One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes

J.B. Stielow (1) , C.A. Lévesque (2) , K.A. Seifert (2) , Wieland Meyer (3, 4) , L. Irinyi (3, 4) , D. Smits (1) , R. Renfurm (1) , G.J.M. Verkley (1) , M. Groenewald (1) , Delphine Chaduli (5) , Anne Lomascolo (5) , S. Welti (6) , Laurence Lesage-Meessen (5) , Anne Favel (5) , A.M.S. Al-Hatmi (1, 7, 8) , U. Damm (1, 9) , N. Yilmaz (1, 2) , J. Houbraken (1) , L. Lombard (1) , W. Quaedvlieg (1) , M. Binder (1) , L.A.I. Vaas (1, 10) , D. Vu (1) , A. Yurkov (11) , D. Begerow (12) , O. Roehl (13) , M. Guerreiro (14) , A. Fonseca (14) , K. Samerpitak (1, 15, 8) , A.D. van Diepeningen (1) , S. Dolatabadi (1, 8) , L.F. Moreno (1, 8, 16) , Serge Casaregola (17) , Sandrine Mallet (17) , Jacques Nicolas (17) , L. Roscini (18) , E. Egidi (19, 20) , C. Bizet (21, 22) , D. Garcia-Hermoso (21, 22) , M.P. Martín (23) , S. Deng (24) , J.Z. Groenewald (1) , T. Boekhout (1, 25) , Z.W. de Beer (26) , I. Barnes (27) , T.A. Duong (27) , M.J. Wingfield (26) , G.S. de Hoog (1, 8, 28) , P.W. Crous (1, 26) , C.T. Lewis (2) , S. Hambleton (2) , T.A.A. Moussa (29, 30) , H.S. Al-Zahrani (28, 30) , O.A. Almaghrabi (28, 30) , G. Louis-Seize (2) , R. Assabgui (2) , W. Mccormick (2) , G. Omer (1) , K. Dukik (1) , G. Cardinali (31) , U. Eberhardt (32, 33) , M. de Vries (1) , V. Robert (1)

1 CBS-KNAW Fungal Biodiversity Centre
2 Biodiversity (Mycology and Microbiology)
3 The Westmead Institute for Medical Research
4 CIDM - Centre for Infectious Disease and Microbiology
5 BBF - Biodiversité et Biotechnologie Fongiques
6 Laboratoire des Sciences Végétales et Fongi- ques, UFR Pharmacie
7 Directorate General of Health Services, Ibri Hospital
8 Institute for Biodiversity and Ecosystem Dynamics
9 Senckenberg Natural History Museum
10 Frauenhofer Institute for Molecular Biotechnology
11 DSMZ - Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH / Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures
12 Evolution and Biodiversity of Plants, Geobotany
13 RUB - Ruhr University Bochum = Ruhr-Universität Bochum
14 CREM - Centro de Recursos Microbiológicos
15 Department of Microbiology, Faculty of Medicine
16 Basic Pathology Department
17 MICALIS - MICrobiologie de l'ALImentation au Service de la Santé
18 Department of Pharmaceutical Sciences
19 Dipartimento di Scienze Ecologiche e Biologiche
20 Department of Physiology Anatomy and Microbiology, School of Life Sciences
21 CNRMA - Centre National de Référence des Mycoses invasives et antifongiques - Mycologie moléculaire
22 CNRS - Centre National de la Recherche Scientifique
23 Departamento de Micología
24 Shanghai Institute of Medical Mycology, Changzheng Hospital
25 Shanghai Institute of Medical Mycology, Changzheng Hospital
26 Department of Microbiology and Plant Pathology, Forestry Agricultural Biotechnology Institute (FABI)
27 Department of Genetics, Forestry Agricultural Biotechnology Institute (FABI)
28 Biological Sciences Department, Faculty of Science
29 Biological Sciences Department, Faculty of Science,
30 Botany and Microbiology Department, Faculty of Science
31 Department of Pharmaceutical Sciences, Microbiology
32 Abteilung Botanik
33 WE11 Biology

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.

Mots clés: species identification; universal primers; species identification; ITS supplement; DNA barcoding; molecular taxonomy; phylogeny

Domaines: Sciences du Vivant [q-bio]

J.B. Stielow, C.A. Lévesque, K.A. Seifert, Wieland Meyer, L. Irinyi, et al.. One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes. Persoonia, 2015, 35 (1), pp.242-263. ⟨10.3767/003158515X689135⟩. ⟨hal-01594853⟩