Identification of DNA molecular markers by comparison of Pinus densiflora and Pinus sylvestris chloroplast genomes
Identifying and characterizing genetic variation can clarify the molecular basis of biological phenomena in plants. In particular, related or morphologically similar species can be distinguished by molecular markers. Pinus densiflora Siebold & Zucc. is a species that is distributed in the Korean peninsula, the Japanese archipelago, and China's Shandong and Manchu Provinces and has long been harvested for timber. However, it is difficult to distinguish P. densiflora from Pinus sylvestris L. both
... sylvestris L. both morphologically and phylogenetically. The complete chloroplast genome of P. densiflora has not yet been reported. In this study, we sequenced the P. densiflora chloroplast genome in order to identify the molecular markers that can be used to distinguish this species from P. sylvestris. Methods: Genomic DNA was extracted from P. densiflora samples obtained from the clone bank of the National Forest Seed Variety Center and was sequenced on an Ion Torrent platform. Filtered sequences were assembled with P. sylvestris sequences used as a reference and gene annotation was performed. The chloroplast genome sequences of the two species were aligned and the number and location of forward, reverse, complement and palindromic matches were determined. Single nucleotide polymorphisms (SNPs) and insertion/deletion mutations (Indels) were identified and analyzed by PCR. Results: The P. densiflora chloroplast genome consisted of circular double-stranded DNA with 119,835 bp compared to 119,758 bp for P. sylvestris. Between the two Pinus chloroplast genomes, we identified 73 SNPs and 171 Indels; two gene regions with amplification products ≤ 300 bp (rpoC1 and trnM-trnV) were validated as molecular markers. Discussion: PCR restriction fragment length polymorphism analysis revealed differences between P. sylvestris and P. densiflora at the molecular level. These differences can be used to distinguish between these two species, which is not possible by microscopy-based morphological examination.