BioProject

‘Pulsechip’, a boutique cDNA microarray, generated from a set of chickpea (Cicer arietinum L.) unigenes, grasspea (Lathyrus sativus L.) ESTs and lentil (Lens culinaris Med.) resistance gene analogs, was employed to generate an expression profile of chickpea accessions tolerant and susceptible to drought stress. Two groups of a tolerant and susceptible accession were challenged with drought stress. The experiments were performed in three biological replications. The experiments were conducted in reference design where respective tissues from unstressed plants served as control. The leaf and flower/bud tissues were collected and used for hybridization to measure changes in RNA abundance of treatment vs. control. The tissues from five experimental replicate plants per biological replication were pooled together (leaf and flower tissues separate) before RNA extraction. This RNA was used to prepare cDNA targets for expression analysis using microarray. The microarray had six technical replicate spots per EST. The transcript level for each EST/cDNA was firstly calculated as the average intensity of the six technical replicates and then the average intensity of three biological replicates. Data analysis included LOWESS normalization (LOcally WEighted polynomial regreSSion) to adjust for differences in quantity of initial RNA, labeling and detection efficiencies. A dye swap in one biological replicate adjusted dye bias, if any. The Differentially Expressed (DE) ESTs were identified as those with a 95% confidence interval for mean fold change (FC) that extended beyond the two-fold cut-off and also passed the Students t test (P<0.05) and FDR correction. These cut-offs translate into induced ESTs having a log2 ratio > 1 and repressed ESTs a ratio of < -1. The analysis consisted of three fold comparison. Firstly, the ESTs that were differentially expressed between treatment and control plants of each accession were detected. Then the ESTs that were similarly expressed by tolerant and susceptible accessions were then eliminated by comparison. This included a two-way comparison, where tolerant and susceptible genotypes were compared within and between groups. Lastly, ESTs that were consensually differentially expressed between tolerant and susceptible accessions of the two batches were identified. The hypothesis was that if a putative gene was consistently expressed only in tolerant or susceptible genotype for a particular stress, it might be a candidate for tolerance/susceptibility for that stress. Globally, the level of 114 transcripts was affected in response to drought stress in all the genotypes and tissue types studied. The DE transcripts in response to drought stress coded for various functional and regulatory proteins, most of which were repressed. Protein and other solute transport was repressed in susceptible-1 flowers but induced in tolerant-2 flowers, as evidenced by the induction of a protein-transport facilitation protein (DY475074) in flowers of tolerant-2 and repression of a aquaporin-like membrane channel protein (DY396334) and DNA-J like protein involved in intracellular protein transport (DY475488) in flowers of susceptible-1. Two putative auxin-repressed proteins (DY396289, DY396359) were highly repressed in flowers and leaves of tolerant-2, whilst being induced in flowers and leaves of susceptible-1. Defence-related genes including pathogenesis-related proteins (DY396305 and DY396343), nematode-resistance protein (CV793603), Cf-9 gene cluster (DY396352) and disease resistance response proteins (DY396265 and DY396276) were repressed in flowers of tolerant and susceptible genotypes. A Pea (pi230) disease resistance response protein (DY396390) and a multi-resistance protein ABC transporter (CV793605) were induced in flowers of both tolerant genotypes. Some genes involved in energy metabolism (DY396279 and DY475316) were repressed in leaves and flowers of tolerant and susceptible genotypes. Additionally, some transcripts related to senescence, including auxin responsive protein IAA9 (DY396315), senescence-associated protein DIN 1 (DY396338), and dehydration stress-induced protein (DY396321) were repressed in leaves and flowers of the tolerant genotypes. Of the 114 DE transcripts expressed in drought tolerant and susceptible genotypes, only two were consistently expressed. These included a cytosolic fructose 1,6-bisphosphatase (DY475548) and a gene with unknown function, which were repressed in flowers of both susceptible genotypes. Keywords: Chickpea, Drought stress, Tolerant, Susceptible, cDNA microarray Overall design: Total RNA was extracted from separately pooled leaf and flower tissues for each genotype {BG 1103 (Tolerant1), Kaniva (Susceptible1), BG 362 (Tolerant2) and Genesis 508 (Susceptible2)} at each time-point (including control samples) using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA). The quantity and quality of the total RNA samples were assessed by OD260/OD280 ratios and gel electrophoresis respectively. Fluorescent-labeled targets were prepared and hybridized to array slides as described [Coram, TE. and Pang, ECK. 2006. Expression profiling of Chickpea genes differentially regulated during a resistance response to Ascochyta rabiei. Plant Biotechnology Journal. 4(6), 647–666]. All hybridizations were performed with six technical replicates and three biological replicates, incorporating dye-swapping (i.e. reciprocal labelling of Cy3 and Cy5) to eliminate any dye bias. Overall, 288 images were analyzed from 48 genotype x tissue type x treatment/control x biological replication condition. Slides were scanned at 532 nm (Cy3 green laser) and 660 nm (Cy5 red laser) at 10 µm resolution using an Affymetrix® 428™ array scanner (Santa Clara, CA), and captured with the Affymetrix® Jaguar™ software (v. 2.0, Santa Clara, CA). Image analysis was performed using Imagene™ 5 (BioDiscovery, Marina Del Rey, CA) software. Quantified spot data was then compiled and transformed using GeneSight™ 3 (BioDiscovery, Marina Del Rey, CA). Data transformations consisted of a local background correction (mean intensity of background was subtracted from mean signal intensity for each spot), omitting flagged spots, LOWESS normalisation of the entire population, creating a Cy5/Cy3 mean signal ratio, taking a shifted log (base 2), and combination of duplicated spot data. To identify differentially expressed (DE) genes, expression ratio results were filtered to eliminate genes whose 95% confidence interval for mean fold change (FC) did not extend to 2-fold up or down, followed by Students t test with False Discovery Rate (FDR) multiple testing correction to retain only genes in which expression changes versus untreated control were significant at P < 0.05.