Genetic differentiation of morphologically similar polyploid wheat species

Background Wheat is a globally important polyploid crop, with hexaploid bread wheat ( Triticum aestivum L.) and tetraploid durum wheat ( T. turgidum subsp . durum (Desf.) Husn.) as its main cultivated forms. Despite distinct end-use properties, these species are morphologically similar, making their identification difficult. Traditional phenotypic approaches often fail to resolve closely related polyploid wheats, emphasizing the need for a reliable molecular diagnostic and wheat barcoding strategy. Method This study developed and validated a multilocus molecular diagnostic framework for the discrimination of polyploid wheat species. The approach integrates plastid ( rbcL , matK ), nuclear ribosomal ( ITS2 , IGS ), and nuclear-coding markers ( Glu-1 and XDuPw167 ), all amplified using the Polymerase Chain Reaction. Validation was performed using ten experimental samples and 203 reference sequences retrieved from the NCBI GenBank database. We developed and validated a multilocus molecular diagnostic method for the reliable discrimination of wheat species. Results Plastid loci showed limited variation, whereas the IGS region contained a diagnostic 71 bp insertion linked to the D genome, clearly distinguishing hexaploids from tetraploids. The Glu-1 and XDuPw167 loci exhibited genome-specific polymorphisms that further differentiated the two species. The multilocus diagnostic method achieved over 95% amplification success and consistent sequence profiles across replicates, confirming its accuracy and reproducibility. Conclusions The proposed molecular diagnostic method provides a reproducible, cost-effective, and high-resolution molecular diagnostic tool for reliable wheat species identification. By combining genome-specific nuclear and expressed sequence tag-simple sequence repeat (EST-SSR) markers, this approach establishes a robust and scalable system applicable to species authentication, seed purity testing, germplasm characterization, and genetic resource management.