|12-208 Project Manager: D. C. Jones|
GENOME WIDE ANALYSES OF WATER DEFICIT STRESS RESPONSIVE COTTON GENES
Todd Campbell, USDA-ARS
Genetic research to improve the productivity of cotton under water deficit stress is needed to minimize water use while maximizing production potential. Plant response to water deficit stress is complex and involves a cascade of gene families and biochemical pathways. During 2012, our work focused on generating a whole transcriptome profile of water deficit stress responsive genes. Previously, a putatively drought tolerant cultivar (Siokra L23) was grown in the field under well watered and water deficit stress conditions. On a weekly basis during the flowering period, three plants (biological replicates) within the two water treatments were evaluated for their leaf water status. At the same time, leaf and root samples from the same plants were harvested and immediately frozen in liquid nitrogen. On each date, frozen plant samples (12 total on a given date) were taken back to the laboratory. Based upon leaf water potential data, samples from a single date with large differences between the two water treatments were selected for further analysis. RNA was extracted from each of the 12 samples, quantified, quality control checked, and sent to Dr. Joshua Udall at Brigham Young University.
Dr. Udall's lab constructed barcoded, genome reduced cDNA libraries of the 12 samples in preparation for high throughput genome sequencing. The barcoded libraries were sent to the University of Utah Huntsman Cancer Institute for single end, 50-bp Illumina Hi-seq transcriptome sequencing. In April 2012, Dr. Wonkeun Park (Clemson University and USDA-ARS Florence) traveled to Dr. Udall's laboratory to initiate transcriptome analysis. Over 300 million total reads were sequenced. After initial trimming and quality assessment, 290 million trimmed reads were generated from which 150 million reads were mapped to the Gossypium raimondii genome sequence reference (Paterson et al., 2012). In total, 148 million reads mapped to annotated genes. In total, we determined the 148 million mapped reads represented approximately 34,000 genes.
These collective data have been used to identify differentially expressed genes in a number of different ways. First, leaf and root data were each analyzed independently to detect genes up- or down-regulated in response to water deficit stress in each tissue. Second, leaf and root data sets were combined so that analyses could be done to also compare tissue specific gene expression patterns in the context of water deficit stress.
Currently, we are focusing attention on genes with tissue and water treatment specific expression. These genes have been assigned to putative biological/functional categories. A set of 140 tissue and water treatment specific genes was selected for further analysis. After aligning sequence to the Gossypium raimondii reference genome and examining putative biological function, a subset of 30 genes have been selected for further evaluation. Gene specific PCR primers have been designed to amplify the 30 genes. PCR products for these 30 genes are currently being amplified, cloned, and re-sequenced to verify their authenticity. Reverse transcript-quantitative PCR assays are ongoing to confirm the differential expression of the selected 30 genes.
A Cotton Incorporated Fellow, Dr. Wonkeun Park, was supported on this project.
|Project Year: 2012|
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