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Nitrogen Starvation, Salt and Heat Stress in Coffee (Coffea arabica L.): Identification and Validation of New Genes for qPCR Normalization

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Abstract

Abiotic stresses are among the most important factors that affect food production. One important step to face these environmental challenges is the transcriptional modulation. Quantitative real-time PCR is a rapid, sensitive, and reliable method for the detection of mRNAs and it has become a powerful tool to mitigate plant stress tolerance; however, suitable reference genes are required for data normalization. Reference genes for coffee plants during nitrogen starvation, salinity and heat stress have not yet been reported. We evaluated the expression stability of ten candidate reference genes using geNorm PLUS, NormFinder, and BestKeeper softwares, in plants submitted to nitrogen starvation, salt and heat stress. EF1, EF1α, GAPDH, MDH, and UBQ10 were ranked as the most stable genes in all stresses and software analyses, while RPL39 and RPII were classified as the less reliable references. For reference gene validation, the transcriptional pattern of a Coffea non-symbiotic hemoglobin (CaHb1) was analyzed using the two new recommended and the most unstable gene references for normalization. The most unstable gene may lead to incorrect interpretation of CaHb1 transcriptional analysis. Here, we recommend two new reference genes in Coffea for use in data normalization in abiotic stresses: MDH and EF1.

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Acknowledgments

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), FINEP, Fundação Araucária and Consórcio Pesquisa Café. KC is the recipient of a scholarship from CAPES/REUNI. LGEV and LFPP are CNPq research fellows.

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Authors and Affiliations

  1. Laboratório de Biotecnologia Vegetal, Instituto Agronômico do Paraná, CP 481, Londrina, PR, CEP 86047-902, Brazil

    Kenia de Carvalho, Tiago Benedito dos Santos, Silvia Graciele Hülse de Souza, Luiz Gonzaga Esteves Vieira, Luis Filipe Protasio Pereira & Douglas Silva Domingues

  2. Programa de Pós-Graduação em Agronomia (Produção Vegetal), Departamento de Fitotecnia e Fitossanitarismo, Universidade Federal do Paraná, Curitiba, PR, CEP 80035-050, Brazil

    Kenia de Carvalho & João Carlos Bespalhok Filho

  3. Embrapa Café, Brasília, DF, CEP 70770-901, Brazil

    Luis Filipe Protasio Pereira

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  1. Kenia de Carvalho
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  2. João Carlos Bespalhok Filho
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  3. Tiago Benedito dos Santos
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Correspondence to Douglas Silva Domingues.

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de Carvalho, K., Bespalhok Filho, J.C., dos Santos, T.B. et al. Nitrogen Starvation, Salt and Heat Stress in Coffee (Coffea arabica L.): Identification and Validation of New Genes for qPCR Normalization. Mol Biotechnol 53, 315–325 (2013). https://doi.org/10.1007/s12033-012-9529-4

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