One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes
2015
Stielow, J.B. | Lévesque, C.A. | Seifert, K.A. | Meyer, Wieland | Irinyi, L. | Smits, D. | Renfurm, R. | Verkley, G.J.M. | Groenewald, M. | Chaduli, Delphine | Lomascolo, Anne | Welti, S. | Lesage-Meessen, Laurence | Favel, Anne | Al-Hatmi, A.M.S. | Damm, U. | Yilmaz, N. | Houbraken, J. | Lombard, L. | Quaedvlieg, W. | Binder, M. | Vaas, L.A.I. | Vu, D. | Yurkov, A. | Begerow, D. | Roehl, O. | Guerreiro, M. | Fonseca, A. | Samerpitak, K. | van Diepeningen, A.D. | Dolatabadi, S. | Moreno, L.F. | Casaregola, Serge | Mallet, Sandrine | Nicolas, Jacques | Roscini, L. | Egidi, E. | Bizet, C. | Garcia-Hermoso, D. | Martín, M.P. | Deng, S. | Groenewald, J.Z. | Boekhout, T. | de Beer, Z.W. | Barnes, I. | Duong, T.A. | Wingfield, M.J. | de Hoog, G.S. | Crous, P.W. | Lewis, C.T. | Hambleton, S. | Moussa, T.A.A. | Al-Zahrani, H.S. | Almaghrabi, O.A. | Louis-Seize, G. | Assabgui, R. | Mccormick, W. | Omer, G. | Dukik, K. | Cardinali, G. | Eberhardt, U. | de Vries, M. | Robert, V. | CBS-KNAW Fungal Biodiversity Centre | Biodiversity (Mycology and Microbiology) ; Agriculture and Agri-Food (AAFC) | The Westmead Institute for Medical Research | Centre for Infectious Disease and Microbiology (CIDM) ; The Westmead Institute for Medical Research | Biodiversité et Biotechnologie Fongiques (BBF) ; Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM) | Laboratoire des Sciences Végétales et Fongi- ques, UFR Pharmacie ; Université de Lille, Droit et Santé | Directorate General of Health Services, Ibri Hospital ; Ministry of Health Oman | Institute for Biodiversity and Ecosystem Dynamics ; Universiteit van Amsterdam = University of Amsterdam (UvA) | Senckenberg Natural History Museum | Frauenhofer Institute for Molecular Biotechnology | Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH / Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ) | Evolution and Biodiversity of Plants, Geobotany ; Ruhr University Bochum = Ruhr-Universität Bochum (RUB) | Ruhr University Bochum = Ruhr-Universität Bochum (RUB) | Centro de Recursos Microbiológicos (CREM) ; Departamento de Ciencias da Vida (DCV) ; Faculdade de Ciências e Tecnologia = School of Science & Technology (FCT NOVA) ; Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Faculdade de Ciências e Tecnologia = School of Science & Technology (FCT NOVA) ; Universidade Nova de Lisboa = NOVA University Lisbon (NOVA)-Universidade Nova de Lisboa = NOVA University Lisbon (NOVA) | Department of Microbiology, Faculty of Medicine ; دانشگاه علوم پزشکی و خدمات بهداشتی - درمانی گیلان = Guilan University of Medical Sciences (GUMS) | Basic Pathology Department ; Universidade Federal do Paraná [Curitiba, Brasil] = Federal University of Paraná [Curitiba, Brazil] = Université fédérale du Paraná [Curitiba, Brésil] (UFPR) | MICrobiologie de l'ALImentation au Service de la Santé (MICALIS) ; Institut National de la Recherche Agronomique (INRA)-AgroParisTech | Department of Pharmaceutical Sciences ; University of Minnesota [Twin Cities] (UMN) ; University of Minnesota System (UMN)-University of Minnesota System (UMN)-College of Pharmacy | Dipartimento di Scienze Ecologiche e Biologiche ; Università degli studi della Tuscia = Tuscia University [Viterbo] (UNITUS) | Department of Physiology Anatomy and Microbiology, School of Life Sciences ; La Trobe University | Centre National de Référence des Mycoses invasives et antifongiques - Mycologie moléculaire (CNRMA) ; Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) | Centre National de la Recherche Scientifique (CNRS) | Departamento de Micología ; Real Jardin Botanico (RJB) | Shanghai Institute of Medical Mycology, Changzheng Hospital ; Second Military Medical University [Shanghai, China] (SMMU) | Shanghai Institute of Medical Mycology, Changzheng Hospital ; Second Military Medical University [Shanghai, China] (SMMU) | Department of Microbiology and Plant Pathology, Forestry Agricultural Biotechnology Institute (FABI) ; University of Pretoria [South Africa] | Department of Genetics, Forestry Agricultural Biotechnology Institute (FABI) ; University of Pretoria [South Africa] | Biological Sciences Department, Faculty of Science ; King Abdulaziz University | Biological Sciences Department, Faculty of Science, ; King Abdulaziz University | Botany and Microbiology Department, Faculty of Science ; Cairo University | Department of Pharmaceutical Sciences, Microbiology ; Università degli Studi di Perugia = University of Perugia (UNIPG) | Abteilung Botanik ; Stuttgart State Museum of Natural History | WE11 Biology ; Universiteit Gent = Ghent University = Université de Gand (UGENT) | Primer development and testing by partners in the European Consortium of Microbial Resource Centres (EMbaRC) was supported through funding of the European Community’s Seventh Framework Programme (FP7, 2007– 2013), Research Infrastructures action, under grant agreement no. FP7-228310. Part of sequencing work in CBS was supported by Fonds Economische Structuurversterking (FES), Dutch Ministry of Education, Culture and Science grant BEK/BPR-2009/137964-U). WM and VR were supported by research grant NH&MRC # APP1031952. Genome mining at CBS and AAFC, and primer development and testing at AAFC, were supported by grants from the A.P. Sloan Foundation Programme on the Microbiology of the Built Environment. We acknowledge the Deanship of Scientific Research (DSR), King Abdulaziz University, under grant No. 1-965/1434 HiCi for technical and financial support. AY was supported by Fundação para a Ciência e a Tecnologia (Portugal), project PTDC/BIA-BIC/4585/2012. MPM was supported by grant CGL2012-359 (Spain). | European Project: 228310,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2008-1,EMBARC(2009) | European Project: 8607092(1986)
The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial beta-tubulin II (TUB2); iv) gamma-actin (ACT); v) translation elongation factor 1-alpha (TEF1 alpha); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1 alpha. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1 alpha, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.
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