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Title: Evaluation of expression stability of candidate references genes among green and yellow pea cultivars (Pisum sativum L.) subjected to abiotic and biotic stress

item SAHA, GOPESH - Washington State University
item Vandemark, George

Submitted to: American Journal of Plant Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2012
Publication Date: 2/1/2012
Citation: Saha, G., Vandemark, G.J. 2012. Evaluation of expression stability of candidate references genes among green and yellow pea cultivars (Pisum sativum L.) subjected to abiotic and biotic stress. American Journal of Plant Sciences. 5:235-242.

Interpretive Summary: Dry peas (Pisum sativum) are commonly grown for human and animal diets throughout the world. Changes in growing conditions due to variable weather conditions and problems with diseases and pests result in a constant need for new and improved varieties. Often how a plant appears is due to a combination of genetic and environmental factors, and plant breeders must be able to identify plants that have beneficial genetic factors (genes) for important traits. RNAs are molecules in plant cells that are used to translate instructions from DNA that result in cellular processes. If different amounts of a given RNA are detected in plants that are different for a trait of interest, then that suggests that the RNA may be involved in trait expression. Scientists must use ‘reference’ genes as controls to verify that other RNAs of interest are present in different amounts. Reference genes should code for RNAs that are present in the same amount under different stages or conditions of plant growth. We tested several possible reference genes in peas exposed to cold temperatures or fungal disease. We identified two genes; translation initiation factor (TIF), involved in making proteins, and B-tubulin, which is part of the basic structure of plant cells, that worked very well as reference genes in several different pea varieties. TIF and B-tubulin worked well for both cold stressed and diseased plants. This work will allow us and other scientists to identify RNA markers for use in developing improved pea varieties.

Technical Abstract: Dry pea (Pisum sativum) is grown as human and animal feed throughout the world. Large yield losses in pea due to biotic and abiotic stresses compel an improved understanding of mechanisms of stress tolerance and genetic determinants conditioning these tolerances. The availability of stably expressed reference genes is a prerequisite for examining differential gene expression. The objective of this study was to examine the expression profile of several candidate reference genes across a broad range of commercial pea cultivars. Expression profiles of five candidate reference genes; 18s rRNA, actin, TIF, ß tubulin-2 and ß tubulin-3 were examined. Relative quantifications of housekeeping genes were estimated from control plants, plants after 48 hr of cold treatment, and plants 24 and 48 hr after inoculation with S. sclerotiorum, the causal agent of white mold disease of pea. qRT-PCR was performed on cDNA synthesized from three food grade spring peas, Ariel, Aragorn, and Sterling, and two spring yellow peas, Delta and Universal, which are used as animal feed. Analysis of variance (ANOVA) of CT values showed that significant variation exists between varieties and treatments under biotic and abiotic conditions and the genes TIF, ß tubulin-2 and ß tubulin-3 are more sensitive to treatment effect under cold and S. sclerotiorum inoculation. The mean CT values for all transcripts under cold and S. sclerotiorum treated conditions ranged from 23.8 to 30.3, except for 18s rRNA transcripts, which had a mean CT ˜ 12.5. These results reflect the greater abundance of 18S rRNA transcripts relative to other quantified transcripts. Under both cold stress and disease pressure the TIF and ß tubulin-3 genes exhibited the highest expression stability and were suitable and sufficient for use as reference genes.[National Sclerotinia Initiative]