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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

Stability values of reference genes in soybean calculated using NormFinder.Stability values are indicated for soybean under stress by A.Bean pod mottle virus (BPMV), B. powdery mildew (PMD), C. soybean aphid (SBA), and D. two‐spotted spider mite (TSSM). Details on tested reference genes and primer sequences are provided in Tables 1 and 2, respectively.
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pone.0134890.g003: Stability values of reference genes in soybean calculated using NormFinder.Stability values are indicated for soybean under stress by A.Bean pod mottle virus (BPMV), B. powdery mildew (PMD), C. soybean aphid (SBA), and D. two‐spotted spider mite (TSSM). Details on tested reference genes and primer sequences are provided in Tables 1 and 2, respectively.

Mentions: Following the BPMV and SBA attack, ABCT and FBOX were found to be most stable in soybean (Fig 3A and 3C). Both these genes have lowest stability value i.e. lowest variation of expression among all genes. In case of PMD and TSSM attack TUA5 and TUA4 were most stable as revealed by NormFinder (Fig 3B and 3D). Similar to geNorm analysis results, the GDPH was the least stable following the BPMV, PMD, and SBA attack whereas RPL30 was the least stable following the TSSM attack (Figs 2 and 3).


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)

Stability values of reference genes in soybean calculated using NormFinder.Stability values are indicated for soybean under stress by A.Bean pod mottle virus (BPMV), B. powdery mildew (PMD), C. soybean aphid (SBA), and D. two‐spotted spider mite (TSSM). Details on tested reference genes and primer sequences are provided in Tables 1 and 2, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134890.g003: Stability values of reference genes in soybean calculated using NormFinder.Stability values are indicated for soybean under stress by A.Bean pod mottle virus (BPMV), B. powdery mildew (PMD), C. soybean aphid (SBA), and D. two‐spotted spider mite (TSSM). Details on tested reference genes and primer sequences are provided in Tables 1 and 2, respectively.
Mentions: Following the BPMV and SBA attack, ABCT and FBOX were found to be most stable in soybean (Fig 3A and 3C). Both these genes have lowest stability value i.e. lowest variation of expression among all genes. In case of PMD and TSSM attack TUA5 and TUA4 were most stable as revealed by NormFinder (Fig 3B and 3D). Similar to geNorm analysis results, the GDPH was the least stable following the BPMV, PMD, and SBA attack whereas RPL30 was the least stable following the TSSM attack (Figs 2 and 3).

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