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Renzi et al. Microbiome Res Rep 2024;3:2 https://dx.doi.org/10.20517/mrr.2023.27 Page 5 of 16

Table 1. List of barcode loci for fungal taxonomic identification
Genomic locus Proposed by Ref.
ITS1-4 (whole region) Schoch et al. [24]
ITS1 as preferred for Basidiomycota identification Bellemain et al. [68]
ITS2 as preferred for Ascomycota identification Bellemain et al. [68]
ITS2 as preferred for human mycobiota identification Hoggard et al. [70]
ITS2 subregion Nilsson et al. [62]
TEF1α James et al. [77]
TOP1 Stielow et al. [76]
PGK Stielow et al. [76]
RPB1 Matheny et al. [78]
RPB2 for environmental fungal communities V trovský et al. [80]
IGS Morrison et al. [81]
β-tubulin Geiser et al. [82]
LSU (D1/D2 region) Kurtzman and Robnett [72]
IGS: Intergenic spacer; LSU: large subunit; PGK: phosphoglycerate kinase; TOP1: topoisomerase I.

Figure 1. Schematic representation of current limitations in culture-independent methods.

region of the LSU gene cluster within the ribosomal DNA (rDNA) has been a longstanding and effective
tool for species identification and strain differentiation, pre-dating the conceptualization of DNA
barcoding [71,72] . In addition, Nilsson et al. propose a set of fungus-specific primers with superior coverage of
the fungal kingdom, targeting the ITS2 sub-region with degenerate forward primers gITS7ngs and a reverse
primer ITS4ng . Besides primers’ choice, length variation among ITS sequences from fungal species,
[61]
spanning from 200 to 800 bp, has a strong impact on PCR efficiency as well as sequencing technologies [73,74] .
Moreover, not only is this region present in multiple copies within one species , but intragenomic
[75]
variation within a single species, such as numerous paralogous or non-orthologous copies, may lead to an
overestimation of global fungal diversity . Since the ITS copy number has highly interspecific variation, an
[24]
accurate determination of fungal abundance is hard to reach, and quantitative comparisons between diverse
species in mixed populations must be made with caution. The lack of universal taxonomic resolution and
the potential presence of non-homologous ITS copies in the genome made the identification of
[76]
supplementary molecular markers necessary. Using in silico pipelines, Stielow et al. confirmed the already
known TEF1α as a secondary barcoding marker for fungi and proposed the genes topoisomerase I
[77]
(TOP1) and phosphoglycerate kinase (PGK) as promising ascomycetes identifiers based on the analysis of
complete sequenced genomes . Other suggested secondary markers for fungal DNA amplification are the
[76]