ArticlePloS one2016
Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae.
Article in PloS one, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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8 citing papers in PubMed.
- Article
- Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data.BMC genomics · 2023Article
- Selection and validation of reference genes for normalizing qRT‒PCR gene expression studies inFrontiers in plant science · 2023Article
- Identification of reference genes and their validation for gene expression analysis in phytopathogenic fungus Macrophomina phaseolina.PloS one · 2022Article
- Comparative proteomic analysis for revealing the advantage mechanisms of salt-tolerant tomato (PeerJ · 2022Article
- Selection of reference genes for quantitative real-time PCR normalization in the plant pathogen Puccinia helianthi Schw.BMC plant biology · 2019Article
- Article
- Identification and evaluation of PCR reference genes for host and pathogen in sugarcane-Sporisorium scitamineum interaction system.BMC genomics · 2018Article
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Abstract
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-C. kahawae 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 C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae 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 C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act 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 C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.
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