**3. Results**

### **3.1 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

Fig. 1. RAPD profiles of the 14 *E. lanceolatus* fish obtained using the primer 115. Lanes M: Bio-100 bp DNA Ladder. Lanes 1-7: *E. lanceolatus* fish wild population (E1, E5, E7, E8, E9, E13, and E14); Lanes 8-14: *E. lanceolatus* fish cultivate population (E2, E3, E4, E6, E10 E11, and E12).

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.

#### **3.2 ISSR method of giant group**

88 Gel Electrophoresis – Advanced Techniques

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,

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

Fig. 1. RAPD profiles of the 14 *E. lanceolatus* fish obtained using the primer 115. Lanes M: Bio-100 bp DNA Ladder. Lanes 1-7: *E. lanceolatus* fish wild population (E1, E5, E7, E8, E9, E13, and E14); Lanes 8-14: *E. lanceolatus* fish cultivate population (E2, E3, E4, E6, E10 E11,

State University of New York, Stony Brook, NY, USA) according to Rohlf (1997).

ISSR19 (CA)6RY 44 ISSR20 (GT)6YR 44 ISSR21 (GT)6AY 43 ISSR22 (ACTG)4 52 ISSR23 (GACA)4 48 ISSR24 (CAC)6 57

**2.4 Genetic distances and phylogenetic analysis** 

Table 3. ISSR primers of PCR amplification

**3.1 RAPD method of giant group**

**3. Results** 

and E12).

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.

Molecular Electrophoretic Technique for Authentication of the Fish Genetic Diversity 91

bands. The primer ISSR UBC809 and RAPD31 were generated 12 to 17 bands and in size

Fig. 5. ISSR profiles of the 14 *R. canadum* fish obtained using the primer UBC 809. Lanes M: Bio-100 bp DNA Ladder. Lanes: 1, 2, 4, 6, 9, 10, 11, 12, 13, and 14 (R1, R2, R4, R6, R9, R10, R11, R12, R13, and R14) *R. canadum* fish wild population; Lanes: 3, 5, 7, and 8 (R3, R5, R7,

Fig. 6. RAPD profiles of the 14 *R. canadum* fish obtained using the primer 31. Lanes M: Bio-100 bp DNA Ladder. Lanes : 1, 2, 4, 6, 9, 10, 11, 12, 13, and 14 (R1, R2, R4, R6, R9, R10, R11, R12, R13, and R14) *R. canadum* fish wild population; Lanes: 3, 5, 7, and 8 (R3, R5, R7, and R8)

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

from 100 to 2000 bp. The results were also shown in Fig 5 and Fig 6.

and R8) *R. canadum* fish cultivate population.

*R. canadum* fish cultivate population.

genetic data in population and stock enhancement studies.

Fig. 3. ISSR profiles of the 14 giant grouper fish obtained using the primer ISSR 868. Lanes M: Bio-100bp DNA Ladder. Lanes 1-7: giant grouper fish wild population (E1, E5, E7, E8, E9, E13, and E14); Lanes 8-14: giant grouper fish cultivated population (E2, E3, E4, E6, E10, E11, and E12).

Fig. 4. UPGMA consensus dendrogram of dissimilarity among individuals analysed using the primer ISSR 868.

#### **3.3 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

Fig. 3. ISSR profiles of the 14 giant grouper fish obtained using the primer ISSR 868. Lanes M: Bio-100bp DNA Ladder. Lanes 1-7: giant grouper fish wild population (E1, E5, E7, E8, E9, E13, and E14); Lanes 8-14: giant grouper fish cultivated population (E2, E3, E4, E6, E10, E11,

Fig. 4. UPGMA consensus dendrogram of dissimilarity among individuals analysed using

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

and E12).

the primer ISSR 868.

**3.3 RAPD and ISSR methods of cobia**

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.

Fig. 5. ISSR profiles of the 14 *R. canadum* fish obtained using the primer UBC 809. Lanes M: Bio-100 bp DNA Ladder. Lanes: 1, 2, 4, 6, 9, 10, 11, 12, 13, and 14 (R1, R2, R4, R6, R9, R10, R11, R12, R13, and R14) *R. canadum* fish wild population; Lanes: 3, 5, 7, and 8 (R3, R5, R7, and R8) *R. canadum* fish cultivate population.

Fig. 6. RAPD profiles of the 14 *R. canadum* fish obtained using the primer 31. Lanes M: Bio-100 bp DNA Ladder. Lanes : 1, 2, 4, 6, 9, 10, 11, 12, 13, and 14 (R1, R2, R4, R6, R9, R10, R11, R12, R13, and R14) *R. canadum* fish wild population; Lanes: 3, 5, 7, and 8 (R3, R5, R7, and R8) *R. canadum* fish cultivate population.

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.

Molecular Electrophoretic Technique for Authentication of the Fish Genetic Diversity 93

Fig. 8. UPGMA consensus dendrogram of dissimilarity among individuals analysed using

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).

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

This research project was supported by the Council of Agriculture (COA) (97AS-4.1.2-AI-I2).

reference resources for species sources and relationships.

the primer ISSR 15.

**5. Conclusion** 

**6. Acknowledgment** 

#### **3.4 ISSR methods of red coral trout**

For screening ISSR primers, the ISSR primer15 (ISSR15: 5'-(AG)8TG -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.

Fig. 7. ISSR profiles of the 14 red coral trout obtained using the primer ISSR 15. Lanes M: Bio-100 bp DNA Ladder. Lanes: 4, 5, 6, 8, 9, 10, and 12 (71411, 71412, 71413, 82011, 91321, 91711, and 91712) red coral trout fish wild population; Lanes: 1, 2, 3, 7, 11, 13, and 14 (71421, 71422, 71423, 81621, 80622, 81623, and 91322) red coral trout fish cultivate population.
