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

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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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Keywords: DNA BARCODING; ITS SUPPLEMENT; MOLECULAR TAXONOMY; PHYLOGENY; SPECIES IDENTIFICATION; UNIVERSAL PRIMERS

Document Type: Research Article

Publication date: 2015-12-23

More about this publication?
  • Persoonia aims to publish papers dealing with molecular phylogeny and evolution of fungi. A further aim is to promote fungal taxonomy by employing a polythetic approach to clarify the true phylogeny and relationships within the kingdom Fungi. The journal publishes high-quality papers elucidating known and novel fungal taxa at the DNA level, and also strives to present novel insights into evolutionary processes and relationships. Papers to be considered include research articles and reviews.

    Papers are published using a Fast Track system. This implies that the papers are immediately published online and freely available through the internet via this website. Volumes appear twice a year (June and December). From Volume 36 onwards (2016) hard copy volumes will only be made available through printing-on-demand. The issues can be ordered separately through the Publication Division of Naturalis Biodiversity Center; e-mail: [email protected]

    Persoonia is a journal published jointly by the Naturalis Biodiversity Center and the Westerdijk Fungal Biodiversity Institute.
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