Identification and characterization of RAPD markers inferring genetic relationships among Pine species

Total genomic DNAs were extracted from several populations of pine species and amplified using oligonucleotides of random sequences. Polymorphism in random amplified polymorphic DNA (RAPD) markers was high and sufficient in distinguishing each of the species. Genetic relationships among eight pine species (Pinus sylvestris, Pinus strobus, Pinus rigida, Pinus resinosa, Pinus nigra, Pinus contorta, Pinus monticola, and Pinus banksiana) from different provenances were analyzed. The degree of band sharing was used to evaluate genetic distance between species and to construct a phylogenetic tree. In general, the dendrogram corroborated the description of relationships based on morphological characteristics and crossability, but also provided new insights into pine taxonomy. RAPD markers specific to some pine species were cloned and sequenced. PCR amplifications using pairs of designed specific primers revealed that all the cloned sequences were likely genus specific because they were not found in spruce or larch. True species-specific sequences were identified using designed primers flanking cloned RAPD fragments. The analysis of RAPD fragment sequences confirmed the genetic relationships among species. A 2281-bp RAPD band called PI-Mt-Stb-23 from P. strobus was used as a probe in restriction fragment length polymorphism (RFLP) analysis and produced distinct banding patterns for each species examined, consistent with the highly polymorphic character of DNA-fingerprinting probes.