PDFStoyanov K., Gevezova M. & Denev I. 2012.
Identification of ISSR markers for studying the biodiversity of Bulgarian representatives of genus Orobanche subsection Minores.
Biotechnology & Biotechnological Equipment, 26(1): 2743-2749.
The family Orobanchaceae consists of parasitic species. One group of taxonomically problematic species is subsection Minores (Beck) Teryokhin genus Orobanche. The species in this subsection (except O. crenata) have been often assigned to aggregate O. minor because of their great morphological similarity. The aim of this study was to find useful molecular makers for characterization of the biodiversity of subsection Minores, Orobanche in Bulgaria and their phylogenetic relationships. Six species of subsection Minores were examined: O. minor Sm.; O. amethystea Thuill.; O. esulae Pančić var. bulgarica T. Georgiev, O. pubescens d’Urv. O. loricata Rchb. and O. crenata Forssk. Genomic DNA was isolated from flowering stems and used as a template for ISSR PCR performed with ten different ISSR primers. The ISSR products were separated on agarose gel and visualized by UV-light. The molecular masses of the polymorphic bands were determined and used to fill Boolean matrices that were subjected to cluster analysis. The suitability of each primer for distinguishing each of the studied species was discussed. The resulting cladogram, based on the average Euclidean distances, displayed grouping by species and by geographic populations. This study confirmed that the assignment of the small-flowered species to agg. O. minor has only practical value and is not genetically justified. The study confirmed the independent taxonomic status of the Balkan endemic species O. esulae.

Acknowledgements
The study was supported by the Scientific Research Center of the Agricultural University – Plovdiv (grant 02-10) and the National Science Fund of Bulgaria grants IFS –B-606, DТK 02/40 and BG051PO001-3.3.04/17, NATO grant CLG 983884
and ERA 117.

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