**2.1 Fish sampling and genomic DNA extraction**

14 giant grouper (*Epinephelus lanceolatus*) and 14 cobia (*Rachycentron canadum*) were collected from Southern Taiwan, as Penghu Island, Kaohsiung, and Pengtung during 2007-2009. Those were selected in different cultured farms and local markets. 14 of red coral trout (*Plectropomus leopardus*) were collected from Penghu Island during 2009-2011. All samples were described in Table 1. All specimens were confirmed in the laboratory.

Approximately 1g of fish muscle tissue samples were cut into small pieces and pulverized in liquid nitrogen. The powdered fish samples were obtained and extracted genomic DNA using the QIAGEN® DNeasy Blood kit (QIAGEN Inc., Valencia, California) according to manufacturer's instructions. The extracted DNA concentration in 200 μl of sterile water and then the quality of DNA were assessed by a QubitTM Fluorometer (invitrogen, USA). The DNAs were stored at -20oC until PCR amplifications.

#### **2.2 PCR-RAPD method**

A total of 95 RAPD primers were used for PCR, which were shown in Table 2. Those sequences were obtained from University of British Columbia Biotechnology Laboratory,


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

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 visualized under UV light.

#### **2.3 PCR-ISSR method**

84 Gel Electrophoresis – Advanced Techniques

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).

stock management.

**2. Methods and material** 

**2.2 PCR-RAPD method** 

**2.1 Fish sampling and genomic DNA extraction** 

DNAs were stored at -20oC until PCR amplifications.

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

14 giant grouper (*Epinephelus lanceolatus*) and 14 cobia (*Rachycentron canadum*) were collected from Southern Taiwan, as Penghu Island, Kaohsiung, and Pengtung during 2007-2009. Those were selected in different cultured farms and local markets. 14 of red coral trout (*Plectropomus leopardus*) were collected from Penghu Island during 2009-2011. All samples

Approximately 1g of fish muscle tissue samples were cut into small pieces and pulverized in liquid nitrogen. The powdered fish samples were obtained and extracted genomic DNA using the QIAGEN® DNeasy Blood kit (QIAGEN Inc., Valencia, California) according to manufacturer's instructions. The extracted DNA concentration in 200 μl of sterile water and then the quality of DNA were assessed by a QubitTM Fluorometer (invitrogen, USA). The

A total of 95 RAPD primers were used for PCR, which were shown in Table 2. Those sequences were obtained from University of British Columbia Biotechnology Laboratory,

were described in Table 1. All specimens were confirmed in the laboratory.

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,

Molecular Electrophoretic Technique for Authentication of the Fish Genetic Diversity 87

**Primer Sequence of primer(5'-3') Tm(**℃**) References**  ISSR1 (GGAC)3A 48 Pazza et al. (2007)

ISSR801 (AT)8T 50 Liu et al. (2006)

SAS1 (GTG)4GC 55 Maltagliati et al.(2006)

ISSR807 (AG)8T 48 UBC Primer Set#9, University ISSR819 (GT) of British Columbia 8A 48

ISSR9 (GAG)5RY 55 Hou *et al*., 2006

ISSR2 (GGAC)3C 48 ISSR3 (GGAC)3T 48 ISSR4 (TGTC)4 48 ISSR5 (GGAC)4 48 ISSR6 (GGAT)4 48 ISSR7 (TAGG)4 48 ISSR8 (GACA)4 48

ISSR817 (CA)8A 48 ISSR825 (AC)8T 48 ISSR842 (CA)8YG 52 ISSR848 (CA)8RG 51 ISSR850 (GT)8YC 51 ISSR855 (AC)8YT 50 ISSR856 (AC)8YA 49 ISSR858 (TG)8RT 48 ISSR859 (TG)8RC 52 ISSR860 (TG)8RA 51 ISSR888 BDB(CA)7 52

SAS3 (GAG)4G 55 UBC809 (AG)8G 55 UBC811 (GA)8C 55 UBC827 (AC)8G 55 IT1 (CA)8GT 55 IT2 (CA)8AC 55 IT3 (GAG)4AG 55 PT1 (GT)8C 55

ISSR822 (TC)8A 48 ISSR831 (AT)8YA 48 ISSR834 (AG)8YT 48 ISSR843 (CT)8RA 48 ISSR852 (TC)8RA 48 ISSR861 (ACC)6 48 ISSR862 (AGC)6 48 ISSR868 (GAA)6 48 ISSR871 (TA)8RG 48 ISSR873 (GACA)4 48 ISSR877 (TGCA)4 48

ISSR10 VBV(CA)8 54 ISSR11 VDV(GT)8 51 ISSR12 HVHT(GT)7 51 ISSR13 (CT)8A 49 ISSR14 (TG)8GT 48 ISSR15 (AG)8TG 54 ISSR16 (TC)8C 52 ISSR17 (TG)8G 53 ISSR18 (TG)6R 45


Table 2. RAPD primers of PCR amplification

extension at 72oC for 30s, and final extension at 72oC for 5 min before analysis by the electrophoresis as described previously.

**primer (5'-3')**

primer 4 AAGAGCCCGT 36

RAPD540 CGGACCGCGT 56 Set#6

**Tm** 

**(**℃**) references** 

Ready-To-Go. RAPD Analysis Kit (Amersham Pharmacia Biotech, Piscataway, N J )

Advanced Biotechnologies)

Portman International (China) Limited, Hong Kong

**primer (5'-3') Tm(**℃**) references Primer Sequence of** 

Primer Set#1, University of British Columbia

RAPD64 GAGGGCGGGA 56 RAPD601 CCGCCCACTG 56 Set#7

RAPD86 GGGGGGAAGG 56 RAPD769 GGGTGGTGGG 56 Set#8

RAPD149 AGCAGCGTGG 56 OPA1 CAGGCCCTTC 56 (Operon,

extension at 72oC for 30s, and final extension at 72oC for 5 min before analysis by the

RAPD17 CCTGGGCCTC 56 RPAD542 CCCATGGCCC 56 RAPD22 CCCTTGGGGG 56 RAPD563 CGCCGCTCCT 56 RAPD23 CCCGCCTTCC 56 RAPD571 GCGCGGCACT 56 RAPD31 CCGGCCTTCC 56 RAPD584 GCGGGCAGGA 56 RAPD34 CCGGCCCCAA 56 RAPD585 CCCGCGAGTC 56 RAPD50 TTCCCCGCGC 56 RAPD592 GGGCGAGTGC 56 RAPD56 TGCCCCGAGC 56 RAPD595 GTCACCGCGC 56 RAPD63 TTCCCCGCCC 56 RAPD598 ACGGGCGCTC 56

RAPD65 AGGGGCGGGA 56 RAPD603 ACCCACCGCG 56 RAPD67 GAGGGCGAGC 56 RAPD606 CGGTCGGCCA 56 RAPD70 GGGCACGCGA 56 RAPD615 CGTCGAGCGG 56 RAPD71 GAGGGCGAGG 56 RAPD620 TTGCGCCCGG 56 RAPD73 GGGCACGCGA 56 RAPD625 CCGCTGCAGC 56 RAPD81 GAGCACGGGG 56 RAPD626 CCAAGCCCGG 56 RAPD83 GGGCTCGTGG 56 RAPD640 CGTGGGGCCT 56 RAPD84 GGGCGCGAGT 56 RAPD647 CCTGTGGGGG 56

RAPD87 GGGGGGAAGC 56 RAPD770 GGGAGGAGGG 56 RAPD88 CGGGGGATGG 56 RAPD771 CCCTCCTCCC 56 RAPD89 GGGGGCTTGG 56 RAPD772 CCCACCACCC 56 RAPD94 GGGGGGAACC 56 primer1 GGTGCGGGAA 36

RAPD95 GGGGGGTTGG 56 primer 2 GTTTCGCTCC 36 RAPD96 GGCGGCATGG 56 primer 3 GTAGACCCGT 36

RAPD106 CGTCTGCCCG 56 primer 5 AACGCGCAAC 36 RAPD115 TTCCGCGGGC 56 primer 6 CCCGTCAGCA 36

RAPD157 CGTGGGCAGG 56 OPA2 TGCCGAGCTG 56 RAPD158 TAGCCGTGGC 56 OPA3 AGTCAGCCAC 56 RAPD173 CAGGCGGCGT 56 OPA4 AATCGGGCTG 56 RAPD174 AACGGGCAGC 56 OPA5 AGGGGTCTTG 56 RAPD190 AGAATCCGCC 56 OPA6 GGTCCCTGAC 56 RAPD196 CTCCTCCCCC 56 OPA7 GAAACGGGTG 56 RAPD198 GCAGGACTGC 56 OPA8 GTGACGTAGG 56 RAPD210 GCACCGAGAG 56 Set#3 OPA9 GGGTAACGCC 56 RAPD211 GAAGCGCGAT 56 OPA10 GTGATCGCAG 56 RAPD218 CTCAGCCCAG 56 OPA11 CAATCGCCGT 56 RAPD241 GCCCGACGCG 56 OPA12 TCGGCGATAG 56 RAPD245 CGCGTGCCAG 56 OPA13 CAGCACCCAC 56 RAPD270 TGCGCGCGGG 56 OPA14 TCTGTGCTGG 56 RAPD286 CGGAGCCGGC 56 S514 CAGGATTCCC 56

RAPD287 CGAACGGCGG 56 S1036 AAGGCACGAC 56 RAPD319 GTGGCCGCGC 56 Set#4 S1040 CCTGTTCCCT 56 RAPD480 GGAGGGGGGA 56 Set#5 S1042 TCGCACAGTC 56 RAPD534 CACCCCCTGC 56 Set#6 S1201 CCATTCCGAG 56

Primer Set#2

**Primer Sequence of** 

RAPD16 GGTGGCGGGA 56 RAPD

RAPD105 CTCGGGTGGG 56 RAPD

RAPD536 GCCCCTCGTC 56

Table 2. RAPD primers of PCR amplification

electrophoresis as described previously.



Molecular Electrophoretic Technique for Authentication of the Fish Genetic Diversity 89

2 polymorphic bands. The sequence and PCR-RAPD condition were listed in Table 2. All primers were generated bands ranging in size from 100 to 3000 bp. The results shown that the ratio of polymorphic bands were between 13.3~66.7% by 21 RAPD primers. For dendrogram analysis, two groups were identified by RAPD 115 primer (Fig 2). E1, E5, E14, E7, E8, and E9 samples were clustered in group I, which were collected from wild population. Group II, which including E13, E10, E2, E3, E4, E6, E11, and E12 samples. For Group II, all samples were belonged to cultivated populations. For giant grouper*,* wild (seven samples) and cultivated (seven samples) populations of giant grouper can be

Fig. 2. UPGMA consensus dendrogram of dissimilarity among individuals analyzed using

Results of ISSR analysis, 59 primers were used in this study. According the results of ISSR method, 17 primers (29%) have polymorphic patterns. Total 166 bands were generated, 58 polymorphic bands (34.9%). The primer ISSR IT3 was got the more diversity than other primers, have 20 bands. The ISSR 15 primer was generated the less bands, only 3 bands. All the polymorphic patterns were ranged between 100~3000 bp. ISSR primer 868 (5'-(GAA)6-3') was better distinguished than other primers. The result was shown in Fig 3. For giant grouper*,* the patterns of ISSR primer868 could discriminate giant grouper between wild and cultivated populations. For dendrogram analysis, four groups were clustered by ISSR primer868 primer (Fig 4). Among those groups, Group I, Group III, and Group IV were collected from wild population. Samples were clustered in Group II were from cultivated populations. We also found that the results of ISSR method have the same tend to RAPD

method. ISSR method was more discriminate ability than RAPD method.

discriminated by RAPD method.

the primer RAPD 115.

**3.2 ISSR method of giant group**

Table 3. ISSR primers of PCR amplification
