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ARS Home » Midwest Area » Wooster, Ohio » Corn, Soybean and Wheat Quality Research » Research » Publications at this Location » Publication #322730
Title: Recommended reference genes for quantitative PCR analysis in soybean have variable stabilities during diverse biotic stresses

Author
item BANSAL, RAMAN - The Ohio State University
item MITTAPELLY, PRIYANKA - The Ohio State University
item CASSONE, BRYAN - The Ohio State University
item MAMIDALA, PRAVEEN - The Ohio State University
item Redinbaugh, Margaret
item MICHEL, ANDREW - The Ohio State University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2015
Publication Date: 8/5/2015
Publication URL: http://handle.nal.usda.gov/10113/61877
Citation: Bansal, R., Mittapelly, P., Cassone, B.J., Mamidala, P., Redinbaugh, M.G., Michel, A. 2015. Recommended reference genes for quantitative PCR analysis in soybean have variable stabilities during diverse biotic stresses. PLoS One. doi:10.1371/journal.pone.0134890.

Interpretive Summary: Real-time reverse transcription PCR (qRT-PCR) is a relatively new technique that can be used to determine how much or how little a specific gene is expressed in an organism. Scientists use the technique to look at the effects of tissue type, environment, disease and genotype on gene expression. qRT-PCR is very sensitive, so we can investigate gene expression in small samples (e.g., part of a flower) and very specific, so we can separately investigate closely related genes. Importantly, the technique is quantitative, so we can determine the levels of gene expression among a number of different samples and treatments. However, accurate quantification requires comparing the expression of test genes to reference genes whose expression is not affected by the experimental treatments. In our research, we need to accurately quantify gene expression expression under a variety of stresses from different insects and pathogens, and this research was carried out to identify good reference genes for our research. We tested ten reference genes previously used in soybean and evaluated their stability under four treatments with three software algorithms. Good reference genes were identified for each treatment, and all but one of the genes tested (GPDH) could be used as a reference in some treatments. Further, our data indicated that using the two most stably expressed reference genes for a given set of treatments would provide accurate and reliable quantification of gene expression. These results can be used by other researchers studying the responses of soybean cultivars to biotic stress.

Technical Abstract: For real-time reverse transcription-PCR (qRT-PCR) in soybean, reference genes in different tissues, developmental stages, various cultivars, and under stress conditions have been suggested but their usefulness for research on soybean under various biotic stresses occurring in North-Central U.S. is not known. Here, we investigated the expression stabilities of ten previously recommended reference genes (ABCT, CYP, EF1A, FBOX, GPDH, RPL30, TUA4, TUB4, TUA5, and UNK2) in soybean under biotic stress from Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two-spotted spider mite (TSSM). BPMV, PMD, SBA, and TSSM are amongst the most common pest problems on soybean in North-Central U.S. and other regions. Reference gene stability was determined using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Reference genes showed variability in their expression as well as stability across various stressors and the best reference genes were stress-dependent. ABCT and FBOX were found to be the most stable in soybean under both BPMV and SBA stress but these genes had only minimal to moderate stability during PMD and TSSM stress. Expression of TUA4 and CYP was found to be most stable during PMD stress; TUB4 and TUA4 were stable under TSSM stress. Under various biotic stresses on soybean analyzed, GPDH expression was found to be consistently unstable. For all biotic stressors on soybean, we obtained pairwise variation (V2/3) values less than 0.15 which suggested that combined use of the two most stable reference genes would be sufficient for normalization. Following of the recommendations from our current study will enable a more accurate and reliable normalization of qRT-PCR data in soybean under biotic stress.