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Reference genes for RT-qPCR analysis in Citrus and Poncirus infected by zoospores of Phytophthora parasitica

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Abstract

Phytophthora species are highly destructive phytopathogens, associated with massive damage in natural ecosystems and agriculture. Citrus production is also affected, mainly by the hemibiothrophic oomycete Phytophthora parasitica, which causes root rot and gummosis. Poncirus trifoliata and Citrus sunki (two rootstocks widely used in citrus orchards) pose a resistance and a susceptible interaction with P. parasitica, respectively, which makes them suitable models to study plant defense mechanisms. Gene expression analysis is a very important tool in this type of study, in particular PCR (RT-qPCR). Hence, it is crucial to use appropriate reference genes for expression normalization. Our aim was to evaluate the stability of several candidate reference genes to determine which set is best suited for normalization in citrus infected with P. parasitica. We evaluated five candidate reference genes selected from the database CitEST. GeNorm and NormFinder algorithms were used to assess the best reference genes. We found that the more stable genes to be used for RT-qPCR analysis in P. trifoliata plants were GAPC2 and F-BOX, while EGIDH and GAPC2 were more suitable to C. sunki. These four genes were found to be excellent normalizers, being stable throughout the infection regardless of pathogen attack or symptom development.

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Acknowledgements

RJDD thanks FAPESP for grant number 2015/14498-6. HJM thanks Anhanguera Educacional and Kroton Group. MAM thanks INCT citrus (Fapesp 2008/57909-2, CNPq 573848/08-4).

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    Authors and Affiliations

    1. Lab. de Biotecnologia, Centro de Citricultura Sylvio Moreira, Instituto Agronômico de Campinas, Cordeirópolis, SP, 13490-970, Brazil

      Heros J. Máximo, Ronaldo J. D. Dalio, Carolina M. Rodrigues, Michèle C. Breton & Marcos A. Machado

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    1. Heros J. Máximo
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    2. Ronaldo J. D. Dalio
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    3. Carolina M. Rodrigues
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    4. Michèle C. Breton
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    5. Marcos A. Machado
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    Correspondence to Marcos A. Machado.

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    Section Editor: Robert Miller

    Heros J. Máximo and Ronaldo J. D. Dalio contributed equally to this work.

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    Máximo, H.J., Dalio, R.J.D., Rodrigues, C.M. et al. Reference genes for RT-qPCR analysis in Citrus and Poncirus infected by zoospores of Phytophthora parasitica . Trop. plant pathol. 42, 76–85 (2017). https://doi.org/10.1007/s40858-017-0134-8

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