Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawaeReport as inadecuate

Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae - Download this document for free, or read online. Document in PDF available to download.

BMC Research Notes

, 6:388

First Online: 28 September 2013Received: 21 May 2013Accepted: 25 September 2013DOI: 10.1186-1756-0500-6-388

Cite this article as: Figueiredo, A., Loureiro, A., Batista, D. et al. BMC Res Notes 2013 6: 388. doi:10.1186-1756-0500-6-388


BackgroundCoffee production in Africa represents a significant share of the total export revenues and influences the lives of millions of people, yet severe socio-economic repercussions are annually felt in result of the overall losses caused by the coffee berry disease CBD. This quarantine disease is caused by the fungus Colletotrichum kahawae Waller and Bridge, which remains one of the most devastating threats to Coffea arabica production in Africa at high altitude, and its dispersal to Latin America and Asia represents a serious concern. Understanding the molecular genetic basis of coffee resistance to this disease is of high priority to support breeding strategies. Selection and validation of suitable reference genes presenting stable expression in the system studied is the first step to engage studies of gene expression profiling.

ResultsIn this study, a set of ten genes S24, 14-3-3, RPL7, GAPDH, UBQ9, VATP16, SAND, UQCC, IDE and β-Tub9 was evaluated to identify reference genes during the first hours of interaction 12, 48 and 72 hpi between resistant and susceptible coffee genotypes and C. kahawae. Three analyses were done for the selection of these genes considering the entire dataset and the two genotypes resistant and susceptible, separately. The three statistical methods applied GeNorm, NormFinder, and BestKeeper, allowed identifying IDE as one of the most stable genes for all datasets analysed, and in contrast GADPH and UBQ9 as the least stable ones. In addition, the expression of two defense-related transcripts, encoding for a receptor like kinase and a pathogenesis related protein 10, were used to validate the reference genes selected.

ConclusionTaken together, our results provide guidelines for reference genes selection towards a more accurate and widespread use of qPCR to study the interaction between Coffea spp. and C. kahawae.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-0500-6-388 contains supplementary material, which is available to authorized users.

Andreia Figueiredo, Andreia Loureiro contributed equally to this work.

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Author: Andreia Figueiredo - Andreia Loureiro - Dora Batista - Filipa Monteiro - Vítor Várzea - Maria Salomé Pais - Elijah K G


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