Rapid molecular authentication of cultured Takifugu rubripes using structural genomic variations

Abstract Takifugu rubripes , a pufferfish species of considerable commercial importance in East Asian cuisine, is widely cultured under controlled aquaculture conditions to produce tetrodotoxin (TTX)-free products. Accurate discrimination between cultured and wild individuals is essential for ensuring food safety and facilitating regulatory compliance, given the potential presence of TTX in wild populations. In this study, we developed a molecular diagnostic method based on structural genomic variations to enable rapid and reliable authentication of cultured T . rubripes . Comparative genomic analyses identified 226 large deletions (> 1 kb) in T . pseudommus relative to the T . rubripes reference genome. Among these, six deletions exhibited consistent amplification exclusively in cultured samples from Korea and Japan ( n = 68), whereas wild individuals of T . rubripes , T . pseudommus , and T . chinensis ( n = 29) either failed to amplify or yielded inconsistent results. These deletion markers were incorporated into a multiplex ultrafast real-time PCR assay capable of distinguishing cultured individuals within 30 min. Furthermore, analysis of the TP7-1 simple sequence repeat locus (GenBank accession no. PP949280) revealed reduced allelic diversity in cultured populations, facilitating additional discrimination from wild counterparts. The developed diagnostic assay provides an accurate and high-throughput platform for identifying TTX-free cultured T. rubripes and differentiating them from wild and closely related toxic species, thereby enhancing traceability and supporting food safety assurance in the global pufferfish trade.