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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 (V 2/3 ) values less than 0.15 which suggested that combined use of the two most stable reference genes would be sufficient for normalization. Further, we demonstrated the utility of normalizing the qRT-PCR data for target genes using the most stable reference genes validated in current study. Following of the recommendations from our current study will enable an accurate and reliable normalization of qRT-PCR data in soybean under biotic stress.

Recommended Reference Genes for Quantitative PCR Analysis in Soybean Have Variable Stabilities during Diverse Biotic Stresses