Identification of chloroplast genome between Pinus densiflora Sieb. et Zucc. and P. thunbergii Parl. based on the polymor- phism in rbcL gene

As there are a lot of cross between Pinus densiflora (Japanese red pine) and P. thunbergii (Japanese black pine) morphologically, it is extremely difficult to identify the two species exactly. In this study, RFLP (restriction fragment length polymorphism), a kind of DNA molecular marker, was used in order to develop the new technique possessing the high reliability. A part of rbcL gene (1,309 bps) in chloroplast DNA was amplified by PCR (polymerase chain reaction), and cleaved with 11 restriction endonucleases which are four-based cutters. In four (Alu I, Cfr13I, Hae III, ScrFI) of 11 endonucleases, the restriction fragment patterns were polymorphic between the two species. It was because of the change of one recognition site in each endonuclease. RFLP analysis of rbcL gene could be used as one of the techniques for the simple identification of these two species. The sequences of the central part of rbcL gene (385 bps) were also examined using Sanger’S Method. It was revealed that six substitutions caused between the two species even though the DNA region examined was very short. The result seems to indicate that the two Pinus species distributed naturally in Japan are not so nearly related phylogenetically.