# Genetic differentiation of morphologically similar polyploid wheat species

## TL;DR

This paper introduces a molecular method to accurately identify closely related wheat species that are hard to tell apart by appearance.

## Contribution

A multilocus molecular diagnostic framework for reliable discrimination of polyploid wheat species is developed and validated.

## Key findings

- The IGS region has a 71 bp insertion linked to the D genome, distinguishing hexaploids from tetraploids.
- Glu-1 and XDuPw167 loci show genome-specific polymorphisms for species differentiation.
- The method achieved over 95% amplification success and consistent results across replicates.

## Abstract

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.

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.

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.

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.