Cereal Research Communications

, Volume 45, Issue 3, pp 381–389 | Cite as

Application of the ITS2 Region for Barcoding Plants of the Genus Triticum L. and Aegilops L.

  • I. Ganopoulos
  • A. Kapazoglou
  • I. Bosmali
  • A. Xanthopoulou
  • I. Nianiou-Obeidat
  • A. Tsaftaris
  • P. MadesisEmail author


Molecular taxonomic studies have been performed in the past in order to identify different wheat species and construct a molecular phylogeny. These were based on universal but sufficiently divergent sequences from both the nuclear and chloroplastic genomes of wheat. They included two short plastid sequences from the plastid genes rbcL and matK which have been proposed as the core “barcode” sequences by the “barcoding” guidelines for general plant identification. Historically, in molecular plant taxonomy, plastidic sequences had been favored over nuclear sequences, due to their uniparental inheritance and consequently lower intra-molecular recombination. However recently, the short nuclear sequence from the internal transcribed spacer 2 (ITS2) has been used successfully for the accurate identification of many medicinal and other plant species. Herein, we have used the plastidic matK, rbcL trnL, and the nuclear ITS2 region for the identification of different wheat species of Triticum L. and goatgrass species of Aegilops L. We have successfully discriminated all species that were examined from both genera, thus, validating the ITS2 region as a ‘barcode tool’ for accurate distinction of plants in the genus Triticum L. and Aegilops L. The success rate of PCR amplification and sequencing of the ITS2 region was 100%. We report also that matK, rbcL and trnL regions could not discriminate all species in contrast to the ITS2 region which demonstrated full discriminatory capacity.


ITS2 Triticum Aegilops DNA barcoding identification 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

42976_2017_4503381_MOESM1_ESM.pdf (204 kb)
Application of the ITS2 Region for Barcoding Plants of the Genus Triticum L. and Aegilops L.


  1. Baum, B.R., Johnson, D.A., Bailey, L.G. 2001. Defining orthologous groups among multicopy genes prior to inferring phylogeny, with special emphasis on the Triticeae (Poaceae). Hereditas 135:123–138.CrossRefGoogle Scholar
  2. Bolson, M., de Camargo Smidt, E., Brotto, M.L., Silva-Pereira, V. 2015. ITS and trnH-psbA as efficient DNA barcodes to identify threatened commercial woody angiosperms from southern Brazilian Atlantic rainforests. PloS one 10:e0143049.CrossRefGoogle Scholar
  3. Boscato, P., Carioni, C., Brandolini, A., Sadori, L., Rottoli, M. 2008. Molecular markers for the discrimination of Triticum turgidum L. subsp. dicoccum (Schrank ex Schübl.) Thell. and Triticum timopheevii (Zhuk.) Zhuk. subsp. timopheevii. J. Archaeological Sci. 35:239–246.CrossRefGoogle Scholar
  4. Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., Ma, X., Gao, T., Pang, X., Luo, K. 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PloS one 5:e8613.CrossRefGoogle Scholar
  5. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791.CrossRefGoogle Scholar
  6. Ganopoulos, I., Madesis, P., Tsaftaris, A. 2012. Universal ITS2 barcoding DNA region coupled with high-resolution melting (HRM) analysis for seed authentication and adulteration testing in leguminous forage and pasture species. Plant Mol. Biol. Reporter 30:1322–1328.CrossRefGoogle Scholar
  7. Gao, T., Yao, H., Song, J., Liu, C., Zhu, Y., Ma, X., Pang, X., Xu, H. Chen, S. 2010. Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2. J. of Ethnopharmacology 130:116–121.CrossRefGoogle Scholar
  8. Golovnina, K.A., Glushkov, S.A., Blinov, A.G., Mayorov, V.I., Adkison, L.R., Goncharov, N.P. 2007. Molecular phylogeny of the genus Triticum L. Plant Systematics and Evolution 264:195–216.CrossRefGoogle Scholar
  9. Goncharov, N.P., Golovnina, K.A., Kondratenko, E.Y. 2009. Taxonomy and molecular phylogeny of natural and artificial wheat species. Breeding Sci. 59:492–498.CrossRefGoogle Scholar
  10. Goryunova, S.V., Chikida, N.N., Gori, M., Kochieva, E.Z. 2005. Analysis of nucleotide sequence polymorphism of internal transcribed spacers of ribosomal genes in diploid Aegilops (L.) species. Mol. Biol. 39:173–176.CrossRefGoogle Scholar
  11. Group, C.P.W., Hollingsworth, P.M., Forrest, L.L., Spouge, J.L., Hajibabaei, M., Ratnasingham, S., van der Bank, M., Chase, M.W., Cowan, R.S., Erickson, D.L., Fazekas, A.J. 2009. A DNA barcode for land plants. Proc. Am. Natl Acad. of Sci. 106:12794–12797.CrossRefGoogle Scholar
  12. Gulbitti-Onarici, S., Sancakz, C., Sumer, S., Ozcan, S. 2009. Phylogenetic relationships of some wild wheat species based on the internal transcribed. Current Science 96:6.Google Scholar
  13. Han, J., Zhu, Y., Chen, X., Liao, B., Yao, H., Song, J., Chen, S., Meng, F. 2013. The short ITS2 sequence serves as an efficient taxonomic sequence tag in comparison with the full-length ITS. BioMed Research International 2013. Article ID 741476, Scholar
  14. Hebert, P.D.N., Cywinska, A., Ball, S.L. 2003. Biological identifications through DNA barcodes. Proc. of the Royal Society of London B: Biological Sciences 270:313–321.CrossRefGoogle Scholar
  15. Hollingsworth, P.M. 2011. Refining the DNA barcode for land plants. Proc. of the National Academy of Sciences 108:19451–19452.CrossRefGoogle Scholar
  16. Kihara, H. 1924. Cytologische und genetische Studien bei wichtigen Getreidearten mit besonderer Rücksicht auf das Verhalten der Chromosomen und die Sterilität in den Bastarden (Cytological and genetic studies in major cereals with particular reference to the behavior of chromosomes and sterility in hybrids). Memoirs of the College of Science. Kyoto Imperial University, Series B, vol. 1. Kyoto, Japan. 200 p. (in German)Google Scholar
  17. Koetschan, C., Hackl, T., Müller, T., Wolf, M., Frank, F., Schultz, J. 2010. ITS2 Database IV: Interactive taxon sampling for internal transcribed spacer 2 based phylogenies. Molecular Phylogenetics and Evolution 63:585–588.CrossRefGoogle Scholar
  18. Kress, W.J., Wurdack, K.J., Zimmer, E.A., Weigt, L.A., Janzen, D.H. 2005. Use of DNA barcodes to identify flowering plants. Proc. Am. Natl Acad. of Sci. 102:8369–8374.CrossRefGoogle Scholar
  19. Kumar, S., Stecher, G., Tamura, K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. and Evol. 33:1870–1874.CrossRefGoogle Scholar
  20. Li, D.-Z., Gao, L.M., Li, H.T., Wang, H., Ge, X.J., Liu, J.Q., Chen, Z.D., Zhou, S.L., Chen, S.L. Yang, J.B. 2011a. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proc. Am. Natl Acad. of Sci. 108:19641–19646.CrossRefGoogle Scholar
  21. Li, D.Z., Liu, J.Q., Chen, Z.D., Wang, H., GE, X.J., Zhou, S.L., Gao, L.M., Fu, C.X., Chen, S.L. 2011b. Plant DNA barcoding in China. J. Systematics and Evol. 49:165–168.CrossRefGoogle Scholar
  22. Librado, P., Rozas, J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451–1452.CrossRefGoogle Scholar
  23. Lilienfeld, F., Kihara, H. 1934. Genomanalyse bei Triticum und Aegilops. Cytologia 6:87–122.CrossRefGoogle Scholar
  24. Madesis, P., Ganopoulos, I., Ralli, P., Tsaftaris, A. 2012. Barcoding the major Mediterranean leguminous crops by combining universal chloroplast and nuclear DNA sequence targets. Genet. and Mol. Res. 11:2548– 2558.CrossRefGoogle Scholar
  25. Meyer, C.P., Paulay, G. 2005. DNA barcoding: error rates based on comprehensive sampling. PLoS Biology 3:e422.CrossRefGoogle Scholar
  26. Newmaster, S., Grguric, M., Shanmughanandhan, D., Ramalingam, S., Ragupathy, S. 2013. DNA barcoding detects contamination and substitution in North American herbal products. BMC Medicine 11:222.CrossRefGoogle Scholar
  27. Rach, J., DeSalle, R., Sarkar, I.N., Schierwater, B., Hadrys, H. 2008. Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata. Proceedings of the Royal Society B: Biological Sciences 275:237–247.CrossRefGoogle Scholar
  28. Saitou, N., Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. and Evol. 4:406–425.Google Scholar
  29. Sakamura, T. 1918: Kurze Mitteilung über die Chromosomenzahlen und die Verwandtschaftsverhältnisse der Triticum-Arten (A brief communication on the chromosome numbers and the relationships of the Triticum species). Bot. Mag. Tokyo 32:151–154. (in German)CrossRefGoogle Scholar
  30. Shi, L.C., Zhang, J., Han, J.P., Song, J.Y., Yao, H., Zhu, Y.J., Li, J.C., Wang, Z.Z., Xiao, W., Lin, Y.L. Xie, C.X. 2011. Testing the potential of proposed DNA barcodes for species identification of Zingiberaceae. J. of Systematics and Evol. 49:261–266.CrossRefGoogle Scholar
  31. Simmons, M.P., Ochoterena, H. 2000. Gaps as characters in sequence-based phylogenetic analyses. Systematic Biol. 49:369–381.CrossRefGoogle Scholar
  32. Tamura, K., Nei, M., Kumar, S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Am. Natl Acad. of Sci. 101:11030–11035.CrossRefGoogle Scholar
  33. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. and Evol. 28:2731–2739.CrossRefGoogle Scholar
  34. Wang, X.C., Liu, C., Huang, L., Bengtsson Palme, J., Chen, H., Zhang, J.H., Cai, D., Li, J.Q. 2015. ITS1: a DNA barcode better than ITS2 in eukaryotes? Molecular Ecol. Resour. 15:573–586.CrossRefGoogle Scholar
  35. Yao, H., Song, J., Liu, C., Luo, K., Han, J., Li, Y., Pang, X., Xu, H., Zhu, Y., Xiao, P., Chen, S. 2010. Use of ITS2 region as the universal DNA barcode for plants and animals. PloS one 5:e13102.CrossRefGoogle Scholar
  36. Zhang, W., Qu, L., Gu, H., Gao, W., Liu, M., Chen, J., Chen, Z. 2002. Studies on the origin and evolution of tetraploid wheats based on the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA. Theor. Appl. Genet. 104:1099–1106.CrossRefGoogle Scholar
  37. Zhang, W.-J., L.-J. Qu, W. Gao, H.-Y. Gu, J. Chen, Chen, Z. L. 1998. ITS1 and ITS2 sequences of four possible donors to bread wheat genome and their phylogenetic relationships. Acta Botanica Sinica 40:994–1000.Google Scholar

Copyright information

© Akadémiai Kiadó, Budapest 2017

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • I. Ganopoulos
    • 2
  • A. Kapazoglou
    • 1
  • I. Bosmali
    • 1
  • A. Xanthopoulou
    • 1
    • 2
  • I. Nianiou-Obeidat
    • 2
  • A. Tsaftaris
    • 1
    • 2
  • P. Madesis
    • 1
    Email author
  1. 1.Institute of Applied BiosciencesCERTH, ThermiThessalonikiGreece
  2. 2.Department of Genetics and Plant Breeding, School of AgricultureAristotle University of ThessalonikiThessalonikiGreece

Personalised recommendations