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Internal transcribed spacer
Internal transcribed spacer
Internal transcribed spacer
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Internal transcribed spacer (ITS) is the spacer DNA situated between the small-subunit ribosomal RNA (rRNA) and large-subunit rRNA genes in the chromosome or the corresponding transcribed region in the polycistronic rRNA precursor transcript.

Across life domains

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In bacteria and archaea, there is a single ITS, located between the 16S and 23S rRNA genes. Conversely, there are two ITSs in eukaryotes: ITS1 is located between 18S and 5.8S rRNA genes, while ITS2 is between 5.8S and 28S (in opisthokonts, or 25S in plants) rRNA genes. ITS1 corresponds to the ITS in bacteria and archaea, while ITS2 originated as an insertion that interrupted the ancestral 23S rRNA gene.[1][2]

Organization

[edit]
Organization of the eukaryotic nuclear ribosomal DNA tandem repeats

In bacteria and archaea, the ITS occurs in one to several copies, as do the flanking 16S and 23S genes. When there are multiple copies, these do not occur adjacent to one another. Rather, they occur in discrete locations in the circular chromosome. It is not uncommon in bacteria to carry tRNA genes in the ITS.[3][4]

In eukaryotes, genes encoding ribosomal RNA and spacers occur in tandem repeats that are thousands of copies long, each separated by regions of non-transcribed DNA termed intergenic spacer (IGS) or non-transcribed spacer (NTS).

Each eukaryotic ribosomal cluster contains the 5' external transcribed spacer (5' ETS), the 18S rRNA gene, the ITS1, the 5.8S rRNA gene, the ITS2, the 26S or 28S rRNA gene, and finally the 3' ETS.[5]

During rRNA maturation, ETS and ITS pieces are excised. As non-functional by-products of this maturation, they are rapidly degraded.[6]

Use in phylogenetic inference

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Sequence comparison of the eukaryotic ITS regions is widely used in taxonomy and molecular phylogeny because of several favorable properties:[7]

  • It is routinely amplified thanks to its small size associated to the availability of highly conserved flanking sequences.
  • It is easy to detect even from small quantities of DNA due to the high copy number of the rRNA clusters.
  • It undergoes rapid concerted evolution via unequal crossing-over and gene conversion. This promotes intra-genomic homogeneity of the repeat units, although high-throughput sequencing showed the occurrence of frequent variations within plant species.[8]
  • It has a high degree of variation even between closely related species. This can be explained by the relatively low evolutionary pressure acting on such non-coding spacer sequences.

For example, ITS markers have proven especially useful for elucidating phylogenetic relationships among the following taxa.

Taxonomic group Taxonomic level Year Authors with references
Asteraceae: Compositae Species (congeneric) 1992 Baldwin et al.[9]
Viscaceae: Arceuthobium Species (congeneric) 1994 Nickrent et al.[10]
Poaceae: Zea Species (congeneric) 1996 Buckler & Holtsford[11]
Leguminosae: Medicago Species (congeneric) 1998 Bena et al.[5]
Orchidaceae: Diseae Genera (within tribes) 1999 Douzery et al.[12]
Odonata: Calopteryx Species (congeneric) 2001 Weekers et al.[13]
Yeasts of clinical importance Genera 2001 Chen et al.[14]
Poaceae: Saccharinae Genera (within tribes) 2002 Hodkinson et al.[15]
Plantaginaceae: Plantago Species (congeneric) 2002 Rønsted et al.[16]
Jungermanniopsida: Herbertus Species (congeneric) 2004 Feldberg et al.[17]
Pinaceae: Tsuga Species (congeneric) 2008 Havill et al.[18]
Chrysomelidae: Altica Genera (congeneric) 2009 Ruhl et al.[19]
Symbiodinium Clade 2009 Stat et al.[20]
Brassicaceae Tribes (within a family) 2010 Warwick et al.[21]
Ericaceae: Erica Species (congeneric) 2011 Pirie et al.[22]
Diptera: Bactrocera Species (congeneric) 2014 Boykin et al.[23]
Scrophulariaceae: Scrophularia Species (congeneric) 2014 Scheunert & Heubl[24]
Potamogetonaceae: Potamogeton Species (congeneric) 2016 Yang et al.[25]

ITS2 is known to be more conserved than ITS1 is. All ITS2 sequences share a common core of secondary structure,[26] while ITS1 structures are only conserved in much smaller taxonomic units. Regardless of the scope of conservation, structure-assisted comparison can provide higher resolution and robustness.[27]

Mycological barcoding

[edit]
Main article: Fungal DNA barcoding

The ITS region is the most widely sequenced DNA region in molecular ecology of fungi[28] and has been recommended as the universal fungal barcode sequence.[29] It has typically been most useful for molecular systematics at the species to genus level, and even within species (e.g., to identify geographic races). Because of its higher degree of variation than other genic regions of rDNA (for example, small- and large-subunit rRNA), variation among individual rDNA repeats can sometimes be observed within both the ITS and IGS regions. In addition to the universal ITS1+ITS4 primers[30][31] used by many labs, several taxon-specific primers have been described that allow selective amplification of fungal sequences (e.g., see Gardes & Bruns 1993 paper describing amplification of basidiomycete ITS sequences from mycorrhiza samples).[32] Despite shotgun sequencing methods becoming increasingly utilized in microbial sequencing, the low biomass of fungi in clinical samples make the ITS region amplification an area of ongoing research.[33][34]

References

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[edit]
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Internal transcribed spacer

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The internal transcribed spacer (ITS) is a region within the eukaryotic nuclear (rRNA) , situated between the encoding the small-subunit (18S) rRNA and large-subunit (28S) rRNA, and consisting of two spacers—ITS1 and ITS2—flanking the conserved 5.8S rRNA . This region is transcribed as part of the pre-rRNA precursor by but is subsequently cleaved and degraded during rRNA maturation, serving no direct functional role in protein synthesis while exhibiting high sequence variability that reflects divergence. In , the ITS is prized for its rapid and species-specific polymorphisms, enabling precise phylogenetic analyses and taxonomic identification across diverse eukaryotes, with particular prominence in fungi where it functions as the official universal DNA marker due to a clear "barcode gap" between intra- and interspecific variation. Applications extend to , protists, and animals, including cryptic species detection in parasites like digeneans and monogeneans, as well as barcoding in nematodes and mosquitoes, supported by extensive sequence databases such as and UNITE containing over 3.8 million fungal ITS sequences, representing approximately 751,000 hypotheses with at least two sequences, as of November 2025. Despite its utility, challenges arise from intragenomic heterogeneity in multicopy rDNA arrays, which can complicate interpretations in groups like basidiomycetes.

Occurrence Across Life Domains

In Eukaryotes

The internal transcribed spacer (ITS) consists of two sequences, ITS1 and ITS2, situated between the genes encoding the small subunit (18S or SSU) , the 5.8S , and the large subunit (28S or LSU) within the nuclear (rDNA) operons of eukaryotes. These spacers are transcribed as part of a single precursor rRNA molecule and subsequently removed during rRNA maturation. ITS regions occur universally in the nuclear rDNA of all major eukaryotic lineages, encompassing animals (Metazoa), (Viridiplantae), fungi, and protists. This conservation reflects the fundamental role of ribosomal genes across eukaryotic diversity, with over 985,000 ITS1 sequences documented as of 2018 spanning more than 130,000 species in these groups (e.g., as of November 2025, fungal ITS sequences alone exceed 3.8 million in the UNITE database). In terms of length, ITS1 typically spans 150–400 base pairs (bp) and ITS2 150–300 bp across eukaryotes, though 91.7% of ITS1 sequences fall within 100–300 bp. Variations are taxon-specific: in vertebrates and other animals, the spacers are generally shorter (ITS2 averaging ~306 bp) and exhibit lower sequence variability, making them more suitable for resolving higher taxonomic levels rather than species delineation. In contrast, fungal ITS regions often range 500–800 bp overall (including the intervening 5.8S gene) and display high interspecific variability, facilitating species-level identification with a success rate of up to 73%. Plant ITS sequences, averaging 221–260 bp for ITS2, show moderate variability that supports phylogenomic analyses and barcoding across dicotyledons, monocotyledons, gymnosperms, ferns, and mosses. Protist ITS lengths align with the broader eukaryotic range of 100–300 bp, contributing to diversity studies in these unicellular eukaryotes.

In Prokaryotes

In prokaryotes, the internal transcribed spacer (ITS) serves as the intergenic region between the 16S and (rRNA) genes in the ribosomal RNA operon, which typically also encompasses the 5S rRNA gene downstream of the 23S gene. This spacer is transcribed as part of the polycistronic pre-rRNA precursor and subsequently removed during rRNA maturation. Unlike the two ITS regions in eukaryotic nuclear rDNA, prokaryotes feature a single ITS between the 16S and 23S genes, reflecting a more streamlined structure evolved for efficient . In , ITS regions exhibit considerable length variability, often ranging from 200 to 1,500 base pairs (bp) depending on the inclusion of tRNA genes, and frequently incorporate one or more tRNA genes such as tRNAIle (isoleucine) and tRNAAla (alanine). These tRNA genes are interspersed within the spacer and processed separately from the rRNAs. Bacterial genomes often contain multiple rRNA operons—ranging from 1 to 15 copies per chromosome, with harboring seven—leading to potential intra-genomic sequence variability in the ITS due to or copy number effects. Although primarily non-coding in the mature , ITS sequences may harbor regulatory elements influencing operon transcription or processing efficiency. Archaea display a comparable ITS organization within their rRNA s, generally following the 16S-ITS-23S-5S arrangement, though with domain-specific sequence motifs that distinguish them from counterparts, such as unique promoter elements or processing signals in euryarchaeotes. Like , archaeal genomes typically possess 1 to 4 operon copies, contributing to similar intra-genomic heterogeneity. A representative bacterial example is found in , where type B operons (rrnA, rrnE, rrnH) feature an ITS containing both tRNAIle and tRNAAla genes, while type A operons (rrnB, rrnC, rrnD, rrnG) have a shorter spacer with only tRNAGlu (glutamate). In mycobacteria, the ITS region's sequence variability has been exploited for strain differentiation, as demonstrated by sequevar analysis in avium isolates, where distinct ITS types correlate with clinical and environmental strain groupings.

Structural Organization

Composition of ITS Regions

The internal transcribed spacer (ITS) regions consist of two non-coding sequences within the (rRNA) precursor transcript: ITS1 and ITS2. ITS1 is located between the small subunit ( gene—18S in eukaryotes and 16S in prokaryotes—and the 5.8S rRNA gene, while ITS2 lies between the 5.8S rRNA gene and the large subunit (LSU) rRNA gene—28S in eukaryotes and 23S in prokaryotes. In prokaryotes, the ITS often includes one or more tRNA genes, such as those encoding tRNAIle and tRNAAla. These spacers are flanked by highly conserved sequences from the adjacent rRNA genes, which define their precise boundaries: the 3' end of the marks the start of ITS1, and the 5' end of the 5.8S rRNA marks its end, whereas the 3' end of the 5.8S rRNA delineates the beginning of ITS2, and the 5' end of the LSU rRNA its conclusion. ITS1 is delimited by the 3' end of the gene and the 5' end of the 5.8S rRNA gene, with conserved sequences serving as recognition sites for endonucleolytic cleavage during rRNA processing. In contrast, ITS2 commonly exhibits a conserved secondary featuring multiple loops, which contribute to its stability and are preserved across eukaryotic lineages despite sequence divergence. These structural motifs, including four helices in eukaryotes, are integral to the spacer's architecture and are identifiable through comparative . The composition of ITS regions is generally AT-rich in many taxa, reflecting a bias toward and bases that facilitates rapid and processing. varies significantly by domain and group; for instance, in fungi, it typically ranges from 40% to 60%, with ITS2 often showing higher GC levels than ITS1 (e.g., means of approximately 54% and 59% in major fungal clades like ). Lengths of these regions also differ across life domains, with prokaryotic ITS regions exhibiting considerable length variation (typically 60–1,500 or more), generally shorter than many eukaryotic ones (which can exceed 1,000 ).

Sequence Variability and Conservation

The internal transcribed spacers (ITS1 and ITS2) exhibit high inter-specific variability, attributed to their rapid evolutionary rates, which makes them particularly suitable for resolving relationships at the species level across eukaryotes. This variability arises from minimal functional constraints on the non-coding spacers, allowing for accumulation of and indels that distinguish closely related taxa, with ITS1 often showing greater heterogeneity and divergence than ITS2 in many lineages such as fungi and . For instance, in fungal species, inter-specific differences in ITS can exceed 20-30% , enabling precise taxonomic discrimination where conserved rRNA genes fail. Despite this variability, ITS regions contain conserved secondary structural elements that facilitate comparative analyses, particularly in ITS2, which typically folds into a four-helix core structure (helices I-IV) with III being the longest and most stable. These helices represent pan-eukaryotic homologies, where base-pairing patterns are more conserved than primary , allowing reliable alignments even among highly divergent taxa such as across metazoans and fungi. Helices I and IV, while variable in length and , contribute to species-specific signatures, whereas the overall scaffold supports phylogenetic by compensating for alignment ambiguities in primary data. In eukaryotes, ITS lengths vary by domain—for example, shorter in fungi (ITS1 ≈ 150-250 bp, ITS2 ≈ 150-200 bp) compared to longer spacers in —but the secondary structure conservation persists. Intra-genomic polymorphism within ITS arrays is generally low due to concerted evolution, a process driven by mechanisms such as unequal crossing-over and gene conversion that homogenize sequences across multiple rDNA copies on chromosomes. This homogenization minimizes intra-individual variation, ensuring that ITS sequences from a single are largely identical and representative for phylogenetic studies. However, exceptions occur in polyploids or hybrid organisms, where incomplete concerted evolution can lead to detectable polymorphisms, reflecting recent genome duplication or inter-species hybridization events that disrupt homogenization. At the boundaries of ITS regions, specific conserved motifs provide anchors for sequence delimitation and evolutionary comparisons; for example, the 5.8S-ITS2 in fungi features a highly conserved restriction site (GAA/TTC) within motif II of the 5.8S gene, which aids in distinguishing fungal sequences from other eukaryotes. This junction also participates in base-pairing interactions during rRNA , underscoring its structural conservation across ascomycetes and basidiomycetes. Such motifs, while short, are invariant in many fungal clades, serving as diagnostic elements amid the surrounding spacer variability.

Biological Role in rRNA Maturation

Transcription and Processing Mechanism

In eukaryotes, the internal transcribed spacers (ITS1 and ITS2) are transcribed as part of a large precursor ribosomal RNA (pre-rRNA) by RNA polymerase I from tandemly repeated ribosomal DNA (rDNA) arrays in the nucleolus. This transcription produces a polycistronic 35S pre-rRNA in yeast (or 47S in mammals), which encompasses the 5' external transcribed spacer (5' ETS), the 18S rRNA, ITS1, the 5.8S rRNA, ITS2, the 25S rRNA (28S in mammals), and the 3' ETS. The process is tightly coupled with nascent pre-rRNA folding and assembly of ribosomal proteins and factors to form the small subunit (SSU) processome. Processing of the pre-rRNA begins co- or post-transcriptionally with endonucleolytic cleavages to remove the spacers, followed by exonucleolytic trimming to generate mature rRNAs. The initial step involves removal of the 5' ETS through cleavages at sites A0 and A1, directed by the U3 small nucleolar ribonucleoprotein (snoRNP), which base-pairs with the pre-rRNA to facilitate access by endonucleases such as Rnt1 in . Subsequent cleavage at site A2 within ITS1, also guided by U3 snoRNP, separates the pre-18S rRNA (20S in ) from the pre-60S intermediates, marking the separation of SSU and large subunit (LSU) biogenesis pathways; the 3' end of the 18S rRNA is later finalized by the endonuclease Nob1 in the . ITS2 excision occurs later in the 27S pre-rRNA (LSU precursor) via endonucleolytic cleavage at site C2, catalyzed by the Las1 endonuclease as part of the Las1-Las2 complex, which generates a 7S pre-5.8S rRNA and a pre-25S rRNA. This is followed by exonucleolytic trimming: the nuclear (including Rrp44, Rrp6, and the helicase Mtr4) degrades the remnant after 5.8S in the 7S species, while the Rat1-Rai1 trims the 3' end of the pre-25S to mature 25S rRNA; additional cytoplasmic like Ngl2 finalize the 5.8S 3' end. These steps ensure precise maturation while degrading the ITS sequences. In prokaryotes, rRNA transcription and processing differ markedly, occurring in the cytoplasm without a dedicated nucleolus and involving RNA polymerase holoenzyme with sigma factors rather than polymerase I. The primary transcript is a 30S precursor polycistron containing 16S rRNA, an intergenic spacer (analogous to ITS1), 23S rRNA, another spacer (analogous to ITS2), and 5S rRNA, often with interspersed tRNA genes. Unlike eukaryotic ITS, these prokaryotic spacers may contain regulatory sequences such as promoters for downstream operons or tRNA processing sites. Processing is rapid and initiated by the double-strand-specific endonuclease RNase III, which cleaves at stem-loop structures flanking the mature rRNA sequences to release precursor 16S and 23S rRNAs, excising the spacers as byproducts. Subsequent exonucleolytic trimming by enzymes like RNase T matures the ends, while any embedded tRNAs are processed separately by RNase P and other factors, enabling quick ribosome assembly under varying growth conditions.

Evolutionary and Functional Significance

The internal transcribed spacers (ITS) in (rDNA) exhibit evolutionary conservation primarily due to their physical linkage with essential rRNA genes, which are critical for and thus under strong purifying selection across taxa. This linkage ensures that the entire pre-rRNA transcript, including ITS regions, is maintained as a functional unit despite sequence divergence in the non-coding spacers. However, ITS sequences evolve more rapidly than the conserved rRNA coding regions, accumulating neutral mutations that reflect lineage-specific divergences without compromising overall rDNA integrity. Such variability arises from relaxed selective pressure in the spacers, allowing them to serve as repositories for neutral genetic changes during . Beyond their role in rRNA processing—where ITS regions form secondary structures essential for endonucleolytic cleavage—rDNA clusters containing ITS serve as hotspots for chromosomal rearrangements, facilitating unequal crossing-over and non-allelic that drive structural polymorphisms and evolution. For instance, in mammals, these hotspots contribute to copy number instability and pericentromeric repositioning, underscoring their impact on plasticity. Inter-domain comparisons highlight distinct organizational strategies: eukaryotic rDNA features tandemly arrayed repeats with multiple ITS copies per locus, enabling concerted evolution through recombination, whereas prokaryotic rRNA operons are typically dispersed across the genome with simpler intergenic spacers lacking the extensive ITS complexity. Horizontal transfer of rDNA units, including ITS, is rare but documented, for example between phylogenetically distinct grass lineages. These events can introduce variant spacers, influencing host ribosomal adaptation. In , ITS-embedded rDNA plays a key role in nucleolar dominance, where epigenetic silencing of select loci ensures balanced rRNA production amid . fluctuations, often exceeding hundreds of units per , are maintained through mechanisms like unequal sister exchange, with ITS sequences homogenizing via concerted to preserve functional uniformity. This dynamic regulation links rDNA variability to broader adaptive processes, such as stress responses and hybrid incompatibility.

Applications in Molecular Identification

Phylogenetic Inference

The internal transcribed spacer (ITS) region functions as an effective marker for phylogenetic inference owing to its moderate evolutionary rate, which lies between the highly conserved ribosomal RNA (rRNA) genes and the more rapidly evolving protein-coding genes, enabling resolution of relationships at intermediate taxonomic scales such as genera and families in fungi. This positioning allows ITS to serve in multi-gene phylogenetic studies, complementing slower-evolving loci for broader evolutionary insights while providing sufficient variability for finer-scale analyses. Phylogenetic reconstruction using ITS typically begins with informed by secondary structure models, which predict folding patterns to improve positional homology and accuracy, particularly for the variable ITS2 subdomain. Tools like 4SALE facilitate this structure-aware alignment, followed by model-based inference methods such as maximum likelihood, often employing the General Time Reversible (GTR) with gamma-distributed rate variation across sites (GTR + G) to account for heterogeneous evolutionary rates and estimate tree topologies. ITS excels in resolving recent evolutionary divergences, such as genus-level relationships in fungal lineages, due to its high sequence variability that distinguishes closely related taxa with a success rate exceeding 70% at the genus rank. However, its utility diminishes for deep phylogenies, where rapid evolution leads to substitution saturation—multiple changes at the same sites that obscure ancient signals and complicate alignments across diverse taxa. The adoption of ITS for fungal began in the 1990s, with early applications in diversity studies and tree-building that revolutionized mycological , amassing over 172,000 sequences in public repositories by the early . Today, it remains a of eukaryotic phylogenomics, exemplified by the UNITE database, which curates reference ITS sequences clustered into hypotheses to support taxonomic communication and evolutionary analyses across fungi and other eukaryotes. As of 2024, it contains nearly 10 million ITS sequences clustered into approximately 2.4 million hypotheses.

Taxonomic Barcoding and Species Delimitation

The Internal Transcribed Spacer (ITS) region serves as the official DNA barcode marker for fungi, formally proposed and adopted in 2012 by the Fungal Barcoding Consortium under the Consortium for the Barcode of Life due to its superior discriminatory power across diverse fungal lineages. This designation was based on comprehensive evaluations showing ITS outperforms other candidates like the large subunit rRNA (LSU) and RNA polymerase II subunit (RPB2) in species identification, with a probability of correct identification (PCI) of approximately 73% in (including 71% in ) and 77% in . These success rates reflect ITS's ability to reveal a clear gap between intraspecific and interspecific variation, enabling reliable discrimination in the two largest fungal phyla, though performance can vary in less-studied groups like early-diverging fungal lineages (such as Mucoromycota). The typical workflow for ITS-based barcoding begins with from fungal specimens, followed by PCR amplification of the core ITS region (ITS1-5.8S-ITS2) using universal primers such as ITS5 and ITS4 or ITS1F and ITS4. Amplified products are then sequenced, often via for targeted identification, and the resulting sequences are queried against public reference databases like the (BOLD) or using tools such as BLAST for similarity matching. This process facilitates rapid species assignment by aligning query sequences to vouchered references, supporting high-throughput identification in field-collected samples. For species delimitation, ITS sequences are analyzed using threshold-based approaches, where genetic similarities of 97-99% are commonly applied to cluster sequences into operational taxonomic units (OTUs), reflecting observed intraspecific variation in many fungal taxa. More sophisticated methods integrate phylogenetic information, such as the model, which recursively partitions sequences based on detected gaps in pairwise distances without requiring a tree, or the model, which uses ultrametric phylogenies to distinguish coalescent processes from events while accounting for intraspecific polymorphism. These integrative techniques enhance resolution of cryptic species, particularly in complexes with low interspecific divergence. ITS barcoding finds extensive application in mycological surveys to catalog fungal in ecosystems like forests and soils, where it aids in documenting overlooked . In , it is pivotal for identifying plant s, enabling early detection and management of diseases caused by fungi such as or Botrytis species through sequence matching to pathogen databases. Beyond fungi, ITS has been extended as a supplementary in select algae, such as dinoflagellates and , where it complements markers like rbcL for resolving closely related taxa, and in certain like nematodes, supporting identification in parasitic or soil-dwelling contexts.

Methodological Considerations

Sequencing Techniques

The sequencing of internal transcribed spacer (ITS) regions begins with DNA extraction from diverse biological sources, such as fresh tissues, herbarium specimens, or environmental samples like soil and water. For herbarium specimens, which often contain degraded DNA due to age and preservation methods, modified cetyltrimethylammonium bromide (CTAB) protocols or commercial kits like the DNeasy Plant Mini Kit are commonly employed to yield sufficient quantities for downstream amplification, with success rates improving when using silicon dioxide-based purification to remove inhibitors. Environmental DNA (eDNA) extraction typically involves kits optimized for low-biomass samples, such as the PowerSoil Kit, followed by concentration steps to enhance recovery from complex matrices. Amplification of ITS regions is achieved through (PCR) using taxon-specific primers. In eukaryotes, particularly fungi, the forward primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and reverse primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3') target the ITS1, 5.8S rRNA, and ITS2 regions, producing amplicons of approximately 600-800 base pairs depending on the species. These primers, originally designed for fungal , exhibit broad universality across eukaryotic groups due to conserved flanking sequences in the small and large subunit rRNA genes. For prokaryotes, primers such as 1406f (5'-TGYACACACCGCCCGT-3') and 23Sr (5'-GGGTTBCCCCATTCRG-3') amplify the 16S-23S ITS region, which varies in length from 200-600 base pairs and includes tRNA genes, enabling species-level resolution in . PCR reactions employ high-fidelity DNA polymerases, such as Phusion or Q5, to minimize errors during amplification, with standard conditions including an initial denaturation at 98°C for 30 seconds, followed by 30-35 cycles of denaturation at 98°C for 10 seconds, annealing at 50-55°C for 30 seconds, and extension at 72°C for 30-60 seconds, concluding with a final extension at 72°C for 5 minutes. Following PCR, sequencing approaches vary by application scale. For targeted sequencing of individual samples or clones, Sanger sequencing is preferred, providing high-accuracy reads up to 1000 base pairs directly from purified PCR products, often using the same ITS1 and ITS4 primers for cycle sequencing. In metagenomic studies, next-generation sequencing (NGS) platforms like the Illumina MiSeq are widely used, generating millions of short reads (250-500 base pairs) from barcoded amplicon libraries to profile diverse communities; demultiplexing is performed post-sequencing using index sequences to assign reads to samples. This NGS method supports high-throughput analysis but requires careful library preparation to avoid biases from primer mismatches in variable ITS regions.

Analysis Challenges and Best Practices

Analysis of internal transcribed spacer (ITS) sequences presents several computational and interpretive challenges that can compromise the accuracy of fungal identification and community profiling. One major issue is the formation of chimeric sequences during PCR amplification, which arise from template switching and can lead to artificial recombinants mimicking novel taxa. These chimeras are particularly prevalent in high-diversity environmental samples and can inflate diversity estimates if undetected. Another challenge is intra-genomic variability within the multi-copy rDNA , where concerted evolution is incomplete, resulting in sequence polymorphisms that may represent true biological variation or sequencing artifacts, complicating species delimitation. Additionally, incompleteness in reference databases, such as UNITE, hinders taxonomic assignment, as many fungal lineages lack representative sequences, leading to unclassified operational taxonomic units (OTUs) or misassignments. To address these challenges, best practices emphasize rigorous preprocessing and . Chimeras can be detected and removed using algorithms like UCHIME, which outperforms earlier methods in sensitivity for de novo and reference-based detection, especially in noisy datasets. Intra-genomic variability is mitigated through of PCR products for or by employing long-read technologies like PacBio or Oxford Nanopore, which resolve full operons and haplotypes without amplification biases. For next-generation sequencing (NGS) data, error correction pipelines such as DADA2 model substitution and indel errors to infer amplicon sequence variants (ASVs) at single-nucleotide resolution, reducing false positives from polymerase errors. Structure-based alignment improves handling of the variable ITS regions; tools like ITSx extract ITS1, 5.8S, and ITS2 subregions using hidden Markov models trained on conserved flanking sequences, enhancing alignment accuracy over generic methods. is then performed with software such as MAFFT, which employs for rapid, high-quality alignments of divergent ITS sequences. Comprehensive pipelines like QIIME 2 integrate these steps, including primer trimming and taxonomic assignment, tailored for fungal ITS via plugins like ITSxpress. For OTU-based approaches, clustering at 97% sequence identity remains a standard threshold for fungal ITS, balancing species-level resolution with over-splitting due to variability, as validated in diverse ascomycete lineages. In , homopolymers—common in ITS2—pose interpretation difficulties due to polymerase slippage, manifesting as ambiguous peaks in chromatograms; these are resolved by sequencing both strands and manually verifying base calls beyond the repeat . Quality metrics ensure reliability: sequences should exceed 500 base pairs in length to cover the full ITS adequately, with Phred scores >Q20 across 80% of bases, and all data linked to vouchered specimens for and validation against morphological traits. Adhering to these practices minimizes artifacts and supports robust downstream applications in fungal and .

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