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Identification of reference genes for gene expression studies during seed germination and seedling establishment in Ricinus communis L.

Published online by Cambridge University Press:  23 September 2014

Paulo R. Ribeiro ,
Bas J. W. Dekkers ,
Luzimar G. Fernandez ,
Renato D. de Castro ,
Wilco Ligterink  and
Henk W. M. Hilhorst
Paulo R. Ribeiro*
Affiliation:
Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708PBWageningen, The Netherlands Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Departamento de Biofunção, Universidade Federal da Bahia, Reitor Miguel Calmon s/n, 40160-100Salvador, Brazil
Bas J. W. Dekkers
Affiliation:
Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708PBWageningen, The Netherlands Molecular Plant Physiology, Utrecht University, Padualaan 8, 3584CH, Utrecht, The Netherlands
Luzimar G. Fernandez
Affiliation:
Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Departamento de Biofunção, Universidade Federal da Bahia, Reitor Miguel Calmon s/n, 40160-100Salvador, Brazil
Renato D. de Castro
Affiliation:
Laboratório de Bioquímica, Biotecnologia e Bioprodutos, Departamento de Biofunção, Universidade Federal da Bahia, Reitor Miguel Calmon s/n, 40160-100Salvador, Brazil
Wilco Ligterink*
Affiliation:
Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708PBWageningen, The Netherlands
Henk W. M. Hilhorst
Affiliation:
Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708PBWageningen, The Netherlands
*
*Correspondence Fax: +31317 418094 E-mails: paulodc3@gmail.com; wilco.ligterink@wur.nl;
*Correspondence Fax: +31317 418094 E-mails: paulodc3@gmail.com; wilco.ligterink@wur.nl;
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Abstract

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available).

Keywords

castor beangene expressionGeNormnormalizationNormFinderquantitative real-time polymerase chain reaction
Type
Research Papers
Information
Seed Science Research , Volume 24 , Issue 4 , December 2014 , pp. 341 - 352
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Copyright
Copyright © Cambridge University Press 2014 

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