Mapping drought-responsive genes in cotton

Structural and functional approaches were used to study cotton (Gossypium) genes implicated in water-deficit stress. A genetic map representing the hypothetical ancestral diploid genome (Consensus Map) was used to map 1,907 of 15,784 tentative consensus sequences (TCs). These TCs represent 25,119 cotton ESTs derived from various tissues under irrigated and water-limited condition. The correspondence of mapped TCs and 42 stress-related quantitative trait loci (QTLs) revealed that 391 of the initial 1907 TCs co-localized within a QTL interval. About 31% of these TCs were annotated as genes involved in plant responses to abiotic stress. By comparison, only 18% of the total annotated TCs mapped on the Consensus map were classified as abiotic stress genes. The enrichment of stress-related TCs that map to stress-related QTLs could not be explained by chance (P = 1.5 x 10-7). Gene expression profiling experiments were carried out using a microarray composed of 12,006 oligonucleotides. Transcriptional responses to imposed water-deficit stress in root and leaf tissue of 8-week old cotton plants revealed 1401 transcripts identified as drought responsive. A total of 158 (84 drought-induced and 74 drought-repressed) genes were mapped, of which 22 (8 induced and 14 repressed) genes co-localized with a QTL. A total of 539 unique genes were identified in the drought-stressed libraries. However, only 91 of these genes are contained on the array. Of these genes, 12 showed significant changes in transcript abundance between stressed and irrigated leaf and root. Forty-five candidate genes implicated in drought-stress response at some level of characterization were identified. Keywords: stress response Overall design: Cotton plants (FiberMax 989) were grown under greenhouse conditions (30ºC/25ºC, day/night) for 8 weeks. Plants were fertilized twice weekly with 50% Hoagland’s solution. Water-deficit stress was imposed by withholding irrigation and monitored based on leaf water potential measured with a pressure bomb. Additionally, leaf level gas-exchange was measured as water-deficit increased using a portable infrared gas analyzer (Li-COR, Model LI-6400, Lincoln, NE, USA). Leaf-to-air vapor pressure deficit (VPD), air temperature, and CO2 concentration (400 µmol mol-1) of the cuvette were set to ambient environmental values for each measurement period and maintained constant for all measurements across plots. Irradiance was set to saturating light conditions (2000 µmol m-2 s-1) using a light-emitting diode (Licor LI-6400-002). Data were logged three times for each leaf and then averaged for each plant to be used as a statistical unit. At the time of leaf and root tissue harvest, leaf water potentials averaged -8.7 Bars (+ 0.37 SE) for fully irrigated control plants and -23.1 Bars (+ 0.28 SE) for stressed plants, and leaf photosynthetic rates had decreased to approximately 60% of the rates seen in the irrigated controls. The youngest, nearly fully expanded leaf (4th leaf from the apical meristem) was sampled from each plant and immediately frozen in liquid nitrogen. Root tissue was harvested by sampling lateral roots in two sequential steps of flash freezing in liquid nitrogen. Gene expression profiling experiments were carried out using the second-generation cotton oligonucleotide microarray. The array is composed of 12,006 oligonucleotides derived from an assembly of more than 180,000 Gossypium ESTs sequenced from 30 cDNA libraries (www.cottonevolution.info/microarray). Expression data were subjected to LOWESS normalization and a Benjamini and Hochberg multiple testing correction. Differential expression was defined as >2 fold change in expression level at p