Isolation and RAPD analysis of DNA from pineneedles of Pinus pinea of three greek populations

Abstract

After the initial work dealing with DNA isolation for plant and especially for conifers, only few efforts were made to develop new techniques. In this work, DNA was isolated using a method that does not require liquid nitrogen. Methodology/Results: The object of this work was examination of genetic diversity of three Greek populations of stone pine. DNA was isolated from healthy pineneedles using the isolation protocol Charge Switch gDNA Plant Kit of Invitrogen. Exsiccation with silicagel was the key for successful results for a resilient plant such as Pinus pinea. For molecular analysis two primers were used in polymerase chain reaction, OPA04 and OPU14. Each DNA segment that was detected constitutes a RAPD marker. Presence or absence of marker was digitally encoded in binary system and represented with unit and zero respectively. According to the results a phylogenetic analysis dendrogram was produced using UPGMA method based on DICE marker. RAPD analysis showed similarities between the populations of Marathonas and Kotuchi, and their divergence from the population of Skiathos. Conclusions: The results can be useful for proposing new methodologies for genetic identification of other species of pine. The modified technique for DNA isolation using exsiccation with silicagel was successfully applied in this study and the RAPD technique may serve as an efficient tool for further molecular studies.