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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

Average expression stability (M) and ranking of reference genes in soybean as calculated by geNorm.M values and rankings 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.g002: Average expression stability (M) and ranking of reference genes in soybean as calculated by geNorm.M values and rankings 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: geNorm algorithm calculated that following BPMV inoculation, the average expression stability values (M) for ABCT and EF1A were the lowest (0.1714) and that for GPDH was highest (0.5283), suggesting that ABCT and EF1A had the most stable expression and that GPDH had the least stable expression (Fig 2A). Nearly identical results were obtained following the SBA attack on soybean; as ABCT and FBox were the most stable (M value 0.1545). GPDH was the least stable during SBA attack as well (M value 0.6820) (Fig 2C). On the other hand, ABCT was among the least stable genes both in case of PMD (M value 0.4928) and TSSM (M value 0.4500) attack whereas TUA4 was the most stable (PMD M value 0.1725 and TSSM M value 0.1154) (Fig 2B and 2D). Besides TUA4, CYP and TUB4 were the most stable following the PMD and TSSM attack, respectively. GPDH (M value 0.5650) remained the least stable following the PMD attack whereas RPL30 (M value 0.5042) was the least stable following the TSSM attack on soybean. The M value calculated for all the reference genes assayed in the current study was less than 1.5, thus suggesting the appropriateness of all genes for normalization consideration in soybean 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)

Average expression stability (M) and ranking of reference genes in soybean as calculated by geNorm.M values and rankings 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.g002: Average expression stability (M) and ranking of reference genes in soybean as calculated by geNorm.M values and rankings 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: geNorm algorithm calculated that following BPMV inoculation, the average expression stability values (M) for ABCT and EF1A were the lowest (0.1714) and that for GPDH was highest (0.5283), suggesting that ABCT and EF1A had the most stable expression and that GPDH had the least stable expression (Fig 2A). Nearly identical results were obtained following the SBA attack on soybean; as ABCT and FBox were the most stable (M value 0.1545). GPDH was the least stable during SBA attack as well (M value 0.6820) (Fig 2C). On the other hand, ABCT was among the least stable genes both in case of PMD (M value 0.4928) and TSSM (M value 0.4500) attack whereas TUA4 was the most stable (PMD M value 0.1725 and TSSM M value 0.1154) (Fig 2B and 2D). Besides TUA4, CYP and TUB4 were the most stable following the PMD and TSSM attack, respectively. GPDH (M value 0.5650) remained the least stable following the PMD attack whereas RPL30 (M value 0.5042) was the least stable following the TSSM attack on soybean. The M value calculated for all the reference genes assayed in the current study was less than 1.5, thus suggesting the appropriateness of all genes for normalization consideration in soybean 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