were confirmed by 5ʹ-RACE assay. Our work provides an inventory of miRNA expression profiles and miRNA-target interactions in rice embryos, and lays a foundation for further studies of miRNA-mediated regulation in initial seed germination.