Development of a Multiplex Polymerase Chain Reaction Method for Rapid and Accurate Identification of Girella punctata and G. leonina (Teleostei: Girellidae)

Abstract

Girella punctata and Girella leonina are economically important species found in the East Sea; along the southern coast of Korea; south of Hokkaido, Japan; around Taiwan; and in the East China sea. In Korea, these two species hold high value, particularly on Jeju Island. These species have similar appearances, and it is challenging to distinguish them, particularly during the seed period. We detected genetic differences in the mtDNA (COI gene) of G. punctata and G. leonina, which are morphologically indistinguishable, and developed species-specific genetic markers for their identification. In total, 16 and 4 haplotypes of the COI genes were obtained from G. punctata (n = 164) and G. leonina (n = 36), respectively. The haplotype diversity (Hd) and nucleotide diversity (Pi, %) of the COI were 0.359 and 0.054 for G. punctata and 0.560 and 0.078 for G. leonina, respectively. We designed a Girella species common primer (control) and species-specific primer sets (experimental) for the two species. Amplicon sizes of 991, 579, and 391 bp were obtained for common primers of the two Girella species G. punctata and G. leonina. To confirm multiple targets in a single reaction, multiplex PCR conditions were optimized to adjust its resolution and accuracy. The detection levels of the multiplex PCR were confirmed to be 0.01 ng/µL for the two Girella species. The multiplex PCR was not associated with cross-reactivity between G. punctata and G. leonina. This multiplex species-specific PCR method provides a simple and rapid technique for the identification of two Girella species, thus increasing the efficiency and quality of Girella species stock management and forensic identification to prevent species misidentification.

1. Introduction

Large-scale blackfish (Girella punctata) and small-scale blackfish (Girella leonina) belong to the Girellidae family (Centrarchiformes) and are among the 20 known species of Girella according to Eschmeyer’s Catalog of Fishes [1]. These species are distributed widely in tropical and subtropical waters, ranging from the Indo-Pacific region to China, Korea’s Jeju Island, and southern parts of Japan [2,3]. Among the 20 species, Girella mezina, G. punctata, and G. leonina coexist and are closely related [3,4]. Taxonomic confusion has arisen concerning Girella melanichthys as it has been incorrectly used as the valid name for G. leonina, resulting in the use of both names [4,5]. Girella punctata and G. leonina are herbivorous species and are often confused with each other due to their similar external morphological characteristics such as number of pored lateral line scales, color of opercular flap, shape of caudal fin, etc. Conversely, G. mezina can be distinguished easily from these other two species based on its morphological characteristics [5,6,7]. Previous studies have explored the phylogenetic relationships among the Girella species and found that G. punctata and G. leonina in East Asia are sister species, occurring sympatrically in overlapping areas [5]. The phylogenetic relationship between G. punctata and G. leonina has received significant attention, particularly with regard to the identification of differences between the species during the juvenile stage. Accurate species identification is challenging as the morphological characteristics of G. punctata and G. leonina cannot be distinguished. Differences in the external characteristics between G. punctata and G. leonina include the number of pored lateral line scales, spines, and soft rays in the dorsal fin and soft rays in the anal fin; the depth of emargination of the caudal fin; and the color of the opercular flap (blackish in G. leonina and “not black” in G. punctata) [4]. G. punctata has a sedentary ecological habit and settles along the coast, while G. leonina exhibits migratory behavior and inhabits areas further away from the coastline. Due to these distinct ecological traits, there are differences in their potential for industrial utilization. Therefore, analytical assays for the clear identification of these two Girella species are required.

In this study, we used a DNA barcode-based approach to classify G. punctata and G. leonina, two species that are challenging to differentiate based on their morphological characteristics. Furthermore, we evaluated the intra- and interspecies genetic diversity of these species and identified species-specific genetic sites in their COI genes. To achieve this, a multiplex species-specific PCR (MSS-PCR) method was developed, which enables the simultaneous discrimination of these two species through a single PCR. This study significantly improves our understanding of the genetic diversity of these two species, as the resulting DNA barcodes can be used not only for species identification but also for monitoring and conserving the diversity of fishery resources.

2. Materials and Methods

2.1. Sample Collection and DNA Extraction

In December 2021, fresh caudal fin tissues from 200 specimens of G. punctata and G. leonina were collected by the Jeju Fisheries Research Institute of the National Institute of Fisheries Science (NIFS). These samples were produced in the Jeju Fisheries Research Institute. The morphological characteristics of the specimens were analyzed. In the field, G. punctata (black) and G. leonina (colorless) were distinguished based on their operculum flap color pattern. Immediately after collection, the tissues were preserved in 95% ethanol until DNA extraction.

Total genomic DNA was extracted from the caudal fin of all 200 individuals of G. punctata and G. leonina using an automated DNA extraction system (KingFisher Flex system; Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. The extracted genomic DNA was quantified using a spectrophotometer (Nanodrop ND-1000; Thermo Fisher Scientific, Waltham, MA, USA) and stored at 4 °C until analysis.

2.2. PCR, Sequencing, Primer Design, and Data Analysis

The mtDNA cytochrome oxidase subunit I (COI) region was amplified, sequenced, and identified for all 200 individuals of G. punctata and G. leonina. The following primers were used for PCR amplification: VF2_t1 (5′-TGT AAA ACG ACG GCC AGT CAA CCA ACC ACA AAG ACA TTG GCA C-3′) and FishR2_t1 (5′-CAG GAA ACA GCT ATG ACA CTT CAG GGT GAC CGA AGA ATC AGA A-3′) [8]. For PCR amplification, we used a 20 µL reaction volume, which consisted of 15.8 µL of ultrapure water; 1 µL of a DNA template (10 ng/μL); 0.4 µL of dNTP (250 μM); and 2 µL of a 1× PCR buffer, with 2 mM MgCl2, 0.8 µL of the forward primer (VF2_t1; 10 pmol), 0.8 µL of the reverse primer (FishR2_t1; 10 pmol), and 0.2 µL of 0.5 U DNA Taq (Anti-HS Taq; TNT Research, Jeonju-si, Republic of Korea). The PCR was conducted using ABI Verity thermal cyclers (Applied Biosystems, Foster City, CA, USA) under the following conditions: 11 min of initial denaturation at 95 °C, followed by 35 cycles of 95 °C for 40 s, 54 °C for 40 s, and 72 °C for 50 s, with final elongation at 72 °C for 7 min.

The acquired bidirectional sequences were assembled using SeqMan Pro 17 (Lasergene 17; DNASTAR, Madison, WI, USA). The obtained COI sequences were compared with COI sequences registered in the GenBank using the NCBI Basic Local Alignment Search Tool (BLAST) web service to exclude potential errors and incomplete sequences. DnaSP (version 5.1) software was used to determine the number of haplotypes, polymorphic sites, haplotype diversity (H), and nucleotide diversity (Pi, %). A minimum spanning network was generated from the COI gene (870 bp) using PopArt v. 1.7 software. The inter- and intraspecies genetic distances were analyzed using MEGA 5 software based on the Kimura two-parameter distance model. The bootstrap support values were derived from 1000 randomized replicate datasets.

To design species-specific primers, we performed sequence alignments using the corresponding sequencing data for G. punctata (AP011060.1) and G. leonina (NC046940.1) registered in GenBank NCBI. The alignments were conducted using the BioEdit (ver. 7.2) and SeqMan Pro 17 (DNASTAR) software to identify inter- and intraspecies variation as well as conserved regions in the mtDNA COI gene. The conserved regions in the aligned sequences were used to design common primers (GR-207F and GR-1181R; 991 bp;

Table 1. Information about common primers in the COI region from mtDNA sequences of the two species of genus Girella.

Oligo Name	Sequence (5′ → 3′)	Mer	Tm (°C)	GC (%)	Product Size
GR-207F	AGT AAT ACC AAT TAT GAT TGG A	22	56	28	991 bp
GR-1181R	ATA GTG GGA ATC AGT GTA	18		39

) for the two species. This approach provided the nucleotide sequence information required for the development of species-specific primers (Figure 1).

For PCR amplification, we used a 20 µL reaction volume, which consisted of 14.8 µL of ultrapure water; 1 µL of a DNA template (10 ng/μL); 0.4 µL of dNTP (250 μM); and 2 µL of a 1× PCR buffer, with 2 mM MgCl2, 0.8 µL of the forward primer (GR-207F; 10 pmol), 0.8 µL of the reverse primer (GR-1181R; 10 pmol), and 0.2 µL 0.5 of U DNA Taq (Anti-HS Taq; TNT Research, Jeonju-si, Korea). The PCR was conducted using ABI Verity thermal cyclers (Applied Biosystems, Foster City, CA, USA) under the following conditions: 10 min of initial denaturation at 95 °C, followed by 35 cycles of 95 °C for 40 s, 56 °C for 40 s, and 72 °C for 50 s, with final elongation at 72 °C for 7 min. The PCR products were visualized using a gel documentation system (AE9000 E-Graph; Atto, Tokyo, Japan) and purified using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany). Fragment lengths were determined by comparing them with a 100 bp DNA ladder (Dynebio, Seongnam-si, Republic of Korea). The PCR products were sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and analyzed using an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA).

2.3. Species-Specific Primer Design

Using SeqMan Pro 17 software, we assembled the forward and reverse sequences obtained from G. punctata and G. leonina using the common primers. After trimming the sequences, a final length of 870 bp was obtained for analysis. The nucleotide sequence of the COI gene region was searched for single nucleotide polymorphisms with specificity between the two species, excluding intraspecies mutations caused by point mutations. We designed species-specific forward primers for the target species in which the species-specific single nucleotide polymorphism was located at the 3′ end to enable PCR amplification for both species. In addition, the PCR product size was considered for clear species identification.

2.4. Multiplex Species-Specific (MSS) PCR

To simultaneously identify the two species, we performed MSS-PCR using two forward primers (GP-825F and GL-639F) and one reverse primer (GR-1181R;

Table 2. Primers used in the multiplex species-specific PCR.

Oligo Name	Sequence (5′ → 3′)	Target Species	Tm (°C)	Product Size (bp)
GP-825F	CTA CAT GGG TAT AGT TTG A	Girella punctata	50	391
GL-639F	AAT ACT TCT CAC AGA CCG A	Girella leonina		579
GR-1181R	ATA GTG GGA ATC AGT GTA			-

). The PCR reaction mixture had a volume of 10 μL and consisted of 5.9 µL of ultrapure water; 1 µL of a DNA template (10 ng/μL); 0.5 µL of dNTP (250 μM); and 1 µL of a 1× PCR buffer, with 2 mM MgCl2, 0.5 µL of the forward primer (GP-825F and GL-639F; 10 pmol), 0.5 µL of the reverse primer (GR-1181R; 10 pmol), and 0.1 µL of 0.5 U DNA Taq (Anti-HS Taq, TNT Research, Jeonju-si, Republic of Korea). The PCR was performed using ABI Verity thermal cyclers (Applied Biosystems) under the following conditions: 10 min of initial denaturation at 95 °C, followed by 30 cycles of 95 °C for 30 s, 48–56 °C for 30 s, and 72 °C for 40 s, with final elongation at 72 °C for 7 min. The PCR products were subjected to 1.8% agarose gel electrophoresis (120 V, 40 min) with 1 × loading star (Dynebio, Seongnam-si, Republic of Korea) and visualized using a gel documentation system (AE9000 E-Graph; Atto, Tokyo, Japan). The fragment lengths were determined by comparing them with a 100 bp DNA ladder (Dynebio, Seongnam-si, Republic of Korea).

2.5. Specificity and Sensitivity of MSS-PCR

To evaluate the specificity of MSS-PCR, the amplified MSS-PCR products were subjected to agarose gel electrophoresis to verify the absence of dimer amplification, nonspecific products, and cross-reaction in the MSS-PCR mixture. An analytical sensitivity test for MSS-PCR was performed by quantifying the DNA of G. punctata and G. leonina with concentrations of 10, 1, 0.1, and 0.01 ng/μL. For both species, the MSS-PCR assay could detect DNA up to a concentration of 0.01 ng/μL.

3. Results and Discussion

Mitochondrial DNA is more easily detected than nuclear DNA due to its greater abundance [9,10]. DNA-based methods for G. punctata and G. leonina identification have previously been reported, such as polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) assays that identify specific mutations at enzyme cut sites [5]. However, PCR-based RFLP assays are labor-intensive and time-consuming and require experienced operators. On the other hand, multiplex PCR methods use a simple PCR machine and a gel documentation system commonly available in most laboratories. Furthermore, these methods are less dependent on the skill of the operators [9,11]. In addition, multiplex PCR requires PCR amplification without adding restriction enzymes. The simultaneous detection of multiple targets in a single reaction makes this method cost-effective and time-efficient, thereby providing a simpler alternative to RFLP. Moreover, species-specific primers can be designed by analyzing differences in nucleotide sequences within species, enabling the supplementation of false-positive and false-negative errors caused by acquired genetic mutations acquired during the analysis [12].

DNA barcoding is a widely used approach based on mitochondrial genes, such as cytochrome oxidase sub-unit I (COI), 16S rRNA, and cytochrome b amplification and profiling. This technique is applied in various biological fields, such as forensic genetics, biodiversity, and fish identification [13,14]. In particular, mitochondrial DNA analysis based on amplification and sequencing of the COI mitochondrial gene enables precise identification of species and strains [15].

3.1. Species Identification Based on mtDNA COI

The collected samples were identified using the sequence analysis of the mtDNA COI gene. The comparison of the 540 bp sequences of G. punctata and G. leonina with sequencing data from the NCBI GenBank identified that more than 99% of the sequences belonged to G. punctata (AP011060.1) and G. leonina (NC046940.1). The degree of similarity was further confirmed using the NCBI database BLAST search.

The obtained COI nucleotide sequences haplotypes were registered in GenBank using the Blast web service of the NCBI to exclude potential errors and incomplete sequences (G. punctate; accession no. OR267414~OR267429, G. leonina; accession no. OR267383~OR267386).

3.2. Sequencing of COI Gene and Genetic Diversity Analysis

The analysis of the COI (870 bp) identified 16 haplotypes (n = 164) and 4 haplotypes (n = 36) among 200 individuals of G. punctata and G. leonina, respectively. The most common haplotypes were present in 80% (n = 131) and 40% (n = 16) of G. punctata and G. leonina individuals, respectively. No haplotype was shared between G. punctata and G. leonina. The haplotype (Hd) and nucleotide (Pi, %) diversity analysis results were 0.357 and 0.054 in G. punctata and 0.560 and 0.078 in G. leonina, indicating that G. leonina had higher numbers of haplotype and nucleotide diversity than G. punctata.

Minimum spanning networks (MSNs) were constructed to investigate the intraspecies relationships of G. punctata and G. leonina. In the haplotype networks, the haplotypes of G. punctata and G. leonina differed by 55 fixed mutational steps; therefore, the two species formed completely different groups. The G. punctata network consisted of the central main haplotype (GP_1, n = 131), including several unique haplotypes (Figure 2). In the G. leonina network, each haplotype was located linear from the central main haplotype (GL_1, n = 21), which had a simple construct because it only has four haplotypes.

To determine the genetic similarity between G. punctata and G. leonina, we compared the distribution of the average inter- and intra-species genetic distances as a percentage of genetic divergence. The intraspecies genetic divergence was 0.2% in G. punctata and G. leonina, which was markedly lower than the interspecies genetic distance (7.2% between G. punctata and G. leonina). Therefore, G. punctata and G. leonina could easily be distinguished.

3.3. Multiplex Species-Specific Primer

The design of species-specific primers is a crucial step for MSS-PCR (Table 2, Figure 3). The MSS primers for multiple targets must contain at least one variable site in each species-specific primer; have a melting temperature ≥ 50 °C; generate PCR products with variable sizes among species; and produce bands with no other reaction products, such as self-dimers and heterodimers. We successfully developed a species-specific primer by identifying variable single nucleotide polymorphism sites with interspecies differences in the sequences obtained from the common primer of the COI gene [14,16,17]. We analyzed the 870 bp sequences from the target species for species-specific variation to enable rapid and simultaneous identification of species. In addition to intraspecies genetic variation, we designed species-specific forward primers based on single nucleotide polymorphisms of species-specific variation among the species (

Table 3. Inter-species variation analysis of COI region from mtDNA sequences of Girella punctata and Girella leonina.

Species	Polymorphic Sites
Base Pair (bp)	288	291	327	336	345	363	369	378	396	429	441
Girella punctata	A	A	T	A	T	A	C	A	T	A	T
Girella leonina	G	G	C	G	C	T	A	G	C	G	C
	475	483	486	495	501	504	534	546	558	564	582
	T	T	T	T	C	C	A	T	G	A	C
	C	A	C	C	T	T	G	C	A	G	A
	583	594	600	621	630	633	636	639	678	696	717
	C	T	T	C	T	G	T	G	T	G	T
	T	C	C	A	C	A	C	A	A	A	C
	756	768	810	816	819	825	849	852	864	867	873
	T	T	C	T	A	A	A	C	G	T	C
	C	C	T	C	G	G	T	T	A	C	T
	966	975	978	984	996	1008	1017	1023	1035	1044	1047
	T	G	T	T	T	G	A	C	T	T	A
	C	A	C	C	C	C	C	T	C	C	G
	1065	1089	1107
	A	G	T
	G	A	C

).

3.4. PCR Specificity and Sensitivity

To verify the specificity and sensitivity of the species-specific primers, gradient PCR was performed using species-specific primer sets. The PCR products obtained from the target species were visualized using agarose gel electrophoresis without nonspecific amplification or cross-reactions. The primer sets GP-825F/GR1181R and GL-639F/GR1181R successfully amplified the specific fragments of the 391 bp for G. punctata and that of the 579 bp for G. leonina, respectively, at an annealing temperature of 48–56 °C (optimal temperature: 50 °C). These results indicate that each species-specific primer could be distinguished using electrophoresis and exhibited high specificity for the target species.

We developed the MSS-PCR by adjusting the reaction components and PCR cycling conditions [14,16,18]. We performed the PCR amplification using mixed species-specific primers (GP-825F, GL-639, and GR-1181R) under different reaction conditions and annealing temperatures. The MSS-PCR products obtained using the same quantities of the forward primers were confirmed using agarose gel electrophoresis, with accurate DNA amplification for each species and clear distinction among the amplified products based on size (Table 1). Sequencing analyses confirmed that the MSS-PCR products of the two species had 100% identity with the expected regions. No primer dimers, nonspecific amplification products, or cross-reactions were observed among the species-specific amplifications (Figure 4). The MSS-PCR sensitivity test showed that the DNA template concentrations of stored products from the two species were 10, 1, 0.1, and 0.01 ng/μL. The sensitivity test showed that the MSS-PCR assay could detect the DNA of G. punctata and G. leonina at a concentration of 0.01 ng/μL (Figure 5).

4. Conclusions

We designed two primer sets to develop an MSS-PCR for simultaneous identification of G. punctata and G. leonina. The MSS-PCR results showed that the species-specific primers successfully detected amplicons of G. punctata (391 bp) and G. leonina (579 bp). The samples from the 200 individuals of the target species were identified correctly. The primer sets for the MSS-PCR were species-specific and sensitive, with a detection limit of 0.01 pg. The MSS-PCR can be used to detect two similar species of Girella and will contribute to fishery management as well as the safety of fishery products via the identification of similar species. In addition, it can be used to confirm the validity of highly degraded, commercially processed products rapidly and at a low cost.