Rice Genetics IV - IRRI books - International Rice Research Institute

Microarray hybridization and data analysisHybridizations were performed overnight at 42 °C in humidified chambers. The slideswere then washed in 1X SSC/0.2% SDS (10 min) and in 0.1X SSC (10 min). Slideswere rinsed for 1 min in 0.01X SSC and dried by centrifugation. The fluorescentsignatures were captured using a ScanArray 3000 (GSI Luminomics) and analyzedusing ImaGene III software (BioDiscovery). Local background was subtracted fromthe value of each spot on the array. Spots covered by dust particles, missing spots,spots with low signal intensity, and spots in high background areas were flagged ascandidates for exclusion after further analysis. Normalization between the Cy3 andCy5 emission channels was achieved by adjusting the signal intensity of exogenouslyadded nonplant control genes. Transcript regulation is expressed as the ratio of intensitiesbetween the stress and control (log ratio, termed LR).Results and discussionThe data selected and outlined here are designed to make several points, mainly onthe variation of transcript profiles in different tissues, the changes that are observedunder stress conditions, and the preliminary microarray analyses destined to provideinformation about likely candidate genes for stress tolerance characteristics.The generation of cDNA libraries in our projects focused almost exclusively ontissues from salinity- and drought-stressed plants, but we included several cDNAlibraries for the unstressed state to obtain EST sequences for comparing expressionprofiles (Table 1). These cDNA libraries were used for EST sequencing. Typically,we collect approximately 1,000–2,000 EST sequences without subtraction. These ESTsare then subtracted from the next set of approximately 2,000 clones and this subtractionprocess can be repeated. However, we have rarely found time to go “deeper” intothese libraries, that is, truly rare messages are presently not represented in our expressionprofiles. This statement is based on the classical DNA/RNA renaturation (cotcurves)experiments (Goldberg et al 1978, Kiper et al 1979, Buffard et al 1982, Kamalayand Goldberg 1984). These analyses had indicated that the complexity in, for example,roots could include 10,000 different expressed genes. At present, our way ofproceeding produced from 2,000 to 4,000 unique transcripts for three grass species,rice, maize, and barley.From the initial nonsubtracted 1,000–2,000 EST sequences, expression profilescan be generated that represent the set of expressed genes in functional categories(Table 2) and also provide an overview of highly transcribed transcripts (Table 3).The category profile for salt-stressed maize roots indicates a large number of unknowntranscripts (42%) and similar percentages have been found for rice (Kawasakiet al 2001). Among the most highly transcribed genes in salt-stressed maize roots areisoforms for a metallothionein-like protein, a glutathione S-transferase, glutaminesynthetase, several different (putative) water channel proteins, and several open readingframes for which no other sequence has been found before (“no hit” category).Isolation of candidate genes for tolerance of abiotic stresses 349