title: One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes.
abstract: 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 β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); 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 TEF1α. 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 TEF1α, 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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authors.lastname: Seifert
authors.affiliation: Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney, Australia; Medical School, Westmead Hospital, The University of Sydney, Westmead Millennium Institute, Westmead, Sydney, Australia.
authors.forename: W
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authors.lastname: Meyer
authors.affiliation: Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney, Australia; Medical School, Westmead Hospital, The University of Sydney, Westmead Millennium Institute, Westmead, Sydney, Australia.
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authors.lastname: Iriny
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
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authors.lastname: Smits
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
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authors.lastname: Renfurm
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
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authors.lastname: Verkley
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authors.affiliation: CIRM-INRA, UMR 1163 BBF, F-13288 Marseille, France.
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authors.lastname: Chaduli
authors.affiliation: CIRM-INRA, UMR 1163 BBF, F-13288 Marseille, France.; Aix-Marseille Université, Polytech, UMR 1163 BBF, F-13288 Marseille, France.
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authors.initials: S
authors.lastname: Welti
authors.affiliation: CIRM-INRA, UMR 1163 BBF, F-13288 Marseille, France.
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authors.lastname: Lesage-Meessen
authors.affiliation: CIRM-INRA, UMR 1163 BBF, F-13288 Marseille, France.; Aix-Marseille Université, Polytech, UMR 1163 BBF, F-13288 Marseille, France.
authors.forename: A
authors.initials: A
authors.lastname: Favel
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Directorate General of Health Services, Ibri Hospital, Ministry of Health Oman, Sultanate of Oman.; Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands.
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authors.initials: AM
authors.lastname: Al-Hatmi
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Senckenberg Museum of Natural History, Görlitz, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Damm
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Biodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
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authors.lastname: Yilmaz
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
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authors.lastname: Houbraken
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
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authors.lastname: Lombard
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authors.lastname: Binder
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Marie Bashir Institute for Infectious Diseases and Biosecurity, Sydney, Australia; Medical School, Westmead Hospital, The University of Sydney, Westmead Millennium Institute, Westmead, Sydney, Australia.; Frauenhofer Institute for Molecular Biotechnology, Hamburg, Germany.
authors.forename: L A I
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authors.lastname: Vaas
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
authors.forename: D
authors.initials: D
authors.lastname: Vu
authors.affiliation: DSMZ-Leibniz Institute German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.
authors.forename: A
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authors.affiliation: Evolution and Biodiversity of Plants, Geobotany, Ruhr-Universität Bochum, Bochum, Germany.
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authors.lastname: Guerreiro
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authors.forename: A
authors.initials: A
authors.lastname: Fonseca
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Department of Microbiology, Faculty of Medicine, Khon Kaen University, Thailand.; Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: K
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authors.lastname: Samerpitak
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
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authors.initials: AD
authors.lastname: van Diepeningen
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: Dolatabadi
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands.; Basic Pathology Department, Federal University of Paraná State, Curitiba, Paraná, Brazil.
authors.forename: L F
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authors.affiliation: INRA UMR 1319, Micalis, CIRM-Levures, AgroParisTech, Thiverval-Grignon, France.
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authors.lastname: Mallet
authors.affiliation: INRA UMR 1319, Micalis, CIRM-Levures, AgroParisTech, Thiverval-Grignon, France.
authors.forename: N
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authors.lastname: Jacques
authors.affiliation: Department of Pharmaceutical Sciences, Microbiology, Università degli Studi di Perugia, Perugia, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Roscini
authors.affiliation: Dipartimento di Scienze Ecologiche e Biologiche, Università degli Studi della Tuscia, Viterbo, Italy.; Department of Physiology Anatomy and Microbiology, School of Life Sciences, La Trobe University, 3083 Bundoora, Melbourne, Australia.
authors.forename: E
authors.initials: E
authors.lastname: Egidi
authors.affiliation: Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses Invasives et Antifongiques, Paris, France.; CNRS URA3012, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Bizet
authors.affiliation: Institut Pasteur, Unité de Mycologie Moléculaire, Centre National de Référence Mycoses Invasives et Antifongiques, Paris, France.; CNRS URA3012, Paris, France.
authors.forename: D
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authors.affiliation: Departamento de Micología, Real Jardín Botánico, RJB-CSIC, Madrid, Spain.
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authors.affiliation: Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
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authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
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authors.lastname: Groenewald
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China.
authors.forename: T
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authors.affiliation: Department of Microbiology and Plant Pathology, Forestry Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.
authors.forename: Z W
authors.initials: ZW
authors.lastname: de Beer
authors.affiliation: Department of Genetics, Forestry Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.
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authors.affiliation: Department of Genetics, Forestry Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.
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authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands.; Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: G S
authors.initials: GS
authors.lastname: de Hoog
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.; Department of Microbiology and Plant Pathology, Forestry Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.; Microbiology, Department of Biology, Utrecht University, Utrecht, The Netherlands.
authors.forename: P W
authors.initials: PW
authors.lastname: Crous
authors.affiliation: Biodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
authors.forename: C T
authors.initials: CT
authors.lastname: Lewis
authors.affiliation: Biodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Hambleton
authors.affiliation: Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.; Botany and Microbiology Department, Faculty of Science, Cairo University, Giza, Egypt.
authors.forename: T A A
authors.initials: TA
authors.lastname: Moussa
authors.affiliation: Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: H S
authors.initials: HS
authors.lastname: Al-Zahrani
authors.affiliation: Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: O A
authors.initials: OA
authors.lastname: Almaghrabi
authors.affiliation: Biodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
authors.forename: G
authors.initials: G
authors.lastname: Louis-Seize
authors.affiliation: Biodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
authors.forename: R
authors.initials: R
authors.lastname: Assabgui
authors.affiliation: Biodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
authors.forename: W
authors.initials: W
authors.lastname: McCormick
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: Omer
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
authors.forename: K
authors.initials: K
authors.lastname: Dukik
authors.affiliation: Department of Pharmaceutical Sciences, Microbiology, Università degli Studi di Perugia, Perugia, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Cardinali
authors.affiliation: Staatliches Museum für Naturkunde Stuttgart, Abteilung Botanik, Stuttgart, Germany.; Ghent University, WE11 Biology, Gent, Belgium.
authors.forename: U
authors.initials: U
authors.lastname: Eberhardt
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: de Vries
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
authors.forename: V
authors.initials: V
authors.lastname: Robert
keywords: DNA barcoding; ITS supplement; molecular taxonomy; phylogeny; species identification; universal primers
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