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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

Usefulness of identified reference genes in detecting target gene expression.Relative expression levels (Mean± Standard Deviation) of two target genes (Glyma.05g184500 and Glyma.06g147100) in soybean under biotic stress by soybean aphid (SBA) are shown. Expression values of both target genes were normalized with respect to the most stable gene pair (ABCT and FBOX) and the least stable gene pair (TUA4 and GPDH) during SBA stress in soybean (see Table 4). The asterisk (*) represents a significant difference (P< 0.05) in SBA-infested soybean compared to uninfested control. N.S: No significant difference (P > 0.05).
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pone.0134890.g005: Usefulness of identified reference genes in detecting target gene expression.Relative expression levels (Mean± Standard Deviation) of two target genes (Glyma.05g184500 and Glyma.06g147100) in soybean under biotic stress by soybean aphid (SBA) are shown. Expression values of both target genes were normalized with respect to the most stable gene pair (ABCT and FBOX) and the least stable gene pair (TUA4 and GPDH) during SBA stress in soybean (see Table 4). The asterisk (*) represents a significant difference (P< 0.05) in SBA-infested soybean compared to uninfested control. N.S: No significant difference (P > 0.05).

Mentions: To demonstrate the utility of identified stable reference genes in validating the gene expression results obtained by high-throughput sequencing, we measured the expression of target genes in soybean under biotic stress. From soybean RNA-Seq data, two target genes, both encoding for WRKY transcription factors were selected. In general, the WRKY transcription factor genes are best known for their role in regulating the plant’s defense response to biotic stresses [37]. Our RNA-Seq experiment showed that following the SBA infestation, both the selected genes have enhanced expression compared to control plants (R. Bansal, unpublished data). For the purpose of comparison, expression values of target genes were normalized with respect to the most stable gene pair (ABCT and FBOX) and the least stable gene pair (TUA4 and GPDH) during SBA stress in soybean. In case of normalization with the most stable gene pair, expression pattern of both target genes concurred with that observed in RNA-Seq analysis (Fig 5). However, in case of normalization with the least stable gene pair, one target gene (Glyma.06g147100) showed enhanced expression during SBA-stress, thus consistent with RNA-Seq results; but the other target gene (Glyma.05g184500) was shown to be unaffected during SBA-stress; thus contradicting with results obtained through the RNA-Seq and through the normalization with most stable gene pair.


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)

Usefulness of identified reference genes in detecting target gene expression.Relative expression levels (Mean± Standard Deviation) of two target genes (Glyma.05g184500 and Glyma.06g147100) in soybean under biotic stress by soybean aphid (SBA) are shown. Expression values of both target genes were normalized with respect to the most stable gene pair (ABCT and FBOX) and the least stable gene pair (TUA4 and GPDH) during SBA stress in soybean (see Table 4). The asterisk (*) represents a significant difference (P< 0.05) in SBA-infested soybean compared to uninfested control. N.S: No significant difference (P > 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134890.g005: Usefulness of identified reference genes in detecting target gene expression.Relative expression levels (Mean± Standard Deviation) of two target genes (Glyma.05g184500 and Glyma.06g147100) in soybean under biotic stress by soybean aphid (SBA) are shown. Expression values of both target genes were normalized with respect to the most stable gene pair (ABCT and FBOX) and the least stable gene pair (TUA4 and GPDH) during SBA stress in soybean (see Table 4). The asterisk (*) represents a significant difference (P< 0.05) in SBA-infested soybean compared to uninfested control. N.S: No significant difference (P > 0.05).
Mentions: To demonstrate the utility of identified stable reference genes in validating the gene expression results obtained by high-throughput sequencing, we measured the expression of target genes in soybean under biotic stress. From soybean RNA-Seq data, two target genes, both encoding for WRKY transcription factors were selected. In general, the WRKY transcription factor genes are best known for their role in regulating the plant’s defense response to biotic stresses [37]. Our RNA-Seq experiment showed that following the SBA infestation, both the selected genes have enhanced expression compared to control plants (R. Bansal, unpublished data). For the purpose of comparison, expression values of target genes were normalized with respect to the most stable gene pair (ABCT and FBOX) and the least stable gene pair (TUA4 and GPDH) during SBA stress in soybean. In case of normalization with the most stable gene pair, expression pattern of both target genes concurred with that observed in RNA-Seq analysis (Fig 5). However, in case of normalization with the least stable gene pair, one target gene (Glyma.06g147100) showed enhanced expression during SBA-stress, thus consistent with RNA-Seq results; but the other target gene (Glyma.05g184500) was shown to be unaffected during SBA-stress; thus contradicting with results obtained through the RNA-Seq and through the normalization with most stable gene pair.

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