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Reference gene identification for real-time PCR analyses in soybean leaves under fungus (Cercospora kikuchii) infection and treatments with salicylic and jasmonic acids

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Abstract

Quantitative PCR (qPCR) is a robust technology for comparing the expression profiles of target genes, however the data produced need normalization with appropriate reference genes. The purpose of this study was to evaluate the expression stability of eight candidate reference genes (EF1A 1a1, EF1A 2a, EF1A 2b, EF1B, ACT11, UKN 1, ACT and SKIP 16) for qPCR assays in soybean leaves under fungus (Cercospora kikuchii) infection and treatments with salicylic and jasmonic acids. Four programs, GeNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stability. The leaves were treated according the following conditions: 1) infected with Cercospora kikuchii (CK); (2) treated with salicylic acid (SA); (3) treated with SA and infected with C. kikuchii; (4) treated with jasmonic acid (JA); and (5) treated with JA and infected with C. kikuchii. For all studied conditions, GeNorm analyses revealed that combinations of six genes were always needed for gene expression normalization. Three EF1A genes (EF1A 2a, EF1A 1a1 and EF1A 2b) were the most stable in all tested conditions and then, they were always included in gene combinations. The other three genes varied according the different conditions. In analyses with other programs, at least two EF1A genes were often ranked among the three best stable genes. The expression of a PR3 (class I chitinase) gene was used to validate the reference genes across the total samples. Our results provide a shortlist of reference genes to normalize qPCR assays in soybean under CK infection and treatments with SA and JA.

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Acknowledgments

This work was supported by National Council for Scientific and Technological Development (CNPq), Ceara State Foundation for the Support of Scientific and Technological Development (FUNCAP) and Coordination of Improvement of Higher Education (CAPES), Brazil.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Ceara, 60440-900, Brazil

    Jose H. Costa, Katia D. C. Saraiva, Vanessa D. Morais, Jose T. A. Oliveira, Daniele O. B. Sousa, Dirce Fernandes de Melo, Janne K. S. Morais & Ilka M. Vasconcelos

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  1. Jose H. Costa
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  2. Katia D. C. Saraiva
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  3. Vanessa D. Morais
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  4. Jose T. A. Oliveira
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  5. Daniele O. B. Sousa
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  7. Janne K. S. Morais
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  8. Ilka M. Vasconcelos
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Correspondence to Ilka M. Vasconcelos.

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Costa, J.H., Saraiva, K.D.C., Morais, V.D. et al. Reference gene identification for real-time PCR analyses in soybean leaves under fungus (Cercospora kikuchii) infection and treatments with salicylic and jasmonic acids. Australasian Plant Pathol. 45, 191–199 (2016). https://doi.org/10.1007/s13313-016-0403-x

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