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: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.
Project description:Microarrays have increasingly become a powerful tool for high throughput gene-expression studies and discovery of novel biomarker genes. Developed for a large number of organisms, including plants, microarrays are commonly performed for species that have sequenced data, for performing gene expression analysis, miRNA profiling, comparative genomic hybridization (CGH), ChIP-on-chip and SNP analysis. Genomic resources are still very limited for chickpea, a very important food legume crop. Here, we report the design and comprehensive validation of Next Generation Sequencing transcriptome data for chickpea through microarray technology to develop a high-throughput resource for studying the expression of all the transcripts in different biological samples to help functional genomics and breeding programs. This microarray design was developed and validated jointly by Genotypic Technology Private Limited and National Institute of Plant Genome Research. First, we designed 400k probes using reads covering 35k assembled contigs and 100k singletons chickpea transcripts. The 400k chip was hybridized with DNA and RNA samples of chickpea and microarray analysis was carried out. A total of 73,922 probes were found to be specific to chickpea transcripts. Best probes were filtered from the analyzed data and a total of 61,659 probes were selected to develop the final microarray design in 60k gene-expression microarray format. The probes represented 51,444 unique transcripts. The probes were annotated based on their corresponding chickpea transcript and similarity with other plants species. Microarray results were concordant with previous results from the NGS studies. The design of custom oligonucleotide probes for microarrays have varied functional genomic applications and this approach represents a valuable resource for chickpea.
Project description:Chickpea (Cicer arietinum L.) seeds are valued for their nutritional scores and limited information on the molecular mechanisms of chickpea fertilization and seed development is available. In the current work, comparative transcriptome analysis was performed on two different stages of chickpea ovules (pre- and post-fertilization) to identify key regulatory transcripts. Two-staged transcriptome sequencing was generated and over 208 million reads were mapped to quantify transcript abundance during fertilization events. Mapping to the reference genome showed that the majority (92.88%) of high-quality illumina reads were aligned to the chickpea genome. Reference-guided genome and transcriptome assembly yielded a total of 28,783 genes. Of these, 3399 genes were differentially expressed after the fertilization event. These involve up-regulated genes including LOC101500970, LOC101506539 and down-regulated genes LOC101493897, LOC101491695 and so on. Transcription factor families including UDP-glucuronyltransferase, NAC transcription factor, heat shock transcription factor, and auxin-responsive transcription factor were also found to be activated after fertilization. Activation of these genes and transcription factors results in the accumulation of carbohydrates and proteins by enhancing their trafficking and biosynthesis. Total 17 differentially expressed genes, were randomly selected for qRT-PCR for validation of transcriptome analysis and showed statistically significant correlations with the transcriptome data. Our findings provide insights into the regulatory mechanisms underlying changes in fertilized chickpea ovules. This work may come closer to a comprehensive understanding of the mechanisms that initiate developmental events in chickpea seeds.