**1. Introduction**

82 Gel Electrophoresis – Advanced Techniques

Weeden N. F., Reisch B. I., Martens M. H. E. (1988): Genetic Analysis of Isozyme

Wolfe W. H (1976): Identification of grape varieties by isozyme banding patterns. American

113 (5): 765-769.

Journal of Enology and Viticulture 27: 68-73.

Polimorohism in Grape. Journal of the American Society for Horticultural Sciences

Cobia (*Rachycentron canadum*) is the sole representative of their family, the Rachycentridae They are distributed worldwide in tropical and subtropical seas, as the Atlantic and Pacific Oceans (Miao, et al., 2009). There are several species, including cobia, seabream, red progy, snappers, scads and groupers that are raised by cage culture in Taiwan. Among these cagecultured fishes, cobia certainly takes a leading distribution in both annual total production (81.9%) and total value production (75.4%) as compared to the rest in Taiwan (Fisheries Agency 2006).

Giant grouper (*Epinephelus lanceolatus*) are also found in tropical and subtropical waters from the Indo-Western Pacific Ocean. It is one of the two largest species of groupers in the world. Due to its fast growth and high price, giant grouper currently is regarded as a favorite species for marine culture in Taiwan (Hseu, et al., 2004).

Red coral trout (*Plectropomus leopardus*) a reef-associated fish in Western Pacific, distributed from southern Japan to Australia and eastward to the Caroline Islands (Zhang, et al., 2010). Only few studies concerning population genetics of *Plectropomus leopardus* has been reported.

All of cobia, giant grouper, and red coral trout are high-valued fish market in Taiwan and neighboring countries, including China, Japan, and Vietnam. For the globalization of the seafood industry, seafood authentication and food safety are very important. We must know that the source of fish or accurately species of the fish. Traditional method to distinguish the fish species was observed the external traits. It can cause the error judgment. Today, DNAbased methods are also more frequently employed for food authentication (Lockley and Bardsley, 2000). It has proven to be reliable, sensitive and fast for many aspects of fish species and food authentication. Asensio et al. (2009) were suggesting that the species-specific PCR method could be potentially used by regulatory agencies as routine control assay for the commercial grouper fillets authentication. PCR-based methods commonly used for fish species identification include PCR-sequencing, random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR). Those methods are simplicity, specificity and sensitivity.

Molecular Electrophoretic Technique for Authentication of the Fish Genetic Diversity 85

**No. Species Sampling** 

**1** *E. lanceolatus* Wild **R1** *R. canadum* Wild **71411** *P. leopardus* Wild **E2** *E. lanceolatus* Cultivated **R2** *R. canadum* Wild **71412** *P. leopardus* Wild **E3** *E. lanceolatus* Cultivated **R3** *R. canadum* Cultivated **71413** *P. leopardus* Wild **E4** *E. lanceolatus* Cultivated **R4** *R. canadum* Wild **82011** *P. leopardus* Wild **E5** *E. lanceolatus* Wild **R5** *R. canadum* Cultivated **91321** *P. leopardus* Wild **E6** *E. lanceolatus* Cultivated **R6** *R. canadum* Wild **91711** *P. leopardus* Wild **E7** *E. lanceolatus* Wild **R7** *R. canadum* Cultivated **91712** *P. leopardus* Wild **E8** *E. lanceolatus* Wild **R8** *R. canadum* Cultivated **71421** *P. leopardus* Cultivated **E9** *E. lanceolatus* Wild **R9** *R. canadum* Wild **71422** *P. leopardus* Cultivated **E10** *E. lanceolatus* Cultivated **R10** *R. canadum* Wild **71423** *P. leopardus* Cultivated **E11** *E. lanceolatus* Cultivated **R11** *R. canadum* Wild **81621** *P. leopardus* Cultivated **E12** *E. lanceolatus* Cultivated **R12** *R. canadum* Wild **80622** *P. leopardus* Cultivated **E13** *E. lanceolatus* Wild **R13** *R. canadum* Wild **81623** *P. leopardus* Cultivated **E14** *E. lanceolatus* Wild **R14** *R. canadum* Wild **91322** *P. leopardus* Cultivated

**sources** 

**No. Species Sampling** 

**sources** 

Table 1. Specimens of *E. lanceolatus, R. canadum, and P. leopardus* fish analyzed and locality

RAPD Analysis Kit (Amersham Pharmacia Biotech, Piscataway, NJ), and Operon primer kit (Operon, Advanced Biotechnologies). DNA amplification was performed in a final volume of 25 μl in the "Gene Amp PCR System 2720" thermal cycler (Applied Biosystems Inc., USA). The reaction mix contained 20 mM Tris-HCl, pH8.0, 50 mM KCl, 2 mM MgCl2, 10 mM dNTPs each (dATP, dCTP, dGTP, dTTP), 20 μM of primer, 2.5 U *Taq*-polymerase (Promega, Co., Wisconsin, USA) and 1 μl of the 10 ng extracted DNA. The preamplification PCR procedure was: treatment at 94oC for 5 min, followed by 35 cycles of denaturation at 94oC for 30s, annealing at a primer-specific annealing temperature as table 2 for 30s and extension at 72oC for 30s, and final extension at 72oC for 10 min. The annealing temperature of Cobia was 36oC. A 10 μl of the PCR product were analyzed in a 2 % agarose gel in 0.5 X TBE. The electrophoresis was performed at a constant voltage of 150 V for 150 min and 250 V for 1 min. The gel was stained with ethidium bromide and

ISSR primers of this study were listed in the Table 3. A total 59 primers were screened. Preamplification PCR reaction was conducted in 25 μl reaction containing 12.5 μl PCR master mix (Promega, Co., Wisconsin, USA), 1μl each primer, 1 μl of the 10 ng extracted DNA, and 10.5 μl dH2O. Then, the mixtures were subjected to 94oC for 5 min, followed by 35 cycles of denaturation at 94oC for 30s, 30s at a primer-specific annealing temperature as table 3,

where they were collected

**No. Species Sampling** 

**sources** 

visualized under UV light.

**2.3 PCR-ISSR method** 

Recently, many researchers have reported for the assessment of genetic structure of aquaculture species such as red mullet (Mullus barbatus), Tropical abalone (Haliotis asinina), and suminoe oyster (Crassostrea ariakensis) using several kinds of molecular markers (Garoia et al., 2004; Tang et al., 2004; Zhang et al., 2005; Maltagliati, et al., 2006), including RAPD, ISSR, AFLP, and RFLP methods. Many molecular methods are available for studying various aspects of wild populations, captive broodstocks and interactions between wild cultured stocks of fish and other aquatic species (Okumus and Ciftci, 2003). Among those methods, RAPD and ISSR technology were cheaper, simple, and fast. And just only one primer could obtain the different profiles for genomic analysis (Welsh and McClelland, 1990). RAPD is simple, rapid and cheap, it have high polymorphism. RAPD analysis has been used to evaluate genetic diversity for species, subspecies and population identification in common carp (Bártfai, et al., 2003), Indian major carps (Barman, et al., 2003). The microsatellite method was already used to study of genetic diversity of other grouper (Antoro, et al., 2006; Ramirez, et al., 2006; Wang, et al., 2007). Zeng, et al. (2008) have report that genetic analysis of Malaysia and Taiwan wild populations of giant grouper by microsatellite method. Their results were shown polymorphic loci in those populations, but they didn't discriminate the wild and cultivated populations of giant grouper. Beside, genetic markers can be suitable for assessing the differences between culture stocks and wild population and monitoring the changes in the genetic variation (Okumuş, et al., 2003). Monitoring the genetic diversity of natural populations and fish raised in fish hatcheries is fundamentally important for species conservation. Molecular markers can be very useful in this context (Povh et al., 2008).

In our study, we try to identify the seafood products, including cobia, giant grouper, and red coral trout from cultivated and wild populations by molecular markers, and provide the fish population genetic diversity for seafood management and good monitoring for brood stock management.
