Small RNAs in rice seed
Ontology highlight
ABSTRACT:
ORGANISM(S): Oryza sativa
SUBMITTER: Hong-Wei Xue
PROVIDER: E-GEOD-11974 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Characterization and expression profiles of miRNAs in rice seeds.
Xue Liang-Jiao LJ Zhang Jing-Jing JJ Xue Hong-Wei HW
Nucleic acids research 20081222 3
Small RNAs (sRNAs) are common and effective modulators of gene expression in eukaryotic organisms. To characterize the sRNAs expressed during rice seed development, massively parallel signature sequencing (MPSS) was performed, resulting in the obtainment of 797,399 22-nt sequence signatures, of which 111,161 are distinct ones. Analysis on the distributions of sRNAs on chromosomes showed that most sRNAs originate from interspersed repeats that mainly consist of transposable elements, suggesting t ...[more]
Similar Datasets
Project description:Small RNAs are common and effective modulators of gene expression in eukaryotic organisms. To characterize the small RNAs expressed during rice seed development, massively parallel signature sequencing (MPSS) was performed, resulting in the obtainment of 22-nt sequence signatures. Through integrative analysis,novel miRNAs were identified mostly based on the miRNA* accumulation.
2009-02-02 | GSE11974 | GEO
Project description:Background The set of all mRNA molecules present in a cell constitute the transcriptome. The transcriptome varies depending on cell type as well as in response to internal and external stimuli during development. Chili pepper is an economically and culturally important horticultural crop as well as a good model for the study of secondary metabolism during fruit development. Here we present a study of the changes that occur in the transcriptome of chili pepper fruit during development and ripening. Results RNA-Seq was used to obtain transcriptomes of whole Serrano-type chili pepper fruits (Capsicum annuum L.; 'Tampiqueno 74') collected at 10, 20, 40 and 60 days after anthesis (DAA). 15,550,468 Illumina MiSeq reads were assembled de novo into 34,066 chili genes. We classified the expression patterns of individual genes as well as genes grouped into Biological Process ontologies and Metabolic Pathway categories using statistical criteria. For the analyses of gene groups we added the weighted expression of individual genes. This method was effective in interpreting general patterns of expression changes and increased the statistical power of the analyses. Subsets of genes were expressed only at a single time point sampled (1,278, 1,596, 1,519 and 1,583 genes at 10, 20, 40 and 60 DAA, respectively). We also estimated the variation in diversity and specialization of the transcriptome during chili pepper development. Approximately 17% of genes exhibited a significant change of expression in at least one of the intervals sampled. In contrast, significant differences in approximately 63% of the Biological Processes and 80% of the Metabolic Pathways studied were detected in at least one interval. Confirming previous reports, genes related to capsaicinoid and ascorbic acid biosynthesis were significantly upregulated at 20 DAA while those related to carotenoid biosynthesis were highly expressed in the last period of fruit maturation (40-60 DAA). Our RNA-Seq data was validated by examining the expression of nine genes involved in carotenoid biosynthesis by qRT-PCR. Conclusions In general, more profound changes in the chili fruit transcriptome were observed in the intervals between 10 to 20 and 40 to 60 DAA. The last interval, between 40 to 60 DAA, included 49% of all significant changes detected, and was characterized predominantly by a global decrease in gene expression. This period signals the end of maturation and the beginning of senescence of chili pepper fruit. The transcriptome at 60 DAA was the most specialized and least diverse of the four states sampled. RNA-seq libraries of whole chili pepper (Capsicum annuum Serrano 'Tampiqueno 74' landrace) were prepared from fruits at 10, 20, 40 and 60 days after anthesis (DAA). Two biological replicates of each fruit state were prepared (8 libraries in total). Libraries were sequenced in the Illumina MiSeq platform. The title of the libraries are: <DAA>repbio<#> where <DAA> are the days after anthesis (10, 20, 40 or 60) and <#> is the number of biological replicate, 1 or 2. Details follow. Biological material and RNA extraction Capsicum annuum Serrano 'Tampiqueno 74' seeds were germinated and the seedlings were cultivated until maturity under greenhouse conditions in a completely randomized experimental design at Cinvestav-Unidad Irapuato (Guanajuato, Mexico). The plants were grown during the spring and summer, and individual flowers were tagged at anthesis. Chili pepper fruits were randomly collected from different plants at 10, 20, 40 and 60 DAA. After sampling, the fruits were cleaned with ethanol immediately frozen in liquid nitrogen and stored at -80C until further use. For total RNA extractions, 10 fruits at the 10 DAA developmental stage and 5 fruits from each of the 20, 40 and 60 DAA stages were randomly selected from the pool of all harvested fruits. Whole fruits (pericarp, placenta and seeds) were ground in liquid nitrogen with a mortar and pestle to form a fine uniform powder. Samples were mixed vigorously and 100 mg aliquots were measured for each RNA extraction. The process was repeated with different sets of fruits in order to obtain two independent biological replicates at each developmental stage. A NucleoSpin RNA Plant kit (Macherey-Nagel) was used for total RNA extraction and contaminating genomic DNA was removed by DNase I (Macherey-Nagel) treatment following the manufacturers' protocols. Total RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer (Nano-Drop, Wilmington, DE, USA) and RNA quality was evaluated by gel electrophoresis on 1.0% denaturing agarose gels. In addition, aliquots of RNA were run on an Agilent 2100 Bioanalyzer using RNA 6000 chips (Agilent, Santa Clara, CA, USA) to test the RNA integrity number (RIN). All eight samples had RIN values higher than 8.7. Thirty ?g of total RNA from each of the eight samples (two biological replicates of four fruit developmental stages) was used for cDNA library preparation. Library construction and sequencing The eight total RNA samples (two biological replicates from chili pepper fruits at 10, 20, 40 and 60 DAA) were prepared for RNA-Seq using the Illumina TruSeq RNA Sample Preparation v2 Guide following the manufacturer's instructions. Briefly, mRNA was purified from 20?g of total RNA using poly-T oligo- attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, mRNA was fragmented using divalent cations under elevated temperature and primed for cDNA synthesis. The cleaved RNA fragments were primed with random hexamers and reverse transcribed into single-stranded cDNA using reverse transcriptase. In the next step, the RNA template was removed and the complementary cDNA strand was synthesized using RNAse H and DNA polymerase I, respectively. The overhangs that resulted from fragmentation were polished into blunt ends using an End Repair Mix (consisting of T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase). A single T nucleotide was added on the 3' end of the adapter for ligating the adapter to the cDNA fragments. Indexing adapters were ligated to the ends of the cDNAs using T4 DNA ligase, preparing them for hybridization onto a flow cell. Finally, the DNA fragments with adapter molecules at both ends were amplified by PCR to enrich the amount of DNA in the library. PCR was performed with a PCR primer cocktail that anneals to the ends of the adapters. Quantity and quality of the DNA libraries was assessed using Agilent DNA-1000 chips on an Agilent 2100 Bioanalyzer. The 8 cDNA libraries were pooled and simultaneously sequenced from both 5’ and 3’ ends using the Illumina MiSeq System platform according to the manufacturer's instructions. We performed three sequencing runs (technical replicates) with the aim of increase the sequencing depth. 150 bp paired-end reads were obtained in each sequencing run. Fluorescent image processing, base-calling and quality value calculations for each of the three runs were performed using Illumina MiSeq Control Software. Data were deposited at the NCBI (GEO database under series record GSE54123).
2014-02-21 | E-GEOD-54123 | biostudies-arrayexpress
Project description:High-throughput sequencing of small RNAs in chicken somite embryos Dissected tissue white leghorn chicken embryos was disaggregated and RNA extracted using the miRVana kit (Ambion). Small RNA fraction between 19 and 24 nt was isolated from 15% denaturing polyacrylamide gel and 15 micro gram was ligated to Solexa adaptors (Illumina) without de-phosphorylating and re-phosphorylating. The short RNAs were converted to DNA by RT-PCR following the Illumina protocol.
2009-04-27 | E-GEOD-15513 | biostudies-arrayexpress
Project description:Transcriptomic analyses are particularly powerful in research when they are based on detailed underlying knowledge of developmental processes, morphological/anatomical features and biochemical/metabolic processes. Our work is based on our detailed knowledge of grain development and provides an invaluable resource for both Brachypodium grain development specifically and for comparative analyses to other species, including cereal crops. Grain development includes several transition points in addition to being preceded and succeeded by the fundamental developmental switches of fertilization and germination. Such complex process requires a larger sets of genes expressed in a highly-controlled manner. We have selected eight stages of developmental in Brachypodium distachyon encompassing these transitions and conducted comprehensive transcriptomic analyses to generate a valuable resource of the developmental transitions and the distinctive biological processes that are activated and/or repressed during grain development and germination. Comparison of the data generated in this project to other grasses, including the important crop species, and beyond will also shed light on the conservation and/or diversification in gene expression and function in an evolutionary context. Brachypodium distachyon grains (Bd-21 accession) were germinated on moist filter paper after stratification at 4°C for 48h. Five-day old seedlings were transferred to 9cm square pots with a 2:2:1 multipurpose compost: vermiculite: sand mix and grown under controlled environment conditions with a 18h photoperiod at 20°-22°C and light intensity of 180-200 umol/m/s. Tissue samples were collected from eight distinct developmental stages; pre-anthesis ovaries, young grains (1-3 DAA), middle length grains (3-8 DAA), full length grains (8-15 DAA), mature grains (15-20 DAA), mature grains without embryo, germinating grains and seedlings at 3-4 days after germination (Fig.1). Tissues were frozen in liquid nitrogen immediately after collection and were kept at -80°C until further analyses. Total RNA was isolated from frozen tissue samples using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) following the manufacturer’s instructions. Three biological replicates were used for each stage of development. The RNA samples were treated with DNase I (New England Biolabs) in order to eliminate DNA contamination and their concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific).
2019-01-31 | E-MTAB-7607 | biostudies-arrayexpress
Project description:Salivary glands or larval ovaries were isolated from transgenic flies expressing RNAi targeting Nautilus (control) or linker histone H1 using a Tub-Gal4 driver. ~200 larvae were used to isolate salivary glands or ovaries, independently. Total RNA was isolated using Trizol reagent following manufacturer's guidelines. Then 5 M-BM-5g of total RNA was separated on a polyacrylamide gel, and 18-29 nt small RNAs were isolated for cloning.
2013-04-22 | E-GEOD-44399 | biostudies-arrayexpress
Project description:Identification of all expressed transcripts in a sequenced complex genome is technically challenging, but essential for systems biology and genome analysis. We used the transcriptional profiling technology called ‘massively parallel signature sequencing’ (MPSS) to develop a comprehensive expression atlas of rice (Oryza sativa cv Nipponbare). A total of 46,971,553 mRNA transcripts from 22 libraries, and 2,953,855 small RNAs from three libraries were sequenced. The data demonstrated widespread transcription throughout the genome, including expression for up to 25,500 annotated genes and antisense expression for nearly 9,000 annotated genes. An additional set of ~15,000 mRNA signatures mapped to unannotated genomic regions. The majority of the small RNA data represented lower abundance small-interfering RNAs (siRNAs) that match repetitive sequences, intergenic regions, and genes. Among these, numerous clusters of highly-regulated small RNAs were readily observed. We developed a genome browser (http://mpss.udel.edu/rice) for public access to the transcriptional profiling data for this important crop plant. Keywords: MPSS, mRNA, small RNA, transcriptome, rice
2007-02-23 | GSE7107 | GEO
Project description:Identification of all expressed transcripts in a sequenced genome is essential both for genome analysis and for realization of the goals of systems biology. We used the transcriptional profiling technologies like ‘massively parallel signature sequencing (MPSS)’ and ‘Sequencing by Synthesis’ (SBS) to develop a comprehensive expression atlas of rice (Oryza sativa cv Nipponbare). Illumina’s SBS technology can generate large amounts of sequence data in a short time at low cost compared to traditional Sanger sequencing based methods. Using the MPSS technology, we previously analyzed the transcriptomes of 72 rice tissues. To validate the sequencing results from MPSS technology, we employed SBS technology and constructed SBS libraries from 32 rice tissues (47 libraries including replications). For SBS library construction, we used the same mRNA samples and same restriction enzyme (DpnII) that were used for the construction of the MPSS libraries. These libraries include six abiotic-stress libraries, eight pathogen-infected libraries, five insect-damaged libraries, three developing seed libraries, and 10 untreated rice tissue libraries. This study was carried out with the following objectives; a) Identification and quantification of expressed genes in rice at all developmental stages of plant growth, response to biotic and abiotic stresses, and developing seeds; b) Compare SBS signatures with rice genomic sequence to identify novel transcripts; c) To validate the transcriptional data obtained through MPSS technology; and To create query and analysis tools to facilitate public use of and access to rice MPSS and SBS data and to display abundance and chromosomal locations of rice MPSS and SBS signatures. The SBS data will be available at http://mpss.udel.edu/rice_sbs/.
2011-06-22 | GSE25596 | GEO
Project description:Total RNA was isolated from different tissues (leaf, stem and flesh, rind and placenta of the fruits) using TRIzol reagent and small RNA libraries were generated from four cucurbit species: bottle gourd (Lagenaria siceraria (accession Grif 1617 collection from India)), Cucurbita moschata (accession Grif 14244 Early Butternut) Cucurbita pepo (accession NSL98075 Table King), and watermelon (Citrullus lanatus var. lanatus) (PI 438676 Charleston Grey) by pooling equimolar amounts of total RNA from the aforesaid tissues. Construction of small RNA libraries from size fractionated RNA was carried out as described previously. In brief, small RNA fractions of 18–28 nt were isolated from 15% denaturing polyacrylamide gels and sequentially ligated to 5′ and 3′ RNA adapters. Small RNAs ligated with adapters were converted to DNA by RT-PCR following Solexa protocol. The final PCR product was gel purified and sequenced by Genome Analyser II (Illumina).
2012-08-03 | GSE38176 | GEO
Project description:Small RNAs (21-24 nt) are involved in gene regulation through translation inhibition, mRNA cleavage, or directing chromatin modifications. In rice, currently ~240 miRNAs have been annotated. We sequenced more than four million small RNAs from rice and identified another 24 miRNA genes. Among these, we found a novel class of miRNAs that derive from natural cis-antisense transcript pairs. This configuration generates miRNAs that can perfectly match their targets. We provide evidence that the miRNAs function by inducing mRNA cleavage in the middle of their complementary site. Their production requires DCL1 activity, which is essential for canonical miRNA biogenesis. All of the nat-miRNAs identified in this study have large introns in their precursors that appear critical for nat-miRNA evolution and for the formation of functional miRNA loci. These findings suggest that other natural cis-antisense loci with similar exon-intron arrangements could be another source of miRNA genes Keywords: small RNA sequencing by MPSS
2008-02-15 | GSE10523 | GEO
Project description:Transcriptional profiling of wheat embryos of developing seed comparing seeds grown at low temperature:13˚C with seeds grown at high temperature:25˚C during seed development using wheat 2 cultivars: Norin61 (N61) and Shiroganekomugi (SK). Goal was to determine the effects of temperature on global gene expression. Two-condition experiment, Low(13˚C) vs. high (25˚C) temperatures during seed development. Time course experiments along with days after anthesis (DAA).
2011-09-07 | E-GEOD-22786 | biostudies-arrayexpress