We developed and validated a rapid, cost-effective multiplex PCR assay targeting mitochondrial cytochrome c oxidase subunit I (COX1) to discriminate three commercially important sea-squirt species, Halocynthia roretzi, H. aurantium and H. hilgendorfi ritteri. Species-specific forward primers were designed from interspecific single-nucleotide polymorphisms within the barcode region and combined with a common reverse primer in a single reaction. Specificity was confirmed in all tested individuals (n = 7 per species) without cross-amplification. Sensitivity tests demonstrated consistent amplification down to 0.1 ng of template DNA, matching or surpassing detection limits reported for other food-authentication markers. Because the entire reaction including DNA extraction can be completed within three hours and requires only basic laboratory equipment, the method is well suited for quality control laboratories, border inspections and routine monitoring of processed products. The COX1 multiplex PCR set proposed here provides a reliable tool to enhance traceability, protect consumer choice, and support regulatory enforcement in the sea-squirt supply chain.

Processed bivalve products typically lack diagnostic shell characters, making DNA-based assays essential for reliable species authentication in routine testing. We developed and validated a conventional, COX1-based multiplex PCR for the simultaneous identification of six commercially important food bivalves: Argopecten irradians, Mimachlamys crassicostata, Mizuhopecten yessoensis, Scaeochlamys farreri, Atrina pectinata, and Swiftopecten swiftii. The assay yielded robust, single-band amplification across an annealing window of 54–59 °C and maintained reproducible performance over template inputs from 0.1 to 50 ng per reaction. This work provides a practical marker set for simultaneous identification of six bivalve species in freeze foods or tissues where morphological traits are not preserved.

The starry flounder (Platichthys stellatus) and the spotted halibut (Verasper variegatus) are economically important flatfish species in Korea. The hybrid P. stellatus (♀) × V. variegatus (♂) has attracted attention in aquaculture. In this study, we report the first complete mitochondrial genome sequence of this hybrid flatfish, obtained using next-generation sequencing. The mitogenome is 16,874 bp in length and comprises 13 protein-coding genes, 22 tRNAs, and two rRNAs. Phylogenetic analysis revealed that the hybrid clusters with its maternal species, P. stellatus, confirming strict maternal inheritance of mitochondrial DNA. These findings provide genomic resources for phylogenetics and hybrid flatfish breeding.

Acanthogobius lactipes is a demersal, euryhaline fish belonging to the suborder Gobiodei. This study sequenced and described the complete mitochondrial genome of A. lactipes for the first time. The circular genome of A. lactipes is 16,592 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. The overall A, C, G, and T contents were 27.78, 27.31, 17.52, and 27.39%, respectively. Based on the 13 protein-coding genes, the phylogenetic tree showed that A. lactipes formed a well-supported cluster with the genus Acanthogobius and rooted with other family Oxudercidae species.

Triploid oysters are widely used in aquaculture, but their commercial species designation may not accurately represent their nuclear genomic composition, particularly when interspecific or polyploid broodstocks are involved. In this study, we combined whole-genome sequencing with a mitochondrial COI-based multiplex PCR assay to characterize commercial triploid Crassostrea gigas, C. angulata, and C. hongkongensis. Whole-genome allele-frequency analysis confirmed triploidy in all 15 individuals examined, comprising five individuals each from the A3N, G3N, and H3N groups. Cross-species ancestry dosage showed that G3N and H3N were predominantly assigned to their nominal species, whereas A3N exhibited mixed C. angulata–C. gigas nuclear ancestry, with approximately 2.2 and 0.7 haploid-equivalent copies assigned to these species, respectively. The minor C. hongkongensis assignment was interpreted as background cross-assignment rather than evidence of an additional ancestral contribution. Local-ancestry analysis further revealed chromosome-scale mosaics of zero, one, and two C. gigas-derived copies, with copy-weighted C. gigas contributions of approximately 0.69–0.88 haploid-equivalent copies among A3N individuals. This interspecific ancestry was supported by convergent evidence from complementary analyses of discrete dosage-state distributions, D-statistics with two alternative outgroups, and mapping-independent C. gigas-diagnostic 21-mers. All A3N individuals nevertheless carried C. angulata COI haplotypes, consistent with a C. angulata maternal lineage. The breeding history underlying the nuclear ancestry mosaics could not be resolved because the actual parental broodstock was unavailable for genomic analysis. A three-species multiplex PCR assay generated diagnostic products of 212, 359, and 169 bp for C. gigas, C. angulata, and C. hongkongensis, respectively, with no detectable cross-amplification at an optimized annealing temperature of 62 °C. Diagnostic amplification remained detectable with 1 ng of template DNA and was successfully applied to specimens from multiple Korean production sites and Dalian, China. These findings demonstrate complex nuclear ancestry in the five A3N individuals examined that was not captured by their nominal species designation or mitochondrial lineage alone. Although the observed ancestry proportions cannot be generalized to commercial A3N stocks as a whole, the results highlight the complementary roles of genome-wide ancestry analysis and rapid mitochondrial lineage authentication.

Two generations were hatched after artificially crossing Paralichthys olivaceus and Verasper variegatus, commercially valuable species of Pleuronectidae. Here, we report the complete mitochondrial genome and phylogenetic relationships of P. olivaceus (♀) × V. variegatus (♂). The mitogenome is 16,946 bp long and contains 13 protein-coding genes (PCGs), 22 tRNA genes, and 2 rRNA genes. Phylogenetically, it clustered with P. olivaceus within Pleuronectidae, providing a justification for the lack of information on flounder hybrids. Analysis of genetic information and genetic mapping studies of individuals with intermediate traits between the parents support previous research suggesting that it has economic value.