Internal transcribed spacer

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.

ITS across life domains

In bacteria and archaea, there is a single ITS, located between the 16S and 23S rRNA genes. Contrastingly, 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

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.

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.[3]

During rRNA maturation, ETS and ITS pieces are excised. As non-functional by-products of this maturation, they are rapidly degraded.[4]

Use in phylogeny

Sequence comparison of the ITS region is widely used in taxonomy and molecular phylogeny because of several favorable properties:[5]

• 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.[6]
• 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.[7]
Viscaceae: Arceuthobium	Species (congeneric)	1994	Nickrent et al.[8]
Poaceae: Zea	Species (congeneric)	1996	Buckler & Holtsford[9]
Leguminosae: Medicago	Species (congeneric)	1998	Bena et al.[3]
Orchidaceae: Diseae	Genera (within tribes)	1999	Douzery et al.[10]
Odonota: Calopteryx	Species (congeneric)	2001	Weekers et al.[11]
Yeasts of clinical importance	Genera	2001	Chen et al.[12]
Poaceae: Saccharinae	Genera (within tribes)	2002	Hodkinson et al.[13]
Plantaginaceae: Plantago	Species (congeneric)	2002	Rønsted et al.[14]
Jungermanniopsida: Herbertus	Species (congeneric)	2004	Feldberg et al.[15]
Pinaceae: Tsuga	Species (congeneric)	2008	Havill et al.[16]
Chrysomelidae: Altica	Genera (congeneric)	2009	Ruhl et al.[17]
Symbiodinium	Clade	2009	Stat et al.[18]
Brassicaceae	Tribes (within a family)	2010	Warwick et al.[19]
Ericaceae: Erica	Species (congeneric)	2011	Pirie et al.[20]
Diptera: Bactrocera	Species (congeneric)	2014	Boykin et al.[21]
Scrophulariaceae: Scrophularia	Species (congeneric)	2014	Scheunert & Heubl[22]
Potamogetonaceae: Potamogeton	Species (congeneric)	2016	Yang et al.[23]

Mycological barcoding

Main article: Fungal DNA barcoding

The ITS region is the most widely sequenced DNA region in molecular ecology of fungi[24] and has been recommended as the universal fungal barcode sequence.[25] 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[26] 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).[27] 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.[28][29]

References

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3. Bena, Gilles; Jubier, Marie-France; Olivieri, Isabelle; Lejeune, Bernard (1998). "Ribosomal External and Internal Transcribed Spacers: Combined Use in the Phylogenetic Analysis of Medicago (Leguminosae)". Journal of Molecular Evolution. 46 (3): 299–306. Bibcode:1998JMolE..46..299B. doi:10.1007/PL00006306. ISSN 0022-2844. PMID 9502673.
4. Michot, Bernard; Bachellerie, Jean-Pierre; Raynal, Francoise (1983-05-25). "Structure of mouse rRNA precursors. Complete sequence and potential folding of the spacer regions between 18S and 28S rRNA". Nucleic Acids Research. 11 (10): 3375–3391. doi:10.1093/nar/11.10.3375. ISSN 0305-1048. PMC 325970. PMID 6304630.
5. Baldwin, Bruce G.; Sanderson, Michael J.; Porter, J. Mark; Wojciechowski, Martin F.; Campbell, Christopher S.; Donoghue, Michael J. (1995-01-01). "The ITS Region of Nuclear Ribosomal DNA: A Valuable Source of Evidence on Angiosperm Phylogeny". Annals of the Missouri Botanical Garden. 82 (2): 247–277. doi:10.2307/2399880. JSTOR 2399880.
6. Song, Jingyuan; Shi, Linchun; Li, Dezhu; Sun, Yongzhen; Niu, Yunyun; Chen, Zhiduan; Luo, Hongmei; Pang, Xiaohui; Sun, Zhiying (2012-08-30). "Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA". PLOS ONE. 7 (8): e43971. Bibcode:2012PLoSO...743971S. doi:10.1371/journal.pone.0043971. ISSN 1932-6203. PMC 3431384. PMID 22952830.
7. Baldwin, B.G. (1992). "Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: An example from the Compositae". Molecular Phylogenetics and Evolution. 1 (1): 3–16. doi:10.1016/1055-7903(92)90030-K. PMID 1342921.
8. Nickrent, Daniel L.; Schuette, Kevin P.; Starr, Ellen M. (1994-01-01). "A Molecular Phylogeny of Arceuthobium (Viscaceae) Based on Nuclear Ribosomal DNA Internal Transcribed Spacer Sequences". American Journal of Botany. 81 (9): 1149–1160. doi:10.2307/2445477. JSTOR 2445477.
9. Buckler, E. S.; Holtsford, T. P. (1996-04-01). "Zea systematics: ribosomal ITS evidence". Molecular Biology and Evolution. 13 (4): 612–622. doi:10.1093/oxfordjournals.molbev.a025621. ISSN 0737-4038. PMID 8882504.
10. Douzery, Emmanuel J. P.; Pridgeon, Alec M.; Kores, Paul; Linder, H. P.; Kurzweil, Hubert; Chase, Mark W. (1999-06-01). "Molecular phylogenetics of Diseae (Orchidaceae): a contribution from nuclear ribosomal ITS sequences". American Journal of Botany. 86 (6): 887–899. doi:10.2307/2656709. ISSN 0002-9122. JSTOR 2656709. PMID 10371730.
11. Weekers, Peter H. H.; De Jonckheere, Johan F.; Dumont, Henri J. (2001-07-01). "Phylogenetic Relationships Inferred from Ribosomal ITS Sequences and Biogeographic Patterns in Representatives of the Genus Calopteryx (Insecta: Odonata) of the West Mediterranean and Adjacent West European Zone". Molecular Phylogenetics and Evolution. 20 (1): 89–99. doi:10.1006/mpev.2001.0947. PMID 11421650.
12. Chen, Y-C, J. D. Eisner, M. M. Kattar, S. L. Rassoulian-Barrett, K. Lafe, A. P. Limaye, and B. T. Cookson (2001). "Polymorphic Internal Transcribed Spacer Region 1 DNA Sequences Identify Medically Important Yeasts". J. Clin. Microbiol. 39 (11): 4042–4051. doi:10.1128/JCM.39.11.4042-4051.2001. PMC 88485. PMID 11682528.CS1 maint: multiple names: authors list (link)
13. Hodkinson, Trevor R.; Chase, Mark W.; Lledó, Dolores M.; Salamin, Nicolas; Renvoize, Stephen A. (2002). "Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers". Journal of Plant Research. 115 (5): 381–392. doi:10.1007/s10265-002-0049-3. ISSN 0918-9440. PMID 12579363.
14. Rønsted, Nina; Chase, Mark W.; Albach, Dirk C.; Bello, Maria Angelica (2002-08-01). "Phylogenetic relationships within Plantago (Plantaginaceae): evidence from nuclear ribosomal ITS and plastid trnL-F sequence data". Botanical Journal of the Linnean Society. 139 (4): 323–338. doi:10.1046/j.1095-8339.2002.00070.x. ISSN 1095-8339.
15. Feldberg, K.; Groth, H.; Wilson, R.; Schäfer-Verwimp, A.; Heinrichs, J. (2004-11-04). "Cryptic speciation in Herbertus (Herbertaceae, Jungermanniopsida): Range and morphology of Herbertus sendtneri inferred from nrITS sequences". Plant Systematics and Evolution. 249 (3–4): 247–261. doi:10.1007/s00606-004-0221-4. ISSN 0378-2697.
16. Havill, Nathan P.; Campbell, Christopher S.; Vining, Thomas F.; LePage, Ben; Bayer, Randall J.; Donoghue, Michael J. (2008-07-01). "Phylogeny and Biogeography of Tsuga (Pinaceae) Inferred from Nuclear Ribosomal ITS and Chloroplast DNA Sequence Data". Systematic Botany. 33 (3): 478–489. doi:10.1600/036364408785679770.
17. Ruhl, Michael W.; Wolf, Matthias; Jenkins, Tracie M. (2010). "Compensatory base changes illuminate morphologically difficult taxonomy". Molecular Phylogenetics and Evolution. 54 (2): 664–669. doi:10.1016/j.ympev.2009.07.036.
18. Stat, Michael; Pochon, Xavier (2008-07-02). "Specificity in communities of Symbiodinium in corals from Johnston Atoll" (PDF). Marine Ecology Progress Series. 386: 83–96. doi:10.3354/meps08080.
19. Warwick, Suzanne I.; Mummenhoff, Klaus; Sauder, Connie A.; Koch, Marcus A.; Al-Shehbaz, Ihsan A. (2010-04-13). "Closing the gaps: phylogenetic relationships in the Brassicaceae based on DNA sequence data of nuclear ribosomal ITS region". Plant Systematics and Evolution. 285 (3–4): 209–232. doi:10.1007/s00606-010-0271-8. ISSN 0378-2697.
20. Pirie, Michael D.; Oliver, E. G. H.; Bellstedt, Dirk U. (2011-11-01). "A densely sampled ITS phylogeny of the Cape flagship genus Erica L. suggests numerous shifts in floral macro-morphology". Molecular Phylogenetics and Evolution. 61 (2): 593–601. doi:10.1016/j.ympev.2011.06.007. PMID 21722743.
21. Boykin, L. M.; Schutze, M. K.; Krosch, M. N.; Chomič, A.; Chapman, T. A.; Englezou, A.; Armstrong, K. F.; Clarke, A. R.; Hailstones, D. (2014-05-01). "Multi-gene phylogenetic analysis of south-east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy". Journal of Applied Entomology. 138 (4): 235–253. doi:10.1111/jen.12047. ISSN 1439-0418.
22. Scheunert, Agnes; Heubl, Günther (2014-01-01). "Diversification of Scrophularia (Scrophulariaceae) in the Western Mediterranean and Macaronesia – Phylogenetic relationships, reticulate evolution and biogeographic patterns". Molecular Phylogenetics and Evolution. 70: 296–313. doi:10.1016/j.ympev.2013.09.023. PMID 24096055.
23. Yang, Tao; Zhang, Tian-lei; Guo, You-hao; Liu, Xing (2016-11-17). "Identification of Hybrids in Potamogeton: Incongruence between Plastid and ITS Regions Solved by a Novel Barcoding Marker PHYB". PLOS ONE. 11 (11): e0166177. Bibcode:2016PLoSO..1166177Y. doi:10.1371/journal.pone.0166177. ISSN 1932-6203. PMC 5113904. PMID 27855191.
24. Peay K.G.; Kennedy P.G.; Bruns T.D. (2008). "Fungal community ecology: a hybrid beast with a molecular master". BioScience. 58 (9): 799–810. doi:10.1641/b580907.
25. Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W., Bolchacova, E., Voigt, K., Crous, P.W.; et al. (2012). "Nuclear Ribosomal Internal Transcribed Spacer (ITS) Region as a Universal DNA Barcode Marker for Fungi". PNAS. 109 (16): 6241–6246. doi:10.1073/pnas.1117018109. PMC 3341068. PMID 22454494.CS1 maint: multiple names: authors list (link)
26. White, T.J., Bruns, T., Lee, S., and Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: a Guide to Methods and Applications 18, 315–322.
27. Gardes, M.; Bruns, T.D. (1993). "ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhiza and rusts". Molecular Ecology. 2 (2): 113–118. doi:10.1111/j.1365-294X.1993.tb00005.x. PMID 8180733.
28. Usyk, Mykhaylo; Zolnik, Christine P.; Patel, Hitesh; Levi, Michael H.; Burk, Robert D. (2017-12-13). Mitchell, Aaron P. (ed.). "Novel ITS1 Fungal Primers for Characterization of the Mycobiome". mSphere. 2 (6): e00488–17, /msphere/2/6/mSphere0488–17.atom. doi:10.1128/mSphere.00488-17. ISSN 2379-5042. PMC 5729218. PMID 29242834.
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External links

• University of Washington Laboratory Medicine: Molecular Diagnosis | Yeast Sequencing