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    A novel high-resolution melting (HRM)-based approach to differentiate ancient wheat species from common bread wheat via custom primer design
    (Springer, 2025) Hurkan, Yasemin Kemec; Gocer, Emine Uygur; Hurkan, Kaan
    Ancient wheat species such as Triticum monococcum L. (einkorn), Triticum dicoccum Schrank. (emmer), and Triticum spelta L. (spelt) are attracting increasing attention for their nutritional qualities, environmental adaptability, and cultural importance. However, ensuring the authenticity of these heritage grains is critical to prevent adulteration with modern bread wheat (Triticum aestivum L.), which may compromise both market value and genetic conservation. In this study, we developed and validated a high-resolution melting (HRM) assay targeting polymorphic regions of the internal transcribed spacer (ITS) locus to differentiate ancient wheat species from T. aestivum. Newly designed HRM-compatible primers generated distinct species-specific melt curve profiles, enabling reliable discrimination among the studied wheat species. The assay successfully detected admixtures containing as little as 0.2% (1/500 w/w) T. aestivum in ancient wheat flours. Principal component analysis (PCA) of HRM difference curve data enhanced resolution and revealed clear clustering patterns separating pure ancient wheat samples from adulterated ones. Additionally, ITS-based phylogenetic analysis confirmed the correct taxonomic placement of all tested species, reflecting their evolutionary relationships from diploid T. monococcum to tetraploid T. dicoccum and hexaploid T. spelta and T. aestivum. These findings demonstrate that ITS-HRM analysis offers a rapid, sensitive, and cost-effective molecular tool for authenticating ancient wheat products, supporting regulatory traceability, food labeling accuracy, and the conservation of agrobiodiversity.