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Fungal DNA barcoding is the process of identifying species of the biological kingdom Fungi through the amplification and sequencing of specific DNA sequences and their comparison with sequences deposited in a DNA barcode database such as the ISHAM reference database,^[1] or the Barcode of Life Data System (BOLD). In this attempt, DNA barcoding relies on universal genes that are ideally present in all fungi with the same degree of sequence variation. The interspecific variation, i.e., the variation between species, in the chosen DNA barcode gene(s) should exceed the intraspecific (within-species) variation.^[2]

A fundamental problem in fungal systematics is the existence of teleomorphic and anamorphic stages in their life cycles. These morphs usually differ drastically in their phenotypic appearance, preventing a straightforward association of the asexual anamorph with the sexual teleomorph. Moreover, fungal species can comprise multiple strains that can vary in their morphology or in traits such as carbon- and nitrogen utilisation, which has often led to their description as different species, eventually producing long lists of synonyms.^[3] Fungal DNA barcoding can help to identify and associate anamorphic and teleomorphic stages of fungi, and through that to reduce the confusing multitude of fungus names. For this reason, mycologists were among the first to spearhead the investigation of species discrimination by means of DNA sequences,^[3]^[4]^[5]^[6]^[7]^[8] at least 10 years earlier than the DNA barcoding proposal for animals by Paul D. N. Hebert and colleagues in 2003, who popularised the term "DNA barcoding".^[9]^[10]

The success of identification of fungi by means of DNA barcode sequences stands and falls with the quantitative (completeness) and qualitative (level of identification) aspect of the reference database. Without a database covering a broad taxonomic range of fungi, many identification queries will not result in a satisfyingly close match. Likewise, without a substantial curatorial effort to maintain the records at a high taxonomic level of identification, queries – even when they might have a close or exact match in the reference database – will not be informative if the closest match is only identified to phylum or class level.^[11]^[12]

Another crucial prerequisite for DNA barcoding is the ability to unambiguously trace the provenance of DNA barcode data back to the originally sampled specimen, the so-called voucher specimen. This is common practice in biology along with the description of new taxa, where the voucher specimens, on which the taxonomic description is based, become the type specimens. When the identity of a certain taxon (or a genetic sequence in the case of DNA barcoding) is in doubt, the original specimen can be re-examined to review and ideally solve the issue. Voucher specimens should be clearly labelled as such, including a permanent voucher identifier that unambiguously connects the specimen with the DNA barcode data derived from it. Furthermore, these voucher specimens should be deposited in publicly accessible repositories like scientific collections or herbaria to preserve them for future reference and to facilitate research involving the deposited specimens.^[13]

Barcode DNA markers

Internal Transcribed Spacer (ITS) – the primary fungal barcode

In fungi, the Internal transcribed spacer (ITS) is a roughly 600 basepairs (bp) long region in the ribosomal tandem repeat gene cluster of the nuclear genome. The region is flanked by the DNA sequences for the ribosomal small subunit (SSU) or 18S subunit at the 5‘ end, and by the large subunit (LSU) or 28S subunit at the 3‘ end.^[14]^[15] The Internal Transcribed Spacer itself consists of two parts, ITS1 and ITS2, which are separated from each other by the 5.8S subunit nested between them. Like the flanking 18S and 28S subunits, the 5.8S subunit contains a highly conserved DNA sequence, as they code for structural parts of the ribosome, which is a key component in intracellular protein synthesis.

Due to several advantages of ITS (see below) and a comprehensive amount of sequence data accumulated in the 1990s and early 2000s, Begerow et al. (2010) and Schoch et al. (2012) proposed the ITS region as primary DNA barcode region for the genetic identification of fungi.^[12]^[2]

Primers

The conserved flanking regions of 18S and 28S serve as anchor points for the primers used for PCR amplification of the ITS region.^[16] Moreover, the conserved nested 5.8S region allows for the construction of "internal" primers, i.e., primers attaching to complementary sequences within the ITS region. White et al. (1990) proposed such internal primers, named ITS2 and ITS3, along with the flanking primers ITS1 and ITS4 in the 18S and the 28S subunit, respectively.^[16] Due to their almost universal applicability to ITS sequening in fungi, these primers are still in wide use today. Optimised primers specifically for ITS sequencing in Dikarya (comprising Basidiomycota and Ascomycota) have been proposed by Toju et al. (2012).^[17]

For the majority of fungi, the ITS primers proposed by White et al. (1990) have become the standard primers used for PCR amplification. These primers are:^[16]

Forward primers:

ITS1: 5'-TCCGTAGGTGAACCTGCGG-3'
ITS2: 5'-GCTGCGTTCTTCCATCGATGC-3'
ITS5: 5'-GGAAGTAAAAGTCGTAACAAG G-3'

Reverse primers:

ITS3: 5'-GCATCGATGAAGAACGCAGC-3'
ITS4: 5'-TCCTCCGCTTATTGATATGC-3'

Advantages and shortcomings

A major advantage of using the ITS region as molecular marker and fungal DNA barcode is that the entire ribosomal gene cluster is arranged in tandem repeats, i.e., in multiple copies.^[15] This allows for its PCR amplification and Sanger sequencing even from small material samples (given the DNA is not fragmented due to age or other degenerative influences).^[14] Hence, a high PCR success rate is usually observed when amplifying ITS. However, this success rate varies greatly among fungal groups, from 65% in non-Dikarya (including the now paraphyletic Mucoromycotina, the Chytridiomycota and the Blastocladiomycota) to 100% in Saccharomycotina and Basidiomycota^[2] (with the exception of very low success in Pucciniomycotina).^[18] Furthermore, the choice of primers for ITS amplification can introduce biases towards certain taxonomic fungus groups.^[19] For example, the "universal" ITS primers^[16] fail to amplify about 10% of the tested fungal specimens.^[18]

The tandem repeats of the ribosomal gene cluster cause the problem of significant intragenomic sequence heterogeneity observed among ITS copies of several fungal groups.^[20]^[21]^[22] In Sanger sequencing, this will cause ITS sequence reads of different lengths to superpose each other, potentially rendering the resulting chromatograph unreadable. Furthermore, because of the non-coding nature of the ITS region that can lead to a substantial amount of indels, it is impossible to consistently align ITS sequences from highly divergent species for further bigger-scale phylogenetic analyses.^[9]^[14] The degree of intragenomic sequence heterogeneity can be investigated in more detail through molecular cloning of the initially PCR-amplified ITS sequences, followed by sequencing of the clones. This procedure of initial PCR amplification, followed by cloning of the amplicons and finally sequencing of the cloned PCR products is the most common approach of obtaining ITS sequences for DNA metabarcoding of environmental samples, in which a multitude of different fungal species can be present simultaneously. However, this approach of sequencing after cloning was rarely done for the ITS sequences that make up the reference libraries used for DNA barcode-aided identification, thus potentially giving an underestimate of the existing ITS sequence variation in many samples.^[23]

The weighted arithmetic mean of the intraspecific (within-species) ITS variability among fungi is 2.51%. This variability, however, can range from 0% for example in Serpula lacrymans (n=93 samples) over 0.19% in Tuber melanosporum (n=179) up to 15.72% in Rhizoctonia solani (n=608), or even 24.75% in Pisolithus tinctorius (n=113). In cases of high intraspecific ITS variability, the application of a threshold of 3% sequence variability – a canonical upper value for intraspecific variation – will therefore lead to a higher estimate of operational taxonomic units (OTUs), i.e., putative species, than there actually are in a sample.^[24] In the case of medically relevant fungal species, a more strict threshold of 2.5% ITS variability allows only around 75% of all species to be accurately identified to the species level.^[1]

On the other hand, morphologically well-defined, but evolutionarily young species complexes or sibling species may only differ (if at all) in a few nucleotides of the ITS sequences. Solely relying on ITS barcode data for the identification of such species pairs or complexes may thus obscure the actual diversity and might lead to misidentification if not accompanied by the investigation of morphological and ecological features and/or comparison of additional diagnostic genetic markers.^[18]^[23]^[25]^[26] For some taxa, ITS (or its ITS2 part) is not variable enough as fungal DNA barcode, as for example has been shown in Aspergillus, Cladosporium, Fusarium and Penicillium.^[27]^[28]^[29]^[30] Efforts to define a univerally applicable threshold value of ITS variability that demarcates intraspecific from interspecific (between-species) variability thus remain futile.^[24]

Nonetheless, the probability of correct species identification with the ITS region is high in the Dikarya, and especially so in Basidiomycota, where even the ITS1 part is often sufficient to identify the species.^[31] However, its discrimination power is partly superseded by that of the DNA-directed RNA polymerase II subunit RPB1 (see also below).^[2]

Due to the shortcomings of ITS' as primary fungal DNA barcode, the necessity of establishing a second DNA barcode marker was expressed.^[9] Several attempts were made to establish other genetic markers that could serve as additional DNA barcodes,^[18]^[32]^[33] similar to the situation in plants, where the plastidial genes rbcL, matK and trnH‐psbA, as well as the nuclear ITS are often used in combination for DNA barcoding.^[34]

Translational elongation factor 1α (TEF1α) – the secondary fungal barcode

The translational elongation factor 1α is part of the eucaryotic elongation factor 1 complex, whose main function is to facilitate the elongation of the amino acid chain of a polypeptide during the translation process of gene expression.^[35]

Stielow et al. (2015) investigated the TEF1α gene, among a number of others, as potential genetic marker for fungal DNA barcoding. The TEF1α gene coding for the translational elongation factor 1α is generally considered to have a slow mutation rate, and it is therefore generally better suited for investigating older splits deeper in the phylogenetic history of an organism group. Despite this, the authors conclude that TEF1α is the most promising candidate for an additional DNA barcode marker in fungi as it also features sequence regions of higher mutation rates.^[18] Following this, a quality-controlled reference database was established and merged with the previously existing ISHAM-ITS database for fungal ITS DNA barcodes^[1] to form the ISHAM database.^[36]

TEF1α has been successfully used to identify a new species of Cantharellus from Texas and distinguish it from a morphologically similar species.^[37] In the genera Ochroconis and Verruconis (Sympoventuriaceae, Venturiales), however, the marker does not allow distinction of all species.^[38] TEF1α has also been used in phylogenetic analyses at the genus level, e.g. in the case of Cantharellus^[39] and the entomopathogenic Beauveria,^[40] and for the phylogenetics of early-diverging fungal lineages.^[41]

Primers

TEF1α primers used in the broad-scale screening of the performance of DNA barcode gene candidates of Stielow et al. (2015) were the forward primer EF1-983F with the sequence 5'-GCYCCYGGHCAYCGTGAYTTYAT-3', and the reverse primer EF1-1567R with the sequence 5'-ACHGTRCCRATACCACCRATCTT-3'.^[40] In addition, a number of new primers was developed, with the primer pair in bold resulting in a high average amplification success of 88%:^[18]

Forward primers:

EF1-1018F: 5'-GAYTTCATCAAGAACATGAT-3'
EF1-1002F: 5'-TTCATCAAGAACATGAT-3'
Al33_alternative_f: 5'-GARTTYGARGCYGGTATCTC-3'
EF1_alterantive_3f: 5'-TTYGARGCYGGTATCTC-3'

Reverse primers:

EF1-1620R: 5'-GACGTTGAADCCRACRTTGTC-3'
EF1-1688R: 5'-GCTATCATCACAATGGACGTTCTTGGAG-3'
EF1_alternative_3r: 5'-GAVACRTTCTTGACGTTGAA-3'

Primers used for the investigation of Rhizophydiales and especially Batrachochytrium dendrobatidis, a pathogen of amphibia, are the forward primer tef1F with the nucleotide sequence 5′-TACAARTGYGGTGGTATYGACA-3′, and the reverse primer tef1R with the sequence 5′-ACNGACTTGACYTCAGTRGT-3′.^[42] These primers also successfully amplified the majority of Cantharellus species investigated by Buyck et al. (2014), with the exception of a few species for which more specific primers were developed: the forward primer tef-1Fcanth with the sequence 5'-AGCATGGGTDCTYGACAAG-3', and the reverse primer tef-1Rcanth with the sequence 5'-CCAATYTTRTAYACATCYTGGAG-3'.^[39]

D1/D2 domain of the LSU ribosomal RNA

The D1/D2 domain is part of the nuclear large subunit (28S) ribosomal RNA, and it is therefore located in the same ribosomal tandem repeat gene cluster as the Internal Transcribed Spacer (ITS). But unlike the non-coding ITS sequences, the D1/D2 domain contains coding sequence. With about 600 bp it is about the same nucleotide sequence length as ITS,^[43] which makes amplification and sequencing rather straightforward, an advantage that has led to the accumulation of an extensive amount of D1/D2 sequence data especially for yeasts.^[3]^[7]^[43]

Regarding the molecular identification of basidiomycetous yeasts, D1/D2 (or ITS) can be used alone.^[43] However, Fell et al. (2000) and Scorzetti et al. (2002) recommend the combined analysis of the D1/D2 and ITS regions,^[3]^[43] a practice that later became the standard required information for describing new taxa of asco- and basidiomycetous yeasts.^[14] When attempting to identify early diverging fungal lineages, the study of Schoch et al. (2012), comparing the identification performance of different genetic markers, showed that the large subunit (as well as the small subunit) of the ribosomal RNA performs better than ITS or RPB1.^[2]

Primers

For basidiomycetous yeasts, the forward primer F63 with the sequence 5’-GCATATCAATAAGCGGAGGAAAAG-3’, and the reverse primer LR3 with the sequence 5’-GGTCCGTGTTTCAAGACGG-3’ have been successfully used for PCR amplification of the D1/D23 domain.^[3] The D1/D2 domain of ascomycetous yeasts like Candida can be amplified with the forward primer NL-1 (sequence: 5'-GCATATCAATAAGCGGAGGAAAAG-3') and the reverse primer NL-4 (sequence: 5'-GGTCCGTGTTTCAAGACGG-3').^[6]

RNA polymerase II subunit RPB1

The RNA polymerase II subunit RPB1 is the largest subunit of the RNA polymerase II. In Saccharomyces cerevisiae, it is encoded by the RPO21 gene.^[45] PCR amplification success of RPB1 is very taxon-dependent, ranging from 70-80% in Ascomycota to 14% in early diverging fungal lineages.^[2] Apart from the early diverging lineages, RPB1 has a high rate of species identification in all fungal groups. In the species-rich Pezizomycotina it even outperforms ITS.^[2]

In a study comparing the identification performance of four genes, RPB1 was among the most effective genes when combining two genes in the analysis: combined analysis with either ITS or with the large subunit ribosomal RNA yielded the highest identification success.^[2]

Other studies also used RPB2, the second-largest subunit of the RNA polymerase II, e.g. for studying the phylogenetic relationships among species of the genus Cantharellus^[39] or for a phylogenetic study shedding light on the relationships among early-diverging lineages in the fungal kingdom.^[41]

Primers

Primers successfully amplifying RPB1 especially in Ascomycota are the forward primer RPB1-Af with the sequence 5′-GARTGYCCDGGDCAYTTYGG-3′, and the reverse primer RPB1-Ac-RPB1-Cr with the sequence 5′-CCNGCDATNTCRTTRTCCATRTA-3′.^[2]

Intergenic Spacer (IGS) of ribosomal RNA genes

The Intergenic Spacer (IGS) is the region of non-coding DNA between individual tandem repeats of the ribosomal gene cluster in the nuclear genome, as opposed to the Internal Transcribed Spacer (ITS) that is situated within these tandem repeats.

IGS has been successfully used for the differentiation of strains of Xanthophyllomyces dendrorhous^[46] as well as for species distinction in the psychrophilic genus Mrakia (Cystofilobasidiales).^[47] Due to these results, IGS has been recommended as a genetic marker for additional differentiation (along with D1/D2 and ITS) of closely related species and even strains within one species in basidiomycete yeasts.^[3]

Other genetic markers

The cytochrome c oxidase subunit I (COI) gene outperforms ITS in DNA barcoding of Penicillium (Ascomycota) species, with species-specific barcodes for 66% of the investigated species versus 25% in the case of ITS. Furthermore, a part of the β-Tubulin A (BenA) gene exhibits a higher taxonomic resolution in distinguishing Penicillium species as compared to COI and ITS.^[48] In the closely related Aspergillus niger complex, however, COI is not variable enough for species discrimination.^[49] In Fusarium, COI exhibits paralogues in many cases, and homologous copies are not variable enough to distinguish species.^[50]

COI also performs poorly in the identification of basidiomycote rusts of the order Pucciniales due to the presence of introns. Even when the obstacle of introns is overcome, ITS and the LSU rRNA (28S) outperform COI as DNA barcode marker.^[51] In the subdivision Agaricomycotina, PCR amplification success was poor for COI, even with multiple primer combinations. Successfully sequenced COI samples also included introns and possible paralogous copies, as reported for Fusarium.^[50]^[52] Agaricus bisporus was found to contain up to 19 introns, making the COI gene of this species the longest recorded, with 29,902 nucleotides.^[53] Apart from the substantial troubles of sequencing COI, COI and ITS generally perform equally well in distinguishing basidiomycote mushrooms.^[52]

Topoisomerase I (TOP1) was investigated as additional DNA barcode candidate by Lewis et al. (2011) based on proteome data, with the devloped universal primer pair^[32] being susequently tested on actual samples by Stielow et al. (2015). The forward primer TOP1_501-F with the sequence 5'-TGTAAAACGACGGCCAGT-ACGAT-ACTGCCAAGGTTTTCCGTACHTACAACGC-3' (where the first section marks the universal M13 forward primer tail, the second part consisting of ACGAT a spacer, and the third part the actual primer) and reverse the primer TOP1_501-R with 5'-CAGGAAACAGCTATGA-CCCAGTCCTCGTCAACWGACTTRATRGCCCA-3' (the first section marking the universal M13 reverse primer tail, the second part the actual TOP1 reverse primer) amplify a fragment of approximately 800 base pairs (bp).^[18]

TOP1 was found to be a promising DNA barcode candidate marker for ascomycetes, where it can distinguish species in Fusarium and Penicillium – genera, in which the primary ITS barcode performs poorly. However, poor amplification success with the TOP1 universal primers is observed in early-diverging fungal lineages and basidiomycetes except Pucciniomycotina (where ITS PCR success is poor).^[18]

Like TOP1, the Phosphoglycerate kinase (PGK) was among the genetic markers investigated by Lewis et al. (2011) and Stielow et al. (2015) as potential additional fungal DNA barcodes. A number of universal primers was developed,^[32] with the PGK533 primer pair, amplifying a circa 1,000 bp fragment, being the most successful in most fungi except Basidiomycetes. Like TOP1, PGK is superior to ITS in species differentiation in ascomycete genera like Penicillium and Fusarium, and both PGK and TOP1 perform as good as TEF1α in distinguishing closely related species in these genera.^[18]

Applications

Fungal DNA barcoding has been sucessfully applied to the investigation of foxing phenomena, a major concern in the conservation of paper documents. Sequeira et al. (2019) sequenced ITS from foxing stains and found Chaetomium globosum, Ch. murorum, Ch. nigricolor, Chaetomium sp., Eurotium rubrum, Myxotrichum deflexum, Penicillium chrysogenum, P. citrinum, P. commune, Penicillium sp. and Stachybotrys chartarum to inhabit the investigated paper stains.^[54]

Another study investigated fungi that act as biodeteriorating agents in the Old Cathedral of Coimbra, part of the University of Coimbra, a UNESCO world heritage site. Sequencing the ITS barcode of ten samples with classical Sanger as well as with Illumina next-generation sequencing techniques, they identified 49 fungal species. Aspergillus versicolor, Cladosporium cladosporioides, C. sphaerospermum, C. tenuissimum, Epicoccum nigrum, Parengyodontium album, Penicillium brevicompactum, P. crustosum, P. glabrum, Talaromyces amestolkiae and T. stollii were the most common species isolated from the samples.^[55]

Another study concerning objects of cultural heritage investigated the fungal diversity on a canvas painting by Paula Rego using the ITS2 subregion of the ITS marker. Altogether, 387 OTUs (putative species) in 117 genera of 13 different classes of fungi were observed.^[56]

References

^ ^a ^b ^c Irinyi L, Serena C, Garcia-Hermoso D, Arabatzis M, Desnos-Ollivier M, Vu D, et al. (May 2015). "International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi". Medical Mycology. 53 (4): 313–37. doi:10.1093/mmy/myv008. PMID 25802363.
^ ^a ^b ^c ^d ^e ^f ^g ^h ⁱ Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W (April 2012). "Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi" (PDF). Proceedings of the National Academy of Sciences of the United States of America. 109 (16): 6241–6. doi:10.1073/pnas.1117018109. PMID 22454494.
^ ^a ^b ^c ^d ^e ^f Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A (May 2000). "Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis" (PDF). International Journal of Systematic and Evolutionary Microbiology. 50 Pt 3: 1351–1371. doi:10.1099/00207713-50-3-1351. PMID 10843082.
^ Cadez N, Raspor P, de Cock AW, Boekhout T, Smith MT (January 2002). "Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera". FEMS Yeast Research. 1 (4): 279–89. doi:10.1146/annurev.es.22.110191.002521. PMID 12702331.
^ Messner, Robert; Prillinger, Hansjörg; Ibl, Martin; Himmler, Gottfried (1995). "Sequences of ribosomal genes and internal transcribed spacers move three plant parasitic fungi, Eremothecium ashbyi, Ashbya gossypii, and Nematospora coryli, towards Saccharomyces cerevisiae". The Journal of General and Applied Microbiology. 41: 31–42. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ ^a ^b Kurtzman CP, Robnett CJ (May 1997). "Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene" (PDF). Journal of Clinical Microbiology. 35 (5): 1216–23. PMID 9114410.
^ ^a ^b Kurtzman CP, Robnett CJ (May 1998). "Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences". Antonie Van Leeuwenhoek. 73 (4): 331–71. doi:10.1023/a:1001761008817. PMID 9850420.
^ Kurtzman CP, Robnett CJ (October 1998). "Three new insect-associated species of the yeast genus Candida". Canadian Journal of Microbiology. 44 (10): 965–73. PMID 9933915.
^ ^a ^b ^c Seifert KA (May 2009). "Progress towards DNA barcoding of fungi". Molecular Ecology Resources. 9 Suppl s1 (Suppl. 1): 83–9. doi:10.1111/j.1755-0998.2009.02635.x. PMID 21564968.
^ Hebert PD, Cywinska A, Ball SL, deWaard JR (February 2003). "Biological identifications through DNA barcodes". Proceedings. Biological Sciences. 270 (1512): 313–21. doi:10.1098/rspb.2002.2218. PMC 1691236. PMID 12614582.
^ Nilsson RH, Ryberg M, Abarenkov K, Sjökvist E, Kristiansson E (July 2009). "The ITS region as a target for characterization of fungal communities using emerging sequencing technologies". FEMS Microbiology Letters. 296 (1): 97–101. doi:10.1111/j.1574-6968.2009.01618.x. PMID 19459974.
^ ^a ^b Begerow D, Nilsson H, Unterseher M, Maier W (June 2010). "Current state and perspectives of fungal DNA barcoding and rapid identification procedures" (PDF). Applied Microbiology and Biotechnology. 87 (1): 99–108. doi:10.1007/s00253-010-2585-4. PMID 20405123.
^ Agerer R, Ammirati J, Baroni TJ, Blanz P, Courtecuisse RE, Desjardin DE, et al. (2000). "Open letter to the scientific community of mycologists". Applied Soil Ecology. 15 (3): 295–298. doi:10.1016/S0929-1393(00)00076-7.
^ ^a ^b ^c ^d Xu J (November 2016). "Fungal DNA barcoding". Genome. 59 (11): 913–932. doi:10.1139/gen-2016-0046. PMID 27829306.
^ ^a ^b Wurzbacher C, Larsson E, Bengtsson-Palme J, Van den Wyngaert S, Svantesson S, Kristiansson E, et al. (January 2019). "Introducing ribosomal tandem repeat barcoding for fungi". Molecular Ecology Resources. 19 (1): 118–127. doi:10.1111/1755-0998.12944. PMID 30240145.
^ ^a ^b ^c ^d White TJ, Bruns T, Lee SJ, Taylor J (1990). "Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics". In Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds.). PCR Protocols: A Guide to Methods and Applications. New York: Academic Press, Inc. pp. 315–322.
^ Toju H, Tanabe AS, Yamamoto S, Sato H (2012). "High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples". PloS One. 7 (7): e40863. doi:10.1371/journal.pone.0040863. PMC 3395698. PMID 22808280.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ ^a ^b ^c ^d ^e ^f ^g ^h ⁱ Stielow JB, Lévesque CA, Seifert KA, Meyer W, Iriny L, Smits D, et al. (December 2015). "One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes". Persoonia. 35: 242–63. doi:10.3767/003158515X689135. PMID 26823635.
^ Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H (July 2010). "ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases". BMC Microbiology. 10 (189): 189. doi:10.1186/1471-2180-10-189. PMC 2909996. PMID 20618939.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ Smith ME, Douhan GW, Rizzo DM (December 2007). "Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland" (PDF). Mycorrhiza. 18 (1): 15–22. doi:10.1007/s00572-007-0148-z. PMID 17710446.
^ Lindner DL, Banik MT (2011). "Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus". Mycologia. 103 (4): 731–40. doi:10.3852/10-331. PMID 21289107.
^ Kovács GM, Balázs TK, Calonge FD, Martín MP (2011). "The diversity of Terfezia desert truffles: new species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity" (PDF). Mycologia. 103 (4): 841–53. doi:10.3852/10-312. PMID 21289106.
^ ^a ^b Kiss L (July 2012). "Limits of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences as species barcodes for Fungi" (PDF). Proceedings of the National Academy of Sciences of the United States of America. 109 (27): E1811, author reply E1812. doi:10.1073/pnas.1207143109. PMID 22715287.
^ ^a ^b Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson KH (May 2008). "Intraspecific ITS variability in the kingdom fungi as expressed in the international sequence databases and its implications for molecular species identification". Evolutionary Bioinformatics Online. 4: 193–201. doi:10.4137/EBO.S653. PMID 19204817.
^ Xu J, Vilgalys R, Mitchell TG (October 2000). "Multiple gene genealogies reveal recent dispersion and hybridization in the human pathogenic fungus Cryptococcus neoformans" (PDF). Molecular Ecology. 9 (10): 1471–81. doi:10.1046/j.1365-294x.2000.01021.x. PMID 11050543.
^ Stockinger H, Krüger M, Schüssler A (July 2010). "DNA barcoding of arbuscular mycorrhizal fungi". The New Phytologist. 187 (2): 461–74. doi:10.1111/j.1469-8137.2010.03262.x. PMID 20456046.
^ Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA (2007). "The current status of species recognition and identification in Aspergillus". Studies in Mycology. 59: 1–10. doi:10.3114/sim.2007.59.01. PMID 18490947.
^ Schubert K, Groenewald JZ, Braun U, Dijksterhuis J, Starink M, Hill CF, et al. (2007). "Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales), with standardisation of methods for Cladosporium taxonomy and diagnostics". Studies in Mycology. 58: 105–56. doi:10.3114/sim.2007.58.05. PMID 18490998.
^ O'Donnell K, Cigelnik E (February 1997). "Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous". Molecular Phylogenetics and Evolution. 7 (1): 103–16. doi:10.1006/mpev.1996.0376. PMID 9007025.
^ Skouboe P, Frisvad JC, Taylor JW, Lauritsen D, Boysen M, Rossen L (1999). "Phylogenetic analysis of nucleotide sequences from the ITS region of terverticillate Penicillium species". Mycological Research. 103 (7): 873–881. doi:10.1017/S0953756298007904.
^ Osmundson TW, Robert VA, Schoch CL, Baker LJ, Smith A, Robich G, et al. (2013). "Filling gaps in biodiversity knowledge for macrofungi: contributions and assessment of an herbarium collection DNA barcode sequencing project". PloS One. 8 (4): e62419. doi:10.1371/journal.pone.0062419. PMID 23638077.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ ^a ^b ^c Lewis CT, Bilkhu S, Robert V, Eberhardt U, Szoke S, Seifert KA, Lévesque CA (2011). "Identification of fungal DNA barcode targets and PCR primers based on Pfam protein families and taxonomic hierarchy" (PDF). The Open Applied Informatics Journal. 5 (suppl. 1-M5): 30–44. doi:10.2174/1874136301005010030.
^ Vincent Robert L, Szöke S, Eberhardt U, Cardinali G, Meyer W, Seifert KA, Lévesque CA, Lewis CT (2011). "The quest for a general and reliable fungal DNA barcode" (PDF). The Open Applied Informatics Journal. 5 (suppl. 1-M6): 45–61. doi:10.2174/1874136301005010045.
^ Kress, W. John (2017). "Plant DNA barcodes: Applications today and in the future". Journal of Systematics and Evolution. 55 (4): 291–307. doi:10.1111/jse.12254. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ Sasikumar AN, Perez WB, Kinzy TG (2012). "The many roles of the eukaryotic elongation factor 1 complex". Wiley Interdisciplinary Reviews. RNA. 3 (4): 543–55. doi:10.1002/wrna.1118. PMC 3374885. PMID 22555874.
^ Meyer W, Irinyi L, Hoang MT, Robert V, Garcia-Hermoso D, Desnos-Ollivier M, et al. (March 2019). "1". Genome. 62 (3): 160–169. doi:10.1139/gen-2018-0083. PMID 30465691.
^ Buyck B, Cruaud C, Couloux A, Hofstetter V (2011). "Cantharellus texensis sp. nov. from Texas, a southern lookalike of C. cinnabarinus revealed by tef-1 sequence data". Mycologia. 103 (5): 1037–46. doi:10.3852/10-261. PMID 21558500.
^ Samerpitak K, Gerrits van den Ende BH, Stielow JB, Menken SB, de Hoog GS (February 2016). "Barcoding and species recognition of opportunistic pathogens in Ochroconis and Verruconis" (PDF). Fungal Biology. 120 (2): 219–30. doi:10.1016/j.funbio.2015.08.010. PMID 26781378.
^ ^a ^b ^c Buyck, Bart; Kauff, Frank; Eyssartier, Guillaume; Couloux, André; Hofstetter, Valérie (2014). "A multilocus phylogeny for worldwide Cantharellus (Cantharellales, Agaricomycetidae)" (PDF). Fungal Diversity. 64: 101–121. doi:10.1007/s13225-013-0272-3. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ ^a ^b Rehner SA, Buckley E (2005). "A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs". Mycologia. 97 (1): 84–98. doi:10.1080/15572536.2006.11832842. PMID 16389960.
^ ^a ^b James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V, Cox CJ, et al. (October 2006). "Reconstructing the early evolution of Fungi using a six-gene phylogeny". Nature. 443 (7113): 818–22. doi:10.1038/nature05110. PMID 17051209.
^ Morehouse EA, James TY, Ganley AR, Vilgalys R, Berger L, Murphy PJ, Longcore JE (February 2003). "Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently emerged clone". Molecular Ecology. 12 (2): 395–403. doi:10.1046/j.1365-294X.2003.01732.x. PMID 12535090.
^ ^a ^b ^c ^d Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A (December 2002). "Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions". FEMS Yeast Research. 2 (4): 495–517. doi:10.1111/j.1567-1364.2002.tb00117.x. PMID 12702266.
^ Armache KJ, Mitterweger S, Meinhart A, Cramer P (February 2005). "Structures of complete RNA polymerase II and its subcomplex, Rpb4/7" (PDF). The Journal of Biological Chemistry. 280 (8): 7131–4. doi:10.2210/pdb1wcm/pdb. PMID 15591044.
^ Strathern J, Malagon F, Irvin J, Gotte D, Shafer B, Kireeva M, et al. (January 2013). "The fidelity of transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in S. cerevisiae" (PDF). The Journal of Biological Chemistry. 288 (4): 2689–99. doi:10.1074/jbc.M112.429506. PMID 23223234.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ Fell, Jack W.; Blatt, G. M. (1999). "Separation of strains of the yeasts Xanthophyllomyces dendrorhous and Phaffia rhodozyma based on rDNA IGS and ITS sequence analysis". Journal of Industrial Microbiology and Biotechnology. 23 (1): 677–681. doi:10.1038/sj.jim.2900681. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ Diaz, Mara R.; Fell, Jack W. (2000). "Molecular analyses of the IGS & ITS regions of rDNA of the psychrophilic yeasts in the genus Mrakia". Antonie van Leeuwenhoek. 77 (1): 7–12. doi:10.1023/A:1002048008295. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ Seifert KA, Samson RA, Dewaard JR, Houbraken J, Lévesque CA, Moncalvo JM, et al. (March 2007). "Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case" (PDF). Proceedings of the National Academy of Sciences of the United States of America. 104 (10): 3901–6. doi:10.1073/pnas.0611691104. PMID 17360450.
^ Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA (2007). "The current status of species recognition and identification in Aspergillus". Studies in Mycology. 59: 1–10. doi:10.3114/sim.2007.59.01. PMC 2275194. PMID 18490947.
^ ^a ^b Gilmore SR, Gräfenhan T, Louis-Seize G, Seifert KA (May 2009). "Multiple copies of cytochrome oxidase 1 in species of the fungal genus Fusarium". Molecular Ecology Resources. 9 Suppl s1 (Suppl. 1): 90–8. doi:10.1111/j.1755-0998.2009.02636.x. PMID 21564969.
^ Vialle A, Feau N, Allaire M, Didukh M, Martin F, Moncalvo JM, Hamelin RC (May 2009). "Evaluation of mitochondrial genes as DNA barcode for Basidiomycota". Molecular Ecology Resources. 9 Suppl s1 (Suppl. 1): 99–113. doi:10.1111/j.1755-0998.2009.02637.x. PMID 21564970.
^ ^a ^b Dentinger BT, Didukh MY, Moncalvo JM (2011). "Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina)". PloS One. 6 (9): e25081. Bibcode:2011PLoSO...625081D. doi:10.1371/journal.pone.0025081. PMC 3178597. PMID 21966418.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ Férandon C, Moukha S, Callac P, Benedetto JP, Castroviejo M, Barroso G (November 2010). "The Agaricus bisporus cox1 gene: the longest mitochondrial gene and the largest reservoir of mitochondrial group i introns". PloS One. 5 (11): e14048. doi:10.1371/journal.pone.0014048. PMC 2987802. PMID 21124976.{{cite journal}}: CS1 maint: unflagged free DOI (link)
^ Sequeira, Sílvia Oliveira; de Carvalho, Hugo Paiva; Mesquita, Nuno; Portugal, António; Macedo, Maria Filomena (2019). "Fungal stains on paper: is what you see what you get?" (PDF). Conservar Património. 32: 18–27. doi:10.14568/cp2018007. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ Trovão, João; Portugal, António; Soares, Fabiana; Paiva, Diana Sofia; Mesquita, Nuno; Coelho, Catarina; Pinheiro, Ana Catarina; Catarino, Lídia; Gil, Francisco; Tiago, Igor (2019). "Fungal diversity and distribution across distinct biodeterioration phenomena in limestone walls of the old cathedral of Coimbra, UNESCO World Heritage Site". International Biodeterioration & Biodegradation. 142: 91–102. doi:10.1016/j.ibiod.2019.05.008. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)
^ Paiva de Carvalho, Hugo; Oliveira Sequeira, Sílvia; Pinho, Diogo; Trovão, João; Fernandes da Costa, Ricardo Manuel; Egas, Conceição; Macedo, Maria Filomena; Portugal, António (2019). "Combining an innovative non-invasive sampling method and highthroughput sequencing to characterize fungal communities on a canvas painting". International Biodeterioration & Biodegradation. 145: 1–9. doi:10.1016/j.ibiod.2019.104816. {{cite journal}}: Unknown parameter |name-list-format= ignored (|name-list-style= suggested) (help)

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 {{short description|Identification of fungal species thanks to specific DNA sequences}}
-'''Fungal DNA barcoding''' is the process of identifying species of the [[kingdom (biology)|biological kingdom]] [[fungus|Fungi]] through the [[Polymerase chain reaction|amplification]] and [[DNA sequencing|sequencing]] of specific DNA sequences and their comparison with sequences deposited in a DNA barcode database such as the ISHAM reference database,<ref name=Irinyi-eal-2015>{{cite journal |last1=Irinyi |first1=Laszlo |last2=Serena |first2=Carolina |last3=Garcia-Hermoso |first3=Dea |last4=Arabatzis |first4=Michael |last5=Desnos-Ollivier |first5=Marie |last6=Vu |first6=Duong |last7=Cardinali |first7=Gianluigi |last8=Arthur |first8=Ian |last9=Normand |first9=Anne-Cécile |last10=Giraldo |first10=Alejandra |last11=da Cunha |first11=Keith Cassia |last12=Sandoval-Denis |first12=Marcelo |last13=Hendrickx |first13=Marijke |last14=Nishikaku |first14=Angela Satie |last15=de Azevedo Melo |first15=Analy Salles |last16=Bellinghausen Merseguel |first16=Karina |last17=Khan |first17=Aziza |last18=Parente Rocha |first18=Juliana Alves |last19=Sampaio |first19=Paula |last20=da Silva Briones |first20=Marcelo Ribeiro |last21=e Ferreira |first21=Renata Carmona |last22=de Medeiros Muniz |first22=Mauro |last23=Castañón-Olivares |first23=Laura Rosio |last24=Estrada-Barcenas |first24=Daniel |last25=Cassagne |first25=Carole |last26=Mary |first26=Charles |last27=Duan |first27=Shu Yao |last28=Kong |first28=Fanrong |last29=Sun |first29=Annie Ying |last30=Zeng |first30=Xianyu |last31=Zhao |first31=Zuotao |last32=Gantois |first32=Nausicaa |last33=Botterel |first33=Françoise |last34=Robbertse |first34=Barbara |last35=Schoch |first35=Conrad |last36=Gams |first36=Walter |last37=Ellis |first37=David |last38=Halliday |first38=Catriona |last39=Chen \first39=Sharon |last40=Sorrell |first40=Tania C. |last41=Piarroux |first41=Renaud |last42=Colombo |first42=Arnaldo L. |last43=Pais |first43=Célia |last44=de Hoog |first44=Sybren |last45=Zancopé-Oliveira |first45=Rosely Maria |last46=Taylor |first46=Maria Lucia |last47=Toriello |first47=Conchita |last48=de Almeida Soares |first48=Célia Maria |last49=Delhaes |first49=Laurence |last50=Stubbe |first50=Dirk |last51=Dromer |first51=Françoise |last52=Ranque |first52=Stéphane |last53=Guarro |first53=Josep |last54=Cano-Lira |first54=Jose F. |last55=Robert |first55=Vincent |last56=Velegraki |first56=Aristea |last57=Meyer |first57=Wieland |date=2015 |title=International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database—the quality controlled standard tool for routine identification of human and animal pathogenic fungi |journal=Medical Mycology |volume=53 |issue=4 |pages=313–337 |doi=10.1093/mmy/myv008 |url=https://academic.oup.com/mmy/article/53/4/313/977005 }}</ref> or the [[Barcode of Life Data System]] (BOLD). In this attempt, DNA barcoding relies on universal genes that are ideally present in all fungi with the same degree of sequence variation. The interspecific variation, i.e., the variation between species, in the chosen DNA barcode gene(s) should exceed the intraspecific (within-species) variation.<ref name=Schoch-eal-2012>{{cite journal |last1=Schoch |first1=Conrad L. |last2=Seifert |first2=Keith A. |last3=Huhndorf |first3=Sabine |last4=Robert |first4=Vincent |last5=Spouge |first5= John L. |last6=Levesque |first6=C. André |last7=Chen |first7=Wen |author8=Fungal Barcoding Consortium |date=2012 |title=Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi |journal=Proceedings of the National Academy of Sciences |volume=109 |issue=16 |pages=6241–6246 |doi=10.1073/pnas.1117018109 |url=https://www.pnas.org/content/pnas/109/16/6241.full.pdf }}</ref>
+'''Fungal DNA barcoding''' is the process of identifying species of the [[kingdom (biology)|biological kingdom]] [[fungus|Fungi]] through the [[Polymerase chain reaction|amplification]] and [[DNA sequencing|sequencing]] of specific DNA sequences and their comparison with sequences deposited in a DNA barcode database such as the ISHAM reference database,<ref name=Irinyi-eal-2015>{{cite journal | vauthors = Irinyi L, Serena C, Garcia-Hermoso D, Arabatzis M, Desnos-Ollivier M, Vu D, Cardinali G, Arthur I, Normand AC, Giraldo A, da Cunha KC, Sandoval-Denis M, Hendrickx M, Nishikaku AS, de Azevedo Melo AS, Merseguel KB, Khan A, Parente Rocha JA, Sampaio P, da Silva Briones MR, e Ferreira RC, de Medeiros Muniz M, Castañón-Olivares LR, Estrada-Barcenas D, Cassagne C, Mary C, Duan SY, Kong F, Sun AY, Zeng X, Zhao Z, Gantois N, Botterel F, Robbertse B, Schoch C, Gams W, Ellis D, Halliday C, Chen S, Sorrell TC, Piarroux R, Colombo AL, Pais C, de Hoog S, Zancopé-Oliveira RM, Taylor ML, Toriello C, de Almeida Soares CM, Delhaes L, Stubbe D, Dromer F, Ranque S, Guarro J, Cano-Lira JF, Robert V, Velegraki A, Meyer W | display-authors = 6 | title = International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database--the quality controlled standard tool for routine identification of human and animal pathogenic fungi | journal = Medical Mycology | volume = 53 | issue = 4 | pages = 313–37 | date = May 2015 | pmid = 25802363 | doi = 10.1093/mmy/myv008 | url = https://academic.oup.com/mmy/article/53/4/313/977005 | first49 = Laurence | first48 = Célia Maria | first41 = Renaud | first40 = Tania C. | first43 = Célia | first42 = Arnaldo L. | first45 = Rosely Maria | first44 = Sybren | first47 = Conchita | first46 = Maria Lucia | first56 = Aristea | first57 = Wieland | first54 = Jose F. | first55 = Vincent | first52 = Stéphane | first53 = Josep | first50 = Dirk | first51 = Françoise }}</ref> or the [[Barcode of Life Data System]] (BOLD). In this attempt, DNA barcoding relies on universal genes that are ideally present in all fungi with the same degree of sequence variation. The interspecific variation, i.e., the variation between species, in the chosen DNA barcode gene(s) should exceed the intraspecific (within-species) variation.<ref name=Schoch-eal-2012>{{cite journal | vauthors = Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W | title = Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 16 | pages = 6241–6 | date = April 2012 | pmid = 22454494 | doi = 10.1073/pnas.1117018109 | url = https://www.pnas.org/content/pnas/109/16/6241.full.pdf | author8 = Fungal Barcoding Consortium }}</ref>
-A fundamental problem in fungal systematics is the existence of [[Teleomorph, anamorph and holomorph|teleomorphic and anamorphic]] stages in their life cycles. These morphs usually differ drastically in their [[phenotype|phenotypic]] appearance, preventing a straightforward association of the asexual anamorph with the sexual teleomorph. Moreover, fungal species can comprise multiple strains that can vary in their morphology or in traits such as carbon- and nitrogen utilisation, which has often led to their description as different species, eventually producing long lists of synonyms.<ref name=Fell-eal-2000>{{cite journal |last1=Fell |first1=Jack W. |last2=Boekhout |first2=Teun |last3=Fonseca |first3=Alvaro |last4=Scorzetti |first4=Gloria |last5=Statzell-Tallman |first5=Adele |date=2000 |title=Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis |journal=International Journal of Systematic and Evolutionary Microbiology |volume=50 |pages=1351–1371 |doi=10.1099/00207713-50-3-1351 |url=https://pdfs.semanticscholar.org/7d0c/18fda4516beb432599134c690324c38df858.pdf }}</ref> Fungal DNA barcoding can help to identify and associate anamorphic and teleomorphic stages of fungi, and through that to reduce the confusing multitude of fungus names. For this reason, mycologists were among the first to spearhead the investigation of species discrimination by means of DNA sequences,<ref name=Fell-eal-2000/><ref name=Bruns-eal-1991>{{cite journal |last1=Bruns |first1=Thomas D. |last2=White |first2=Thomas J. |last3=Taylor |first3=John W. |date=1991 |title=Fungal molecular systematics |journal=Annual Review of Ecology and Systematics |volume=22 |pages=525–564 |url=https://www.annualreviews.org/doi/pdf/10.1146/annurev.es.22.110191.002521 }}</ref><ref name=Messner-eal-1995>{{cite journal |last1=Messner |first1=Robert |last2=Prillinger |first2=Hansjörg |last3=Ibl |first3=Martin |last4=Himmler |first4=Gottfried |date=1995 |title=Sequences of ribosomal genes and internal transcribed spacers move three plant parasitic fungi, ''Eremothecium ashbyi'', ''Ashbya gossypii'', and ''Nematospora coryli'', towards ''Saccharomyces cerevisiae'' |journal=The Journal of General and Applied Microbiology |volume=41 |pages=31–42 |url=https://www.jstage.jst.go.jp/article/jgam1955/41/1/41_1_31/_pdf }}</ref><ref name=Kurtzman-Robnett-1997>{{cite journal |last1=Kurtzman |first1=Cletus P. |last2=Robnett |first2=Christie J. |date=1997 |title=Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5’ end of the Large-Subunit (26S) ribosomal DNA gene |journal=Journal of Clinical Microbiology |volume=35 |issue=5 |pages=1216–1223 |url=https://jcm.asm.org/content/jcm/35/5/1216.full.pdf }}</ref><ref name=Kurtzmann-Robnett-1998a>{{cite journal |last1=Kurtzman |first1=Cletus P. |last2=Robnett |first2=Christie J. |date=1998 |title=Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences |journal=Antonie van Leeuwenhoek |volume=73 |pages=331–371 |url=https://pubag.nal.usda.gov/download/102/PDF }}</ref><ref name=Kurtzmann-Robnett-1998b>{{cite journal |last1=Kurtzman |first1=Cletus P. |last2=Robnett |first2=Christie J. |date=1998 |title=Three new insect-associated species of the yeast genus ''Candida'' |journal=Canadian Journal of Microbiology |volume=44 |pages=965–973 |url=https://pubag.nal.usda.gov/download/24858/PDF }}</ref> at least 10 years earlier than the DNA barcoding proposal for animals by [[Paul D. N. Hebert]] and colleagues in 2003, who popularised the term "DNA barcoding".<ref name=Seifert-2009>{{cite journal |last=Seifert |first=Keith A. |date=2009 |title=Progress towards DNA barcoding of fungi |journal=Molecular Ecology Resources |volume=9 |issue=Suppl. 1 |pages=83–89 |doi=10.1111/j.1755-0998.2009.02635.x |url=https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1755-0998.2009.02635.x }}</ref><ref name=Hebert-eal-2003>{{cite journal |last=Hebert |first=Paul D. N. |author1-link=Paul D. N. Hebert |last2=Cywinska |first2=Alina |last3=Ball |first3=Shelley L. |last4=deWaard |first4=Jeremy R. |date=2003 |title=Biological identifications through DNA barcodes |journal=Proceedings of the Royal Society of London. Series B: Biological Sciences |volume=270 |issue=1512 |pages=313–321 |doi=10.1098/rspb.2002.2218 |issn=1471-2954 |pmc=1691236 |pmid=12614582 |url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1691236/pdf/12614582.pdf }}</ref>
+A fundamental problem in fungal systematics is the existence of [[Teleomorph, anamorph and holomorph|teleomorphic and anamorphic]] stages in their life cycles. These morphs usually differ drastically in their [[phenotype|phenotypic]] appearance, preventing a straightforward association of the asexual anamorph with the sexual teleomorph. Moreover, fungal species can comprise multiple strains that can vary in their morphology or in traits such as carbon- and nitrogen utilisation, which has often led to their description as different species, eventually producing long lists of synonyms.<ref name=Fell-eal-2000>{{cite journal | vauthors = Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A | title = Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis | journal = International Journal of Systematic and Evolutionary Microbiology | volume = 50 Pt 3 | pages = 1351–1371 | date = May 2000 | pmid = 10843082 | doi = 10.1099/00207713-50-3-1351 | url = https://pdfs.semanticscholar.org/7d0c/18fda4516beb432599134c690324c38df858.pdf }}</ref> Fungal DNA barcoding can help to identify and associate anamorphic and teleomorphic stages of fungi, and through that to reduce the confusing multitude of fungus names. For this reason, mycologists were among the first to spearhead the investigation of species discrimination by means of DNA sequences,<ref name=Fell-eal-2000/><ref name=Bruns-eal-1991>{{cite journal | vauthors = Cadez N, Raspor P, de Cock AW, Boekhout T, Smith MT | title = Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera | journal = FEMS Yeast Research | volume = 1 | issue = 4 | pages = 279–89 | date = January 2002 | pmid = 12702331 | doi = 10.1146/annurev.es.22.110191.002521 }}</ref><ref name=Messner-eal-1995>{{cite journal |last1=Messner |first1=Robert |last2=Prillinger |first2=Hansjörg |last3=Ibl |first3=Martin |last4=Himmler |first4=Gottfried | name-list-format = vanc |date=1995 |title=Sequences of ribosomal genes and internal transcribed spacers move three plant parasitic fungi, ''Eremothecium ashbyi'', ''Ashbya gossypii'', and ''Nematospora coryli'', towards ''Saccharomyces cerevisiae'' |journal=The Journal of General and Applied Microbiology |volume=41 |pages=31–42 |url=https://www.jstage.jst.go.jp/article/jgam1955/41/1/41_1_31/_pdf }}</ref><ref name=Kurtzman-Robnett-1997>{{cite journal | vauthors = Kurtzman CP, Robnett CJ | title = Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene | journal = Journal of Clinical Microbiology | volume = 35 | issue = 5 | pages = 1216–23 | date = May 1997 | pmid = 9114410 | url = https://jcm.asm.org/content/jcm/35/5/1216.full.pdf }}</ref><ref name=Kurtzmann-Robnett-1998a>{{cite journal | vauthors = Kurtzman CP, Robnett CJ | title = Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences | journal = Antonie Van Leeuwenhoek | volume = 73 | issue = 4 | pages = 331–71 | date = May 1998 | pmid = 9850420 | doi = 10.1023/a:1001761008817 | url = https://pubag.nal.usda.gov/download/102/PDF }}</ref><ref name=Kurtzmann-Robnett-1998b>{{cite journal | vauthors = Kurtzman CP, Robnett CJ | title = Three new insect-associated species of the yeast genus Candida | journal = Canadian Journal of Microbiology | volume = 44 | issue = 10 | pages = 965–73 | date = October 1998 | pmid = 9933915 | url = https://pubag.nal.usda.gov/download/24858/PDF }}</ref> at least 10 years earlier than the DNA barcoding proposal for animals by [[Paul D. N. Hebert]] and colleagues in 2003, who popularised the term "DNA barcoding".<ref name=Seifert-2009>{{cite journal | vauthors = Seifert KA | title = Progress towards DNA barcoding of fungi | journal = Molecular Ecology Resources | volume = 9 Suppl s1 | issue = Suppl. 1 | pages = 83–9 | date = May 2009 | pmid = 21564968 | doi = 10.1111/j.1755-0998.2009.02635.x | url = https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1755-0998.2009.02635.x }}</ref><ref name=Hebert-eal-2003>{{cite journal | vauthors = Hebert PD, Cywinska A, Ball SL, deWaard JR | title = Biological identifications through DNA barcodes | journal = Proceedings. Biological Sciences | volume = 270 | issue = 1512 | pages = 313–21 | date = February 2003 | pmid = 12614582 | pmc = 1691236 | doi = 10.1098/rspb.2002.2218 | author1-link = Paul D. N. Hebert }}</ref>
-The success of identification of fungi by means of DNA barcode sequences stands and falls with the quantitative (completeness) and qualitative (level of identification) aspect of the reference database. Without a database covering a broad taxonomic range of fungi, many identification queries will not result in a satisfyingly close match. Likewise, without a substantial curatorial effort to maintain the records at a high taxonomic level of identification, queries &ndash; even when they might have a close or exact match in the reference database &ndash; will not be informative if the closest match is only identified to [[phylum]] or [[class (biology)|class]] level.<ref name=Nilsson-eal-2009>{{cite journal |last1=Nilsson |first1=Rolf Henrik |last2=Ryberg |first2=Martin |last3=Abarenkov |first3=Kessy |last4=Sjökvist |first4=Elisabet |last5=Kristiansson |first5=Erik |date=2009 |title=The ITS region as a target for characterization of fungal communities using emerging sequencing technologies |journal=FEMS Microbiology Letters |volume=296 |issue=1 |pages=97–101 |doi=10.1111/j.1574-6968.2009.01618.x |url=https://academic.oup.com/femsle/article/296/1/97/485832 }}</ref><ref name=Begerow-eal-2010>{{cite journal |last1=Begerow |first1=Dominik |last2=Nilsson |first2=Henrik |last3=Unterseher |first3=Martin |last4=Maier |first4= Wolfgang |date=2010 |title=Current state and perspectives of fungal DNA barcoding and rapid identification procedures |journal=Applied Microbiology and Biotechnology |volume=87 |issue=1 |pages=99–108 |doi=10.1007/s00253-010-2585-4 |url=https://s3.amazonaws.com/academia.edu.documents/45722522/Current_state_and_perspectives_of_fungal20160517-2657-1li7gw9.pdf?response-content-disposition=inline%3B%20filename%3DCurrent_state_and_perspectives_of_fungal.pdf&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWOWYYGZ2Y53UL3A%2F20200305%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20200305T161825Z&X-Amz-Expires=3600&X-Amz-SignedHeaders=host&X-Amz-Signature=e9e605c37b6c9bdb8f6511a6eaaba20a10fc1380b8647ced7d852a49b0dbf337}}</ref>
+The success of identification of fungi by means of DNA barcode sequences stands and falls with the quantitative (completeness) and qualitative (level of identification) aspect of the reference database. Without a database covering a broad taxonomic range of fungi, many identification queries will not result in a satisfyingly close match. Likewise, without a substantial curatorial effort to maintain the records at a high taxonomic level of identification, queries &ndash; even when they might have a close or exact match in the reference database &ndash; will not be informative if the closest match is only identified to [[phylum]] or [[class (biology)|class]] level.<ref name=Nilsson-eal-2009>{{cite journal | vauthors = Nilsson RH, Ryberg M, Abarenkov K, Sjökvist E, Kristiansson E | title = The ITS region as a target for characterization of fungal communities using emerging sequencing technologies | journal = FEMS Microbiology Letters | volume = 296 | issue = 1 | pages = 97–101 | date = July 2009 | pmid = 19459974 | doi = 10.1111/j.1574-6968.2009.01618.x | url = https://academic.oup.com/femsle/article/296/1/97/485832 }}</ref><ref name=Begerow-eal-2010>{{cite journal | vauthors = Begerow D, Nilsson H, Unterseher M, Maier W | title = Current state and perspectives of fungal DNA barcoding and rapid identification procedures | journal = Applied Microbiology and Biotechnology | volume = 87 | issue = 1 | pages = 99–108 | date = June 2010 | pmid = 20405123 | doi = 10.1007/s00253-010-2585-4 | url = https://s3.amazonaws.com/academia.edu.documents/45722522/Current_state_and_perspectives_of_fungal20160517-2657-1li7gw9.pdf?response-content-disposition=inline%3B%20filename%3DCurrent_state_and_perspectives_of_fungal.pdf&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWOWYYGZ2Y53UL3A%2F20200305%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20200305T161825Z&X-Amz-Expires=3600&X-Amz-SignedHeaders=host&X-Amz-Signature=e9e605c37b6c9bdb8f6511a6eaaba20a10fc1380b8647ced7d852a49b0dbf337 }}</ref>
-Another crucial prerequisite for DNA barcoding is the ability to unambiguously trace the provenance of DNA barcode data back to the originally sampled specimen, the so-called voucher specimen. This is common practice in biology along with the description of new [[taxon|taxa]], where the voucher specimens, on which the taxonomic description is based, become the [[Type (biology)#Type specimen|type specimens]]. When the identity of a certain taxon (or a genetic sequence in the case of DNA barcoding) is in doubt, the original specimen can be re-examined to review and ideally solve the issue. Voucher specimens should be clearly labelled as such, including a permanent voucher identifier that unambiguously connects the specimen with the DNA barcode data derived from it. Furthermore, these voucher specimens should be deposited in publicly accessible repositories like [[scientific collection]]s or [[herbarium|herbaria]] to preserve them for future reference and to facilitate research involving the deposited specimens.<ref name=Agerer-eal-2000>{{cite journal |last1=Agerer |first1=Reinhard |last2=Ammirati |first2=Joe |last3=Blanz |first3=Paul |last4=Courtecuisse |first4=Régis |last5=Desjardin |first5=Dennis E. |last6=Gams |first6=Walter |last7=Hallenberg |first7=Nils |last8=Halling |first8=Roy |last9=Hawksworth |first9=David L. |last10=Horak |first10=Egon |last11=Korf |first11=Richard P. |last12=Mueller |first12=Greg M. |last13=Oberwinkler |first13=Franz |last14=Rambold |first14=Gerhard |last15=Summerbell |first15=Richard C. |last16=Triebel |first16=Dagmar |last17=Watling |first17=Roy |date=2000 |title=Open letter to the scientific community of mycologists |journal=Applied Soil Ecology |volume=15 |issue=3 |pages=295–298 |doi=10.1016/S0929-1393(00)00076-7 |url=https://reader.elsevier.com/reader/sd/pii/S0929139300000767?token=C590FD403AA59D5AE704E466B116B3846667B8491E04A5D23D71F83669B4F491CCBE074974F3B622A015F1680AD71660 }}</ref>
+Another crucial prerequisite for DNA barcoding is the ability to unambiguously trace the provenance of DNA barcode data back to the originally sampled specimen, the so-called voucher specimen. This is common practice in biology along with the description of new [[taxon|taxa]], where the voucher specimens, on which the taxonomic description is based, become the [[Type (biology)#Type specimen|type specimens]]. When the identity of a certain taxon (or a genetic sequence in the case of DNA barcoding) is in doubt, the original specimen can be re-examined to review and ideally solve the issue. Voucher specimens should be clearly labelled as such, including a permanent voucher identifier that unambiguously connects the specimen with the DNA barcode data derived from it. Furthermore, these voucher specimens should be deposited in publicly accessible repositories like [[scientific collection]]s or [[herbarium|herbaria]] to preserve them for future reference and to facilitate research involving the deposited specimens.<ref name=Agerer-eal-2000>{{cite journal | vauthors = Agerer R, Ammirati J, Baroni TJ, Blanz P, Courtecuisse RE, Desjardin DE, Gams W, Hallenberg N, Halling R, Hawksworth DL, Horak E | display-authors = 6 |date=2000 |title=Open letter to the scientific community of mycologists |journal=Applied Soil Ecology |volume=15 |issue=3 |pages=295–298 |doi=10.1016/S0929-1393(00)00076-7 |url=https://reader.elsevier.com/reader/sd/pii/S0929139300000767?token=C590FD403AA59D5AE704E466B116B3846667B8491E04A5D23D71F83669B4F491CCBE074974F3B622A015F1680AD71660 }}</ref>
 ==Barcode DNA markers==
 ===Internal Transcribed Spacer (ITS) &ndash; the primary fungal barcode===
 [[File:Eucaryot rdna.png|thumb|400px|[[Tandem repeat]]s of the [[eukaryote|eukaryotic]] rDNA [[Metabolic gene cluster|gene cluster]] containing the genetic sequences for the 18S, 5.8S, and 28S subunits of the [[ribosome]]. ETS – external transcribed spacer, ITS – internal transcribed spacers 1 and 2, numbered from 5' end; NTS – nontranscribed spacer.]]
-In fungi, the [[Internal transcribed spacer]] (''ITS'') is a roughly 600 basepairs (bp) long region in the [[ribosome|ribosomal]] [[tandem repeat]] [[Metabolic gene cluster|gene cluster]] of the [[nuclear DNA|nuclear genome]]. The region is flanked by the DNA sequences for the ribosomal [[SSU rRNA|small subunit (SSU)]] or [[18S ribosomal RNA|18S]] subunit at the 5‘ end, and by the [[LSU rRNA|large subunit (LSU)]] or [[28S ribosomal RNA|28S]] subunit at the 3‘ end.<ref name=Xu-2016>{{cite journal |last=Xu |first=Jianping |date=2016 |title=Fungal DNA barcoding |journal=Genome |volume=59 |pages=913–932 |doi=10.1139/gen-2016-0046 |url=https://www.nrcresearchpress.com/doi/pdfplus/10.1139/gen-2016-0046 }}</ref><ref name=Wurzbacher-eal-2018>{{cite journal |last1=Wurzbacher |first1=Christian |last2=Larsson |first2=Ellen |last3=Bengtsson Palme |first3= Johan |last4=Van den Wyngaert |first4=Silke |last5=Svantesson |first5=Sten |last6=Kristiansson |first6=Erik |last7=Kagami |first7=Maiko |last8=Nilsson |first8=R. Henrik |date=2018 |title=Introducing ribosomal tandem repeat barcoding for fungi |journal=Molecular Ecology Resources |volume=19 |pages=118–127 |doi=10.1111/1755-0998.12944 |url=https://onlinelibrary.wiley.com/doi/pdf/10.1111/1755-0998.12944 }}</ref> The Internal Transcribed Spacer itself consists of two parts, ''ITS1'' and ''ITS2'', which are separated from each other by the [[5.8S ribosomal RNA|5.8S]] subunit nested between them. Like the flanking 18S and 28S subunits, the 5.8S subunit contains a highly conserved DNA sequence, as they code for structural parts of the [[ribosome]], which is a key component in intracellular [[translation (biology)|protein synthesis]].
+In fungi, the [[Internal transcribed spacer]] (''ITS'') is a roughly 600 basepairs (bp) long region in the [[ribosome|ribosomal]] [[tandem repeat]] [[Metabolic gene cluster|gene cluster]] of the [[nuclear DNA|nuclear genome]]. The region is flanked by the DNA sequences for the ribosomal [[SSU rRNA|small subunit (SSU)]] or [[18S ribosomal RNA|18S]] subunit at the 5‘ end, and by the [[LSU rRNA|large subunit (LSU)]] or [[28S ribosomal RNA|28S]] subunit at the 3‘ end.<ref name=Xu-2016>{{cite journal | vauthors = Xu J | title = Fungal DNA barcoding | journal = Genome | volume = 59 | issue = 11 | pages = 913–932 | date = November 2016 | pmid = 27829306 | doi = 10.1139/gen-2016-0046 | url = https://www.nrcresearchpress.com/doi/pdfplus/10.1139/gen-2016-0046 }}</ref><ref name=Wurzbacher-eal-2018>{{cite journal | vauthors = Wurzbacher C, Larsson E, Bengtsson-Palme J, Van den Wyngaert S, Svantesson S, Kristiansson E, Kagami M, Nilsson RH | display-authors = 6 | title = Introducing ribosomal tandem repeat barcoding for fungi | journal = Molecular Ecology Resources | volume = 19 | issue = 1 | pages = 118–127 | date = January 2019 | pmid = 30240145 | doi = 10.1111/1755-0998.12944 | url = https://onlinelibrary.wiley.com/doi/pdf/10.1111/1755-0998.12944 }}</ref> The Internal Transcribed Spacer itself consists of two parts, ''ITS1'' and ''ITS2'', which are separated from each other by the [[5.8S ribosomal RNA|5.8S]] subunit nested between them. Like the flanking 18S and 28S subunits, the 5.8S subunit contains a highly conserved DNA sequence, as they code for structural parts of the [[ribosome]], which is a key component in intracellular [[translation (biology)|protein synthesis]].
 Due to several advantages of ''ITS'' (see below) and a comprehensive amount of sequence data accumulated in the 1990s and early 2000s, Begerow et al. (2010) and Schoch et al. (2012) proposed the ''ITS'' region as primary [[DNA barcode]] region for the genetic identification of [[fungus|fungi]].<ref name=Begerow-eal-2010/><ref name=Schoch-eal-2012/>
 ====Primers====
-The conserved flanking regions of 18S and 28S serve as anchor points for the [[primer (molecular biology)|primers]] used for [[polymerase chain reaction|PCR]] amplification of the ''ITS'' region.<ref name=White-eal-1990>{{cite book|last1=White |first1=T. J. |last2=Bruns |first2=T. |last3=Lee |first3=S. |last4=Taylor |first4=J. W. |date=1990 |chapter=Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics |pages=315–322 |editor-last1=Innis |editor-first1=M. A. |editor-last2=Gelfand |editor-first2=D. H. |editor-last3=Sninsky |editor-first3=J. J. |editor-last4=White |editor-first4=T. J. |title=PCR Protocols: A Guide to Methods and Applications |publisher=Academic Press, Inc. |place=New York |url=https://www.researchgate.net/publication/262687766_Amplification_and_Direct_Sequencing_of_Fungal_Ribosomal_RNA_Genes_for_Phylogenetics }}</ref> Moreover, the conserved nested 5.8S region allows for the construction of "internal" primers, i.e., primers attaching to complementary sequences within the ITS region. White et al. (1990) proposed such internal primers, named ITS2 and ITS3, along with the flanking primers ITS1 and ITS4 in the 18S and the 28S subunit, respectively.<ref name=White-eal-1990/> Due to their almost universal applicability to ITS sequening in fungi, these primers are still in wide use today. Optimised primers specifically for ITS sequencing in [[Dikarya]] (comprising [[Basidiomycota]] and [[Ascomycota]]) have been proposed by Toju et al. (2012).<ref name=Toju-eal-2012>{{cite journal |last1=Toju |first1=Hirokazu |last2=Tanabe |first2=Akifumi S. |last3=Yamamoto |first3=Satoshi |last4=Sato |first4=Hirotoshi |date=2012 |title=High-coverage ITS primers for the DNA-based identification of Ascomycetes and Basidiomycetes in environmental samples |journal=Plos ONE |volume=7 |issue=7 |pages=e40863 |doi=10.1371/journal.pone.0040863 |url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3395698/pdf/pone.0040863.pdf }}</ref>
+The conserved flanking regions of 18S and 28S serve as anchor points for the [[primer (molecular biology)|primers]] used for [[polymerase chain reaction|PCR]] amplification of the ''ITS'' region.<ref name=White-eal-1990>{{cite book| vauthors = White TJ, Bruns T, Lee SJ, Taylor J |date=1990 |chapter=Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics |pages=315–322 | veditors = Innis MA, Gelfand DH, Sninsky JJ, White TJ |title=PCR Protocols: A Guide to Methods and Applications |publisher=Academic Press, Inc. |place=New York |url=https://www.researchgate.net/publication/262687766_Amplification_and_Direct_Sequencing_of_Fungal_Ribosomal_RNA_Genes_for_Phylogenetics }}</ref> Moreover, the conserved nested 5.8S region allows for the construction of "internal" primers, i.e., primers attaching to complementary sequences within the ITS region. White et al. (1990) proposed such internal primers, named ITS2 and ITS3, along with the flanking primers ITS1 and ITS4 in the 18S and the 28S subunit, respectively.<ref name=White-eal-1990/> Due to their almost universal applicability to ITS sequening in fungi, these primers are still in wide use today. Optimised primers specifically for ITS sequencing in [[Dikarya]] (comprising [[Basidiomycota]] and [[Ascomycota]]) have been proposed by Toju et al. (2012).<ref name=Toju-eal-2012>{{cite journal | vauthors = Toju H, Tanabe AS, Yamamoto S, Sato H | title = High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples | journal = PloS One | volume = 7 | issue = 7 | pages = e40863 | date = 2012 | pmid = 22808280 | pmc = 3395698 | doi = 10.1371/journal.pone.0040863 }}</ref>
 For the majority of fungi, the ITS primers proposed by White et al. (1990) have become the standard primers used for PCR amplification. These primers are:<ref name=White-eal-1990/>
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 ====Advantages and shortcomings====
-A major advantage of using the ITS region as molecular marker and fungal [[DNA barcode]] is that the entire ribosomal gene cluster is arranged in tandem repeats, i.e., in multiple copies.<ref name=Wurzbacher-eal-2018/> This allows for its PCR amplification and [[Sanger sequencing]] even from small material samples (given the DNA is not fragmented due to age or other [[DNA damage (naturally occurring)|degenerative influences]]).<ref name=Xu-2016/> Hence, a high PCR success rate is usually observed when amplifying ''ITS''. However, this success rate varies greatly among fungal groups, from 65% in non-Dikarya (including the now [[paraphyly|paraphyletic]] [[Mucoromycotina]], the [[Chytridiomycota]] and the [[Blastocladiomycota]]) to 100% in [[Saccharomycotina]] and [[Basidiomycota]]<ref name=Schoch-eal-2012/> (with the exception of very low success in [[Pucciniomycotina]]).<ref name=Stielow-eal-2015>{{cite journal |last1=Stielow |first1=J. B. |last2=Lévesque |first2=C. A. |last3=Seifert |first3=K. A. |last4=Meyer |first4=W. |last5=Irinyi |first5=L. |last6=Smits |first6=D. |last7=Renfurm |first7=R. |last8=Verkley |first8=G. J. M. |last9=Groenewald |first9=M. |last10=Chaduli |first10=D. |last11=Lomascolo |first11=A. |last12=Welti |first12=S. |last13=Lesage-Meessen |first13=L. |last14=Favel |first14=A. |last15=Al-Hatmi |first15=A. M. S. |last16=Damm |first16=U. |last17=Yilmaz |first17=N. |last18=Houbraken |first18=J. |last19=Lombard |first19=L. |last20=Quaedvlieg |first20=W. |last21=Binder |first21=M. |last22=Vaas |first22=L. A. I. |last23=Vu |first23=D. |last24=Yurkov |first24=A. |last25=Begerow |first25=D. |last26=Roehl |first26=O. |last27=Guerreiro |first27=M. |last28=Fonseca |first28=A. |last29=Samerpitak |first29=K. |last30=van Diepeningen |first30=A. D. |last31=Dolatabadi |first31=S. |last32=Moreno |first32=L. F. |last33=Casaregola |first33=S. |last34=Mallet |first34=S. |last35=Jacques |first35=N. |last36=Roscini |first36=L. |last37=Egidi |first37=E. |last38=Bizet |first38=C. |last39=Garcia-Hermoso |first39=D. |last40=Martín |first40=M. P. |last41=Deng |first41=S. |last42=Groenewald |first42=J. Z. |last43=Boekhout |first43=T. |last44=de Beer |first44=Z. W. |last45=Barnes |first45=I. |last46=Duong |first46=T. A. |last47=Wingfield |first47=M. J. |last48=de Hoog |first48=G. S. |last49=Crous |first49=P. W. |last50=Lewis |first50=C. T. |last51=Hambleton |first51=S. |last52=Moussa |first52=T. A. A. |last53=Al-Zahrani |first53=H. S. |last54=Almaghrabi |first54=O. A. |last55=Louis-Seize |first55=G. |last56=Assabgui |first56=R. |last57=McCormick |first57=W. |last58=Omer |first58=G. |last59=Dukik |first59=K. |last60=Cardinali |first60=G. |last61=Eberhardt |first61=U. |last62=de Vries |first62=M. |last63=Robert |first63=V. |date=2015 |title=One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes |journal=Persoonia |volume=35 |pages=242–263 |doi=10.3767/003158515X689135 |url=https://www.ingentaconnect.com/content/nhn/pimj/2015/00000035/00000001/art00011?crawler=true }}</ref> Furthermore, the choice of primers for ''ITS'' amplification can introduce biases towards certain [[taxonomy|taxonomic]] fungus groups.<ref name=Bellemain-eal-2010>{{cite journal |last1=Bellemain |first1=Eva |last2=Carlsen |first2=Tor |last3=Brochmann |first3=Christian |last4=Coissac |first4=Eric |last5=Taberlet |first5=Pierre |last6=Kauserud |first6=Håvard |date=2010 |title=ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases |journal=BMC Microbiology |volume=10 |issue=189 |pages=1–9 |doi=10.1186/1471-2180-10-189 |url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909996/pdf/1471-2180-10-189.pdf }}</ref> For example, the "universal" ''ITS'' primers<ref name=White-eal-1990/> fail to amplify about 10% of the tested fungal specimens.<ref name=Stielow-eal-2015/>
+A major advantage of using the ITS region as molecular marker and fungal [[DNA barcode]] is that the entire ribosomal gene cluster is arranged in tandem repeats, i.e., in multiple copies.<ref name=Wurzbacher-eal-2018/> This allows for its PCR amplification and [[Sanger sequencing]] even from small material samples (given the DNA is not fragmented due to age or other [[DNA damage (naturally occurring)|degenerative influences]]).<ref name=Xu-2016/> Hence, a high PCR success rate is usually observed when amplifying ''ITS''. However, this success rate varies greatly among fungal groups, from 65% in non-Dikarya (including the now [[paraphyly|paraphyletic]] [[Mucoromycotina]], the [[Chytridiomycota]] and the [[Blastocladiomycota]]) to 100% in [[Saccharomycotina]] and [[Basidiomycota]]<ref name=Schoch-eal-2012/> (with the exception of very low success in [[Pucciniomycotina]]).<ref name=Stielow-eal-2015>{{cite journal | vauthors = Stielow JB, Lévesque CA, Seifert KA, Meyer W, Iriny L, Smits D, Renfurm R, Verkley GJ, Groenewald M, Chaduli D, Lomascolo A, Welti S, Lesage-Meessen L, Favel A, Al-Hatmi AM, Damm U, Yilmaz N, Houbraken J, Lombard L, Quaedvlieg W, Binder M, Vaas LA, Vu D, Yurkov A, Begerow D, Roehl O, Guerreiro M, Fonseca A, Samerpitak K, van Diepeningen AD, Dolatabadi S, Moreno LF, Casaregola S, Mallet S, Jacques N, Roscini L, Egidi E, Bizet C, Garcia-Hermoso D, Martín MP, Deng S, Groenewald JZ, Boekhout T, de Beer ZW, Barnes I, Duong TA, Wingfield MJ, de Hoog GS, Crous PW, Lewis CT, Hambleton S, Moussa TA, Al-Zahrani HS, Almaghrabi OA, Louis-Seize G, Assabgui R, McCormick W, Omer G, Dukik K, Cardinali G, Eberhardt U, de Vries M, Robert V | display-authors = 6 | title = One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes | journal = Persoonia | volume = 35 | pages = 242–63 | date = December 2015 | pmid = 26823635 | doi = 10.3767/003158515X689135 | url = https://www.ingentaconnect.com/content/nhn/pimj/2015/00000035/00000001/art00011?crawler=true }}</ref> Furthermore, the choice of primers for ''ITS'' amplification can introduce biases towards certain [[taxonomy|taxonomic]] fungus groups.<ref name=Bellemain-eal-2010>{{cite journal | vauthors = Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P, Kauserud H | title = ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases | journal = BMC Microbiology | volume = 10 | issue = 189 | pages = 189 | date = July 2010 | pmid = 20618939 | pmc = 2909996 | doi = 10.1186/1471-2180-10-189 }}</ref> For example, the "universal" ''ITS'' primers<ref name=White-eal-1990/> fail to amplify about 10% of the tested fungal specimens.<ref name=Stielow-eal-2015/>
-The tandem repeats of the ribosomal gene cluster cause the problem of significant intragenomic sequence heterogeneity observed among ''ITS'' copies of several fungal groups.<ref name=Smith-eal-2007>{{cite journal |last1=Smith |first1=Matthew E. |last2=Douhan |first2=Greg W. |last3=Rizzo |first3=David M. |date=2007 |title=Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a ''Quercus'' woodland |journal=Mycorrhiza |volume=18 |pages=15–22 |doi=10.1007/s00572-007-0148-z |url=https://www.researchgate.net/profile/Greg_Douhan/publication/6130396_Intra-specific_and_intra-sporocarp_ITS_variation_of_ectomycorrhizal_fungi_as_assessed_by_rDNA_sequencing_of_sporocarps_and_pooled_ectomycorrhizal_roots_from_a_Quercus_woodland/links/0046351c8936e23c4e000000/Intra-specific-and-intra-sporocarp-ITS-variation-of-ectomycorrhizal-fungi-as-assessed-by-rDNA-sequencing-of-sporocarps-and-pooled-ectomycorrhizal-roots-from-a-Quercus-woodland.pdf }}</ref><ref name=Lindner-Banik-2011>{{cite journal |last1=Lindner |first1=Daniel L. |last2=Banik |first2=Mark T. |date=2011 |title=Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus ''Laetiporus'' |journal=Mycologia |volume=103 |issue=4 |pages=731–740 |doi=10.3852/10-331 |url=https://www.tandfonline.com/doi/abs/10.3852/10-331?journalCode=umyc20 }}</ref><ref name=Kovacs-eal-2011>{{cite journal |last1=Kovács |first1=Gábor M. |last2=Balázs |first2=Tímea K. |last3=Calonge |first3=Francisco D. |last4=Martín |first4=María P. |date=2011 |title=The diversity of ''Terfezia'' desert truffles: new species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity |journal=Mycologia |volume=203 |issue=4 |pages=841–853 |doi=10.3852/10-312 |url=https://digital.csic.es/bitstream/10261/78739/1/diversity_MYCOLOGIA_2011.pdf }}</ref> In Sanger sequencing, this will cause ''ITS'' sequence reads of different lengths to superpose each other, potentially rendering the resulting chromatograph unreadable. Furthermore, because of the non-coding nature of the ''ITS'' region that can lead to a substantial amount of [[indel]]s, it is impossible to consistently align ''ITS'' sequences from highly [[genetic divergence|divergent]] species for further bigger-scale phylogenetic analyses.<ref name=Seifert-2009/><ref name=Xu-2016/> The degree of intragenomic sequence heterogeneity can be investigated in more detail through [[molecular cloning]] of the initially PCR-amplified ITS sequences, followed by sequencing of the clones. This procedure of initial PCR amplification, followed by cloning of the [[amplicon]]s and finally sequencing of the cloned PCR products is the most common approach of obtaining ''ITS'' sequences for [[DNA barcoding#DNA metabarcoding|DNA metabarcoding]] of environmental samples, in which a multitude of different fungal species can be present simultaneously. However, this approach of sequencing after cloning was rarely done for the ''ITS'' sequences that make up the reference libraries used for DNA barcode-aided identification, thus potentially giving an underestimate of the existing ''ITS'' sequence variation in many samples.<ref name=Kiss-2012>{{cite journal |last=Kiss |first=Levente |date=2012 |title=Limits of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences as species barcodes for Fungi |journal=Proceedings of the National Academy of Sciences |volume=109 |issue=27 |pages=E1811 |doi=10.1073/pnas.1207143109 |url=https://www.pnas.org/content/pnas/109/27/E1811.full.pdf }}</ref>
+The tandem repeats of the ribosomal gene cluster cause the problem of significant intragenomic sequence heterogeneity observed among ''ITS'' copies of several fungal groups.<ref name=Smith-eal-2007>{{cite journal | vauthors = Smith ME, Douhan GW, Rizzo DM | title = Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland | journal = Mycorrhiza | volume = 18 | issue = 1 | pages = 15–22 | date = December 2007 | pmid = 17710446 | doi = 10.1007/s00572-007-0148-z | url = https://www.researchgate.net/profile/Greg_Douhan/publication/6130396_Intra-specific_and_intra-sporocarp_ITS_variation_of_ectomycorrhizal_fungi_as_assessed_by_rDNA_sequencing_of_sporocarps_and_pooled_ectomycorrhizal_roots_from_a_Quercus_woodland/links/0046351c8936e23c4e000000/Intra-specific-and-intra-sporocarp-ITS-variation-of-ectomycorrhizal-fungi-as-assessed-by-rDNA-sequencing-of-sporocarps-and-pooled-ectomycorrhizal-roots-from-a-Quercus-woodland.pdf }}</ref><ref name=Lindner-Banik-2011>{{cite journal | vauthors = Lindner DL, Banik MT | title = Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus | journal = Mycologia | volume = 103 | issue = 4 | pages = 731–40 | date = 2011 | pmid = 21289107 | doi = 10.3852/10-331 | url = https://www.tandfonline.com/doi/abs/10.3852/10-331?journalCode=umyc20 }}</ref><ref name=Kovacs-eal-2011>{{cite journal | vauthors = Kovács GM, Balázs TK, Calonge FD, Martín MP | title = The diversity of Terfezia desert truffles: new species and a highly variable species complex with intrasporocarpic nrDNA ITS heterogeneity | journal = Mycologia | volume = 103 | issue = 4 | pages = 841–53 | date = 2011 | pmid = 21289106 | doi = 10.3852/10-312 | url = https://digital.csic.es/bitstream/10261/78739/1/diversity_MYCOLOGIA_2011.pdf }}</ref> In Sanger sequencing, this will cause ''ITS'' sequence reads of different lengths to superpose each other, potentially rendering the resulting chromatograph unreadable. Furthermore, because of the non-coding nature of the ''ITS'' region that can lead to a substantial amount of [[indel]]s, it is impossible to consistently align ''ITS'' sequences from highly [[genetic divergence|divergent]] species for further bigger-scale phylogenetic analyses.<ref name=Seifert-2009/><ref name=Xu-2016/> The degree of intragenomic sequence heterogeneity can be investigated in more detail through [[molecular cloning]] of the initially PCR-amplified ITS sequences, followed by sequencing of the clones. This procedure of initial PCR amplification, followed by cloning of the [[amplicon]]s and finally sequencing of the cloned PCR products is the most common approach of obtaining ''ITS'' sequences for [[DNA barcoding#DNA metabarcoding|DNA metabarcoding]] of environmental samples, in which a multitude of different fungal species can be present simultaneously. However, this approach of sequencing after cloning was rarely done for the ''ITS'' sequences that make up the reference libraries used for DNA barcode-aided identification, thus potentially giving an underestimate of the existing ''ITS'' sequence variation in many samples.<ref name=Kiss-2012>{{cite journal | vauthors = Kiss L | title = Limits of nuclear ribosomal DNA internal transcribed spacer (ITS) sequences as species barcodes for Fungi | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 27 | pages = E1811; author reply E1812 | date = July 2012 | pmid = 22715287 | doi = 10.1073/pnas.1207143109 | url = https://www.pnas.org/content/pnas/109/27/E1811.full.pdf }}</ref>
-The [[weighted arithmetic mean]] of the intraspecific (within-species) ''ITS'' variability among fungi is 2.51%. This variability, however, can range from 0% for example in ''[[Serpula lacrymans]]'' (n=93 samples) over 0.19% in ''[[Tuber melanosporum]]'' (n=179) up to 15.72% in ''[[Rhizoctonia solani]]'' (n=608), or even 24.75% in ''[[Pisolithus tinctorius]]'' (n=113). In cases of high intraspecific ''ITS'' variability, the application of a threshold of 3% sequence variability &ndash; a canonical upper value for intraspecific variation &ndash; will therefore lead to a higher estimate of [[operational taxonomic unit]]s (OTUs), i.e., putative species, than there actually are in a sample.<ref name=Nielsson-eal-2008>{{cite journal |last1=Nilsson |first1=R. Henrik |last2=Kristiansson |first2=Erik |last3=Ryberg |first3=Martin |last4=Hallenberg |first4=Nils |last5=Larsson |first5=Karl-Henrik |date=2008 |title=Intraspecific ITS variability in the kingdom Fungi as expressed in the International Sequence Databases and its implications for molecular species identification |journal=Evolutionary Bioinformatics |volume=4 |pages=193–201 |doi=10.4137/EBO.S653 |url=https://journals.sagepub.com/doi/pdf/10.4137/EBO.S653 }}</ref> In the case of medically relevant fungal species, a more strict threshold of 2.5% ''ITS'' variability allows only around 75% of all species to be accurately identified to the species level.<ref name=Irinyi-eal-2015/>
+The [[weighted arithmetic mean]] of the intraspecific (within-species) ''ITS'' variability among fungi is 2.51%. This variability, however, can range from 0% for example in ''[[Serpula lacrymans]]'' (n=93 samples) over 0.19% in ''[[Tuber melanosporum]]'' (n=179) up to 15.72% in ''[[Rhizoctonia solani]]'' (n=608), or even 24.75% in ''[[Pisolithus tinctorius]]'' (n=113). In cases of high intraspecific ''ITS'' variability, the application of a threshold of 3% sequence variability &ndash; a canonical upper value for intraspecific variation &ndash; will therefore lead to a higher estimate of [[operational taxonomic unit]]s (OTUs), i.e., putative species, than there actually are in a sample.<ref name=Nielsson-eal-2008>{{cite journal | vauthors = Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson KH | title = Intraspecific ITS variability in the kingdom fungi as expressed in the international sequence databases and its implications for molecular species identification | journal = Evolutionary Bioinformatics Online | volume = 4 | pages = 193–201 | date = May 2008 | pmid = 19204817 | doi = 10.4137/EBO.S653 | url = https://journals.sagepub.com/doi/pdf/10.4137/EBO.S653 }}</ref> In the case of medically relevant fungal species, a more strict threshold of 2.5% ''ITS'' variability allows only around 75% of all species to be accurately identified to the species level.<ref name=Irinyi-eal-2015/>
-On the other hand, morphologically well-defined, but evolutionarily young [[species complex]]es or [[Species complex#Sibling species|sibling species]] may only differ (if at all) in a few nucleotides of the ''ITS'' sequences. Solely relying on ''ITS'' barcode data for the identification of such species pairs or complexes may thus obscure the actual diversity and might lead to misidentification if not accompanied by the investigation of morphological and ecological features and/or comparison of additional diagnostic [[genetic marker]]s.<ref name=Stielow-eal-2015/><ref name=Kiss-2012/><ref name=Xu-eal-2000>{{cite journal|last1=Xu|first1=Jianping|last2=Vilgalys|first2=Rytas|last3=Mitchell|first3=Thomas G.|date=2000|title=Multiple gene genealogies reveal recent dispersion and hybridization in the human pathogenic fungus ''Cryptococcus neoformans''|journal=Molecular Ecology|volume=9|pages=1471–1481|url=https://pdfs.semanticscholar.org/acc3/5d99b68a155ea562aa6ae09c374affe3276e.pdf}}</ref><ref name=Stockinger-eal-2010>{{cite journal|last1=Stockinger|first1=Herbert|last2=Krüger|first2=Manuela|last3=Schüßler|first3=Arthur|date=2010|title=DNA barcoding of arbuscular myrorrhizal fungi|journal=New Phytologist|volume=187|pages=461–474|doi=10.1111/j.1469-8137.2010.03262.x|url=https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1469-8137.2010.03262.x}}</ref> For some taxa, ''ITS'' (or its ''ITS2'' part) is not variable enough as fungal DNA barcode, as for example has been shown in ''[[Aspergillus]]'', ''[[Cladosporium]]'', ''[[Fusarium]]'' and ''[[Penicillium]]''.<ref name=Geisler-eal-2007>{{cite journal |last1=Geiser |first1=D. M. |last2=Klich |first2=M. A. |last3=Frisvad |first3=J. C. |last4=Peterson |first4=S. W. |last5=Varga |first5=J. |last6=Samson |first6=R. A. |date=2007 |title=The current status of species recognition and identification in ''Aspergillus'' |journal=Studies in Mycology |volume=59 |pages=1–10 |doi=10.3114/sim.2007.59.01 |url=https://reader.elsevier.com/reader/sd/pii/S016606161460169X?token=D3E5FE8E14E3AEFAE99DD1B917424EEE26F84D76CF7D3A5BF74FF0FABE3C45CE5EA88C3D35E85FFF4DB608EC6535ADEF }}</ref><ref name=Schubert-eal-2007>{{cite journal |last1=Schubert |first1=K. |last2=Groenewald |first2=Z. |last3=Braun |first3=U. |last4=Dijksterhuis |first4=J. |last5=Starink |first5=M. |last6=Hill |first6=C.F. |last7=Zalar |first7=P. |last8=de Hoog |first8=G.S. |last9=Crous |first9=P.W. |date=2007 |title=Biodiversity in the ''Cladosporium herbarum'' complex (Davidiellaceae, Capnodiales), with standardisation of methods for ''Cladosporium'' taxonomy and diagnostics |journal=Studies in Mycology |volume=58 |pages=105–156 |doi=10.3114/sim.2007.58.05 |url=https://reader.elsevier.com/reader/sd/pii/S0166061614601214?token=8CB23689722047DE79ACD4831B32D95F29A3B13FE5E26D6532D61E98A9284A93B27C59E322334772EC85FB7390C600CF }}</ref><ref name=ODonnell-Cigelnik-1997>{{cite journal |last1=O’Donnell |first1=Kerry |last2=Cigelnik |first2=Elizabeth |date=1997 |title=Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus ''Fusarium'' are nonorthologous |journal=Molecular Phylogenetics and Evolution |volume=7 |pages=103–116 |doi=10.1006/mpev.1996.0376 |url=https://reader.elsevier.com/reader/sd/pii/S1055790396903760?token=AF906D22D14589B0F80FE0C58C1AC6DC7F3D13B8C3FA40B281D5DAC2AF17273A6D69817A3162AF0E0132E5DD4D84B545 }}</ref><ref name=Skouboe-eal-1999>{{cite journal |last1=Skouboe |first1=Pernille |last2=Frisvad |first2=Jens C. |last3=Taylor |first3=John W. |last4=Lauritsen |first4=Dorte |last5=Boysen |first5=Marianne |last6=Rossen |first6=Lone |date=1999 |title=Phylogenetic analysis of nucleotide sequences from the ITS region of terverticillate ''Penicillium'' species |journal=Mycological Research |volume=103 |issue=7 |pages=873–881 |doi=10.1017/S0953756298007904 |url=https://www.cambridge.org/core/journals/mycological-research/article/phylogenetic-analysis-of-nucleotide-sequences-from-the-its-region-of-terverticillate-penicillium-species/66C0A327F03E6CD934767AB13F7A82DF }}</ref> Efforts to define a univerally applicable threshold value of ''ITS'' variability that demarcates intraspecific from interspecific (between-species) variability thus remain futile.<ref name=Nielsson-eal-2008/>
+On the other hand, morphologically well-defined, but evolutionarily young [[species complex]]es or [[Species complex#Sibling species|sibling species]] may only differ (if at all) in a few nucleotides of the ''ITS'' sequences. Solely relying on ''ITS'' barcode data for the identification of such species pairs or complexes may thus obscure the actual diversity and might lead to misidentification if not accompanied by the investigation of morphological and ecological features and/or comparison of additional diagnostic [[genetic marker]]s.<ref name=Stielow-eal-2015/><ref name=Kiss-2012/><ref name=Xu-eal-2000>{{cite journal | vauthors = Xu J, Vilgalys R, Mitchell TG | title = Multiple gene genealogies reveal recent dispersion and hybridization in the human pathogenic fungus Cryptococcus neoformans | journal = Molecular Ecology | volume = 9 | issue = 10 | pages = 1471–81 | date = October 2000 | pmid = 11050543 | doi = 10.1046/j.1365-294x.2000.01021.x | url = https://pdfs.semanticscholar.org/acc3/5d99b68a155ea562aa6ae09c374affe3276e.pdf }}</ref><ref name=Stockinger-eal-2010>{{cite journal | vauthors = Stockinger H, Krüger M, Schüssler A | title = DNA barcoding of arbuscular mycorrhizal fungi | journal = The New Phytologist | volume = 187 | issue = 2 | pages = 461–74 | date = July 2010 | pmid = 20456046 | doi = 10.1111/j.1469-8137.2010.03262.x }}</ref> For some taxa, ''ITS'' (or its ''ITS2'' part) is not variable enough as fungal DNA barcode, as for example has been shown in ''[[Aspergillus]]'', ''[[Cladosporium]]'', ''[[Fusarium]]'' and ''[[Penicillium]]''.<ref name=Geisler-eal-2007>{{cite journal | vauthors = Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA | title = The current status of species recognition and identification in Aspergillus | journal = Studies in Mycology | volume = 59 | pages = 1–10 | date = 2007 | pmid = 18490947 | doi = 10.3114/sim.2007.59.01 | url = https://reader.elsevier.com/reader/sd/pii/S016606161460169X?token=D3E5FE8E14E3AEFAE99DD1B917424EEE26F84D76CF7D3A5BF74FF0FABE3C45CE5EA88C3D35E85FFF4DB608EC6535ADEF }}</ref><ref name=Schubert-eal-2007>{{cite journal | vauthors = Schubert K, Groenewald JZ, Braun U, Dijksterhuis J, Starink M, Hill CF, Zalar P, de Hoog GS, Crous PW | display-authors = 6 | title = Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales), with standardisation of methods for Cladosporium taxonomy and diagnostics | journal = Studies in Mycology | volume = 58 | pages = 105–56 | date = 2007 | pmid = 18490998 | doi = 10.3114/sim.2007.58.05 | url = https://reader.elsevier.com/reader/sd/pii/S0166061614601214?token=8CB23689722047DE79ACD4831B32D95F29A3B13FE5E26D6532D61E98A9284A93B27C59E322334772EC85FB7390C600CF }}</ref><ref name=ODonnell-Cigelnik-1997>{{cite journal | vauthors = O'Donnell K, Cigelnik E | title = Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous | journal = Molecular Phylogenetics and Evolution | volume = 7 | issue = 1 | pages = 103–16 | date = February 1997 | pmid = 9007025 | doi = 10.1006/mpev.1996.0376 | url = https://reader.elsevier.com/reader/sd/pii/S1055790396903760?token=AF906D22D14589B0F80FE0C58C1AC6DC7F3D13B8C3FA40B281D5DAC2AF17273A6D69817A3162AF0E0132E5DD4D84B545 }}</ref><ref name=Skouboe-eal-1999>{{cite journal | vauthors = Skouboe P, Frisvad JC, Taylor JW, Lauritsen D, Boysen M, Rossen L |date=1999 |title=Phylogenetic analysis of nucleotide sequences from the ITS region of terverticillate ''Penicillium'' species |journal=Mycological Research |volume=103 |issue=7 |pages=873–881 |doi=10.1017/S0953756298007904 |url=https://www.cambridge.org/core/journals/mycological-research/article/phylogenetic-analysis-of-nucleotide-sequences-from-the-its-region-of-terverticillate-penicillium-species/66C0A327F03E6CD934767AB13F7A82DF }}</ref> Efforts to define a univerally applicable threshold value of ''ITS'' variability that demarcates intraspecific from interspecific (between-species) variability thus remain futile.<ref name=Nielsson-eal-2008/>
-Nonetheless, the probability of correct species identification with the ''ITS'' region is high in the [[Dikarya]], and especially so in [[Basidiomycota]], where even the ''ITS1'' part is often sufficient to identify the species.<ref name=Osmundsen-eal-2013>{{cite journal |last1=Osmundson |first1=Todd W. |last2=Robert |first2=Vincent A. |last3=Schoch |first3=Conrad L. |last4=Baker |first4=Lydia J. |last5=Smith |first5=Amy |last6=Robich |first6=Giovanni |last7=Mizzan |first7=Luca |last8=Garbeletto |first8= Matteo M. |date=2013 |title=Filling gaps in biodiversity knowledge of Macrofungi: Contributions and assessment of an herbarium collection DNA Barcode sequencing project |journal=Plos ONE |volume=8 |issue=4 |pages=e62419 |doi=10.1371/journal.pone.0062419 |url=https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0062419&type=printable }}</ref> However, its discrimination power is partly superseded by that of the [[POLR2A|DNA-directed RNA polymerase II subunit ''RPB1'']] (see also below).<ref name=Schoch-eal-2012/>
+Nonetheless, the probability of correct species identification with the ''ITS'' region is high in the [[Dikarya]], and especially so in [[Basidiomycota]], where even the ''ITS1'' part is often sufficient to identify the species.<ref name=Osmundsen-eal-2013>{{cite journal | vauthors = Osmundson TW, Robert VA, Schoch CL, Baker LJ, Smith A, Robich G, Mizzan L, Garbelotto MM | display-authors = 6 | title = Filling gaps in biodiversity knowledge for macrofungi: contributions and assessment of an herbarium collection DNA barcode sequencing project | journal = PloS One | volume = 8 | issue = 4 | pages = e62419 | date = 2013 | pmid = 23638077 | doi = 10.1371/journal.pone.0062419 | url = https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0062419&type=printable }}</ref> However, its discrimination power is partly superseded by that of the [[POLR2A|DNA-directed RNA polymerase II subunit ''RPB1'']] (see also below).<ref name=Schoch-eal-2012/>
-Due to the shortcomings of ''ITS''' as primary fungal DNA barcode, the necessity of establishing a second DNA barcode marker was expressed.<ref name=Seifert-2009/> Several attempts were made to establish other genetic markers that could serve as additional DNA barcodes,<ref name=Stielow-eal-2015/><ref name=Lewis-eal-2011>{{cite journal |last1=Lewis |first1=Christopher T. |last2=Bilkhu |first2=Satpal |last3=Robert |first3=Vincent |last4=Eberhardt |first4=Ursula |last5=Szoke |first5=Szaniszlo |last6=Seifert |first6=Keith A. |last7=Lévesque |first7=C. André |date=2011 |title=Identification of fungal DNA barcode targets and PCR primers based on Pfam protein families and taxonomic hierarchy |journal=The Open Applied Informatics Journal |volume=5 |issue=suppl. 1-M5 |pages=30–44 |doi=10.2174/1874136301005010030 |url=https://benthamopen.com/contents/pdf/TOAINFOJ/TOAINFOJ-5-30.pdf }}</ref><ref name=Robert-eal-2011>{{cite journal |last1=Robert |first1=Vincent |last2=Szöke |first2=Szaniszlo |last3=Eberhardt |first3=Ursula |last4=Cardinali |first4=Gianluigi |last5=Meyer |first5=Wieland |last6=Seifert |first6=Keith A. |last7=Lévesque |first7=C. Andre |last8=Lewis |first8=Chris T. |date=2011 |title=The quest for a general and reliable fungal DNA barcode |journal=The Open Applied Informatics Journal |volume=5 |issue=suppl. 1-M6 |pages=45–61 |doi=10.2174/1874136301005010045 |url=https://benthamopen.com/contents/pdf/TOAINFOJ/TOAINFOJ-5-45.pdf }}</ref> similar to the situation in [[plant]]s, where the [[chloroplast DNA|plastidial genes]] [[RuBisCO#structure|''rbcL'']], [[Maturase K|''matK'']] and ''trnH‐psbA'', as well as the nuclear ''ITS'' are often used in combination for DNA barcoding.<ref name=Kress-2017>{{cite journal |last=Kress |first=W. John |date=2017 |title=Plant DNA barcodes: Applications today and in the future |journal=Journal of Systematics and Evolution |volume=55 |issue=4 |pages=291–307 |doi=10.1111/jse.12254 |url=https://onlinelibrary.wiley.com/doi/epdf/10.1111/jse.12254 }}</ref>
+Due to the shortcomings of ''ITS''' as primary fungal DNA barcode, the necessity of establishing a second DNA barcode marker was expressed.<ref name=Seifert-2009/> Several attempts were made to establish other genetic markers that could serve as additional DNA barcodes,<ref name=Stielow-eal-2015/><ref name=Lewis-eal-2011>{{cite journal | vauthors = Lewis CT, Bilkhu S, Robert V, Eberhardt U, Szoke S, Seifert KA, Lévesque CA |date=2011 |title=Identification of fungal DNA barcode targets and PCR primers based on Pfam protein families and taxonomic hierarchy |journal=The Open Applied Informatics Journal |volume=5 |issue=suppl. 1-M5 |pages=30–44 |doi=10.2174/1874136301005010030 |url=https://benthamopen.com/contents/pdf/TOAINFOJ/TOAINFOJ-5-30.pdf }}</ref><ref name=Robert-eal-2011>{{cite journal | vauthors = Vincent Robert L, Szöke S, Eberhardt U, Cardinali G, Meyer W, Seifert KA, Lévesque CA, Lewis CT |date=2011 |title=The quest for a general and reliable fungal DNA barcode |journal=The Open Applied Informatics Journal |volume=5 |issue=suppl. 1-M6 |pages=45–61 |doi=10.2174/1874136301005010045 |url=https://benthamopen.com/contents/pdf/TOAINFOJ/TOAINFOJ-5-45.pdf }}</ref> similar to the situation in [[plant]]s, where the [[chloroplast DNA|plastidial genes]] [[RuBisCO#structure|''rbcL'']], [[Maturase K|''matK'']] and ''trnH‐psbA'', as well as the nuclear ''ITS'' are often used in combination for DNA barcoding.<ref name=Kress-2017>{{cite journal |last=Kress |first=W. John | name-list-format = vanc |date=2017 |title=Plant DNA barcodes: Applications today and in the future |journal=Journal of Systematics and Evolution |volume=55 |issue=4 |pages=291–307 |doi=10.1111/jse.12254 |url=https://onlinelibrary.wiley.com/doi/epdf/10.1111/jse.12254 }}</ref>
 ===Translational elongation factor 1α (TEF1α) &ndash; the secondary fungal barcode===
-The translational elongation factor 1α is part of the [[EEF-1|eucaryotic elongation factor 1]] complex, whose main function is to facilitate the elongation of the [[amino acid]] chain of a [[polypeptide]] during the [[translation (biology)|translation]] process of [[gene expression]].<ref name=Sasikumar-eal-2012>{{cite journal |last1=Sasikumar |first1=Arjun N. |last2=Perez |first2=Winder B. |last3=Kinzy |first3=Terri Goss |date=2012 |title=The many roles of the eukaryotic Elongation Factor 1 Complex |journal=Wiley Interdiscip Rev RNA |volume=3 |issue=4 |pages=543–555 |doi=10.1002/wrna.1118 |url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3374885/pdf/nihms-369746.pdf }}</ref>
+The translational elongation factor 1α is part of the [[EEF-1|eucaryotic elongation factor 1]] complex, whose main function is to facilitate the elongation of the [[amino acid]] chain of a [[polypeptide]] during the [[translation (biology)|translation]] process of [[gene expression]].<ref name=Sasikumar-eal-2012>{{cite journal | vauthors = Sasikumar AN, Perez WB, Kinzy TG | title = The many roles of the eukaryotic elongation factor 1 complex | journal = Wiley Interdisciplinary Reviews. RNA | volume = 3 | issue = 4 | pages = 543–55 | date = 2012 | pmid = 22555874 | pmc = 3374885 | doi = 10.1002/wrna.1118 }}</ref>
-Stielow et al. (2015) investigated the ''TEF1α'' gene, among a number of others, as potential [[genetic marker]] for fungal DNA barcoding. The ''TEF1α'' gene coding for the translational elongation factor 1α is generally considered to have a slow [[mutation rate]], and it is therefore generally better suited for investigating older splits deeper in the phylogenetic history of an organism group. Despite this, the authors conclude that ''TEF1α'' is the most promising candidate for an additional DNA barcode marker in fungi as it also features sequence regions of higher mutation rates.<ref name=Stielow-eal-2015/> Following this, a quality-controlled reference database was established and merged with the previously existing ISHAM-ITS database for fungal ITS DNA barcodes<ref name=Irinyi-eal-2015/> to form the ISHAM database.<ref name=Meyer-eal-2018>{{cite journal |last1=Meyer |first1=Wieland |last2=Irinyi |first2=Laszlo |last3=Hoang |first3=Minh Thuy Vi |last4=Robert |first4=Vincent |last5=Garcia-Hermoso |first5=Dea |last6=Desnos-Ollivier |first6=Marie |last7=Yurayart |first7=Chompoonek |last8=Tsang |first8=Chi-Ching |last9=Lee |first9=Chun-Yi |last10=Woo |first10=Patrick C.Y. |last11=Pchelin |first11=Ivan Mikhailovich |last12=Uhrlaß |first12=Silke |last13=Nenoff |first13=Pietro |last14=Chindamporn |first14=Ariya |last15=Chen |first15=Sharon |last16=Hebert |first16=Paul D. N. |author16-link=Paul D. N. Hebert |last17=Sorrell |first17=Tania C. |author18=ISHAM barcoding of pathogenic fungi working group |date=2018 |title=Database establishment for the secondary fungal DNA barcode ''translational elongation factor 1α'' (''TEF1α'') |journal=Genome |volume=62 |pages=160–169 |doi=10.1139/gen-2018-0083 |url=https://www.nrcresearchpress.com/doi/pdf/10.1139/gen-2018-0083 }}</ref>
+Stielow et al. (2015) investigated the ''TEF1α'' gene, among a number of others, as potential [[genetic marker]] for fungal DNA barcoding. The ''TEF1α'' gene coding for the translational elongation factor 1α is generally considered to have a slow [[mutation rate]], and it is therefore generally better suited for investigating older splits deeper in the phylogenetic history of an organism group. Despite this, the authors conclude that ''TEF1α'' is the most promising candidate for an additional DNA barcode marker in fungi as it also features sequence regions of higher mutation rates.<ref name=Stielow-eal-2015/> Following this, a quality-controlled reference database was established and merged with the previously existing ISHAM-ITS database for fungal ITS DNA barcodes<ref name=Irinyi-eal-2015/> to form the ISHAM database.<ref name=Meyer-eal-2018>{{cite journal | vauthors = Meyer W, Irinyi L, Hoang MT, Robert V, Garcia-Hermoso D, Desnos-Ollivier M, Yurayart C, Tsang CC, Lee CY, Woo PC, Pchelin IM, Uhrlaß S, Nenoff P, Chindamporn A, Chen S, Hebert PD, Sorrell TC | display-authors = 6 | title = 1 | journal = Genome | volume = 62 | issue = 3 | pages = 160–169 | date = March 2019 | pmid = 30465691 | doi = 10.1139/gen-2018-0083 | url = https://www.nrcresearchpress.com/doi/pdf/10.1139/gen-2018-0083 | author16-link = Paul D. N. Hebert | author18 = ISHAM barcoding of pathogenic fungi working group }}</ref>
-''TEF1α'' has been successfully used to identify a new species of ''[[Cantharellus]]'' from [[Texas]] and distinguish it from a morphologically similar species.<ref name=Buyck-eal-2011>{{cite journal |last1=Buyck |first1=Bart |last2=Cruaud |first2=C. |last3=Couloux |first3=André |last4=Hofstetter |first4=Valérie |date=2011 |title=''Cantharellus texensis'' sp. nov. from Texas, a southern lookalike of ''C. cinnabarinus'' revealed by tef-1 sequence data |journal=Mycologia |volume=103 |issue=5 |pages=1037–1046 |doi=10.3852/10-261 }}</ref> In the genera ''[[Ochroconis]]'' and ''[[Verruconis]]'' (Sympoventuriaceae, Venturiales), however, the marker does not allow distinction of all species.<ref name=Samerpitak-eal-2016>{{cite journal |last1=Samerpitak |first1=Kittipan |last2=Gerrits van den Ende |first2=Bert H. G. |last3=Stielow |first3=J. Benjamin |last4=Menken |first4=Steph B. J. |last5=de Hoog |first5=G. Sybren |date=2016 |title=Barcoding and species recognition of opportunistic pathogens in ''Ochroconis'' and ''Verruconis'' |journal=Fungal Biology |volume=120 |issue=2 |pages=219–230 |doi=10.1016/j.funbio.2015.08.010 |url=https://pure.uva.nl/ws/files/2588741/168953_Samerpitak_thesis_complete.pdf#page=159 }}</ref> ''TEF1α'' has also been used in phylogenetic analyses at the genus level, e.g. in the case of ''[[Cantharellus]]''<ref name=Buyck-eal-2014/> and the entomopathogenic ''[[Beauveria]]'',<ref name=Rehner-Buckley-2005>{{cite journal |last1=Rehner |first1=Stephen A. |last2=Buckley |first2=Ellen |date=2005 |title=A ''Beauveria'' phylogeny inferred from nuclear ITS and EF1-α sequences: evidence for cryptic diversification and links to ''Cordyceps'' teleomorphs |journal=Mycologia |volume=97 |issue=1 |pages=84–98 |doi=10.1080/15572536.2006.11832842 }}</ref> and for the phylogenetics of early-diverging fungal lineages.<ref name=James-eal-2006/>
+''TEF1α'' has been successfully used to identify a new species of ''[[Cantharellus]]'' from [[Texas]] and distinguish it from a morphologically similar species.<ref name=Buyck-eal-2011>{{cite journal | vauthors = Buyck B, Cruaud C, Couloux A, Hofstetter V | title = Cantharellus texensis sp. nov. from Texas, a southern lookalike of C. cinnabarinus revealed by tef-1 sequence data | journal = Mycologia | volume = 103 | issue = 5 | pages = 1037–46 | date = 2011 | pmid = 21558500 | doi = 10.3852/10-261 }}</ref> In the genera ''[[Ochroconis]]'' and ''[[Verruconis]]'' (Sympoventuriaceae, Venturiales), however, the marker does not allow distinction of all species.<ref name=Samerpitak-eal-2016>{{cite journal | vauthors = Samerpitak K, Gerrits van den Ende BH, Stielow JB, Menken SB, de Hoog GS | title = Barcoding and species recognition of opportunistic pathogens in Ochroconis and Verruconis | journal = Fungal Biology | volume = 120 | issue = 2 | pages = 219–30 | date = February 2016 | pmid = 26781378 | doi = 10.1016/j.funbio.2015.08.010 | url = https://pure.uva.nl/ws/files/2588741/168953_Samerpitak_thesis_complete.pdf#page=159 }}</ref> ''TEF1α'' has also been used in phylogenetic analyses at the genus level, e.g. in the case of ''[[Cantharellus]]''<ref name=Buyck-eal-2014/> and the entomopathogenic ''[[Beauveria]]'',<ref name=Rehner-Buckley-2005>{{cite journal | vauthors = Rehner SA, Buckley E | title = A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs | journal = Mycologia | volume = 97 | issue = 1 | pages = 84–98 | date = 2005 | pmid = 16389960 | doi = 10.1080/15572536.2006.11832842 }}</ref> and for the phylogenetics of early-diverging fungal lineages.<ref name=James-eal-2006/>
 ====Primers====
@@ Line 69: / Line 69: @@
 {{col-end}}
-Primers used for the investigation of [[Rhizophydiales]] and especially ''[[Batrachochytrium dendrobatidis]]'', a pathogen of amphibia, are the forward primer ''tef1F'' with the nucleotide sequence 5′-TACAARTGYGGTGGTATYGACA-3′, and the reverse primer ''tef1R'' with the sequence 5′-ACNGACTTGACYTCAGTRGT-3′.<ref name=Morehouse-eal-2003>{{cite journal |last1=Morehouse |first1=Erica A. |last2=James |first2=Timothy Y. |last3=Ganley |first3=Austen R. D. |last4=Vilgalys |first4=Rytas |last5=Berger |first5=Lee |last6=Murphy |first6=Peter J. |last7=Longcore |first7=Joyce E. |date=2003 |title=Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently emerged clone |journal=Molecular Ecology |volume=12 |pages=395–403 |doi=10.1046/j.1365-294X.2003.01732.x }}</ref> These primers also successfully amplified the majority of ''[[Cantharellus]]'' species investigated by Buyck et al. (2014), with the exception of a few species for which more specific primers were developed: the forward primer ''tef-1Fcanth'' with the sequence <tt>5'-AGCATGGGTDCTYGACAAG-3'</tt>, and the reverse primer ''tef-1Rcanth'' with the sequence <tt>5'-CCAATYTTRTAYACATCYTGGAG-3'</tt>.<ref name=Buyck-eal-2014/>
+Primers used for the investigation of [[Rhizophydiales]] and especially ''[[Batrachochytrium dendrobatidis]]'', a pathogen of amphibia, are the forward primer ''tef1F'' with the nucleotide sequence 5′-TACAARTGYGGTGGTATYGACA-3′, and the reverse primer ''tef1R'' with the sequence 5′-ACNGACTTGACYTCAGTRGT-3′.<ref name=Morehouse-eal-2003>{{cite journal | vauthors = Morehouse EA, James TY, Ganley AR, Vilgalys R, Berger L, Murphy PJ, Longcore JE | title = Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently emerged clone | journal = Molecular Ecology | volume = 12 | issue = 2 | pages = 395–403 | date = February 2003 | pmid = 12535090 | doi = 10.1046/j.1365-294X.2003.01732.x }}</ref> These primers also successfully amplified the majority of ''[[Cantharellus]]'' species investigated by Buyck et al. (2014), with the exception of a few species for which more specific primers were developed: the forward primer ''tef-1Fcanth'' with the sequence <tt>5'-AGCATGGGTDCTYGACAAG-3'</tt>, and the reverse primer ''tef-1Rcanth'' with the sequence <tt>5'-CCAATYTTRTAYACATCYTGGAG-3'</tt>.<ref name=Buyck-eal-2014/>
-<!-- <ref name=>{{cite journal |last1=Carbone |first1=Ignazio |last2=Kohn |first2=Linda M. |date=1999 |title=A method for designing primer sets for speciation studies in filamentous ascomycetes |journal=Mycologia |volume=91 |issue=3 |pages=553-556 |doi=10.1080/00275514.1999.12061051 }}</ref>
+<!-- <ref name=>{{cite journal | vauthors = Carbone I, Kohn LM |date=1999 |title=A method for designing primer sets for speciation studies in filamentous ascomycetes |journal=Mycologia |volume=91 |issue=3 |pages=553-556 |doi=10.1080/00275514.1999.12061051 }}</ref>
 -->
 ===D1/D2 domain of the LSU ribosomal RNA===
-The D1/D2 domain is part of the nuclear [[LSU rRNA|large subunit]] ([[28S ribosomal RNA|28S]]) [[ribosome|ribosomal]] RNA, and it is therefore located in the same ribosomal tandem repeat gene cluster as the Internal Transcribed Spacer (''ITS''). But unlike the non-coding ITS sequences, the D1/D2 domain contains coding sequence. With about 600 bp it is about the same nucleotide sequence length as ''ITS'',<ref name=Scorzetti-eal-2002>{{cite journal |last1=Scorzetti |first1=Gloria |last2=Fell |first2=Jack W. |last3=Fonseca |first3=Alvaro |last4=Statzell-Tallman |first4=Adele |date=2002 |title=Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions |journal=FEMS Yeast Research |volume=2 |pages=495–517 |doi=10.1111/j.1567-1364.2002.tb00117.x |url=https://academic.oup.com/femsyr/article/2/4/495/551274 }}</ref> which makes amplification and sequencing rather straightforward, an advantage that has led to the accumulation of an extensive amount of ''D1/D2'' sequence data especially for [[yeast]]s.<ref name=Fell-eal-2000/><ref name=Kurtzmann-Robnett-1998a/><ref name=Scorzetti-eal-2002/>
+The D1/D2 domain is part of the nuclear [[LSU rRNA|large subunit]] ([[28S ribosomal RNA|28S]]) [[ribosome|ribosomal]] RNA, and it is therefore located in the same ribosomal tandem repeat gene cluster as the Internal Transcribed Spacer (''ITS''). But unlike the non-coding ITS sequences, the D1/D2 domain contains coding sequence. With about 600 bp it is about the same nucleotide sequence length as ''ITS'',<ref name=Scorzetti-eal-2002>{{cite journal | vauthors = Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A | title = Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions | journal = FEMS Yeast Research | volume = 2 | issue = 4 | pages = 495–517 | date = December 2002 | pmid = 12702266 | doi = 10.1111/j.1567-1364.2002.tb00117.x | url = https://academic.oup.com/femsyr/article/2/4/495/551274 }}</ref> which makes amplification and sequencing rather straightforward, an advantage that has led to the accumulation of an extensive amount of ''D1/D2'' sequence data especially for [[yeast]]s.<ref name=Fell-eal-2000/><ref name=Kurtzmann-Robnett-1998a/><ref name=Scorzetti-eal-2002/>
 Regarding the molecular identification of basidiomycetous yeasts, ''D1/D2'' (or ''ITS'') can be used alone.<ref name=Scorzetti-eal-2002/> However, Fell et al. (2000) and Scorzetti et al. (2002) recommend the combined analysis of the ''D1/D2'' and ''ITS'' regions,<ref name=Fell-eal-2000/><ref name=Scorzetti-eal-2002/> a practice that later became the standard required information for describing new taxa of asco- and basidiomycetous yeasts.<ref name=Xu-2016/> When attempting to identify early diverging fungal lineages, the study of Schoch et al. (2012), comparing the identification performance of different genetic markers, showed that the [[LSU rRNA|large subunit]] (as well as the [[SSU rRNA|small subunit]]) of the ribosomal RNA performs better than ''ITS'' or ''RPB1''.<ref name=Schoch-eal-2012/>
@@ Line 82: / Line 82: @@
 ===RNA polymerase II subunit RPB1===
-[[File:Eukaryotic RNA-polymerase II structure 1WCM.png|thumb|300px|right|Eukaryotic RNA-polymerase II of ''[[Saccharomyces cerevisiae]]'',<ref name=Armache-eal-2019>{{cite journal |last1=Armache |first1=Karim-Jean |last2=Mitterweger |first2=Simone |last3=Meinhart |first3=Anton |last4=Cramer |first4=Patrick |date=2019 |title=Structures of complete RNA polymerase II and its subcomplex, Rpb4/7 |journal=Journal of Biological Chemistry |volume=280 |issue=8 |pages=7131–1734 |doi=10.2210/pdb1wcm/pdb |pmid=15591044 |url=https://www.jbc.org/content/280/8/7131.full.pdf }}</ref> with the <span style="color:red"> RPB1 subunit coloured in red</span>. Other subunits: <span style="color:orange"> RPB3 – orange </span>, <span style="color:gold"> RPB11 – yellow </span>, <span style="color:peru"> RPB2 – wheat</span>, <span style="color:pink"> RPB6 – pink</span>; the remaining seven subunits are in grey colour.]]
+[[File:Eukaryotic RNA-polymerase II structure 1WCM.png|thumb|300px|right|Eukaryotic RNA-polymerase II of ''[[Saccharomyces cerevisiae]]'',<ref name=Armache-eal-2019>{{cite journal | vauthors = Armache KJ, Mitterweger S, Meinhart A, Cramer P | title = Structures of complete RNA polymerase II and its subcomplex, Rpb4/7 | journal = The Journal of Biological Chemistry | volume = 280 | issue = 8 | pages = 7131–4 | date = February 2005 | pmid = 15591044 | doi = 10.2210/pdb1wcm/pdb | url = https://www.jbc.org/content/280/8/7131.full.pdf }}</ref> with the <span style="color:red"> RPB1 subunit coloured in red</span>. Other subunits: <span style="color:orange"> RPB3 – orange </span>, <span style="color:gold"> RPB11 – yellow </span>, <span style="color:peru"> RPB2 – wheat</span>, <span style="color:pink"> RPB6 – pink</span>; the remaining seven subunits are in grey colour.]]
-The [[POLR2A|RNA polymerase II subunit ''RPB1'']] is the largest subunit of the [[RNA polymerase II]]. In ''[[Saccharomyces cerevisiae]]'', it is encoded by the ''RPO21'' gene.<ref name=Strathern-eal-2013>{{cite journal |last1=Strathern |first1=Jeffrey |last2=Malagon |first2=Francisco |last3=Irvin |first3=Jordan |last4=Gotte |first4=Deanna |last5=Shafer |first5=Brenda |last6=Kireeva |first6=Maria |last7=Lubkowska |first7=Lucyna |last8=Jin |first8=Ding Jun |last9=Kashlev |first9=Mikhail |date=2013 |title=The Fidelity of Transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in ''S. cerevisiae'' |journal=Journal of Biological Chemistry |volume=288 |issue= 4 |pages=2689–2699 |doi=10.1074/jbc.M112.429506 |url=https://www.jbc.org/content/288/4/2689.full.pdf }}</ref> [[polymerase chain reaction|PCR]] amplification success of ''RPB1'' is very taxon-dependent, ranging from 70-80% in [[Ascomycota]] to 14% in early diverging fungal lineages.<ref name=Schoch-eal-2012/> Apart from the early diverging lineages, ''RPB1'' has a high rate of species identification in all fungal groups. In the species-rich [[Pezizomycotina]] it even outperforms ITS.<ref name=Schoch-eal-2012/>
+The [[POLR2A|RNA polymerase II subunit ''RPB1'']] is the largest subunit of the [[RNA polymerase II]]. In ''[[Saccharomyces cerevisiae]]'', it is encoded by the ''RPO21'' gene.<ref name=Strathern-eal-2013>{{cite journal | vauthors = Strathern J, Malagon F, Irvin J, Gotte D, Shafer B, Kireeva M, Lubkowska L, Jin DJ, Kashlev M | display-authors = 6 | title = The fidelity of transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in S. cerevisiae | journal = The Journal of Biological Chemistry | volume = 288 | issue = 4 | pages = 2689–99 | date = January 2013 | pmid = 23223234 | doi = 10.1074/jbc.M112.429506 | url = https://www.jbc.org/content/288/4/2689.full.pdf }}</ref> [[polymerase chain reaction|PCR]] amplification success of ''RPB1'' is very taxon-dependent, ranging from 70-80% in [[Ascomycota]] to 14% in early diverging fungal lineages.<ref name=Schoch-eal-2012/> Apart from the early diverging lineages, ''RPB1'' has a high rate of species identification in all fungal groups. In the species-rich [[Pezizomycotina]] it even outperforms ITS.<ref name=Schoch-eal-2012/>
 In a study comparing the identification performance of four genes, ''RPB1'' was among the most effective genes when combining two genes in the analysis: combined analysis with either ''ITS'' or with the [[LSU rRNA|large subunit]] ribosomal RNA yielded the highest identification success.<ref name=Schoch-eal-2012/>
-Other studies also used [[POLR2B|RPB2]], the second-largest subunit of the RNA polymerase II, e.g. for studying the phylogenetic relationships among species of the genus ''[[Cantharellus]]''<ref name=Buyck-eal-2014>{{cite journal |last1=Buyck |first1=Bart |last2=Kauff |first2=Frank |last3=Eyssartier |first3=Guillaume |last4=Couloux |first4=André |last5=Hofstetter |first5=Valérie |date=2014 |title=A multilocus phylogeny for worldwide ''Cantharellus'' (Cantharellales, Agaricomycetidae) |journal=Fungal Diversity |volume=64 |pages=101–121 |doi=10.1007/s13225-013-0272-3 |url=https://link.springer.com/content/pdf/10.1007/s13225-013-0272-3.pdf }}</ref> or for a phylogenetic study shedding light on the relationships among early-diverging lineages in the fungal kingdom.<ref name=James-eal-2006>{{cite journal |last1=James |first1=Timothy Y. |last2=Kauff |first2=Frank |last3=Schoch |first3=Conrad L. |last4=Matheny |first4=P. Brandon |last5=Hofstetter |first5=Valérie |last6=Cox |first6=Cymon J. |last7=Celio |first7=Gail |last8=Gueidan |first8=Cécile |last9=Fraker |first9=Emily |last10=Miadlikowska |first10=Jolanta |last11=Lumbsch |first11=H. Thorsten |last12=Rauhut |first12=Alexandra |last13=Reeb |first13=Valérie |last14=Arnold |first14=A. Elizabeth |last15=Amtoft |first15=Anja |last16=Stajich |first16=Jason E. |last17=Hosaka |first17=Kentaro |last18=Sung |first18=Gi-Ho |last19=Johnson |first19=Desiree |last20=O’Rourke |first20=Ben |last21=Crockett |first21=Michael |last22=Binder |first22=Manfred |last23=Curtis |first23=Judd M. |last24=Slot |first24=Jason C. |last25=Wang |first25=Zheng |last26=Wilson |first26=Andrew W. |last27=Schüßler |first27=Arthur |last28=Longcore |first28=Joyce E. |last29=O’Donnell |first29=Kerry |last30=Mozley-Standridge |first30=Sharon |last31=Porter |first31=David |last32=Letcher |first32=Peter M. |last33=Powell |first33=Martha J. |last34=Taylor |first34=John W. |last35=White |first35=Merlin M. |last36=Griffith |first36=Gareth W. |last37=Davies |first37=David R. |last38=Humber |first38=Richard A. |last39=Morton |first39=Joseph B. |last40=Sugiyama |first40=Junta |last41=Rossman |first41=Amy Y. |last42=Rogers |first42=Jack D. |last43=Pfister |first43=Don H. |last44=Hewitt |first44=David |last45=Hansen |first45=Karen |last46=Hambleton |first46=Sarah |last47=Shoemaker |first47=Robert A. |last48=Kohlmeyer |first48=Jan |last49=Volkmann-Kohlmeyer |first49=Brigitte |last50=Spotts |first50=Robert A. |last51=Serdani |first51=Maryna |last52=Crous |first52=Pedro W. |last53=Hughes |first53=Karen W. |last54=Matsuura |first54=Kenji |last55=Langer |first55=Ewald |last56=Langer |first56=Gitta |last57=Untereiner |first57=Wendy A. |last58=Lücking |first58=Robert |last59=Büdel |first59=Burkhard |last60=Geiser |first60=David M. |last61=Aptroot |first61=André |last62=Diederich |first62=Paul |last63=Schmitt |first63=Imke |last64=Schultz |first64=Matthias |last65=Yahr |first65=Rebecca |last66=Hibbett |first66=David S. |last67=Lutzoni |first67=François |last68=McLaughlin |first68=David J. |last69=Spatafora |first69=Joseph W. |last70=Vilgalys |first70=Rytas |date=2006 |title=Reconstructing the early evolution of Fungi using a six-gene phylogeny |journal=Nature |volume=443 |pages=818–822 |doi=10.1038/nature05110 |url=https://pubag.nal.usda.gov/download/8595/PDF }}</ref>
+Other studies also used [[POLR2B|RPB2]], the second-largest subunit of the RNA polymerase II, e.g. for studying the phylogenetic relationships among species of the genus ''[[Cantharellus]]''<ref name=Buyck-eal-2014>{{cite journal |last1=Buyck |first1=Bart |last2=Kauff |first2=Frank |last3=Eyssartier |first3=Guillaume |last4=Couloux |first4=André |last5=Hofstetter |first5=Valérie | name-list-format = vanc |date=2014 |title=A multilocus phylogeny for worldwide ''Cantharellus'' (Cantharellales, Agaricomycetidae) |journal=Fungal Diversity |volume=64 |pages=101–121 |doi=10.1007/s13225-013-0272-3 |url=https://link.springer.com/content/pdf/10.1007/s13225-013-0272-3.pdf }}</ref> or for a phylogenetic study shedding light on the relationships among early-diverging lineages in the fungal kingdom.<ref name=James-eal-2006>{{cite journal | vauthors = James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V, Cox CJ, Celio G, Gueidan C, Fraker E, Miadlikowska J, Lumbsch HT, Rauhut A, Reeb V, Arnold AE, Amtoft A, Stajich JE, Hosaka K, Sung GH, Johnson D, O'Rourke B, Crockett M, Binder M, Curtis JM, Slot JC, Wang Z, Wilson AW, Schüssler A, Longcore JE, O'Donnell K, Mozley-Standridge S, Porter D, Letcher PM, Powell MJ, Taylor JW, White MM, Griffith GW, Davies DR, Humber RA, Morton JB, Sugiyama J, Rossman AY, Rogers JD, Pfister DH, Hewitt D, Hansen K, Hambleton S, Shoemaker RA, Kohlmeyer J, Volkmann-Kohlmeyer B, Spotts RA, Serdani M, Crous PW, Hughes KW, Matsuura K, Langer E, Langer G, Untereiner WA, Lücking R, Büdel B, Geiser DM, Aptroot A, Diederich P, Schmitt I, Schultz M, Yahr R, Hibbett DS, Lutzoni F, McLaughlin DJ, Spatafora JW, Vilgalys R | display-authors = 6 | title = Reconstructing the early evolution of Fungi using a six-gene phylogeny | journal = Nature | volume = 443 | issue = 7113 | pages = 818–22 | date = October 2006 | pmid = 17051209 | doi = 10.1038/nature05110 | url = https://pubag.nal.usda.gov/download/8595/PDF }}</ref>
 ====Primers====
@@ Line 96: / Line 96: @@
 The [[Spacer DNA|Intergenic Spacer]] (''IGS'') is the region of non-coding DNA between individual [[tandem repeat]]s of the [[ribosome|ribosomal]] [[Metabolic gene cluster|gene cluster]] in the [[nuclear DNA|nuclear genome]], as opposed to the Internal Transcribed Spacer (ITS) that is situated within these tandem repeats.
-''IGS'' has been successfully used for the differentiation of [[strain (biology)|strains]] of ''Xanthophyllomyces dendrorhous''<ref name=Fell-Blatt-1999>{{cite journal |last1=Fell |first1=Jack W. |last2=Blatt |first2=G. M. |date=1999 |title=Separation of strains of the yeasts ''Xanthophyllomyces dendrorhous'' and ''Phaffia rhodozyma'' based on rDNA IGS and ITS sequence analysis |journal=Journal of Industrial Microbiology and Biotechnology |volume=23 |issue=1 |pages=677–681 |url=https://link.springer.com/content/pdf/10.1038/sj.jim.2900681.pdf }}</ref> as well as for species distinction in the [[psychrophile|psychrophilic]] genus ''Mrakia'' ([[Cystofilobasidiales]]).<ref name=Diaz-Fell-2000>{{cite journal |last1=Diaz |first1=Mara R. |last2=Fell |first2=Jack W. |date=2000 |title=Molecular analyses of the IGS & ITS regions of rDNA of the psychrophilic yeasts in the genus ''Mrakia'' |journal=Antonie van Leeuwenhoek |volume=77 |issue=1 |pages=7–12 |url=https://link.springer.com/content/pdf/10.1023/A:1002048008295.pdf }}</ref> Due to these results, ''IGS'' has been recommended as a genetic marker for additional differentiation (along with D1/D2 and ''ITS'') of closely related species and even strains within one species in basidiomycete yeasts.<ref name=Fell-eal-2000/>
+''IGS'' has been successfully used for the differentiation of [[strain (biology)|strains]] of ''Xanthophyllomyces dendrorhous''<ref name=Fell-Blatt-1999>{{cite journal |last1=Fell |first1=Jack W. |last2=Blatt |first2=G. M. | name-list-format = vanc |date=1999 |title=Separation of strains of the yeasts ''Xanthophyllomyces dendrorhous'' and ''Phaffia rhodozyma'' based on rDNA IGS and ITS sequence analysis |journal=Journal of Industrial Microbiology and Biotechnology |volume=23 |issue=1 |pages=677–681 |doi = 10.1038/sj.jim.2900681 }}</ref> as well as for species distinction in the [[psychrophile|psychrophilic]] genus ''Mrakia'' ([[Cystofilobasidiales]]).<ref name=Diaz-Fell-2000>{{cite journal |last1=Diaz |first1=Mara R. |last2=Fell |first2=Jack W. | name-list-format = vanc |date=2000 |title=Molecular analyses of the IGS & ITS regions of rDNA of the psychrophilic yeasts in the genus ''Mrakia'' |journal=Antonie van Leeuwenhoek |volume=77 |issue=1 |pages=7–12 |doi = 10.1023/A:1002048008295 }}</ref> Due to these results, ''IGS'' has been recommended as a genetic marker for additional differentiation (along with D1/D2 and ''ITS'') of closely related species and even strains within one species in basidiomycete yeasts.<ref name=Fell-eal-2000/>
 ===Other genetic markers===
-The '''[[cytochrome c oxidase subunit I]] (''COI'')''' gene outperforms ''ITS'' in DNA barcoding of ''[[Penicillium]]'' (Ascomycota) species, with species-specific barcodes for 66% of the investigated species versus 25% in the case of ''ITS''. Furthermore, a part of the [[Tubulin#β-Tubulin|β-Tubulin]] A (''BenA'') gene exhibits a higher taxonomic resolution in distinguishing ''Penicillium'' species as compared to ''COI'' and ''ITS''.<ref name=Seifert-eal-2007>{{cite journal |last1=Seifert |first1=Keith A. |last2=Samson |first2=Robert A. |last3=deWaard |first3=Jeremy R. |last4=Houbraken |first4=Jos |last5=Lévesque |first5=C. André |last6=Moncalvo |first6=Jean-Marc |last7=Louis-Seize |first7=Gerry |last8=Hebert |first8=Paul D. N. |author8-link=Paul D. N. Hebert |date=2007 |title=Prospects for fungus identification using CO1 DNA barcodes, with ''Penicillium'' as a test case |journal=PNAS |volume=104 |issue=10 |pages=3901–3906 |doi=10.1073/pnas.0611691104 |url=http://pnas.org/content/pnas/104/10/3901.full.pdf }}</ref> In the closely related ''[[Aspergillus niger]]'' complex, however, ''COI'' is not variable enough for species discrimination.<ref name=Geiser-eal-2007>{{cite journal |last1=Geiser |first1=D. M. |last2=Klich |first2=M. A. |last3=Frisvad |first3=J. C. |last4=Peterson |first4=S. W. |last5=Varga |first5=J |last6=Samson |first6=R. A. |date=2007 |title=The current status of species recognition and identification in ''Aspergillus'' |journal=Studies in Mycology |volume=59 |pages=1–10 |doi=10.3114/sim.2007.59.01 |pmc=2275194 |pmid=18490947 }}</ref> In ''[[Fusarium]]'', ''COI'' exhibits [[Sequence homology#Paralogy|paralogues]] in many cases, and [[Sequence homology|homologous]] copies are not variable enough to distinguish species.<ref name=Gilmore-eal-2009>{{cite journal |last1=Gilmore |first1=Scott R. |last2=Gräfenhan |first2=Tom |last3=Louis-Seize |first3=Gerry |last4=Seifert |first4=Keith A. |date=2009 |title=Multiple copies of cytochrome oxidase 1 in species of the fungal genus ''Fusarium'' |journal=Molecular Ecology Resources |volume=9 |issue=Suppl. 1 |pages=90–98 |doi=10.1111/j.1755-0998.2009.02636.x |url=https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1755-0998.2009.02636.x }}</ref>
+The '''[[cytochrome c oxidase subunit I]] (''COI'')''' gene outperforms ''ITS'' in DNA barcoding of ''[[Penicillium]]'' (Ascomycota) species, with species-specific barcodes for 66% of the investigated species versus 25% in the case of ''ITS''. Furthermore, a part of the [[Tubulin#β-Tubulin|β-Tubulin]] A (''BenA'') gene exhibits a higher taxonomic resolution in distinguishing ''Penicillium'' species as compared to ''COI'' and ''ITS''.<ref name=Seifert-eal-2007>{{cite journal | vauthors = Seifert KA, Samson RA, Dewaard JR, Houbraken J, Lévesque CA, Moncalvo JM, Louis-Seize G, Hebert PD | display-authors = 6 | title = Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 104 | issue = 10 | pages = 3901–6 | date = March 2007 | pmid = 17360450 | doi = 10.1073/pnas.0611691104 | url = http://pnas.org/content/pnas/104/10/3901.full.pdf | author8-link = Paul D. N. Hebert }}</ref> In the closely related ''[[Aspergillus niger]]'' complex, however, ''COI'' is not variable enough for species discrimination.<ref name=Geiser-eal-2007>{{cite journal | vauthors = Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA | title = The current status of species recognition and identification in Aspergillus | journal = Studies in Mycology | volume = 59 | pages = 1–10 | date = 2007 | pmid = 18490947 | pmc = 2275194 | doi = 10.3114/sim.2007.59.01 }}</ref> In ''[[Fusarium]]'', ''COI'' exhibits [[Sequence homology#Paralogy|paralogues]] in many cases, and [[Sequence homology|homologous]] copies are not variable enough to distinguish species.<ref name=Gilmore-eal-2009>{{cite journal | vauthors = Gilmore SR, Gräfenhan T, Louis-Seize G, Seifert KA | title = Multiple copies of cytochrome oxidase 1 in species of the fungal genus Fusarium | journal = Molecular Ecology Resources | volume = 9 Suppl s1 | issue = Suppl. 1 | pages = 90–8 | date = May 2009 | pmid = 21564969 | doi = 10.1111/j.1755-0998.2009.02636.x | url = https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1755-0998.2009.02636.x }}</ref>
-''COI'' also performs poorly in the identification of [[Basidiomycota|basidiomycote]] [[rust (fungus)|rusts]] of the [[order (biology)|order]] [[Pucciniales]] due to the presence of [[intron]]s. Even when the obstacle of introns is overcome, ''ITS'' and the [[LSU rRNA]] ([[28S ribosomal RNA|28S]]) outperform ''COI'' as DNA barcode marker.<ref name=Vialle-eal-2009>{{cite journal |last1=Vialle |first1=Agathe |last2=Feau |first2=Nicolas |last3=Allaire |first3=Mathieu |last4=Didukh |first4=Maryna |last5=Martin |first5=Francis |last6=Moncalvo |first6=Jean-Marc |last7=Hamelin |first7=Richard C. |date=2009 |title=In silico evaluation of mitochondrial genes as DNA barcode for Basidiomycota |journal=Molecular Ecology Resources |volume=9 |issue=Suppl. 1 |pages=99–113 |doi=10.1111/j.1755-0998.2009.02637.x |url=https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1755-0998.2009.02637.x }}</ref> In the subdivision [[Agaricomycotina]], PCR amplification success was poor for ''COI'', even with multiple primer combinations. Successfully sequenced ''COI'' samples also included introns and possible paralogous copies, as reported for ''Fusarium''.<ref name=Gilmore-eal-2009/><ref name=Dentinger-eal-2011>{{cite journal |last1=Dentinger |first1=Bryn T. M. |last2=Didukh |first2=Maryna Y. |last3=Moncalvo |first3=Jean-Marc |date=2011 |title=Comparing COI and ITS as DNA Barcode Markers for Mushrooms and Allies (Agaricomycotina) |journal=PLOS ONE |volume=6 |issue=9 |pages=e25081 |doi=10.1371/journal.pone.0025081 |issn=1932-6203 |pmc=3178597 |pmid=21966418 |bibcode=2011PLoSO...625081D }}</ref> ''[[Agaricus bisporus]]'' was found to contain up to 19 introns, making the ''COI'' gene of this species the longest recorded, with 29,902 nucleotides.<ref name=Férandon-eal-2010>{{cite journal |last1=Férandon |first1=Cyril |last2=Moukha |first2=Serge |last3=Callac |first3=Philippe |last4=Benedetto |first4=Jean-Pierre |last5=Castroviejo |first5=Michel |last6=Barroso |first6=Gérard |date=2011 |title=The Agaricus bisporus cox1 gene: The longest mitochondrial gene and the largest reservoir of mitochondrial group I introns |journal=PLoS One |volume=5 |pages=e14048 |doi= |url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2987802/pdf/pone.0014048.pdf }}</ref> Apart from the substantial troubles of sequencing ''COI'', ''COI'' and ''ITS'' generally perform equally well in distinguishing basidiomycote mushrooms.<ref name=Dentinger-eal-2011/>
+''COI'' also performs poorly in the identification of [[Basidiomycota|basidiomycote]] [[rust (fungus)|rusts]] of the [[order (biology)|order]] [[Pucciniales]] due to the presence of [[intron]]s. Even when the obstacle of introns is overcome, ''ITS'' and the [[LSU rRNA]] ([[28S ribosomal RNA|28S]]) outperform ''COI'' as DNA barcode marker.<ref name=Vialle-eal-2009>{{cite journal | vauthors = Vialle A, Feau N, Allaire M, Didukh M, Martin F, Moncalvo JM, Hamelin RC | title = Evaluation of mitochondrial genes as DNA barcode for Basidiomycota | journal = Molecular Ecology Resources | volume = 9 Suppl s1 | issue = Suppl. 1 | pages = 99–113 | date = May 2009 | pmid = 21564970 | doi = 10.1111/j.1755-0998.2009.02637.x | url = https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1755-0998.2009.02637.x }}</ref> In the subdivision [[Agaricomycotina]], PCR amplification success was poor for ''COI'', even with multiple primer combinations. Successfully sequenced ''COI'' samples also included introns and possible paralogous copies, as reported for ''Fusarium''.<ref name=Gilmore-eal-2009/><ref name=Dentinger-eal-2011>{{cite journal | vauthors = Dentinger BT, Didukh MY, Moncalvo JM | title = Comparing COI and ITS as DNA barcode markers for mushrooms and allies (Agaricomycotina) | journal = PloS One | volume = 6 | issue = 9 | pages = e25081 | date = 2011 | pmid = 21966418 | pmc = 3178597 | doi = 10.1371/journal.pone.0025081 | bibcode = 2011PLoSO...625081D }}</ref> ''[[Agaricus bisporus]]'' was found to contain up to 19 introns, making the ''COI'' gene of this species the longest recorded, with 29,902 nucleotides.<ref name=Férandon-eal-2010>{{cite journal | vauthors = Férandon C, Moukha S, Callac P, Benedetto JP, Castroviejo M, Barroso G | title = The Agaricus bisporus cox1 gene: the longest mitochondrial gene and the largest reservoir of mitochondrial group i introns | journal = PloS One | volume = 5 | issue = 11 | pages = e14048 | date = November 2010 | pmid = 21124976 | pmc = 2987802 | doi = 10.1371/journal.pone.0014048 }}</ref> Apart from the substantial troubles of sequencing ''COI'', ''COI'' and ''ITS'' generally perform equally well in distinguishing basidiomycote mushrooms.<ref name=Dentinger-eal-2011/>
 '''[[Topoisomerase]] I (''[[TOP1]]'')''' was investigated as additional DNA barcode candidate by Lewis et al. (2011) based on [[proteome]] data, with the devloped universal primer pair<ref name=Lewis-eal-2011/> being susequently tested on actual samples by Stielow et al. (2015). The forward primer ''TOP1_501-F'' with the sequence <tt>5'-TGTAAAACGACGGCCAGT-ACGAT-ACTGCCAAGGTTTTCCGTACHTACAACGC-3'</tt> (where the first section marks the universal M13 forward primer tail, the second part consisting of ACGAT a spacer, and the third part the actual primer) and reverse the primer ''TOP1_501-R'' with <tt>5'-CAGGAAACAGCTATGA-CCCAGTCCTCGTCAACWGACTTRATRGCCCA-3'</tt> (the first section marking the universal M13 reverse primer tail, the second part the actual TOP1 reverse primer) amplify a fragment of approximately 800 base pairs (bp).<ref name=Stielow-eal-2015/>
@@ Line 112: / Line 112: @@
 ==Applications==
-Fungal DNA barcoding has been sucessfully applied to the investigation of [[foxing]] phenomena, a major concern in the [[Conservation and restoration of books, manuscripts, documents and ephemera|conservation of paper documents]]. Sequeira et al. (2019) sequenced ''ITS'' from foxing stains and found ''[[Chaetomium globosum]]'', ''[[Chaetomium murorum|Ch. murorum]]'', ''[[Chaetomium nigricolor|Ch. nigricolor]]'', ''[[Chaetomium]]'' sp., ''[[Eurotium rubrum]]'', ''[[Myxotrichum deflexum]]'', ''[[Penicillium chrysogenum]]'', ''[[Penicillium citrinum|P. citrinum]]'', ''[[Penicillium commune|P. commune]]'', ''[[Penicillium]]'' sp. and ''[[Stachybotrys chartarum]]'' to inhabit the investigated paper stains.<ref name=Sequeira-eal-2019>{{cite journal |last1=Sequeira |first1=Sílvia Oliveira |last2=de Carvalho |first2=Hugo Paiva |last3=Mesquita |first3=Nuno |last4=Portugal |first4=António |last5=Macedo |first5=Maria Filomena |date=2019 |title=Fungal stains on paper: is what you see what you get? |journal=Conservar Património |volume=32 |pages=18–27 |doi=10.14568/cp2018007 |url=http://revista.arp.org.pt/pdf/2018007.pdf }}</ref>
+Fungal DNA barcoding has been sucessfully applied to the investigation of [[foxing]] phenomena, a major concern in the [[Conservation and restoration of books, manuscripts, documents and ephemera|conservation of paper documents]]. Sequeira et al. (2019) sequenced ''ITS'' from foxing stains and found ''[[Chaetomium globosum]]'', ''[[Chaetomium murorum|Ch. murorum]]'', ''[[Chaetomium nigricolor|Ch. nigricolor]]'', ''[[Chaetomium]]'' sp., ''[[Eurotium rubrum]]'', ''[[Myxotrichum deflexum]]'', ''[[Penicillium chrysogenum]]'', ''[[Penicillium citrinum|P. citrinum]]'', ''[[Penicillium commune|P. commune]]'', ''[[Penicillium]]'' sp. and ''[[Stachybotrys chartarum]]'' to inhabit the investigated paper stains.<ref name=Sequeira-eal-2019>{{cite journal |last1=Sequeira |first1=Sílvia Oliveira |last2=de Carvalho |first2=Hugo Paiva |last3=Mesquita |first3=Nuno |last4=Portugal |first4=António |last5=Macedo |first5=Maria Filomena | name-list-format = vanc |date=2019 |title=Fungal stains on paper: is what you see what you get? |journal=Conservar Património |volume=32 |pages=18–27 |doi=10.14568/cp2018007 |url=http://revista.arp.org.pt/pdf/2018007.pdf }}</ref>
-Another study investigated fungi that act as biodeteriorating agents in the [[Old Cathedral of Coimbra]], part of the [[University of Coimbra]], a [[UNESCO]] [[world heritage site]]. Sequencing the ''ITS'' barcode of ten samples with classical [[Sanger sequencing|Sanger]] as well as with [[Illumina dye sequencing|Illumina]] [[Massive parallel sequencing|next-generation sequencing]] techniques, they identified 49 fungal species. ''[[Aspergillus versicolor]]'', ''[[Cladosporium cladosporioides]]'', ''[[Cladosporium sphaerospermum|C. sphaerospermum]]'', ''[[Cladosporium tenuissimum|C. tenuissimum]]'', ''[[Epicoccum nigrum]]'', ''[[Parengyodontium album]]'', ''[[Penicillium brevicompactum]]'', ''[[Penicillium crustosum|P. crustosum]]'', ''[[Penicillium glabrum|P. glabrum]]'', ''[[Talaromyces amestolkiae]]'' and ''[[Talaromyces stollii|T. stollii]]'' were the most common species isolated from the samples.<ref name=Trovao-eal-2019>{{cite journal |last1=Trovão |first1=João |last2=Portugal |first2=António |last3=Soares |first3=Fabiana |last4=Paiva |first4=Diana Sofia |last5=Mesquita |first5=Nuno |last6=Coelho |first6=Catarina |last7=Pinheiro |first7=Ana Catarina |last8=Catarino |first8=Lídia |last9=Gil |first9=Francisco |last10=Tiago |first10=Igor |date=2019 |title=Fungal diversity and distribution across distinct biodeterioration phenomena in limestone walls of the old cathedral of Coimbra, UNESCO World Heritage Site |journal=International Biodeterioration & Biodegradation |volume=142 |pages=91–102 |doi=10.1016/j.ibiod.2019.05.008 }}</ref>
+Another study investigated fungi that act as biodeteriorating agents in the [[Old Cathedral of Coimbra]], part of the [[University of Coimbra]], a [[UNESCO]] [[world heritage site]]. Sequencing the ''ITS'' barcode of ten samples with classical [[Sanger sequencing|Sanger]] as well as with [[Illumina dye sequencing|Illumina]] [[Massive parallel sequencing|next-generation sequencing]] techniques, they identified 49 fungal species. ''[[Aspergillus versicolor]]'', ''[[Cladosporium cladosporioides]]'', ''[[Cladosporium sphaerospermum|C. sphaerospermum]]'', ''[[Cladosporium tenuissimum|C. tenuissimum]]'', ''[[Epicoccum nigrum]]'', ''[[Parengyodontium album]]'', ''[[Penicillium brevicompactum]]'', ''[[Penicillium crustosum|P. crustosum]]'', ''[[Penicillium glabrum|P. glabrum]]'', ''[[Talaromyces amestolkiae]]'' and ''[[Talaromyces stollii|T. stollii]]'' were the most common species isolated from the samples.<ref name=Trovao-eal-2019>{{cite journal |last1=Trovão |first1=João |last2=Portugal |first2=António |last3=Soares |first3=Fabiana |last4=Paiva |first4=Diana Sofia |last5=Mesquita |first5=Nuno |last6=Coelho |first6=Catarina |last7=Pinheiro |first7=Ana Catarina |last8=Catarino |first8=Lídia |last9=Gil |first9=Francisco |last10=Tiago |first10=Igor | name-list-format = vanc |date=2019 |title=Fungal diversity and distribution across distinct biodeterioration phenomena in limestone walls of the old cathedral of Coimbra, UNESCO World Heritage Site |journal=International Biodeterioration & Biodegradation |volume=142 |pages=91–102 |doi=10.1016/j.ibiod.2019.05.008 }}</ref>
-Another study concerning objects of [[cultural heritage]] investigated the fungal diversity on a canvas painting by [[Paula Rego]] using the ''ITS2'' subregion of the ''ITS'' marker. Altogether, 387 [[operational taxonomic unit|OTUs]] (putative species) in 117 genera of 13 different classes of fungi were observed.<ref name=Paiva-eal-2019>{{cite journal |last1=Paiva de Carvalho |first1=Hugo |last2=Oliveira Sequeira |first2=Sílvia |last3=Pinho |first3=Diogo |last4=Trovão |first4=João |last5=Fernandes da Costa |first5=Ricardo Manuel |last6=Egas |first6=Conceição |last7=Macedo |first7=Maria Filomena |last8=Portugal |first8=António |date=2019 |title=Combining an innovative non-invasive sampling method and highthroughput sequencing to characterize fungal communities on a canvas painting |journal=International Biodeterioration & Biodegradation |volume=145 |pages=1–9 |doi=10.1016/j.ibiod.2019.104816 }}</ref>
+Another study concerning objects of [[cultural heritage]] investigated the fungal diversity on a canvas painting by [[Paula Rego]] using the ''ITS2'' subregion of the ''ITS'' marker. Altogether, 387 [[operational taxonomic unit|OTUs]] (putative species) in 117 genera of 13 different classes of fungi were observed.<ref name=Paiva-eal-2019>{{cite journal |last1=Paiva de Carvalho |first1=Hugo |last2=Oliveira Sequeira |first2=Sílvia |last3=Pinho |first3=Diogo |last4=Trovão |first4=João |last5=Fernandes da Costa |first5=Ricardo Manuel |last6=Egas |first6=Conceição |last7=Macedo |first7=Maria Filomena |last8=Portugal |first8=António | name-list-format = vanc |date=2019 |title=Combining an innovative non-invasive sampling method and highthroughput sequencing to characterize fungal communities on a canvas painting |journal=International Biodeterioration & Biodegradation |volume=145 |pages=1–9 |doi=10.1016/j.ibiod.2019.104816 }}</ref>
-==See also==
+== See also ==
 * [[DNA barcoding]]
@@ Line 126: / Line 126: @@
 * [[Consortium for the Barcode of Life]]
-==References==
+== References ==
 {{Reflist}}
-==Further reading==
+== Further reading ==
 * {{cite journal |last=Eberhardt |first=Ursula |date=2010 |title=A constructive step towards selecting a DNA barcode for fungi |journal=New Phytologist |volume=187 |pages=265–268 |url=https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1469-8137.2010.03329.x }}