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Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis

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Abstract

The recent publication of the grapevine genome sequence facilitates the use of qRT-PCR to study gene expression changes. For this approach, reference genes are commonly used to normalize data and their stability of expression should be systematically validated. Among grapevine defenses is the production of the antimicrobial stilbenic phytoalexins, notably the highly fungitoxic pterostilbene, which plays a crucial role in grapevine interaction with Plasmopara viticola and Botrytis cinerea. As a resveratrol O-methyltransferase (ROMT) gene involved in pterostilbene synthesis was recently identified, we investigated the accumulation of the corresponding transcripts to those of two other stilbene biosynthesis related genes phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) in response to pathogen infection. Using three computer-based statistical methods and C t values or LRE method generated values as input data, we have first identified two reference genes (VATP16 and 60SRP) suitable for normalization of qPCR expression data obtained in grapevine leaves and berries infected by P. viticola and B. cinerea, respectively. Next, we have highlighted that the expression of ROMT is induced in P. viticola-infected leaves and also in B. cinerea-infected berries, confirming the involvement of pterostilbene in grapevine defenses.

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Acknowledgments

This work was funded by the Conseil Régional de Bourgogne, Bureau Interprofessionnel des Vins de Bourgogne, Comité Interprofessionnel des Vins de Champagne, Comité National des Interprofessions des Vins à Appellation d’Origine and the ANR Génoplante Safegrape Project (O8-GENO-148G). The authors thank Olivier Lamotte (UMR Plante Microbe Environnement, INRA Dijon, France), Patrick Frettinger (Laboratory of plant-pathogen fungi functional genomics, Bayer CropScience, Lyon, France), and Dominique Garmyn (UMR Microbiologie des Sols et de l’Environnement, Dijon, France) for helpful discussions and Jean Roudet and Marc Fermaud (UMR Santé végétale, INRA-ENITA Bordeaux, France) for assistance with berry experiments. Many thanks also to Alberto Ferrarini (University of Verona, Italy), Grégoire Aubert, and Myriam Sanchez (UMR LEG, INRA Dijon, France) for technical support.

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  1. Unité Mixte de Recherche INRA 1088/CNRS 5184, Université de Bourgogne Plante-Microbe-Environnement, 17 rue Sully, BP 86510, 21065, Dijon cedex, France

    Magdalena Gamm, Marie-Claire Héloir, Jani Kelloniemi, Benoît Poinssot, David Wendehenne & Marielle Adrian

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  1. Magdalena Gamm
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  2. Marie-Claire Héloir
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  3. Jani Kelloniemi
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Correspondence to Marielle Adrian.

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Communicated by S. Hohmann.

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Gamm, M., Héloir, MC., Kelloniemi, J. et al. Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis. Mol Genet Genomics 285, 273–285 (2011). https://doi.org/10.1007/s00438-011-0607-2

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