**1. Introduction**

*Cheilinus undulatus*, known as Maori, Napoleon, humphead wrasse, and so-mei [1], belongs to the order Perciformes [2]. The species is found in reefs and nearshore habitats with seagrass beds and mangroves distributed in tropical waters of the Pacific and Indian Oceans [3]. Its abundance is usually very low, and it feeds on mollusks, small fish, sea urchins, and crustaceans [4]. *C. undulatus* is one of the most valuable and expensive fish species in coral reefs [5], and the large coral triangle is the main distribution area [6]. Due to the heavy exploitation of the live reef fish trade (LRFT), it is classified as "vulnerable" in the IUCN 1996 Red Data Book [1]. International regulations treat *C. undulatus* as a wild fish that can be traded within a limited quota [7]. Human activities are a major cause of biodiversity decline, and marine animal extinctions began to accelerate in the 1970s, when fisheries harvesting peaked and began to linger. The marine animals under threat are mainly large animals at the top of the food chain. The populations of *C. undulatus*, a large fish in coral reefs, have rapidly declined because of heavy fishing and habitat destruction [8].

Fluctuations in population size can affect genetic diversity [9], and very small populations tend to cause inbreeding within the population [10], which can lead to a reduction in population fitness. Inbreeding decline is less pronounced in better environments but is readily apparent in harsh environments [11]. Current scientific studies have identified

**Citation:** Zhao, F.; Guo, L.; Zhang, N.; Zhu, K.; Yang, J.; Liu, B.; Guo, H.; Zhang, D. Establishment and Application of Microsatellite Multiplex PCR System for *Cheilinus undulatus*. *J. Mar. Sci. Eng.* **2022**, *10*, 2000. https://doi.org/10.3390/ jmse10122000

Academic Editor: Nguyen Hong Nguyen

Received: 21 October 2022 Accepted: 12 December 2022 Published: 15 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

a dramatic decline in the size of *C. undulatus* populations [12]; however, the role of coral reef destruction and human fishing is not clear. Assessing changes in genetic diversity and inbreeding levels is a practical monitoring tool [13]. Current molecular markers used to monitor genetic diversity include single-nucleotide polymorphisms (SNPs) [14] and microsatellites, which are also called simple sequence repeats [15]. For monitoring specific populations, microsatellite markers have the advantages of abundant alleles at individual loci [16], low typing cost [17], and mature technology [18]. They have a wide range of applications in genetic mapping, population structure analysis, genetic diversity studies, and germplasm conservation studies [19–22]. Multiplex polymerase chain reaction (multiplex PCR) refers to the simultaneous amplification of multiple target sequences by adding two or more pairs of primers to the same PCR reaction system [23]. Multiplex PCR can increase the number of microsatellite markers detected in a single run, simplifying the test procedure and reducing the cost and amount of DNA used in the sample [24,25]. In this study, 12 polymorphic microsatellite markers were selected, and one 7-plex PCR amplification system and one 5-plex PCR amplification system were successfully constructed. Then, the two systems were used to examine the genetic diversity of 30 *C. undulatus* specimens. This is the first multiplex PCR amplification system for the *C. undulatus*, providing technical support for germplasm identification, genetic diversity analysis and assessment of the effects of accretion and release in this species.
