Abstract
Quantitative real-time PCR (qPCR) is a sensitive method used to investigate relevant changes in gene expression during somatic embryogenesis. Understanding its regulatory network might be helpful to the process of induction of embryos and facilitate the development of efficient plant regeneration procedures. In this study, a set of 12 genes was selected and their stability was assessed in different tissues of somatic embryogenesis-related cultures of Coffea arabica. Analyses of gene expression stability were performed using the RefFinder tool that integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms. Among the all candidate reference genes studied, APRT/EF1a, UBQ/ACT, ACT/24S, RPL39/24S, PP2A/RPL39, PP2A/AP47, emerged as the most stable for normalization of qPCR analyses of embryogenic cell suspensions, non-embryogenic calli, embryogenic calli, combined embryogenic and non-embryogenic calli, somatic embryos and plantlet, respectively. A combination of two genes, 24S and PP2A, was identified as most suitable reference genes across all samples for the C. arabica tissues studied. The commonly employed reference gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found to be inappropriate as a reference gene for embryogenic tissues of C. arabica. In addition, Baby boom (BBM) gene expression was investigated to confirm the validity of the selected reference genes, the transcript levels of gene were overestimated when unsuitable reference genes were used for normalization. The results shown herein will permit a more precise and reliable normalization of qPCR in experiments involving somatic embryogenesis of C. arabica.
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Acknowledgements
We thank especially MSc Wesley Pires Flausino Máximo for helpful comments on this manuscript. The authors also would like to thank Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their financial support. These funding programs did not play a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Conceived and designed the experiments NCF, HGB, LVP, ACJ. Performed the experiments NCF, CNF, ROM. Analyzed and interpreted the data NCF, CNF. Contributed with reagents and materials LVP, ACJ. Wrote the paper NCF. Made critical revisions of the content of the paper LVP, HGB, ACJ.
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Freitas, N.C., Barreto, H.G., Fernandes-Brum, C.N. et al. Validation of reference genes for qPCR analysis of Coffea arabica L. somatic embryogenesis-related tissues. Plant Cell Tiss Organ Cult 128, 663–678 (2017). https://doi.org/10.1007/s11240-016-1147-6
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DOI: https://doi.org/10.1007/s11240-016-1147-6