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:Flowering pathways are accelerated for rapid production of flowers and seeds in response to drought in certain varieties of sorghum (Sorghum bicolor (L.) Moench). The objective of the present study was to identify potential drought responsive genes that affect flowering time in sorghum under drought stress. Sorghum germplasm accessions representing early, intermediate, and late flowering groups were selected, and drought stress was administered on 25-day old seedlings of the Drought-Stressed group (DS) by withdrawing water whilst the control group of plants were well-watered (WW). At anthesis, with the initiation of pollen shedding, flag leaf tissues were harvested, and total RNA was separately isolated from samples. Transcription profiles consisting of 60 base pairs, paired end reads from total RNA of each sample were explored using Illumina Genome Analyzer deep sequencing method. An average of 66,059,932 clean reads were mapped. Among 10,468 differentially expressed genes, a set of 126 genes was up-regulated, and a set of 61 genes was down-regulated in all comparisons. Pathway enrichment analysis revealed de novo purine biosynthesis and lipoate biosynthesis pathways and Wnt signaling pathway affecting differentially expressed sorghum genes in response to drought. Transcriptome level differences among early, intermediate and late flowering groups of sorghum under WW and DS conditions were efficiently explored in the present study using RNA sequence analysis tools. Candidate genes and pathways that might be used to improve drought tolerance in sorghum were identified. Findings of the present study would lead to new targets for enhancing drought stress tolerance in sorghum.
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.