A Combinatorial and Tubulin-Based Polymorphism (TBP) Approach Helps in Discriminating Species.

The simple and straightforward recognition of species is not an easy task due to their complex genetic origins. To provide a recommendation, we have compared the performance of different PCR-based methods relying on the discrimination ability of the and () genes, as well as TBP (Tubulin-Based Polymorphism), a method based on the multiple amplification of genes of the β-tubulin family. Among these approaches, the PCR-RFLP (Restriction Fragment Length Polymorphism) assay based on a single-nucleotide polymorphism (SNP) present in the gene is the only one capable of fully discerning hexaploid spelt and common wheat species, while both and TBP fail with similar error frequencies. The assay results in the attainment of either a single fragment or a doublet, depending on the presence of a suitable restriction site, which is affected by the mutation. This dual pattern of resolution limits both the diagnostic effectiveness, when additional species are assayed and compared to each other, and its usefulness, when commercially available flours are analyzed. These limitations are overtaken by flanking the assay with the TBP analysis. In this way, almost all of the species can be accurately identified.