# 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

## TL;DR

A new multiplex PCR method was developed to quickly and accurately identify five species of onchidiid slugs in the South China Sea for better conservation and management.

## Contribution

The first multiplex PCR method for simultaneous identification of five onchidiid slug species using mitochondrial COI gene sequences.

## Key findings

- The multiplex PCR method successfully identified five onchidiid slug species in a single reaction.
- The method is rapid, economical, and does not require sequencing equipment.
- It overcomes limitations of traditional morphological identification and DNA sequencing.

## Abstract

The species of Onchidiidae boast a global distribution, with a particularly high concentration of diversity in Southeast Asia. In this review, based on the mitochondrial COI gene sequence, a multiplex PCR method was successfully established for the first time to simultaneously identify five species of Onchidiidae. By designing specific primer combinations, this method can produce species-specific and different-length amplified fragments in a single reaction, and achieve accurate and stable identification of O. stuxbergi, P. verruculata, O. reevesii, P. martensi and P. tumidus, and no intraspecific variation was observed. The establishment of this technology effectively overcomes the application limitations of traditional morphological identification in larvae or characteristic missing samples, as well as the shortcomings of DNA sequencing technology, such as high cost, long time-consuming and strong equipment dependence. With its strong specificity, simple operation, rapid and economical characteristics, this method provides an efficient and reliable technical tool for large-scale screening, on-site rapid identification and resource monitoring of snail species, and has important practical significance for germplasm resource management, artificial breeding and ecological protection of Onchidiidae.

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: Onchidium stuxbergi, Peronia verruculata, Onchidium reevesii, Platevindex martensi and Paromoionchis tumidus. 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.