Molecular Electrophoretic Technique for Authentication of the Fish Genetic Diversity

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).


Introduction
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.

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 Qubit TM Fluorometer (invitrogen, USA).The DNAs were stored at -20 o C until PCR amplifications.

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 MgCl 2 , 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 94 o C for 5 min, followed by 35 cycles of denaturation at 94 o C for 30s, annealing at a primer-specific annealing temperature as table 2 for 30s and extension at 72 o C for 30s, and final extension at 72 o C for 10 min.The annealing temperature of Cobia was 36 o C. 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.

PCR-ISSR method
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 dH 2 O.Then, the mixtures were subjected to 94 o C for 5 min, followed by 35 cycles of denaturation at 94 o C for 30s, 30s at a primer-specific annealing temperature as

Genetic distances and phylogenetic analysis
Patterns from RAPD and ISSR methods were scored for the presence (1) or absence (0) of clear bands to analyze genetic similarities using the Dice coefficient of similarity.Similarity matrix cluster and phylogenetic analysis was used to reveal association among strains based on the unweighted pair group method with arithmetic averages (UPGMA) using the NTSYSpc software (Numerical taxonomy and multivariate analysis system, version 2.01b, State University of New York, Stony Brook, NY, USA) according to Rohlf (1997).

RAPD method of giant group
A total of 14 giant grouper including cultivate and wild were obtained.Analysis on species of giant grouper in cultivate and wild.For RAPD method amplification, the species, For RAPD method, total 95 of RAPD oligonucletide primers were used to screening the genetic diversity of giant grouper.There are 21 RAPD primers (22.1%) have polymorphic bands.Total 279 bands were generated by those primers and 86 polymorphic bands (31%).The primer RAPD 115 (5'-TTCCGCGGGC-3') was got the more diversity than other primers, have 8 polymorphic bands (Fig 1).The RAPD 245 primer was generated the less bands, only 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 discriminated by RAPD method.
Fig. 2. UPGMA consensus dendrogram of dissimilarity among individuals analyzed using the primer RAPD 115.

ISSR method of giant group
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.

RAPD and ISSR methods of cobia
Ninety-five RAPD primers and 59 ISSR primers were used for PCR amplification.The results were shown that all the cobia samples were the same patterns and no polymorphic   Sequence variability of mitochondrial DNA regions was low between the six cobia (Garnet, et al., 2002).These results were also similar in our study.Both RAPD and ISSR methods have no different patterns.Hence, this could provide more useful information of molecular genetic data in population and stock enhancement studies.

Discussions
RAPD and ISSR methods were generally used for genetic diversity and populations study; those methods also could be used to analyze the breeding relationship.For species identification and genetic resource/diversity analysis, RAPD and microsatellitsa method were recommended (Liu & Cordes, 2004).The RAPD techniques has been used for discrimination of populations of species of the genus Barbus, grouper, Nile perch and wreck fish, salmonids, among others (Partis & Wells, 1996;Callejas & Ochando, 2001;Asensio et al., 2002;Jin, Cho, Seong, Park, Kong & Hong, 2006).Genetic analysis with RAPD markers is relatively easy, fast, and efficient.RAPD analysis, however, may not be practical for identifying interbreed species (Martinez, Elvevoll & Haug, 1997).SSRs are inherited in a codominant fashion.This allows one to discriminate between homo-and heterozygous state, and increases the efficiency of genetic mapping and population genetic studies.ISSR markers have recently been used successfully for genetic analysis in hatchery and wild Paralichthys olivaceus strains.It indicates that molecular marker systems contribute greater levels of capability for the detection of polymorphism, and provide a better solution for the assessment of genetic variations (Shikano, 2005;Liu, Chen, Li & Li, 2006).According to our results, RAPD and ISSR method can be effectively discriminate giant grouper from different sources, such as cultivate and wild, even if fish are from different cultivate farms.But cobia and red coral trout were less discriminate ability.The study found that ISSR and RAPD methods were positively high correlations.Giant grouper species have highly genetic diversity.A comparison of RAPD and ISSR patterns in 14 giant grouper samples, ISSR primers have higher polymorphism and fewer bands than those of RAPD primers.It could provide simple and convenient method to discriminate genetic variation of giant grouper samples.In this study, ISSR method could distinguish genetic variation within specie and different populations.Some reports also have suggested that ISSR may reveal a much higher numbers of polymorphic fragments per primer than those of RAPD (Esselman, et al., 1999).Among these markers, microsatellite DNAs have revolutionized the use of molecular genetic markers in the applications mentioned before, and the markers are destined to dominate this type of studies in the coming years (Asensio, 2007).It also has been revealed as important tools in studies regarding the genetic structure of populations, phylogeographic relations and phylogenetic reconstruction in fish (Antunes, et al., 2010).

Conclusion
We developed DNA molecular marker techniques which could be used to generate information for fish genetic diversity, species identification, trace genetic variation between different individuals in aquaculture, authenticate fish, fishery products and provide good reference resources for species sources and relationships.
bands.The primer ISSR UBC809 and RAPD31 were generated 12 to 17 bands and in size from 100 to 2000 bp.The results were also shown in Fig 5 and Fig 6.
methods of red coral trout For screening ISSR primers, the ISSR primer15 (ISSR15: 5'-(AG) 8 TG -3') was better distinguished than other primers.The result was shown in Fig 7. The primer ISSR 15 was generated 10 to 16 bands and in size from 200 to 2000 bp.For dendrogram analysis, three groups were identified.Group I, the 71412, 71413, 71422, and 82011 were clustered in the group.Group II, including 81621, 91321, 71411, and 91712 samples; group III including 71423, 80622, 91322, 71421, and 81623 samples.All the nodes of the dendrograms ranged from 90 to 100%.The result was shown in Fig 8.

Table 2 .
RAPD primers of PCR amplification extension at 72 o C for 30s, and final extension at 72 o C for 5 min before analysis by the electrophoresis as described previously.