Multiplex Species-Specific PCR Identification of Native Onchidiid Slugs in the South China Sea: A Useful Tool for Application in Onchidiid Slug Stock Management and Germplasm Conservation

To address the need for accurate onchidiid slug species identification, the present study engineered a multiplex PCR-based assay to facilitate the efficient, economical, and discrimination of five morphologically similar onchidiid slug species endemic to the South China Sea: <i>Onchidium stuxbergi</i>, <i>Peronia verruculata</i>, <i>Onchidium reevesii</i>, <i>Platevindex martensi</i> and <i>Paromoionchis tumidus</i>. A diagnostic multiplex PCR assay was designed based on a partial fragment of the mitochondrial cytochrome oxidase subunit I. It utilizes a shared forward primer and five taxon-specific reverse primers to simultaneously produce amplicons of varying sizes, each indicative of a particular species. Five species of Onchidiidae can be identified in a single reaction, which provides technical support for molecular identification of Onchidiidae. The development of a simple, effective and rapid multiple PCR identification method is of great significance for stock management and germplasm conservation of Onchidiidae.