Project description:In this work, we performed high throughput sequencing of small RNA libraries in maize (Zea mays ssp. mays) and teosinte (Zea mays ssp. parviglumis) to investigate the response mediated by miRNAs in these plants under control conditions, submergence, drought and alternated drought-submergence or submergence-drought stress. After Illumina sequencing of 8 small RNA libraries, we obtained from 16,139,354 to 46,522,229 raw reads across the libraries. Bioinformatic analysis identified 88 maize miRNAs and 76 miRNAs from other plants differentially expressed in maize and/or in teosinte in response to at least one of the treatments, and revealed that a larger set of miRNAs were regulated in maize than in teosinte in response to submergence and drought stress.
Project description:To identify novel microRNAs that are associated with drought tolerance in two different cowpea genotypes, we generated small RNA sequences from adult cowpea plants under control and dought stress treatments. Over 79 million raw reads were generated and numerous novel microRNAs are identified, including some associated with drought tolerance.
Project description:Purpose: We aimed to identify ZAT18 target genes and characterize functions of ZAT18 during plant drought tolerance Methods: A total amount of 3 μg RNA was used for generation of sequencing libraries using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. Clean reads were obtained by removing low quality reads, reads containing adapter and ploy-N from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. Index of the Arabidopsis genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM (Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of drought stress versus control condition was performed using the DESeq R package (1.18.0). Results:In total, eight samples with two biological replicates per genotype/treatment combination were used for RNA sequencing analysis. At least 2 G clean bases were generated for each sample. Comparative analysis revealed that 1777 genes were transcriptionally affected by AtZAT18 trasngene or drought treatment. The results showed that overexpression of AtZAT18 modulated expression level changes of 423 and 561genes under control and drought stress conditions, respectively. Drought stress treatment changed expression of 971 genes with 768 up-regulated and 203 down-regulated.
Project description:In this study, we aim to present a global view of transcriptome dynamics during drought stress in different chickpea genotypes. We generated about 800 million high-quality reads from 14 libraries (control and stress samples for two chickpea genotypes for drought stress at two developmental stages) using Illumina high-throughput sequencing platform. We mapped the reads to the kabuli chickpea genome for estimation of their transcript abundance in different tissue samples. The transcriptome dynamics was studied by differential gene expression analyses between stress treatment and control sample for each genotype.
Project description:The present study is expected to reveal differentially expressed genes under drought stress of Sorghum bicolor. The seeds of Sorghum genotype drought tolerant (DT) were grown at 28-32°C day/night temperature with 12/12 h light/dark period in the phytotron glass house. The fully opened uppermost leaves from control and drought stressed seedlings were sampled and stored at -80°C. For RNA-Seq libraries, one microgram of total RNA was extracted with Trizol reagent (Invitrogen, USA) and mRNA libraries were produced using the TruSeq mRNA-Seq library kit (Illumina) according to manufacturer’s instructions. The libraries generated were quantitated using an Agilent Bioanalyzer DNA 1000 chip. (Agilent Technologies, Santa Clara, CA) and a 2x101 cycle paired end sequencing (sequenced by Sandor Pvt. Ltd., Hyderabad, India) was performed using an Illumina HiScanSQ sequencer (Illumina Inc.). Initially, raw reads were processed by NGSQC toolkit (http://59.163.192.90:8080/ngsqctoolkit/) and high quality reads were subjected to de-novo assembly using Trinity assembler (Patel and Jain, 2012). Assembled transcripts were quantified by standard pipeline (Trinity→RSEM→R→DESeq) and those transcripts were removed which has zero FPKM in all four samples (Anders, 2010; Grabherr, et al., 2011; Li and Dewey, 2011). These transcripts were further processed by transdecoder tool to retrieve full length coding sequence and subsequent annotated by FastAnnotator (http://fastannotator.cgu.edu.tw/index.php) (Chen, et al., 2012). Pathway enrichment analysis was performed for the predicted transcripts by KEGG Automatic Annotation Server (KAAS; www.genome.jp/tools/kaas/) for the classification of spatial and temporally governed pathways.
Project description:To identify novel microRNAs that are associated with drought tolerance in two different cowpea genotypes, we generated small RNA sequences from adult cowpea plants under control and dought stress treatments. Over 79 million raw reads were generated and numerous novel microRNAs are identified, including some associated with drought tolerance. Sequencing of small RNAs in two cowpea genotypes under control and drought stress conditions.
Project description:In this study, genome-wide transcriptome profiling was used to understand molecular genetic mechanism of drought tolerance in rice. Illumina High-Seq 2000 platform was used for sequencing RNA from leaf tissue of rice plants exposed to controlled drought stress and well-watered conditions. The differentially expressed genes were used to identify biological process and cis-regulatory elements enriched under drought stress compared to well-watered conditions. Oryza sativa ssp. japonica cv. Nipponbare plants were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.
Project description:Heat shock factors (Hsfs) are known to regulate heat and drought stress response by controlling the expression of heat shock proteins and oxidative stress responsive genes. Loss-of-function of OsHSFA2e gene resulted in increased sensitivity of rice plants to drought and heat stress. To identify the targets of OsHSFA2e and dissect the stress response pathway regulated by it, we performed transcriptome profiling of Oshsfa2e mutant plants under drought stress as well as well-watered conditions by RNA-sequencing. OsHSFA2e loss-of-function rice plants (Oryza sativa ssp. japonica cv. Nipponbare) were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.
Project description:The members of bHLH transcription factor superfamily are known to play key role in plant development and abiotic stress response. Loss-of-function of OsbHLH148 gene resulted in increased sensitivity of rice plants to drought stress. To identify the targets of OsbHLH148 and dissect the drought stress response pathway regulated by it, we performed transcriptome profiling of Osbhlh148 mutant plants under drought stress as well as well-watered conditions by RNA-sequencing. OsbHLH148 loss-of-function rice plants (Oryza sativa ssp. japonica cv. Nipponbare) were exposed to controlled drought stress and well-watered conditions at the vegetative stage. Controlled drought (DR) stress was applied on 45 day old plants following gravimetric approach. The soil water content was brought down to 40% field capacity over a period of 3-4 days and plants were maintained at that level for 10 days by weighing the pots daily at a fixed time of the day and replenishing the water lost through evapotranspiration. Another set of plants were maintained at 100% FC as well-watered (WW) condition. Total RNA isolated from leaf tissue was used for RNA-sequencing. Two biological replicates per sample were sequenced. cDNA library was constructed using TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina). Sequencing was carried out on each library to generate 50 bp SE reads using Illumina High-Seq 2000 platform. The transcriptome reads were mapped to the rice reference genome sequence (MSU 7.0) with tophat1.3.1 using the program’s default parameters (http://tophat.cbcb.umd.edu). Mapped RNA-Seq reads were assembled into transcripts by Cufflinks (http://cufflinks.cbcb.umd.edu/) and differentially expressed genes were identified by using Cuffdiff.
Project description:Purpose: The goals of this study are studies the response of annual Zea mays ssp. mexicana L. under cold and drought stress Methods: The seedlings of zea may ssp. mexicana L. were generated by Illumina HiSeq2500 deep-sequencing. In order to generate a global overview of Zea mexicana transcriptome data, 3 of complement DNA (cDNA) libraries were prepared from RNA isolated from root, stem, and leave mixed tissues of Zea Mexicana from Control (24℃), Cold (4℃) and Drought (PEG2000, 20%) treatments and each teatment has two repetitions. The sequence reads that passed quality filters were merged and de novo to generate all transcripts set by Trinity with default parameter, which will be treated as reference genome. The number of paired-reads of each sample were mapped to reference genome by Bowtie software v1.1.1 and the number of mapped reads were calculated by RSEM. qRT-PCR validation was performed using BIO-RAD CFX96 sequence detection system and SYBR Green assays. Results: Using RNA-Seq technology with the Trinity assembled method, we generated a seedling plant transcriptome at a sequencing size of 51.78Gb of Zea mays ssp. mexicana L. from pooled RNA samples which included control (CK), cold (4℃) and drought (PEG2000, 20%) stressed plant samples. A total of 414,232,462 high quality clean reads were used to conduct de novo assembly and annotation of genes without reference genome information. All of these reads were assembled into 251,145 transcripts (N50 = 1,269 bp) and 184,280 unigenes (N50 = 923 bp). A total of 3,504 up-regulated and 1,220 down-regulated genes were detected under cold stress and 532 up-regulated and 82 down-regulated genes were detected under drought stress. A Venn diagram indicated that 208 genes were affected by both cold and drought stresses. 3 cold stress pathways and 5 drought related pathways showed significant KEGG pathways. Functional enrichment analyses identified many common or specific biological processes and gene sets in response to drought and cold stresses. The ABA dependent pathway, trehalose synthetic pathway and CBF6 gene of ICE1-CBF pathway may play important roles in the DEGs co-up-regulated by both stresses of Zea mays ssp. mexicana L. Conclusions: We analyzed transcriptome data and gene expression profile information from seedlings of Zea mays ssp. mexicana L. under cold and drought stresses. Together these data provides the most comprehensive sequence study available for Zea mays ssp. mexicana L. and provides some important functional genes and molecular mechanism information for improving the quality characteristic of maize in the future.