Project description:Comparative transcriptome profiles of cotton (G. hirsutum L. cv. Bikaneri narma) during boll development stages (0, 2, 5 and 10 dpa) under bollworm infested biotic stress. Cotton is one of the most commercially important fibre crops in the world and used as a source for natural textile fibre and cottonseed oil. The biotic stress is one of the major constraints for crop production. Cotton bollworm (Helicoverpa armigera) is one the major insect pest in cotton and drastically damages the cotton boll. To decipher the molecular mechanisms involved in cotton boll/fibre cell development, transcriptome analysis has been carried out by comparing G. hirsutum L cv. Bikaneri narma cotton boll samples induced by biotic stress (bollworm infested) and that their respective control cotton bolls collected under field conditions. Cotton bolls were collected at fibre initiation (0, 2 dpa/days post anthesis) and elongation (5, 10 dpa) stages for both control and biotic stress condition and gene expression profiles were analyzed by Affymetrix cotton GeneChip Genome array. Cotton plants (G. hirsutum L. cv. Bikaneri narma) were grown under field condition. Helicoverpa armigera (cotton bollworm) second instar larvae was released at the time of flower opening and covered with polythene bag to prevent insect escape and tagged. The infested flowers were collected after 8 hrs infestations and labeled as 0 dpa. Likewise after two and five dayM-bM-^@M-^Ys infestation the bolls were collected and labelled as 2 and 5 dpa, respectively. After five days the insect was removed from bolls and left the bolls for up to 10 days and collected then labeled as 10 dpa. Meanwhile respective control samples also tagged and collected. Total RNA was isolated from control (WT) and biotic stress (bollworm infested) induced samples collected at 0, 2, 5 and 10 dpa boll 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 seeds (Gossypium hirsutum cv. CCRI12) were grown in a growth chamber under 29/25Â°C temperature and a 16:8 h light:dark cycle, and water was added every two days. All plants were used in experiments at the 6-7 fully expanded true leaf stage, which occurred 5-6 weeks after sowing. Cotton bollworm (CBW; Helicoverpa armigera) larvae were reared on an artificial diet and maintained at 27 Â± 2Â°C, 75 Â± 10% relative humidity, and 14:10 h light:dark in the laboratory. For insect treatment, seven H. armigera larvae (third instars) were placed on a group of three plants, which were kept within plastic bags (30 Ã? 40 cm), until time of harvest, with samples for each time point maintained separately. Undamaged plants maintained under the same conditions were used as controls. Cotton leaves from control plants and plants exposed to H. armigera were harvested at 6 h, 12 h, 24 h, and 48 h after onset of herbivory. For each treatment group and time point, cotton leaves were harvested from the three plants per treatment group and flash frozen in liquid nitrogen. For each time point, three replicate treatments and controls were performed. For insect treatment, seven H. armigera larvae (third instars) were placed on a group of three plants, which were kept within plastic bags (30 Ã? 40 cm), until time of harvest, with samples for each time point maintained separately. Undamaged plants maintained under the same conditions were used as controls. Cotton leaves from control plants and plants exposed to H. armigera were harvested at 6 h, 12 h, 24 h, and 48 h after onset of herbivory. For each treatment group and time point, cotton leaves were harvested from the three plants per treatment group and flash frozen in liquid nitrogen. For each time point, three replicate treatments and controls were performed. In this study we present dynamic transcriptome analysis and volatile profiling of cotton plants fed upon by larvae of a leaf-chewing herbivore CBW. Plant transcriptomic changes induced by CBW were analyzed using Affymetrixâ??s Cotton GeneChips. Samples from a time course of six hour to 48 hours following onset of CBW feeding were analyzed to identify target genes and key pathways involved in the activation of herbivory-induced indirect defense and to explore genetic basis of such defense. In addition, we monitored the accumulation of VOCs, which represent changes in cotton plant phenotype, following CBW infestation.

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IlHelArmi9

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