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We aimed to develop a multiplex PCR method for the simultaneous detection of the three most consumed commercial loaches in East Asia: Misgurnus mizolepis, Misgurnus anguillicaudatus, and Paramisgurnus dabryanus. Phylogenetic analysis of genetic divergence and a phylogenetic tree based on the cytochrome oxidase I gene enabled successful genetic identification of the 3 species of loach. Multiplex species-specific PCR (MSS-PCR) primers designed based on single nucleotide polymorphisms were able to differentiate the species and generate PCR products among species that differed in size by at least 150 bp. The lengths of the amplified fragments were 818 bp for P. dabryanus, 669 bp for M. mizolepis, and 486 bp for M. anguillicaudatus, which could be clearly distinguished by gel electrophoresis. The three sets of PCR primers were mixed and applied to simultaneously detect the three loaches. The MSS-PCR markers designed in this study will be useful for the detection and identification of M. mizolepis, M. anguillicaudatus, and P. dabryanus.

Although the mitochondrial genomes of most Acanthuriformes fish have been sequenced, this has not been done for Prionurinae fish yet. The Scalpel sawtail (Prionurus scalprum Valenciennes, 1835) found in all tropical and sub-tropical seas, is an important link between primary producers and higher trophic levels. In this study, the complete mitochondrial genome of the Scalpel sawtail, Prionurus scalprum Valenciennes, 1835 (accession number: OK323243) was sequenced. The complete mitochondrial genome included 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region; the total length was 16,522 bp. The nucleotide composition of the genome was as follows: A, 28.46%; T, 27.62%; G, 16.46%; and C, 27.46%. All genes were encoded on the H-strand, except for the eight tRNA genes and NADH dehydrogenase subunit (ND6). A phylogenetic tree was constructed using the neighbor-joining (NJ) method and the phylogenetic position of the Scalpel sawtail was established. This provides useful data for future research on Acanthuridae fish.

Girella punctata and Girella leonina are economically important species found in the East Sea; along the southern coast of Korea; south of Hokkaido, Japan; around Taiwan; and in the East China sea. In Korea, these two species hold high value, particularly on Jeju Island. These species have similar appearances, and it is challenging to distinguish them, particularly during the seed period. We detected genetic differences in the mtDNA (COI gene) of G. punctata and G. leonina, which are morphologically indistinguishable, and developed species-specific genetic markers for their identification. In total, 16 and 4 haplotypes of the COI genes were obtained from G. punctata (n = 164) and G. leonina (n = 36), respectively. The haplotype diversity (Hd) and nucleotide diversity (Pi, %) of the COI were 0.359 and 0.054 for G. punctata and 0.560 and 0.078 for G. leonina, respectively. We designed a Girella species common primer (control) and species-specific primer sets (experimental) for the two species. Amplicon sizes of 991, 579, and 391 bp were obtained for common primers of the two Girella species G. punctata and G. leonina. To confirm multiple targets in a single reaction, multiplex PCR conditions were optimized to adjust its resolution and accuracy. The detection levels of the multiplex PCR were confirmed to be 0.01 ng/µL for the two Girella species. The multiplex PCR was not associated with cross-reactivity between G. punctata and G. leonina. This multiplex species-specific PCR method provides a simple and rapid technique for the identification of two Girella species, thus increasing the efficiency and quality of Girella species stock management and forensic identification to prevent species misidentification.