title: Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae.
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.
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|Computational Biology and Population Genomics group, cE3c - Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Vieira
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|LEAF-Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Cabral
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|Computational Biology and Population Genomics group, cE3c - Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Fino
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|LEAF-Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Helena G
authors.initials: HG
authors.lastname: Azinheira
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|LEAF-Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Andreia
authors.initials: A
authors.lastname: Loureiro
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|LEAF-Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.|Plant Cell Biology, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Talhinhas
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|Plant Cell Biology, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Ana Sofia
authors.initials: AS
authors.lastname: Pires
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|LEAF-Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Vitor
authors.initials: V
authors.lastname: Varzea
authors.affiliation: Cenicafe, Manizales, Colombia.
authors.forename: Pilar
authors.initials: P
authors.lastname: Moncada
authors.affiliation: LEAF-Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Helena
authors.initials: H
authors.lastname: Oliveira
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|LEAF-Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Maria do Céu
authors.initials: Mdo C
authors.lastname: Silva
authors.affiliation: Computational Biology and Population Genomics group, cE3c - Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Octávio S
authors.initials: OS
authors.lastname: Paulo
authors.affiliation: CIFC-Centro de Investigação das Ferrugens do Cafeeiro, Instituto Superior de Agronomia, Universidade de Lisboa, Oeiras, Portugal.|Computational Biology and Population Genomics group, cE3c - Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal.|LEAF-Linking Landscape, Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Dora
authors.initials: D
authors.lastname: Batista
mesh_terms: D020231:Colletotrichum / Q000235:genetics* / Q000472:pathogenicity; D020869:Gene Expression Profiling / Q000592:standards*; D012331:RNA, Fungal / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D017423:Sequence Analysis, RNA / Q000592:standards*
chemical_list: D012331:RNA, Fungal