Big data-based identification and in situ application of single nucleotide polymorphism markers for geographical origin in aquatic organisms

Abstract

Aquatic organisms such as Scapharca subcrenata, Octopus minor, Chionoecetes opilio and Pagrus major are economically, ecologically and culturally significant resources in East Asia. However, the same species are often farmed in multiple countries or harvested from shared natural habitats. Particularly in countries like Korea, China and Japan, where adjacent sea areas are shared, the origin of harvested species frequently varies depending on the harvester, leading to challenges in origin identification. Currently, there is a lack of definitive methods for determining the origin of seafood, and existing techniques suffer from low accuracy and limited practical applicability. These limitations underscore the urgent need for innovative technologies to address such issues. With the increasing globalization of seafood trade and a rise in cases of origin mislabeling, the precise identification of seafood origin and the establishment of a systematic management framework have become essential. To achieve this, the development of molecular diagnostic methods with high accuracy and practical utility is imperative. This study aimed to address the challenges of seafood origin identification by employing genomic analysis. Single Nucleotide Polymorphism (SNP)-based molecular markers were developed for four species, namely S. subcrenata, O. minor, C. opilio, and P. major, using Genotyping-by-Sequencing (GBS). These markers revealed distinct genetic differentiation between populations. Principal Component Analysis (PCA) and population structure analysis validated the utility of the SNP markers, and allele- specific primers were designed based on the validated SNP. These primers were optimized for SYBR Green and TaqMan-based quantitative PCR (qPCR) analysis, achieving high classification accuracy. Furthermore, a ΔCt-based method incorporating reference primers was developed to ensure consistent results across varying DNA concentrations, thereby enhancing the reliability and reproducibility of the findings. In the case of S. subcrenata, three SNP markers (Insertion1, SNP16, SNP21) were utilized to effectively differentiate Korean and Chinese populations, achieving a classification accuracy of 95%. For O. minor, a single SNP marker (SNP18) distinguished Korean and Chinese populations based on TT, TC, and CC genotypes, with an overall accuracy of 92.2%, a sensitivity of 100%, and a specificity of 84.4%. For C. opilio, two SNP markers (SNP 08-12 and SNP 08-13) were used to successfully differentiate Korean and Russian populations, while for P. major, two SNP markers (SNP001 and SNP008) enabled accurate discrimination between Korean and Japanese populations. Across all species, the qPCR-based methods developed in this study demonstrated an accuracy exceeding 80%, outperforming previously reported approaches. Notably, this study set the ultimate goal of implementing origin identification technologies in in situ systems under field conditions. To realize this, a biomarker kit for field validation was developed based on the SNP18 marker for O. minor, enhancing the practicality of the method. This study provides significant molecular insights into the geographical origin identification of marine organisms. It is expected to contribute to strengthening seafood origin identification and management systems, supporting regulatory compliance and quality assurance. Additionally, the findings offer valuable foundational data for future academic research on geographical origin and enhance the understanding of genetic diversity in marine organisms, serving as a useful resource for genetic studies.