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.

Fungal DNA barcoding

“…Their results suggest that the TEF1 locus was the most informative among the four compared gene fragments. Stielow et al (2015) obtained a similar conclusion on the advantages of TEF1 over ITS for several groups of mushrooms, including the Russula, Gasteromycetes, and Polyporaceae. Due to its single copy nature, TEF1 does not have the problems of potential heterogeneity among copies of ITS within individual mushrooms.…”

“…The main problems include (i) the lack of sequence variation between many closely related sister species (i.e., lack of a barcode gap; e.g., Xu et al 2000); (ii) the inability to obtain clear ITS sequences directly using PCR and Sanger sequencing of PCR products due to intrastrain sequence heterogeneity among copies of the ITS within the ribosomal RNA gene cluster (Chen et al 2016); and (iii) the inability of the "universal primers" (ITS1, ITS2, ITS3, and ITS4) to amplify about 10% of the tested fungal specimens (Schoch et al 2012;Stielow et al 2015). As a result, a number of attempts have been made to identify other loci with suitable barcode characteristics.…”

“…Table 1. Selected top-performing loci and primers as primary and secondary fungal DNA barcodes based on Schoch et al (2012) and Stielow et al (2015).…”

Bretziella, a new genus to accommodate the oak wilt fungus, Ceratocystis fagacearum (Microascales, Ascomycota)

“…These gene regions were the same as those selected and used by De Beer et al (2014) to define generic boundaries in the Ceratocystidaceae. In addition to these, sequences were determined of the ribosomal internal transcribed spacer region (ITS) and translation elongation factor 1-α (TEF1α), respectively the universal DNA barcode (Schoch et al 2012) and secondary barcode (Stielow et al 2015) for fungi, for isolate CBS 138363 = CMW 2656. Total genomic DNA was extracted with PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, California) following the protocols used by Duong et al (2012).…”

Resurrection and emendation of the Hypoxylaceae, recognised from a multigene phylogeny of the Xylariales

“…The studies by Hsieh et al (2005, with focus on Hypoxylon and allies), and Hsieh et al (2010, with focus on the xylarioid clades) provided a better resolution based on the protein-coding genes alpha-actin (ACT) and beta-tubulin (TUB2). Both genes exhibit intron-rich 5′ primed ends and thus met ideal requirements for phylogenetically informative loci, but are -as non-universal regions -only of limited use for phylogenetic analyes (Tang et al 2007;Stielow et al 2015). Interestingly, Hsieh et al (2005) already chose to abandon ITS data from their great experience with comparative morphology as phylogenetically valid criterion at a time when many other mycologists promoted them, and some even proposed that this DNA locus can serve as "sole means of identification of a fungus".…”