Barcode-High Resolution Melting Clearly Distinguishes Durum and Bread Wheat Varieties by Novel Primer Sets

Abstract

Wheat is a vital global staple, with Triticum durum Desf. (durum wheat) primarily used for pasta and couscous, and Triticum aestivum L. (bread wheat) widely cultivated for baking. Differentiating these species, especially in processed forms, poses significant challenges due to their genetic similarity and overlapping end-use applications. This study applied Barcode-High-Resolution Melting (Bar-HRM) analysis to distinguish common durum (Cesare, Ovidio, Tiziana, Zivago) and bread wheat (Adana 99, Ceyhan 99, Dinç, Nizar) varieties in Türkiye. DNA was extracted separately from seeds, flours, and fresh leaves to ensure successful recovery, with seed-derived DNA used for HRM analysis. The Internal Transcribed Spacer (ITS) region was amplified via PCR, revealing two polymorphic sites in ITS1 and 20 in ITS2, with 14 sites differentiating the two species. HRM primer sets targeting these regions achieved genotyping confidence levels exceeding 95%. Sequencing validated the HRM results, and phylogenetic analysis confirmed clear separation between durum and bread wheat. This rapid, reliable, and contamination-resistant method enables accurate wheat discrimination from small samples within three hours, offering significant potential for broader applications in agricultural diagnostics and food authenticity testing

Keywords

• Triticum aestivum
• Triticum durum
• Bar-HRM
• Genotyping
• Internal Transcribed Spacer.

References

1. Abdel-Aal E S M, Hucl P & Sosulski F W (1995). Compositional and nutritional characteristics of spring einkorn and spelt wheats. https://doi.org/10.1006/jcrs.1997.0139
2. Álvarez I J F W& Wendel J F (2003). Ribosomal ITS sequences and plant phylogenetic inference. Molecular Phylogenetics and Evolution 29: 417–434. https://doi.org/10.1016/s1055-7903(03)00208-2
3. Ay N & Hürkan K (2023). A novel high-resolution melting method for detection of adulteration on pistachio (Pistacia vera L.). Brazilian Archives of Biology and Technology 66. https://doi.org/10.1590/1678-4324-2023220550
4. Ceyhan V & Akkaya A (2020). Wheat production and sustainability in Turkey. Journal of Agricultural Sciences Chaves M S, Martinelli J A, Wesp-Guterres C, Graichen FAS, Brammer S P, Scagliusi S M & Chaves, A L S (2013). The importance for food security of maintaining rust resistance in wheat. Food Security 5:157–176. https://doi.org/10.1007/s12571-013-0248-x
5. Cheng J, Ma X, Zeng C, Zhang J & Li Z (2014). Application of high-resolution melting (HRM) for genotyping and species identification. Molecular Plant Breeding 5(2):89–95
6. Druml B & Cichna-Markl M (2014). High-resolution melting (HRM) analysis of DNA: a new molecular tool in food authentication. Food Chemistry 158:245–254. https://doi.org/10.1016/j.foodchem.2014.02.111
7. Dubcovsky J, & Dvorak J (2007). Genome plasticity a key factor in the success of polyploid wheat under domestication. Science, 316(5833), 1862-1866. https://doi.org/10.1126/science.1143986
8. El-Esawi M A, Germaine K, Bourke P, Malone R (2018). Genetic diversity and population structure of wheat species. Plant Biotechnology Journal 16(5): 1089–1100

9. Gadaleta A, Nigro D, Giancaspro A & Blanco A (2011). The glutamine synthetase (GS2) genes in relation to grain protein content of durum wheat. Functional & Integrative Genomics, 11: 665-670. https://doi.org/10.1007/s10142-011-0235-2
10. Hürkan K (2021). Distinguishing the protected designation of origin apricot (Prunus armeniaca L. cv. Şalak) from closely related cultivars by high resolution melting. Commagene Journal Biology 5: 136–142. https://doi.org/10.31594/commagene.891932
11. Karaman H, Ozkan B & Celik A (2018). Enhancing wheat resilience through sustainable practices in Turkey. Sustainable Agriculture Reviews.
12. Kaufman L & Rousseeuw P J (1990). Finding groups in data: an introduction to cluster analysis. Wiley. Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E & Wittwer C T (2004). Genotyping by amplicon melting analysis using LCGreen. Clin Chemistry 50:1156–1164.. https://doi.org/10.1373/clinchem.2004.032136
13. Lv R, Han L, Xiao B, Xiao C, Yang Z, Wang H & Yang C (2019). An extracted tetraploid wheat harbouring the BBAA component of common wheat shows anomalous shikimate and sucrose metabolism. BMC Plant Biology 19: 1–11. https://doi.org/10.1186/s12870-019-1796-9
14. Pastore G, Kernchen S, & Spohn M (2020). Microbial solubilization of silicon and phosphorus from bedrock in relation to abundance of phosphorus-solubilizing bacteria in temperate forest soils. Soil Biology and Biochemistry 151: 108050. https://doi.org/10.1016/j.soilbio.2020.108050
15. Peng J H, Sun D & Nevo E (2011). Domestication evolution, genetics, and genomics in wheat. Molecular Biology and Evolution 28(5):1471 1492. https://doi.org/10.1007/s11032-011-9608-4
16. Rahaie M, Xue G P & Schenk, P M (2013). The role of transcription factors in wheat under different abiotic stresses. Abiotic stress-plant responses and applications in agriculture, 2: 367-385. https://doi.org/10.5772/54795
17. Reed K A, Wittwer C T & Oetjen J (2007). High-resolution melting analysis for mutation scanning and genotyping. Nature Protocols 2: 1761 1772. https://doi.org/10.1038/nprot.2007.247. https://doi.org/10.1038/nprot.2007.10 Rudnóy S, Páldi E, Bratek Z, Szegő D, Rácz I & Lásztity D (2004). ITS regions in hexaploid bread wheat and its supposed progenitors. Cereal Research Communications 32: 423–428. https://doi.org/10.1007/bf03543330 Shewry P R (2009). Wheat. JXB 60(6): 1537-1553 van der
18. Stoep N, van Paridon C D, Janssens T, Krenkova P, Stambergova A, Macek M & Bakker E (2009). Diagnostic guidelines for high resolution melting curve (HRM) analysis: An interlaboratory validation of BRCA1 mutation scanning using the 96‐well LightScanne. Human. Mutant. 30(6): 899-909. https://doi.org/10.1002/humu.21004
19. Vossen R H A M, Aten E, Roos A & den Dunnen J T (2009). High-resolution melting analysis (HRMA): more than just sequence variant screening. Human Mutant 30: 860–866. https://doi.org/10.1002/humu.21019. https://doi.org/10.1002/humu.21019
20. White T J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications. Academic Press, Inc. https://doi.org/10.1016/b978-0-12-372180-8.50042-1
21. Wittwer C T, Reed G H, Gundry C N, Vandersteen J G & Pryor R J (2003a). High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chemistry 49:853–860.. https://doi.org/10.1373/49.6.853
22. Wittwer C T, Ririe K M, Andrew R E & Heiner C R (2003b). High-resolution DNA melting analysis for mutation scanning and genotyping. National Biotechnology 21: 271–275. https://doi.org/10.1038/nbt0303-271
23. Wojdacz T K & Dobrowolski S F (2007). A new approach to high-resolution melting analysis using amplicons shorter than 100 bp. Nucleic Acids Research 35. https://doi.org/10.1093/nar/gkm059
24. Yan J, Warburton M L & Crouch J H (2013). High-resolution melting analysis of single nucleotide polymorphisms in wheat breeding. Journal of Cereal Science 58(3): 405–411
25. Yüce İ, Başkonuş T, Dokuyucu T, Akkaya A, Güngör H & Dumlupinar Z (2022). Bazı ekmeklik buğday (Triticum aestivum L.) çeşitleri ve ileri hatlarının Kahramanmaraş ekolojik şartlarında tarımsal özellikler ve kalite bakımından değerlendirilmesi. YYU Journal of Agricultural Sciences 32(2)
26. Zeven A C (1991). Wheats with purple and blue grains: A review. Euphytica, 56(3): 243–258. https://doi.org/10.1007/BF00042371