Genetic authentication by RFLP versus ARMS? The case of Moldavian dragonhead (Dracocephalum moldavica L.)

Globalisation extends the spectrum of plants entering the European market. Growing consumer awareness for the medical aspects of food, and a general attention on health issues in an ageing society stimulate the introduction of novel foreign plant products often derived from traditional medical or dietary use. These products are mostly located somewhere at the interphase between food supplement and phytomedicine, and only few of them are regulated by the EU Traditional Herbal Medicinal Products Directive. Moreover, only those herbal preparations, where no historical record is documented for Europe, fall under the legislation of the Novel-Food-Regulation of the European Union [1]. For most of these foreign plant products, legislation is therefore far from clear. This problem is not a merely academic: These plants are used, in their country of origin, in the context of a specific cultural and medical tradition that safeguards against undesired side effects. When isolated from this context, unforeseen problems can arise such as adverse interactions, adulteration, and even toxicity [2]. In addition, such market trends can cause short-term limitations in the supply with these plants, creating a situation, Abstract Moldavian dragonhead (Dracocephalum moldavica L.), due to its pleasant lemon scent and medical effects, has acquired increasing impact as functional food. The high diversity within the genus, limited supply not keeping pace with the growing demand, the morphological similarity with other Labiatae, and trading under the common name Turkish Melissa invite adulteration by surrogate species. We have developed several verified reference accessions of D. moldavica L. along with potential surrogate species to compare different approaches of authentication, also in commercial samples. We report on three strategies of authentication—a microscopic method, based on the relative size of epidermal pavement cells versus palisade cells, and two strategies of genetic authentication based on the barcoding marker large subunit of ribulose1,5-bisphosphate carboxylase oxygenase (rbcL). We can detect single-nucleotide exchanges between D. moldavica L. and the potential surrogate species Melissa officinalis L. and Nepeta cataria L. by restriction fragment length polymorphism (RFLP), and we show that we can use this to verify the presence of D. moldavica even in dried and highly fragmented mixtures from commercial samples. We further develop a third strategy derived from the so-called amplification refractory mutation system (ARMS), based on multiplex PCR of the rbcL marker upon addition of specifically designed intermediate primers that will generate a diagnostic second band in case of D. moldavica L., but not for the surrogate species. We demonstrate that this ARMS approach is superior to the RFLP strategy, because it