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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

Pairwise variation (V) analysis of the reference genes in soybean using geNorm.The pairwise variation (Vn/n+1) between the normalization factors NFn and NFn+1 (shown along x-axis) is calculated to determine the optimal number of reference genes for normalization in soybean under stress by Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two‐spotted spider mite (TSSM). Each bar indicates change in normalization when adding reference genes stepwise according to rankings in Fig 2.
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pone.0134890.g004: Pairwise variation (V) analysis of the reference genes in soybean using geNorm.The pairwise variation (Vn/n+1) between the normalization factors NFn and NFn+1 (shown along x-axis) is calculated to determine the optimal number of reference genes for normalization in soybean under stress by Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two‐spotted spider mite (TSSM). Each bar indicates change in normalization when adding reference genes stepwise according to rankings in Fig 2.

Mentions: Though a single reference gene which is stable and has moderate to high expression is sufficient for quantifying mRNA transcript levels, the use of more than one reference gene for effective normalization of gene expression data is suggested [14]. The optimal number of reference genes required for normalization under a given set of experimental conditions can be obtained from the pairwise variation (V). Vandesompele et al. [14] proposed a cutoff V value of 0.15, below which the inclusion of other reference genes are not required. Accordingly in all biotic stressors on soybean, the pairwise variation of V2/3 was less than 0.15 (Fig 4) which means that the addition of third reference gene will not further increase the statistical significance obtained for the first reference gene pair employed. Thus, the combined use of the two most stable reference genes would be enough for normalization.


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)

Pairwise variation (V) analysis of the reference genes in soybean using geNorm.The pairwise variation (Vn/n+1) between the normalization factors NFn and NFn+1 (shown along x-axis) is calculated to determine the optimal number of reference genes for normalization in soybean under stress by Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two‐spotted spider mite (TSSM). Each bar indicates change in normalization when adding reference genes stepwise according to rankings in Fig 2.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134890.g004: Pairwise variation (V) analysis of the reference genes in soybean using geNorm.The pairwise variation (Vn/n+1) between the normalization factors NFn and NFn+1 (shown along x-axis) is calculated to determine the optimal number of reference genes for normalization in soybean under stress by Bean pod mottle virus (BPMV), powdery mildew (PMD), soybean aphid (SBA), and two‐spotted spider mite (TSSM). Each bar indicates change in normalization when adding reference genes stepwise according to rankings in Fig 2.
Mentions: Though a single reference gene which is stable and has moderate to high expression is sufficient for quantifying mRNA transcript levels, the use of more than one reference gene for effective normalization of gene expression data is suggested [14]. The optimal number of reference genes required for normalization under a given set of experimental conditions can be obtained from the pairwise variation (V). Vandesompele et al. [14] proposed a cutoff V value of 0.15, below which the inclusion of other reference genes are not required. Accordingly in all biotic stressors on soybean, the pairwise variation of V2/3 was less than 0.15 (Fig 4) which means that the addition of third reference gene will not further increase the statistical significance obtained for the first reference gene pair employed. Thus, the combined use of the two most stable reference genes would be enough for normalization.

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