Germination of Leafy Spurge (Euphorbia esula) Seeds under Constant and Alternating Temperature

Transcriptome changes were investigated for Euphorbia esula (leafy spurge) seeds with a focus on the effect of constant and diurnal fluctuating temperature on dormancy and germination. Leafy spurge seeds do not germinate when incubated for 21 days at 20°C constant temperatures, but nearly 30% germinate after 21 days under fluctuating temperatures 20:30°C (16:8 h). Incubation at 20°C for 21 followed by 20:30°C resulted in approximately 63% germination in about 10 days. A cDNA microarray representing approximately 22,000 unique sequences was used to profile transcriptome changes. Overall design: Labeled cDNA was prepared from total RNA using the Alexa Fluor cDNA labeling kit (Invitrogen, Carlsbad, CA) according to manufacture's protocols. Labeled cDNAs were hybridized to a custom made 23 K element microarray that contained 19,808 unigenes from the leafy spurge EST database and an additional 4,129 unigenes from a cassava EST database. A rolling circle dye swap hybridization scheme was used to compare gene expression between samples. There were three biological and two technical replications for each treatment. Microarray hybridization was visualized using a GenPix 4000B scanner (Axon Instruments/Molecular Devices Corp., Sunnyvale, CA) and spot intensities and background was quantified using GenPix Pro software. Hybridization intensities were log2 transformed, and arrays were centered and normalized against each other.