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

采用结构性和功能性的研究方法，对棉花（Gossypium）基因进行探讨，这些基因与水分亏缺胁迫相关。利用代表假想祖先二倍体基因组（共识图谱）的遗传图谱，对15,784个假定共识序列（TCs）中的1,907个进行了定位。这些TCs代表了在灌溉和水分限制条件下从各种组织中提取的25,119个棉花ESTs。定位的TCs与42个与胁迫相关的数量性状位点（QTLs）的对应关系表明，初始的1,907个TCs中的391个位于QTL区间内。其中约31%的TCs被注释为参与植物对非生物胁迫反应的基因。相比之下，总注释TCs中有18%在共识图谱上定位，并被归类为非生物胁迫基因。映射到与胁迫相关QTLs的胁迫相关TCs的富集现象，无法用偶然性来解释（P = 1.5 x 10^-7）。通过使用由12,006个寡核苷酸组成的微阵列，进行了基因表达谱分析实验。对8周龄棉花植株的根和叶片组织施加水分亏缺胁迫，揭示了1,401个被鉴定为干旱响应的转录本。总共定位了158个基因（其中84个受干旱诱导，74个受干旱抑制），其中22个基因（8个诱导基因和14个抑制基因）与QTL共定位。在干旱胁迫库中鉴定了539个独特的基因，然而，其中只有91个基因包含在阵列中。在这些基因中，12个在受胁迫和灌溉的叶片及根之间的转录本丰度上发生了显著变化。共鉴定了45个在不同程度上涉及干旱胁迫响应的候选基因。关键词：胁迫响应
总体设计：将棉花植株（FiberMax 989）在温室条件下（日/夜温度为30℃/25℃）培养8周。每周用50%的Hoagland溶液施肥两次。通过停止灌溉施加水分亏缺胁迫，并基于压力室测量的叶片水势进行监测。此外，随着水分亏缺的增加，使用便携式红外气体分析仪（Li-COR，型号LI-6400，林肯，NE，美国）测量叶片与空气的蒸气压亏缺（VPD）、空气温度和细胞器腔室中的CO2浓度（400 µmol mol^-1）。对于每个测量周期，将细胞器腔室中的VPD、空气温度和CO2浓度设置为环境值，并保持所有测量中的恒定。使用发光二极管（Licor LI-6400-002）将辐照度设置为饱和光照条件（2000 µmol m^-2 s^-1）。每个叶片的数据记录三次，然后对每株植物的平均值进行平均，用作统计单位。在收获叶片和根组织时，完全灌溉的对照植物的叶片水势平均为-8.7 Bars（+ 0.37 SE），而受胁迫植物的叶片水势平均为-23.1 Bars（+ 0.28 SE），叶片的光合速率降至灌溉对照植物速率的大约60%。从每个植株上采样最年轻的、几乎完全展开的叶片（顶芽原基的第4片叶），并立即在液氮中冷冻。通过在液氮中快速冷冻的两个连续步骤采集侧根来收获根组织。使用第二代棉花寡核苷酸微阵列进行基因表达谱分析实验。该阵列由来自30个cDNA库的超过180,000个Gossypium ESTs组装而成（www.cottonevolution.info/microarray）。表达数据经过LOWESS标准化和Benjamini和Hochberg多重检验校正。差异表达定义为表达水平在p值上大于2倍变化。