**3. Results**

### *3.1. Multiplex PCR System Establishment*

The optimal amplification temperature for 12 microsatellite loci was first determined using gradient PCR amplification, and the results showed that the optimal annealing temperature was 53 ◦C. Two multiplex PCR systems, multiplex PCR1 and multiplex PCR2, were constructed using 12 pairs of microsatellites based on the annealing temperature and amplification length. Information on the combination of sites, primer concentrations used for multiplex PCR and information on the two universal primers and the fluorescent markers compatible with the universal primers is shown in Table 1. The results of PCR amplification and agarose gel electrophoresis of samples from 30 individuals showed that the PCR target bands were clear and did not overlap. The amplification efficiency of each locus was similar, and the amplified fragment sizes were as expected. The capillary electrophoresis diagram is shown in Figures 1 and 2.

#### *3.2. Polymorphism Analysis*

Thirty *C. undulatus* specimens were selected for PCR amplification, capillary electrophoresis, and genotyping of 12 loci in the two multiplex PCR systems, using fluorescently labeled primers. The genetic parameters were calculated and are presented in Table 3. The number of alleles of the 12 microsatellite markers ranged from 2 to 8. Among these, locus Cun148 had the highest number of alleles (8). The mean He was 0.594, and the mean Ho was 0.475. The null allele frequencies ranged from −1.104 to 0.716, with 2 loci having higher frequency invalid alleles (F(null) > 0.2), at Cun752 and Cun27. The polymorphic information content (PIC) ranged from 0.422 to 0.746, with a mean value of 0.557. A total of 9 loci were highly polymorphic (PIC ≥ 0.5) and 3 loci were moderately polymorphic (0.25 ≤ PIC < 0.5). Five loci deviated from Hardy–Weinberg equilibrium (HWE, *p* < 0.05 after Bonferroni correction) The above results show that the multiplex PCR method consisting of 12 primer pairs of microsatellites is stable and accurate in the population typing of *C. undulatus*. This can provide accurate and reliable genetic information for *C. undulatus* germplasm identification, family lineage management, and stocking effect evaluation.

**Figure 1.** The capillary electrophoresis diagram of the seven primer pairs in group G1. (The 1 to 7 in this Figure correspond to alleles loci Cun463, Cun378, Cun500, Cun626, Cun672, Cun586 and Cun148, respectively).

**Figure 2.** The capillary electrophoresis diagram of the five primer pairs in group G2. (The 8 to 12 in this Figure correspond to alleles loci Cun752, Cun230, Cun27, Cun485 and Cun484, respectively).

**Table 3.** Genetic parameters of 12 microsatellite loci in *C.undulatus*.



**Table 3.** *Cont.*

Note: ns = not significant (*p* > 0.05), \*. Significant difference (*p* < 0.05), \*\*. Extremely significant difference (*p* < 0.01).

#### **4. Discussion**

*C. undulatus* is one of the most valuable and expensive fish species in coral reefs [5], and its populations have experienced a dramatic decline [12]; however, the role of coral reef destruction and human fishing remains unclear. Assessing changes in genetic diversity and inbreeding levels is a very valuable monitoring tool [13]. For monitoring specific populations, microsatellites have several advantages and a wide range of applications. Multiplex PCR is a cost-effective and rapid method for obtaining accurate genetic information [31].

Here, we selected 12 polymorphic microsatellites, developed one 7-plex PCR amplification system and one 5-plex PCR amplification system, and used them to examine the genetic diversity of 30 *C. undulatus* specimens. Three main factors, primer combination, primer concentration, and annealing temperature, were optimized, establishing an accurate, rapid, and efficient microsatellite analysis technique for this species. We obtained a total of 12 alleles; 9 of the 12 loci were highly polymorphic, 3 were moderately polymorphic, and overall had a high level of polymorphism. After Hardy–Weinberg equilibrium validation, seven loci showed no significance and five loci deviated from Hardy–Weinberg equilibrium, which may be related to the limited sample size and the presence of invalid alleles. We further calculated the null allele frequencies and two loci had high null alleles. Therefore, it was further speculated that the five loci deviated from Hardy–Weinberg equilibrium mainly due to the limited sample size. The results showed that the multiplex amplification system was stable and reliable, and the loci were highly polymorphic. This is the first multiplex PCR amplification system for the *C. undulatus*.

Microsatellite multiplex PCR technology has been used for parentage analysis of Cirrhinus molitorella [32], and is also used commercially in large-scale selection for breeding [33]. It has also been developed and utilized as a powerful and low-cost parental assignment tool to support company-breeding programs [34]. Furthermore, it has been used to identify paternity assignments in grass carp [35] and in genetic diversity studies of Portunus trituberculatus [36]. Multiplex PCR has also been applied to detect hemolytic strains in fish and fishery products [37] and to assess Lateolabrax japonicus population genetics [38]. These studies illustrate the applicability of multiplex PCR, demonstrating its advantages in terms of performance, accuracy, experimental time, and experimental cost.

The small number of samples is a limitation in this study. More samples could improve the accuracy and reliability of the data. We can be able to take more samples and redesign primers to validate loci that have high frequency of invalid alleles and deviate from Hardy–Weinberg equilibrium to ensure the accuracy and reliability of this multiplex PCR system in the future, and more parental and offspring samples can be collected for parentage identification using the 12 microsatellite loci to elucidate genealogical information for the genetic improvement of *C. undulatus*, and to provide a basis for selective breeding.
