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Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection

Tropical Plant Pathology volume 43, pages 402–412 (2018)Cite this article

Abstract

Quantifying gene expression is essential in most functional genomics experiments. For quantitative PCR (qPCR) assays, reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings for candidate reference genes suitable for expression studies on plant-phytoplasma interactions in plants have not been reported. In the present study, we analyzed the expression patterns of 14 genes in midrib samples of C. aurantifolia plants infected with a ‘Candidatus Phytoplasma aurantifolia’ strain. Using GeNormPlus, NormFinder and BestKeeper algorithms, as well as testing relative expression by REST2009 software, the expression stability of several “classical” reference genes, such as GAPDH, CYCLOPHILIN and 18S rRNA, and of newly identified candidates, was assessed. Our results showed similar performance among GeNormPlus, NormFinder and BestKeeper in evaluating the suitability of reference genes, with few differences among the top five genes. Furthermore, our data showed that some of the widely used reference genes for relative expression normalization in plants, including citrus lineages, were not the most stably expressed transcripts. In conclusion, we provide a list of validated reference genes and their relative primer sequences, usable to conduct reliable qPCR experiments in C. aurantifolia during phytoplasma infection.

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Acknowledgments

This work was funded by Vale S.A. MSA was a Vale Technological Institute postdoctoral fellow. CMC is the recepient of a CNPq research productivity fellowship. We thank Sam Elliot (Dep. de Entomologia, UFV) for critically reviewing the English of the manuscript.

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Authors and Affiliations

  1. Dep. de Fitopatologia, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil

    Murilo S. Alves & Claudine M. Carvalho

  2. Dep. de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Campus do Pici, Fortaleza, CE, 60440-900, Brazil

    Murilo S. Alves

  3. Dep. of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34, Al-khod, 123, Sultanate of Oman

    Abdullah M. Al-Sadi

Authors
  1. Murilo S. Alves
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  2. Abdullah M. Al-Sadi
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  3. Claudine M. Carvalho
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Contributions

MSA and CMC conceived and designed the research. AMA assisted in the collection of infected samples. MSA conducted the experiments. MSA analyzed the data. MSA and CMC wrote the manuscript. All authors read and approved of the manuscript.

Corresponding author

Correspondence to Claudine M. Carvalho.

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Section Editor: Robert Miller

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Alves, M.S., Al-Sadi, A.M. & Carvalho, C.M. Selection of reference genes for quantitative PCR analysis in Citrus aurantifolia during phytoplasma infection. Trop. plant pathol. 43, 402–412 (2018). https://doi.org/10.1007/s40858-018-0224-2

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