Recommended Reference Genes for Quantitative PCR Analysis in Soybean Have Variable Stabilities during Diverse Biotic
Raman Bansal1, Priyanka Mittapelly1, Bryan J Cassone2
1Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH, 44691, United States of America.
Plos One
|August 6, 2015
Summary
Selecting stable reference genes is crucial for accurate soybean qRT-PCR analysis under biotic stress. This study identifies the most reliable genes for normalizing gene expression data in soybean plants infected with common pests and diseases.
Area of Science:
- Plant molecular biology
- Agricultural science
- Genomics
Background:
- Accurate gene expression analysis using quantitative real-time reverse transcription PCR (qRT-PCR) in soybean relies on stable reference genes.
- Previous recommendations for soybean reference genes have not been validated under specific biotic stress conditions prevalent in the North-Central U.S.
Purpose of the Study:
- To evaluate the expression stability of ten commonly recommended reference genes in soybean under four different biotic stresses: Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two-spotted spider mite (TSSM).
- To identify the most suitable reference genes for reliable qRT-PCR data normalization in soybean under these specific biotic stress conditions.
Main Methods:
- Investigated the expression stability of ten candidate reference genes (ABCT, CYP, EF1A, FBOX, GPDH, RPL30, TUA4, TUB4, TUA5, UNK2) across soybean samples subjected to BPMV, PMD, SBA, and TSSM.
- Utilized three statistical software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder) to analyze gene expression data and determine reference gene stability.
Main Results:
- Reference gene stability varied significantly across different biotic stressors.
- ABCT and FBOX were the most stable under BPMV and SBA stress.
- TUA4 and CYP showed highest stability under PMD stress, while TUB4 and TUA4 were stable under TSSM stress.
- GPDH was consistently unstable across all tested biotic stresses.
- Pairwise variation (V2/3) values below 0.15 indicated that using the two most stable reference genes is sufficient for normalization.
Conclusions:
- The optimal reference genes for qRT-PCR in soybean are stress-dependent.
- ABCT, FBOX, TUA4, CYP, and TUB4 are recommended as stable reference genes under specific biotic stresses (BPMV, SBA, PMD, TSSM).
- Accurate normalization of qRT-PCR data in soybean under biotic stress can be achieved by following the validated reference gene recommendations from this study.
