Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in T. aestivum and B. distachyon
ABSTRACT: The small RNA transcriptomes of bread wheat (Triticum aestivum L.) and its emerging model (Brachypodium distachyon (L.) Beauv) were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNA) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of ESTs, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5' RACE experiments demonstrated their capability to cleave predicted target genes including three disease resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in Brachypodium distachyon. Keywords: Small RNA Overall design: One wheat small RNA library (Tae) and two Brachypodium small RNA libraries (BdR and BdV) were sequenced.
INSTRUMENT(S): Illumina Genome Analyzer II (Triticum aestivum)
Project description:The small RNA transcriptomes of bread wheat (Triticum aestivum L.) and its emerging model (Brachypodium distachyon (L.) Beauv) were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNA) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of ESTs, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5' RACE experiments demonstrated their capability to cleave predicted target genes including three disease resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in Brachypodium distachyon. Keywords: Small RNA One wheat small RNA library (Tae) and two Brachypodium small RNA libraries (BdR and BdV) were sequenced.
Project description:The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using Parallel Analysis of RNA Ends (PARE) data is lacking in B. distachyon. B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants. Examination of various tissues and stresses in Brachypodium by high throughput sequencing for small RNA profiling and PARE (Parallel Analysis of RNA Ends)
Project description:Background: MicroRNAs regulate various biological processes in plants. Considerable data are available on miRNAs involved in the development of rice, maize and barley. In contrast, little is known about miRNAs and their functions in the development of wheat. In this study, five small RNA (sRNA) libraries from wheat seedlings, flag leaves, and developing seeds were developed and sequenced to identify miRNAs and understand their functions in wheat development. Results: Twenty-four known miRNAs belonging to 15 miRNA families were identified from 18 MIRNA loci in wheat in the present study, including 15 (9 MIRNA loci) first identified in wheat, 13 miRNA families (16 MIRNA loci) being highly conserved and 2 (2 MIRNAs loci) moderately conserved. In addition, fifty-five novel miRNAs were also identified. The potential target genes for 15 known miRNAs and 37 novel miRNAs were predicted using strict criteria, and these target genes are involved in a wide range of biological functions. Four of the 15 known miRNA families and 22 of the 55 novel miRNAs were preferentially expressed in the developing seeds with logarithm of the fold change of 1.0～7.6, and half of them were seed-specific, suggesting that they participate in regulating wheat seed development and metabolism. From 5 days post-anthesis to 20 days post-anthesis, miR164 and miR160 increased in abundance in developing seeds, whereas miR169 decreased, suggesting their coordinating functions in the different developmental stages of wheat seed. Moreover, eight known miRNA families and 28 novel miRNAs exhibited tissue-biased expression in wheat flag leaves, with the logarithm of the fold changes of 0.5～5.2. The putative targets of these tissue-preferential miRNAs were involved in various metabolism and biological processes, suggesting complexity of the regulatory networks in different tissues. Our data also suggested that wheat flag leaves have more complicated regulatory networks of miRNAs than developing seeds. Conclusions: Our work identified and characterised wheat miRNAs, their targets and expression patterns. This study is the first to elucidate the regulatory networks of miRNAs involved in wheat flag leaves and developing seeds, and provided a foundation for future studies on specific functions of these miRNAs. Overall design: To identify more miRNAs and understand the regulatory roles of miRNAs in wheat flag leaf and developing seed, five sRNA libraries from seedling, flag leaf, 5-d seed, 10-d seed and 20-d seed were sequenced
Project description:We aimed to identify differentially expressed miRNAs during wheat grain development by using high-throughput sequencing approach. Four small RNA libraries were constructed from wheat grains collected at 7, 14, 21 and 28 days post anthesis (DPA). A total of 165 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Examination of 4 different small RNA expression profilings in the 4 developmental stages of wheat grains.
Project description:Purpose: To identify abiotic stress responsive and tissue specific miRNAs at genome wide level in wheat (Triticum aestivum) Results: Small RNA libraries were constructed from four tissues (root, shoot, mature leaf and spikelets) and three stress treatments of wheat seedlings (control, high temperature, salinity and water-deficit). A total of 59.5 million reads were obtained by high throughput sequencing of eight wheat libraries, of which 32.5 million reads were found to be unique. Using UEA sRNA workbench we identified 47 conserved miRNAs belonging to 20 families, 1030 candidate novel and 51 true novel miRNAs. Several of these miRNAs displayed tissue specific expression whereas few were found to be responsive to abiotic stress treatments. Target genes were predicted for miRNAs identified in this study and their grouping into functional categories revealed that the putative targets were involved in diverse biological processes. RLM-RACE of predicted targets of three conserved miRNAs (miR156, miR160 and miR164) confirmed their mRNA cleavage, thus indicating their regulation at post-transcriptional level by corresponding miRNAs. Expression profiling of confirmed target genes of these miRNAs was also performed. Conclusions: This is the first comprehensive study on profiling of miRNAs in a variety of tissues and in response to several abiotic stresses in wheat. Our findings provide valuable resource for better understanding on the role of miRNAs in stress tolerance as well as plant development. Additionally, this information could be utilized for designing wheat plants for enhanced abiotic stress tolerance and higher productivity. Total eight (three stress, one control and four tissue specific small RNA libraries were pepared and sequenced independently [wheat control (WC), wheat high temperature stressed (WHTS), wheat salinity stressed (WSS) and wheat drought stressed (WDS), wheat shoot(WSH), wheat leaf (WLF), wheat flower(WFL), wheat root(WRT)] on Illumina GAIIx
Project description:This study provides a first large-scale cloning and characterization of Porphyra miRNAs and their predicted targets. These miRNAs belong to 224 conserved miRNA families and 7 are novel in Porphyra. These miRNAs add to the growing database of new miRNA and lay the foundation for further understanding of miRNA function in the regulation of Porphyra yezoensis development. We constructed a small RNA library from Porphyra yezoensis.
Project description:This study provides a first large-scale cloning and characterization of Porphyra miRNAs and their predicted targets. These miRNAs belong to 224 conserved miRNA families and 7 are novel in Porphyra. These miRNAs add to the growing database of new miRNA and lay the foundation for further understanding of miRNA function in the regulation of Porphyra yezoensis development. Overall design: We constructed a small RNA library from Porphyra yezoensis.
Project description:Background: MicroRNAs are endogenous small noncoding RNAs that play critical roles in plant abiotic stress responses. The interaction between miRNA-mRNA targets and their regulatory pathways in response to water deficit stress has been investigated in many plant species. However, the miRNA transcriptome of durum wheat (Triticum turgidum L. ssp. durum) is poorly characterised, with little known about miRNA functions related to water deficit stress. Yield loss in durum wheat can be exacerbated due to minimal rainfall in the early reproductive stages of development during Spring in Australia. This study describes genotypic differences in the miRNAome between water deficit tolerant/sensitive durum, using flag leaf and developing head tissue, and more specifically identifies miRNAs associated with water deficit stress. Results: Small RNA libraries (96 in total) were constructed from flag leaf and developing head tissues of four durum genotypes (Tamaroi, Yawa, EGA Bellaroi, Tjilkuri), with or without water deficit stress. Illumina sequencing and subsequent analysis detected 110 conserved miRNAs and 159 novel candidate miRNA hairpins. Statistical analysis of the abundance of sequencing reads revealed 66 conserved miRNAs and five novel miRNA hairpins showing differential expression under water deficit stress. During stress, several conserved and novel miRNAs showed unambiguous inverted regulatory profiles between the durum genotypes studied. Several miRNAs were also identified to have different abundance in the flag leaf compared to the developing head regardless of treatment. Predicted mRNA targets from four novel durum miRNAs were characterised using Gene Ontology (GO) which revealed functions common to stress responses and plant development. Conclusion: For the first time, we present a comprehensive study of the miRNA transcriptome of flag leaf and developing head tissues in different durum genotypes under water deficit stress. The identification of differentially expressed 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 genotype-specific physiological responses leading to stress tolerance capacity. Further functional analysis of specific stress responsive miRNAs identified, and their interaction with mRNA targets is ongoing and will assist in developing future durum wheat varieties with enhanced water deficit stress tolerance. Overall design: A total of 96 small RNA libraries were analysed [4 durum genotypes * 2 tissue types * 2 treatment groups (control and water stress) * 6 biological replicates] in this study. Please see Methods, Liu et al.(2015)
Project description:Two hundreds eighty three and 293 known miRNAs were identified from the control and drought stress libraries, respectively. In addition, 253 potential candidate miRNAs were identified, and among them 48 novel miRNAs/ novel members of known miRNA families whose complementary miRNAs were also detected. Both high-throughput sequencing and RT-qPCR confirmed that 22 members of 4 miRNA families were up-regulated and 10 members of 6 miRNA families were down-regulated in response to drought stress. Among the 48 novel miRNAs, 24 miRNAs were responsive to drought stress with 21 and 3 miRNAs being up- and down-regulated, respectively. The known and predicted targets of these miRNAs in response to drought stress are involved in diverse cellular processes in plants, such as development, transcription, protein degradation, detoxification, nutrient status and cross adaptation. To identify miRNAs in response to drought stress in M. truncatula, control and drought stress shoots were used to construct small RNA libraries, respectively. The date from Solexa sequencer (Illumina) was compared to identify miRNAs in response to drought stress.
Project description:MicroRNAs (miRNAs) are endogenous small RNAs that play important roles in various biological and metabolic processes in plants. Caragana intermedia is an important ecological and economic tree species that is prominent in the desert environment of west and northwest China. To date, no investigation into C. intermedia miRNAs has been reported. In this study, high-throughput sequencing of small RNAs was performed to identify both conserved and novel miRNAs, and also their target mRNA genes in C. intermedia. Based on sequence similarity and hairpin structure prediction, 132 putative conserved miRNAs (12 of which were confirmed to form hairpin precursors) belonging to 31 known miRNA families were identified. Ten novel miRNAs (including the miRNA* sequences of three novel miRNAs) were also discovered. The expression of 12 miRNAs was validated in different tissues, and the 12 miRNAs were further assessed by qRT-PCR after salt treatment. Furthermore, 36 potential target genes of 17 known miRNA families and two potential target genes of one novel miRNA were predicted, and some target genes were also assessed by qRT-PCR after salt treatment. Our study provides a basic catalog of miRNAs and their targets, which will promote further understanding of the important roles of miRNAs in C. intermedia and in other species of the leguminous genus, Caragana. Examination of small RNA expression profiling in three-weeks-old seedings of Caragana intermedia.