An Efficient PCR-RFLP Method for the Rapid Identification of Korean Pyropia Species

The present study utilizes polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using partial plastid rbcL and mitochondrial trnC-trnP gene sequences to distinguish the six representative Pyropia species produced via mariculture in Korea. The rbcL, trnC, and trnP sequences of 15 Pyropia species from the NCBI database were aligned to determine specific restriction enzyme sites of the six Pyropia species. To confirm the presence of restriction sites of eight enzymes, PCR amplicons were digested as follows: a 556 bp fragment within the rbcL region of chloroplast DNA was confirmed in P. yezoensis using BglI, whereas Tth111I, AvaII, BsrI, and BsaAI enzymes produced fragments of 664, 271, 600, and 510 bp, respectively, from the rps11-trnG region of mitochondrial DNA in P. seriata, P. dentata, P. suborbiculata, and P. haitanensis. In the case of P. pseudolinearis, HindIII, SacII, and SphI enzymes each had two cleavage sites, at positions 174 and 825, 788 and 211, and 397 and 602 bp, respectively. All six species were successfully distinguished using these eight restriction enzymes. Therefore, we propose that PCR-RFLP analysis is an efficient tool for the potential use of distinguishing between the six Pyropia species cultivated via mariculture in Korea.