Adoption of RAPD-related methodology for analyses of Hypericum maculatum Crantz growing wild in Lithuania

Abstract

Hypericum genus plants are widespread all over Europe and Asia and they are one of the most important world plants used for medicinal purposes. In Lithuania insufficient attention is paid to the second according to the abundance species of Hypericum genus – H. Maculatum L. Till now research of this species was mainly concentrated on morphology and some chemical parameters. The present study aimed at selection of the conditions for random amplification of polymorphic DNA extracted from H. maculatum growing wild in Lithuania. NucleoSpin Plant II (Macherey-Nagel, Germany) as the most efficient kit was selected for DNA extraction from H. maculatum. For polymerase chain reaction (PCR) 14 nucleotide primers (Biomers.net, Germany) were used and OP-A1, OP-A4, OP-A9, OP-B12, OP-B19, OP-C6a, OP-C7a, OP-C11, OP-C19, OP-D20, OP-Q2 were selected for further studies of genetic diversity of populations of H. maculatum (OP-B13, OP-D19a, OP-Q11 were rejected). DNA amplification program with 40 cycles was chosen from 35, 40 and 45 cycles tested. Among two different composition mixes used for PCR, more efficient was the one consisting of: 0.05 U/μl Taq DNA polymerase; 1 × Taq buffer; 1.5 nM MgCl2; 0.2 mM dNTP; 2 μl primer (10 pmol/μl) and 14.55 μl deionised water (Fermentas, Lithuania). For preparation of above mentioned mix 3 different types of Taq buffer were tested: 1 – Taq buffer with KCl; 2 – Taq buffer with (NH4)2SO4; 3 – Taq buffer without detergent. The best results were achieved with the first buffer. Keywords: imperforate St John’s wort, DNA markers, genetic diversity, populations, Hypericaceae