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

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.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).Peer reviewe