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Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Plant Cell Reports volume 31, pages 827–838 (2012)Cite this article

Abstract

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C q) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (β-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

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Acknowledgments

We greatly thank Dr. Adriane Wendland from the National Rice and Beans Research Center (EMBRAPA), Goiás, Brazil, for providing strains of the fungus; the Center for Phytosanitary Research and Development (IAC), Campinas, Brazil, for the preparation of fungus inoculum; the Center for Analysis and Technological Research of Grain and Fiber Agribusiness (IAC), Campinas, Brazil, for verifying the pathogen race; CNPq (National Council for Scientific and Technological Development) for the scholarship and financial support (Universal-474337/2008-1); and CAPES for the post-doctoral fellowship.

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The authors declare that they have no conflicts of interest.

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  1. Center of Nuclear Energy for Agriculture, Laboratory of Cellular and Molecular Biology, University of São Paulo, P. O. Box 96 CEP, Piracicaba, SP, 13400-970, Brazil

    Aline Borges, Siu Mui Tsai & Danielle Gregorio Gomes Caldas

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  1. Aline Borges
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  2. Siu Mui Tsai
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  3. Danielle Gregorio Gomes Caldas
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Correspondence to Danielle Gregorio Gomes Caldas.

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Communicated by Q. Zhao.

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Borges, A., Tsai, S.M. & Caldas, D.G.G. Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses. Plant Cell Rep 31, 827–838 (2012). https://doi.org/10.1007/s00299-011-1204-x

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  • DOI: https://doi.org/10.1007/s00299-011-1204-x

Keywords

  • Phaseolus vulgaris
  • RT-qPCR
  • Normalizer genes
  • Stress conditions