Project description:In this study, we sequenced small RNA content from seven major tissues/organs employing Illumina technology. More than 154 million reads were generated using Illumina high-throughput sequencing GAII platform, which represented more than 20 million distinct small RNA sequences. After pre-processing, several conserved and novel miRNAs were identified in chickpea. Further, the putative targets of chickpea miRNAs were identified and their functional categorization was analyzed. In addition, we identified miRNAs exhibitng differential and specific expression in various tissues/organs. We collected different tissue samples used in this study and total RNA isolated was subjected to Illumina sequencing. The sequenced data was further filtered using NGS QC Toolkit to obtain high-quality reads. The filtered reads were pre-processed using modified perl script provided in the miRTools software. After quality control, the identical reads were collapsed into a unique read and read count for each sequence was recorded. All the filtered unique reads from each sample were screened stepwise against annotated non-coding RNA sequences, including plant snoRNA, tRNA and rRNA. The remaining reads were screened against repeat sequences from RepBase and chickpea chloroplast sequence. Conserved miRNAs were identified based on similarity with miRBase database and novel miRNAs were identified using miRDeep-P pipeline. For differential expression analysis, the read count for each miRNA was normalized using DESeq software. The genes preferentially and specifically expressed in various tissues/organs were identified.
Project description:In this study, we have elucidated the DNA methylation patterns in different organs of a cultivated chickpea genotype ICC 4958 (leaf, root, flower and young pod) and leaf of wild chickpea PI 489777 using bisulphite sequencing. Approximately 108 million read pairs were analyzed per sample. The extent of methylation along-with the context and genomic location of methylated Cs was identified. Further processing was performed to identify the differentially methylated regions among samples with leaves of ICC 4958 as the reference sample. The high resolution methylome maps of different organs and differentially methylated regions will serve as reference for understanding the epigenetic regulation in chickpea.
Project description:In this study, we aim to present a global view of transcriptome dynamics during flower development in chickpea. We generated around 234 million high-quality reads for eight flower development stages (ranging from 16 to 40 million reads for each stage) and 91 million high-quality reads from three vegetative tissues using Illumina high-throughput sequencing GAII platform. Because of non-availability of reference genome sequence, we mapped the reads to chickpea transcriptome comprised of 34,760 transcripts for estimation of their transcriptional activity in different tissue samples. The transcriptome dynamics was studied by comparison of gene expression during flower development stages with vegetative tissues. We collected different tissue samples used in this study and total RNA isolated was subjected to Illumina sequencing. The sequenced data was further filtered using NGS QC Toolkit to obtain high-quality reads. The filtered reads were mapped to 34760 chickpea transcripts and reads per kilobase per million (RPKM) was calculated for each gene in all the sample to measure their gene expression. Differential expression analysis was performed using DESeq software. The genes preferentially expression during various stages of flower development as compared to vegetative stages and those with speciifc expression were identified.
Project description:We report small RNA data from the leaves of wild chickpea PI 489777. Small RNA library was prepared and sequencing was performed using Illumina platform. A total of 23 million reads were generated, which represented 0.95 million unique reads. These were mapped to the chickpea genome using Bowtie to obtain the non-redundant set of unique small RNA sequences.
Project description:In this study, we performed bisulphite of two stages of seed development in a small-seeded chickpea cultivar (Himchana 1) using Illumina platform. Paired-end reads were generated from 5 libraries. Data obtained in FASTQ files were pre-processed to remove adapters and low-quality reads. We identified methylation level at each cytosine residue covered in sequencing and differentially methylated regions (DMRs) between stages of seed development.
Project description:In this study, we performed bisulphite of five stages of seed development in a large-seeded chickpea cultivar (JGK 3) using Illumina platform. Paired-end reads were generated from 11 libraries. Data obtained in FASTQ files were pre-processed to remove adapters and low-quality reads. We identified methylation level at each cytosine residue covered in sequencing and differentially methylated regions (DMRs) between stages of seed development.
Project description:In this study, we aim to present a global view of transcriptome dynamics during various abiotic stresses in chickpea. We generated about 252 million high-quality reads from eight libraries (control, desiccation, salinity and cold stress samples for roots and shoots) using Illumina high-throughput sequencing GAII platform. We mapped the reads to the desi 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. We collected different tissue samples (root and shoot tissues of 10-day-old seedlings subjected to control (kept in water), desiccation (transferred on folds of tissue paper), salinity (transferred to beaker containing 150 mM NaCl solution) and cold (kept in water at 4 M-BM-1 1M-BM-0C) stress for 5 h. Total RNA isolated from these tissue samples was subjected to Illumina sequencing. The sequenced data was further filtered using NGS QC Toolkit to obtain high-quality reads. The filtered reads were mapped to annotated chickpea genome using TopHat and fragments per exon kilobase per million (FPKM) was calculated using Cufflinks software for each gene in all the sample to measure their gene expression. Differential expression analysis was performed using Cuffdiff software. The differentially expressed genes during various abiotic stress conditions were identified.
Project description:The total RNA were extracted from pooled tissues of leaves and flowers from several plants of chickpea (Cicer arietinum) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then small RNAs ranging in 18–30 nucleotides were size fractionated electrophoretically, isolated from the gel, ligated with the 5′ and 3′ RNA adapters. The ligated product was reverse transcribed and subsequently amplified using 10–12 PCR cycles. The purified PCR product was sequenced using Illumina Genome Analyzer II. The qualified reads were used to predict microRNAs and phased small interfering RNAs from chickpea. Identification of microRNAs and phased small inferfering RNAs in chickpea (Cicer arietinum) by analyzing small RNA sequencing profiles of leaves and flowers using Illumina GAII.
Project description:Chickpea (Cicer arietinum) is the third largest legume grown worldwide and are prone to drought and various pathogen infections. These stresses often occur concurrently in the field conditions. Previous studies in other plant species indicated that plant senses concurrently occurring stresses as new state of stress however, the molecular events in response to that is largely unknown. In the present study, we studied the transcriptome changes in chickpea plants exposed to combination of drought stress and a potential wilt pathogen, Ralstonia solanacearum by microarray analysis. Chickpea plants were exposed to short duration individual drought (SD-drought, soil field capacity, FC-35%), long duration individual drought (LD-drought, FC-30%), short duration individual pathogen stress (SD-pathogen = 2 days pathogen infection), long duration individual pathogen stress (LD-pathogen = 4 days of infection) and short duration and long duration combined stress, SD-combined = 2 days of pathogen infection with progressive drought (FC-40% to FC- 35%), LD combined = 4 days of pathogen infection with progressive drought (FC-35% to 30%).Transcriptome analysis for the leaf samples from above treatment were done by microarray analysis using Agilent ChickpeaGXP_8X60K chip. Result indicated presence of specific molecular events and also some common but tailored events in response to combined stress. Global transcriptional analysis in chickpea leaves exposed to individual and combined drought stress and Ralstonia solanacearum infection.
Project description:‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea accessions tolerant and susceptible to cold stress. Two groups of a tolerant and susceptible accession were challenged with cold stress. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaves and flowers/buds/early pods tissues were collected and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (leaf and flower tissues separate) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1. The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress. Globally, the level of 221 transcripts was affected in response to cold stress in all the genotypes and tissue types studied. The DE transcripts in response to cold stress fell into various functional categories, indicating a broad response. Sixteen out of the 221 DE transcripts were consistently expressed in cold tolerant/susceptible genotypes. All these transcripts were repressed and none was found to be consistently induced in response to cold-stress. Most of the putative genes were identified in leaves of tolerant genotypes, and included a beta-galactosidase (DY475141) transcript that was possibly indicative of disaccharide (e. g. sucrose) retention with the effect of protecting cell membranes during cold stress. Several protein synthesis/modification and energy/metabolism transcripts were also repressed (e.g. DY475282, DY396371 and DY475555), which was likely due to the impairment of photosynthesis and respiration at low temperature. Other consistently repressed transcripts in tolerant genotypes included putative signalling (DY396262, DY475384 and DY396307) and defence-related proteins (CV793589 and DY396343), which may be involved in the repression of cell death mechanisms that are absent in tolerant genotypes. In susceptible genotypes, a putative superoxide dismutase precursor protein (DY475397) and sorting nexin protein (DY475523) were the only known transcripts to be consistently repressed. Keywords: Chickpea, Cold stress, Tolerant, Susceptible, cDNA microarray