Small RNA sequencing identifies miRNA roles in ovule and fiber development
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ABSTRACT: To dissect the roles of miRNAs in fiber development, we sequenced small RNAs from ovules -1 to +1 day post anthesis (DPA) and young leaves. A series of conserved and novel miRNA were identified from cotton EST database and the genome of G.raimondii, many of which were shown to be expressed differentially between ovule and leaf, indicating their potential roles in fiber development or leaf development. Cotton (Gossypium Hirsutum L.) cultivar TM-1 were grown in the greenhouse. Cotton ovules -1 to +1 day post anthesis (DPA) and young leaves were harvested for five biological replicates and immediately frozen in liquid nitrogen. They were then stored at -80M-BM-0C following RNA extraction. Totals RNAs were extracted from each tissue sample using the mirVana miRNA isolation kit (Ambion, Austin, TX) according to the manufacturerM-bM-^@M-^Ys protocol. The small RNA samples extracted from the five biological replicates were pooled together for leaf and ovule, respectively. Finally, the construction of pooled small RNA libraries and sequenced were performed by LC Sciences (Houston, TX) using Illumina high-throughput sequencing platform.
Project description:To dissect the roles of miRNAs in fiber development, we sequenced small RNAs from ovules -1 to +1 day post anthesis (DPA) and young leaves. A series of conserved and novel miRNA were identified from cotton EST database and the genome of G.raimondii, many of which were shown to be expressed differentially between ovule and leaf, indicating their potential roles in fiber development or leaf development.
Project description:Three samples of cotton fiber harvested 3, 7 and 15 days post anthesis (DPA) and a sample of cotton ovule harvestd 0 DPA were used for non-coding RNA extraction and seloxa sequencing experiments, respectively.Cotton plants (Gossypium hirsutum cv. Xuzhou 142) were grown in a soil mixture in fully automated walk-in growth rooms with 300 M-BM-5mol mM-bM-^@M-^S2 sM-bM-^@M-^S1 average light intensity, 60% relative humidity and temperatures set to 30C during the light period and 28C during the dark (12-h light/dark cycle). These conditions were consistent throughout the year. Small RNA molecules under 30 bases were amplified and isolated from an agarose gel. The purified DNA was used directly for cluster generation and sequencing analysis using the Illumina Genome Analyzer IIx according to the manufacturer's instructions. The 38nt sequence tags from sequencing went through data cleaning first, which included getting rid of the low-quality tags and clipping adapter sequences. Three samples of cotton fiber harvested 3, 7 and 15 days post anthesis (DPA) and a sample of cotton ovule harvestd 0 DPA were used for non-coding RNA extraction and seloxa sequencing experiments, respectively.
Project description:Cotton is one of the most commercially important Fiber crops in the world and used as a source for natural textile Fiber and cottonseed oil. The fuzzless-lintless ovules of cotton mutants are ideal source for identifying genes involved in Fiber development by comparing with Fiber bearing ovules of wild-type. To decipher molecular mechanisms involved in Fiber cell development, transcriptome analysis has been carried out by comparing G. hirsutum cv. MCU5 (wild-type) with its fuzzless-lintless mutant (MUT). Cotton bolls were collected at Fiber initiation (0 dpa/days post anthesis), elongation (5, 10 and 15 dpa) and secondary cell wall synthesis stage (20 dpa) and gene expression profiles were analyzed in wild-type and MUT using Affymetrix cotton GeneChip Genome array. Cotton plants were grown under field condition. Flowers were tagged and cotton bolls were collected during Fiber development stages. Total RNA was isolated from Fiber bearing ovules of wild-type (WT) and fuzzless-lintless ovules of mutant (MUT) collected at various (0, 5, 10, 15 and 20 dpa) Fiber development stages using SpectrumTM Plant Total RNA kit (Sigma, USA) according to the manufacturerM-bM-^@M-^Ys protocol. Affymetrix cotton GeneChip Genome array (Affymetrix, USA) having 23,977 probe sets representing 21,854 cotton transcripts was used for transcriptome analysis. Three biological replicates were maintained to test the reproducibility and quality of the chip hybridization. cDNA labeling, array hybridization, staining and washing procedures were carried out as described in the Affymetrix protocols. CEL files having estimated probe intensity values were analyzed with GeneSpring GX-11.5 software (Agilent Technologies, USA) to get differentially expressed transcripts. The Robust Multiarray Average (RMA) algorithm was used for the back ground correction, quantile normalization and median polished probe set summarization to generate single expression value for each probe set. Normalized expression values were log2-transformed and differential expression analysis was performed using unpaired t-test. The p-values were corrected by applying the false discovery rate (FDR) correction (Benjamini and Hochberg, 2000).
Project description:Cotton is one of the most commercially important Fiber crops in the world and used as a source for natural textile Fiber and cottonseed oil. The fuzzless-lintless ovules of cotton mutants are ideal source for identifying genes involved in Fiber development by comparing with Fiber bearing ovules of wild-type. To decipher molecular mechanisms involved in Fiber cell development, transcriptome analysis has been carried out by comparing G. hirsutum cv. MCU5 (wild-type) with its fuzzless-lintless mutant (MUT). Cotton bolls were collected at Fiber initiation (0 dpa/days post anthesis), elongation (5, 10 and 15 dpa) and secondary cell wall synthesis stage (20 dpa) and gene expression profiles were analyzed in wild-type and MUT using Affymetrix cotton GeneChip Genome array.
Project description:This experiment was designed to investigate the molecular basis of cotton fiber cell initiation. 32,000 ESTs were sequenced from Gossypium hirsutum L. TM-1 immature ovules (GH_TMO) and developed cotton oligonucleotide microarrays containing ~23,000 unigenes. Transcriptome analyses were performed to compare gene expression changes in laser capture microdissected fiber cell initials (or epidermis) and inner ovules. The gene expression profiles of the fiber cell initials were compared with those of the inner ovules in each developmental stage prior to, right at, and shortly after the initiation of fiber cells. Many genes in various molecular function or biological processes were over- or under-represented between fibers and non-fiber tissues in each developmental stage, suggesting temporal regulation of gene expression during early stages of fiber development. For gene expression studies using a large set cotton oligo-microarray, 4 developmental stages were chosen. To study differential expression during fiber initiation, ovules at -2 DPA, 0 DPA, and 2 DPA were used. One of the fiber elongation stage tissues (7 DPA) was included. In each developmental stage, epidermis was separated from inner ovules and subjected to the hybridization. In addition, epidermis and ovule comparisons were performed individually with 0 DPA as a control point for comparison.
Project description:Transcriptomes fiber and ovules were compared by applying serial analysis of gene expression (SAGE). Keywords: Tissue Comparison We constructed three SAGE libraries and sequenced 57321, 64188, and 69104 tags from fiber, Xu-142 ovule (ovule) and fl mutant ovules (fl) respectively of Upland Cotton, Gossypium hirsutum L. cv. Xu-142.
Project description:The chip of cotton oligonucleotide microarrrays, which contain ~23,000 UniGenes from our own ESTs sequence project (Gossypium hirsutum L. TM-1 immature ovules (GH_TMO) ) and open resources, was developed. Transcriptome analyses were performed to compare gene expression changes in laser capture microdissected (LCM) fiber cell initials (or epidermis) and inner ovules. The gene expression profiles of the fiber cell initials were compared with those of the inner ovules in each developmental stage prior to (-2DPA), right at (0DPA), and shortly after the initiation of fiber cells (2DAP and 7DPA). Many genes in various molecular functions or biological processes were over- or under-represented between fibers and non-fiber tissues in each developmental stage, suggesting temporal regulation of gene expression during early stages of fiber development. For gene expression studies using a large set cotton oligo-microarray, 4 developmental stages were chosen. To study differential expression during fiber initiation, ovules at -2 DPA, 0 DPA, and 2 DPA were used. One of the fiber elongation stage tissues (7 DPA) was included. In each developmental stage, epidermis was separated from inner ovules and subjected to the hybridization. In addition, epidermis and ovule comparisons were performed individually with 0 DPA as a control point for comparison.
Project description:Transcriptome analysis in cotton during fibre development stages. To study the molecular response of drought stress in cotton under field condition global gene expression analysis was carried out at fibre development stages (0, 5, 10 and 20 dpa/Days post anthesis). Gossypium hirsutum cv. Bikaneri Nerma was used for the gene expression analysis. Cotton plants were subjected to drought stress at peak flowering stage. Samples were collected when the soil moisture content was 19.5% which is 50% of the normal control plots. Gene expression profiles in drought induced and their respective control samples were analyzed using Affymertix cotton Genechip Genome arrays to study the global changes in the expression of genome. Total RNA was isolated from 0 dpa, 5 dpa, fibre bearing ovules of 10 dpa, and fibre bearing ovules of 20 dpa. Samples were collected from both drought induced and control plants. Biotin labeled cRNA was hybridized on Affymertix cotton Genechip Genome array following the Affymetrix protocols. Three biological replicates were maintained.
Project description:Transcriptional profiling of cotton ovule cells. Comparison of expression of genes in cultured ovules (-1 DPA) treated with or without phytohormones (5um IAA and 1um GA) and over a time-course from 0 to 12 hours. The phytohormone incubation periods for unfertilized ovules were 0, 1, 3, 6, and 12 hours (H) with two biological replicates at each time-point.
Project description:RNAs from the upland cotton 9-DPA fibers were compared to the 9-DPA fiber-detached ovule. RNAs from the upland cotton 9-DPA fibers were compared to the 9-DPA fiber-detached ovule.