Nucleotide variation and secondary structures of nuclear ribosomal ITS2 in phylogeny relationships of some wheat species

Document Type : Research paper


Department of Genetics and Plant Breeding, Imam Khomeini International University, P. O. Box: 34148-96818, Qazvin, Iran.


This research aimed to estimate the nucleotide polymorphism and point mutation/substitutions for ITS2 region in 12 different Triticum and Aegilops species, and to demonstrate their phylogenetic relationships. The aligned ITS2 dataset showed a total of 67 SNPs and transitions (75%) predominant over transversions (25%). The C/T transitions were observed at highest level in the studied species. The highest nucleotide substitutions occurred in ITS2 sequences of T. aestivum, T. turgidum, and A. speltoides that showed similar substitutions on the base sites 76, 193, 215, 230 and 241 (TCCAG changed to ATTTA). T. boeticum and T. urartu showed similar substitutions on the base sites 85, 201, 231, 234 and 252 (GTGCC changed to AGATA). Remarkably, the nucleotide of base site 146 was C in all Triticum species, but T in all Aegilops species. The substitution rate in A. neglecta, and A. umbellulata was zero, it was concluded that their ITS2 is conserved. The secondary structures of ITS2 in the studied species was similar to that of the other flowering plants with four helices and the variation in helices length. The helix III had the longest length compared to the other helices. The length ranges of helices I, II, IV varied from 9 (T. turgidum, and T. urartu) to 10, 11 (A. cylindrica) 13 and 6 (A. speltoides) and 10 (T. boeticum) paired bases, respectively. Using pairwise ITS2 nucleotide comparisons, the closeness of A. caudata with A. cylindrica, T. boeticum with T. urartu, A. neglecta with A. umbellulata was observed. Phylogeny trees classified 12 species into four main clades. The highest point mutations occurred in ITS2 sequences related to the species grouped in the first clade.


Aguilar C., and Sanchez J. A. (2007). Phylogenetic hypotheses of Gorgonii doctocorals according to ITS2 and their predicted RNA secondary structures. Molecular Phylogenetics and Evolution, 43: 774–786.
Alnaddaf L. M., Moualla M. Y., and Haider N. (2012). Resolving genetic relationships among Aegilops L. and Triticum L. species using analysis of chloroplast DNA by Cleaved Amplified Polymorphic Sequence (CAPS). Asian Journal of Agricultural Science, 4: 270–279.
Alvarez I., and Wendel J. F. (2003). Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution, 29(3): 417–434.
Ankenbrand M. J., Keller A., Wolf M., Schultz J., and Förster F. (2015). ITS2 database V: Twice as much. Molecular Biology and Evolution, 32(11): 3030–32. DOI: https://doi:10.1093/molbev/msv174.
Balasubramani S. P., Murugan R., Ravikumar K., and Venkatasubramanian P. (2010). Development of ITS sequence based molecular marker to distinguish Tribulu sterrestris L. (Zygophyllaceae) from its adulterants. Fitoterapia, 81(6): 503–508. DOI: 10.1016/j.fitote.2010.01.002.
Banchi E., Ametrano C. G., Greco S., Stankovic D., Muggia L., and Pallavicini A. (2020). PLANiTS: a curated sequence reference dataset for plant ITS DNA metabarcoding, Database, 2020: 1–9. DOI: 10.1093/database/baz155.
Bordbar F., Rahiminejad M. R., Saeidi H., and Blattner F. R. (2011). Phylogeny and genetic diversity of D-Genome species of Aegilops and Triticum (Triticeae, Poaceae) from Iran based on microsatellites, ITS, and trnL-F. Plant Systematics and Evolution, 291: 117–131.
Chen Q. F. (1997). Inquisition about the origin and evolution of wheat genomes. Guihaia, 17: 276–282.
Chen S. L., Yao H., Han J. P., Liu C., and Song J. Y. (2010). Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One, 5(1): e8613. DOI:
Coleman A. W. (2003). ITS2 is a double-edged tool for eukaryote evolutionary comparisons. Trends in Genetics, 19: 370–375.
Coleman A. W. (2007). Pan-eukaryote ITS2 homologies revealed by RNA secondary structure. Nucleic Acids Research, 35: 3322-3329. DOI: 10.1093/nar/gkm233.
Coleman A. W. (2009). Is there a molecular key to the level of ‘‘biological species’’ in eukaryotes? Molecular Phylogenetics and Evolution, 50: 197–203. DOI: 10.1016/j.ympev.2008.10.008.
Coleman A. W., and Mai J. C. (1997). Ribosomal DNA ITS-1 and ITS-2 sequence comparisons as a tool for predicting genetic relatedness. Journal of Molecular Evolution, 45: 168–177.
Coleman A. W., Preparata R. M., Mehrotra B., and Mai J. C. (1998). Derivation of the secondary structure of the ITS-1 transcript in Volvocales and its taxonomical correlations. Protist, 149: 135–146.
Duan H., Wang W., Zeng Y. Gue M., Zhou Y. (2019). The screening and identification of DNA barcode sequences for Rehmannia. Scientific Reports, 9: 17295. DOI:
Dvogak J., Terlizzi P., Zheng H. B., and Resta P. (1993). The evolution of polyploidwheats: identification of the A genome donor species. Genome, 36: 21–30.
Feliner G. N., and Rossello J. A. (2007). Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Molecular Phylogenetics and Evolution, 44(2): 911–919. DOI: 10.1016/j.ympev.2007.01.013.
Goertzen L. R., Cannone J. J., Gutell R. R., and Jansen R. K. (2003). ITS secondary structure derived from comparative analysis: implications for sequence alignment and phylogeny of the Asteraceae. Molecular Phylogenetics Evolution, 29: 216–234.
Golovnina K. A., Kondratenko E. Y., Blinov A. G., and Goncharov N. P. (2009). Phylogeny of the a genomes of wild and cultivated wheat species. Russian Journal of Genetics, 45: 1360–1367.
Gottschling M., Hilger H. H., Wolf M., and Diane N. (2001). Secondary structure of the ITS1 transcript and its application in a reconstruction of the phylogeny of Boraginales. Plant Biology, 3: 629–636.
Gulbitti-Onarici S., Sancak C., Sumer S., and Ozcan S. (2009). Phylogenetic relationships of some wild wheat species based on the internal transcribed spacer sequences of nrDNA. Current Science, 96 (60): 794–800.
Haider N. (2013). The origin of the B genome of bread wheat (Triticum aestivum
L.). Russian Journal of Genetics, 49: 263–274.
Hillis D. M., and Dixon M. T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology, 66: 411–453.
Jones B. L., Lookhart G. L., Mak A., and Cooper D. B. (1982). Sequences of purothionins and their inheritance in diploid, tetraploid, and hexapliod wheats. Journal of Heredity, 73: 143–144.
Joseph N., Krauskopf E., Vera M. I., and Michot B. (1999). Ribosomal internal transcribed spacer 2 (ITS2) exhibits a common core of secondary structure in vertebrates and yeast. Nucleic Acids Research, 27: 4533–4540.
Keller A., Forster F., Muller T., Dandekar T., and Schultz J. (2010). Including RNA secondary structures improves accuracy and robustness in reconstruction of phylogenetic trees. Biology Direct, 5: 4. DOI: 10.1186/1745-6150-5-4. PMID: 20078867.
Keller A., Schleicher T., Forster F., Ruderisch B., Dandekar T., Muller T., and Wolf M. (2008). ITS2 data corroborate a monophyletic chlorophycean group (Sphaeropleales). Biomed Central Evolutionary Biology, 8: 218. DOI:
Kerby K., and Kuspira J. (1987). The phylogeny of polyploidy wheats Triticum aestivum (bread wheat) and Triticum turgidum (macaroni wheat). Genome, 29: 722–737.
Kilian B., Mammen, K. Millet E., Sharma R., Graner A., Salamini F., Hammer K., and Ozkan H. (2011). Aegilops. In: Kole C. (Eds.) Wild Crop Relatives: Genomic and Breeding Resources. Springer, Berlin, Heidelberg, 1–76. DOI:
Konarev V. G. (1983). The nature and origin of wheat genomes on the data of grain protein immunochemistry and electrophoresis. In proceeding of 6th International Wheat Genetics Symposium, Kyoto, Japan, 65–75.
Kumar S., Stecher G., and Tamura K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33: 1870–1874.
Lenaers G., Maroteaux L., Michot B., and Herzog M. (1989). Dinoflagellates in evolution. A molecular phylogenetic analysis of large subunit ribosomal RNA. Journal of Molecular Evolution, 29: 40–51.
Liu J. S., and Schardl C., L. (1994). A conserved sequence in internal transcribed spacer 1 of plant nuclear rRNA genes. Plant Molecular Biology, 26: 775–778.
Luo K., Chen S. L., Chen K. L., Song J. Y., and Yao H. (2010). Assessment of candidate plant DNA barcodes using the Rutaceae family. China Life Sciences, 40: 342–351.
Mai J. C., and Coleman A. W. (1997). The internal transcribed spacer 2 exhibits a common secondary structure in green algae and flowering plants. Journal of Molecular Evolution, 44: 258–271.
Maxted N., and Kell S. (2009). CWR in crop improvement: To what extent are they used? Crop Wild Relative Newsletter, 7: 7–8.
Mehrabi A. A., and safari Z. (2019). Molecular phylogeny of Aegilops L. and Triticum L. species revealed by internal transcribed spacers of ribosomal genes. Journal of Agricultural Science and Technology, 21(3): 699–714.
Meyer C. P., and Paulay G. (2005). DNA Barcoding: error rates based on comprehensive sampling. PLoS Biology, 3: e422. DOI:
Michot B., Joseph N., Sazan S., and Bachellerie J. P. (1999). Evolutionary conserved structural features in the ITS2 of mammalian pre-rRNAs and potential interactions with the snoRNA U8 detected by comparative analysis of new mouse sequences. Nucleic Acids Research, 27: 2271–2282.
Nepolo E., Chimwamurombe P. M., Cullisand C. A., and Kandawa-Schulz M. A. (2010). Determining genetic diversity based on ribosomal intergenic spacer length variation in Marama bean (Tylosemae sculentum) from the Omipanda Area, Eastern Namibia. African Journal of. Plant Science, 4: 368–373.
Petersen G., Seberg O., Yde M., and Berthelsen K. (2006). Phylogenetic relationships of Triticum and Aegilops and evidence for the origin of the A, B, and D genomes of common wheat (Triticum aestivum). Molecular Phylogenetics and Evolution, 39: 70–82.
Piccolo S. L., Alfonzo A., Conigliaro G., Moschetti G., Burruano S., and Barone A. (2012). A simple and rapid DNA extraction method from leaves of grapevine suitable for polymerase chain reaction analysis. African Journal of Biotechnology, 11(45): 10305–10309. DOI: https://doi: 10.5897/AJB11.3023.
Poczai P., and Hyvonen J. (2010). Nuclear ribosomal spacer regions in plant phylogenetics, problems and prospects. Molecular Biology Reports, 37: 1897–1912.
Rzhetsky A., and Nei M. (1992). A simple method for estimating and testing minimum evolution trees. Molecular Biology and Evolution, 9: 945–967.
Saitou N., and Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406–425.
Schultz J., and Wolf M. (2009). ITS2 sequence structure analysis in phylogenetics: a how to manual for molecular systematics. Molecular Phylogenetics and Evolution, 52: 520–523.
Schultz J., Muller T., Achtziger M., Seibel P. N., Dandekar T., and Wolf M. (2006). The internal transcribed spacer 2 database- a web server for (not only) low level phylogenetic analyses. Nucleic Acids Research, 34: 704–707.
Sliai, A. M., and Amer S. A. M. (2011). Contribution of chloroplast DNA in the biodiversity of some Aegilops species. African Journal of. Biotechnology, 10: 2212–2215.
Tamura K., and Nei M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 10:512–526.
Thompson J. D., Higgins D. G., and Gibson T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22: 4673–4680.
Thornhill D. J., Lajeunesse T. C., and Santos S. R. (2007). Measuring rDNA diversity in eukaryotic microbial systems: how intragenomic variation, pseudogenes, and PCR artifacts confound biodiversity estimates. Molecular Ecology, 16: 5326–5340.
Torres R. A., Ganal M., and Hembleden V. (1990). GC balance in the internal transcribed spacers ITS1 and ITS2 of nuclear ribosomal RNA genes. Journal of Molecular Evolution, 30: 170–181.
Wang M., Zhao H. X., and Wang L. (2013). Potential use of DNA barcoding for the identification of Salvia based on cpDNA and nrDNAsequences. Gene, 528: 206–215.
Wheeler W. C. (1994). Sources of ambiguity in nucleic acid sequences alignment. In Molecular Ecologyand Evolution: Approaches and Applications, Schierwater B., Streit B., Wagner G. P., and DeSalle R. (Eds.), Birkhauser Verlag, Basel, 323–354.