Internal Transcribed Spacer (ITS) – the primary fungal barcode s of the
eukaryotic rDNA
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
base pairs 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. 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. is an open
ITS barcoding database for fungi and all other eukaryotes.
Primers The conserved flanking regions of 18S and 28S serve as anchor points for the
primers used for
PCR amplification of the
ITS region. 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. For the majority of fungi, the ITS primers proposed by White et al. (1990) have become the standard primers used for PCR amplification (with the most common pairing being ITS1 + ITS4). These primers are as follows: Furthermore, the choice of primers for
ITS amplification can introduce biases towards certain
taxonomic fungus groups. For example, the "universal"
ITS primers 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. 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. 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. 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. Efforts to define a universally applicable threshold value of
ITS variability that demarcates intraspecific from interspecific (between-species) variability thus remain futile. However, its discrimination power is partly superseded by that of the
DNA-directed RNA polymerase II subunit RPB1 (see also below). 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.
Translational elongation factor 1α (TEF1α) – the secondary fungal barcode The translational elongation factor 1α is part of the
eukaryotic 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. 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.
TEF1α has been successfully used to identify a new species of
Cantharellus from
Texas and distinguish it from a morphologically similar species. In the genera
Ochroconis and
Verruconis (Sympoventuriaceae, Venturiales), however, the marker does not allow distinction of all species.
TEF1α has also been used in phylogenetic analyses at the genus level, e.g. in the case of
Cantharellus and for the phylogenetics of early-diverging fungal lineages. 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 , and the reverse primer
tef-1Rcanth with the sequence . -->
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 base pairs it is about the same nucleotide sequence length as
ITS, 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. with the RPB1 subunit coloured in red. Other subunits: RPB3 – orange , RPB11 – yellow , RPB2 – wheat, RPB6 – pink; the remaining seven subunits are in grey colour. 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.
PCR amplification success of
RPB1 is very taxon-dependent, ranging from 70 to 80% in
Ascomycota to 14% in early diverging fungal lineages. or for a phylogenetic study shedding light on the relationships among early-diverging lineages in the fungal kingdom.
Primers Primers successfully amplifying RPB1 especially in Ascomycota are the forward primer
RPB1-Af with the sequence , and the reverse primer
RPB1-Ac-RPB1-Cr with the sequence . as well as for species distinction in the
psychrophilic genus
Mrakia (
Cystofilobasidiales). 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.
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. In the closely related
Aspergillus niger complex, however,
COI is not variable enough for species discrimination. In
Fusarium,
COI exhibits
paralogues in many cases, and
homologous copies are not variable enough to distinguish species.
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. 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.
Agaricus bisporus was found to contain up to 19 introns, making the
COI gene of this species the longest recorded, with 29,902 nucleotides. Apart from the substantial troubles of sequencing
COI,
COI and
ITS generally perform equally well in distinguishing basidiomycete mushrooms. '
Topoisomerase I (TOP1)' was investigated as additional DNA barcode candidate by Lewis et al. (2011) based on
proteome data, with the developed universal primer pair being subsequently tested on actual samples by Stielow et al. (2015). The forward primer
TOP1_501-F with the sequence (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 (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.
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). 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, with the PGK533 primer pair, amplifying a circa 1,000 base pair 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. ==Applications==