Application of codominant DNA markers for investigation of molecular diversity of Lithuanian reed canary grass populations

Abstract

Reed canary grass (Phalaris arundinacea L.) is a perennial herbaceous plant widespread all over the world, belonging to Poaceae / Gramineae family, Phalaris genus, which includes 15 species. This species is common for the temperate climate zone and is naturally growing in Lithuania. Attempts to use reed canary grass as a forage or decoration increases need for molecular analysis [1]. The objective of this study was to select microsatellite DNA markers appropriate for evaluation of molecular diversity of Lithuania populations of Phalaris arundinacea. DNA was extracted using CTAB method. The quality of DNA was assessed spectrophotometrically and by gel electrophoresis. The quantity of DNA of each sample was standardised to approximately 20 ng/μl. Four SSR primer pairs (PHI071, UMC2185, UMC2272, and UMC2779) developed for Zea mays were tested for amplification of Phalaris arundinacea DNA. Polymerase chain reaction (PCR) was carried out in the volume of 8 μl, containing 20 ng of DNA of reed canary grass, 3.5 μl PCR Master mix (0.05 U/μl Taq DNA Polymerase; 4 mM MgCl2; 0.4 mM each dNTP), 0.5 μl 10 μM of the forward primer, the same was true for the reverse primer, 0.31 μl 20 mg/ml BSA and 0.13 μl 5 M betaine. Amplification was performed following these conditions: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 15 s, annealing at 54–56 °C for 45 s, extension at 72 °C for 2 min and a final extension at 72 °C for 20 min. The amplified DNA products were screened on an agarose (1.5 %) gel and visualized under UV light after staining by ethidium bromide. Three annealing temperatures were tested to get a clear band on an agarose gel. Annealing temperature at 54 °C was suitable for application of the primers PHI071, UMC2185 and UMC2779. For the primer UMC2272 annealing temperatures at 54 °C, 55 °C and 56 °C did not cause reaction of polimerisation.[...]