Studies on the development of molecular markers for the detection of salep

Salep, is a non-cultivable product which is obtained by collecting tubers of the Orchidaceae plants from the wild. Following various processes the tubers are available for consumption as a highly valued food ingredient. Salep is most often marketed in powder form. As the main components in salep are glucomannans and starch, it is a powder material which may be mimicked or adulterated. Original salep finds high market value thus, manufacturers of commercial salep powders or drinks use very little of the original ingredient, adding starch or gums as bulking agents. On the other hand, salep is a product protected under the law (export of salep in any form is strictly prohibited). There is presently no method to test the presence of salep in a food powder or to evaluate salep authenticity. The aim of this study is the determination genetic marker(s) that may be used in detection, determination and authentication of salep. To this end, with a holistic approach, RAPD fragments were obtained for the development of SCAR markers. Additionally, specific primers designed from the ITS2 locus were analyzed. Tuber samples representative of genera used in salep production were used to construct the gene pool. Methods were validated by testing against a collection of salep samples as well as possible adulterants/bulking agents/additives. Sequences of the ITS2 region were utilized in designing a primer set which could be used in real time PCR experiments to obtain a calibration curve that could be applicable for quantification. The sensitivity of the developed methods were validated by testing with powder salep mixtures mixed with predetermined amounts of starch or corn flour in real time PCR set up with designed ITS2a specific primers and 18S rRNA universal primers.