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

Bansal R, Mittapelly P, Cassone BJ, Mamidala P, Redinbaugh MG, Michel A - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH, 44691, United States of America.

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. 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.

No MeSH data available.


Related in: MedlinePlus

Ct values for reference genes in control and infested soybean plants.Details on tested reference genes are provided in Table 1. Each point represents the Mean± Standard Deviation of Ct values for three biological replications in each treatment. BPMV: Bean pod mottle virus; PMD: powdery mildew; SBA: soybean aphid; TSSM: two‐spotted spider mite.
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pone.0134890.g001: Ct values for reference genes in control and infested soybean plants.Details on tested reference genes are provided in Table 1. Each point represents the Mean± Standard Deviation of Ct values for three biological replications in each treatment. BPMV: Bean pod mottle virus; PMD: powdery mildew; SBA: soybean aphid; TSSM: two‐spotted spider mite.

Mentions: The expression profiles of qRT-PCR products for all primer pairs in each experiment are shown in Fig 1. CYP and EF1A were highly expressed with mean Ct values ranging between 17 and 21 whereas UNK2 was the least expressed gene with mean Ct value between 26 and 30. All tested genes showed expression variability across various samples as evident from a wide range of Ct values obtained. Genes such as CYP and TUB4 showed relatively smaller variation (below 2 cycles) in their expression across various samples, while others like GPDH had the highest expression variation (nearly 5 cycles). Our selected genes had the most stable expression and had been recommended for use in qRT-PCR analysis in different soybean tissues, developmental stages, and under stress conditions [13,18–23]; however the variability and the wide range in their expression deemed it necessary to select a suitable reference to normalize soybean gene expression under biotic stress.


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

Bansal R, Mittapelly P, Cassone BJ, Mamidala P, Redinbaugh MG, Michel A - PLoS ONE (2015)

Ct values for reference genes in control and infested soybean plants.Details on tested reference genes are provided in Table 1. Each point represents the Mean± Standard Deviation of Ct values for three biological replications in each treatment. BPMV: Bean pod mottle virus; PMD: powdery mildew; SBA: soybean aphid; TSSM: two‐spotted spider mite.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526470&req=5

pone.0134890.g001: Ct values for reference genes in control and infested soybean plants.Details on tested reference genes are provided in Table 1. Each point represents the Mean± Standard Deviation of Ct values for three biological replications in each treatment. BPMV: Bean pod mottle virus; PMD: powdery mildew; SBA: soybean aphid; TSSM: two‐spotted spider mite.
Mentions: The expression profiles of qRT-PCR products for all primer pairs in each experiment are shown in Fig 1. CYP and EF1A were highly expressed with mean Ct values ranging between 17 and 21 whereas UNK2 was the least expressed gene with mean Ct value between 26 and 30. All tested genes showed expression variability across various samples as evident from a wide range of Ct values obtained. Genes such as CYP and TUB4 showed relatively smaller variation (below 2 cycles) in their expression across various samples, while others like GPDH had the highest expression variation (nearly 5 cycles). Our selected genes had the most stable expression and had been recommended for use in qRT-PCR analysis in different soybean tissues, developmental stages, and under stress conditions [13,18–23]; however the variability and the wide range in their expression deemed it necessary to select a suitable reference to normalize soybean gene expression under biotic stress.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH, 44691, United States of America.

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. 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.

No MeSH data available.


Related in: MedlinePlus