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Comparative validation of conventional and RNA-seq data-derived reference genes for qPCR expression studies of Colletotrichum kahawae

  • Articles in SCI Journals
  • May, 2016

Vieira, A., Cabral, A., Fino, J., Azinheira, H.G., Loureiro, A., Talhinhas, P., Pires, A.S., Varzea, V., Moncada, P., Oliveira, H., Silva, M.C., Paulo, O.S., Batista, D. (2016) Comparative validation of conventional and RNA-seq data-derived reference genes for qPCR expression studies of Colletotrichum kahawae

PLOS One, 11(3), e0150651. DOI:10.1371/journal.pone.0150651 (IF2015 3,057; Q1 Multidisciplinary Sciences)
Summary:

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica—Ckahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of Ckahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of Ckahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For Carabica—Ckahawae interaction samples, the best normalization factor included the combination of PP1Act and ck34620 genes, while for Ckahawae samples the combination of PP1Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in Ckahawae considering its aggressiveness pattern, developmental stage and host interaction.


http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150651