A STUDY ON DNA FINGERPRINTING OF AREOLATED GROUPER ( Epinephelus areolatusl USING RAPD ANALYSTS

This study reports the application of Random Amplified Polymorphic DNA (RAPD) technique in establishment of DNA fingerprinting of areolated grouper, Epinephelus areolatus, an economically tmportant fish species of the subfamily Epinephelinae (popularly known as grouper) The RAPD technique was applied to determine the genetic variability within the species for the purpose of implementing and managing its breeding program. Among 20 screened arbitrary primers, six RAPD primers namely oPA-06, oPA-10, oPA-15, oPA 16, opA-lr7, and opA-'l9 were selected to be used in this study to analyze the RAPD profile, polymorphism, similarity index and genetic distance of E areolatus in the DNA level marker. The results showed that the six primers generated a total of 46 scorable loci (fragments) with 52% polymorphic loci (24 fragments), The RAPD fragment ranged from two to eight fragments with a size range of 350-3000 bp and the number of genotypes of each primer varied from three to flve. Average similarity index among individuals was 060156 t 0.1045 and genetic distance level ranged from 0.1'1 9 to 0462


INTRODUCTION
Areofated grouper, Epinephelus areolatus is one of the i m porta nt com mercial g ro upers (Epi ne phe I us spp./ caught in the coral reef areas of Southeast Asian countries.lt belongs to the subfamily Epinephelinae, and the family Serranidae.Sixtythree species of the genus Epinephelus have been identified by Randall (1 987) and at least 21 of them have been reported to have potential for commercial culture as well as a good source of protein (Shamsudin, 1992).E. areolatusis commonly named as aerolated grouper, yellow-spotted rockcod or squaretail rockcod.The body of this species is covered with brownish yellow spots with usually hexagonal in shape on head, body, and fins (sometimes close-set forming a pale meshwork), bigger in size and lighter in shade and the caudal fin is usually emarginate (Kohno ef a/., 1990).There is little information available on the moleculargenetics of this species.Determination of genetic variation of this species on the DNA level marker is necessary to design future management and conservation for its breed-Ing program.
Detection of genetic variability is essential to a wide range of comparative genetic endeavors.These include studies as diverse as gene mapping, individual identification, parentage determination, population genetic, molecular phylogenetic, conservation and DNA fingerprinting.Williams et al. (1990) described a novel Polymerase Chain Reaction (PCR) based method called Random Amplified Polymorphic DNA (RAPD) fingerprinting.This technique allows detec- tion of DNA polymorphisms by randomly amplifying multiple regions of the genome by PCR using a single arbitrary primerdesigned independent of target DNA sequence.DNA fingerprinting is a relatively newtechnique by which a set of polymorphic markers can be simultaneously detected, resulting in a pattern unique to individual, species, or strain.The RAPD fingerprinting technique has been reported to be extremely efficient for detection of molecular genetic markers in assessment of genetic variation in fish (Dinesh et al.,  1993).This technique also has several advantages overother published methods available: (i) a universal set of primers can be used for genomic analyses in a wide variety of species; (ii) no preliminary work, such as isolation of cloned DNA probes, preparation of filter for hybridization, or nucleotide sequencing, is required; and (iii) each RAPD marker is the equivalent of a sequence target site, which can greatly simplify information transfer in collaborative research programs (Williams et al., 1990).The aim of this present study is to establish DNA fingerprinting of areolated grou- per, E. areolatus using RAPD analysis.
'') co | | ected f rom South S u lawesi waters.Approxi m ately 50 mg of the muscle tissue was taken out from each individual and placed in a 1 .5 mL microcentrifuge tube containing 250 mLTNES-Urea preservation buffer.This buffer consists of 6 M urea; 1OmM Tris-HCl; 125 mM NaCl; 10 mM EDTA; and 1% SDS, at pH 7.5 (Asahida et a|..1996).The samples were transported at ambi- ent temperature from the field and kept at room temperature (25"to 28"C) in the laboratory prior to DNA extraction.

DNA Extraction
Genomic DNA from the muscle tissue was ex- tracted using the Phenol-Chloroform technique.Five hundred microlitres of lysis buffer (0.5 M NaCl, 0.001 M EDTA.1% SDS.0.8% Triton X-100, and 0.1 M Tris- HCl, at pH 9.0) was added to the sample and followed by adding 40 mL SDS 10% and 40 mL Proteinase K (20 mg/mL solution).The sample was incubated at 55'C for 1-3 hours until completely lysed.Samples were then treated with 25 mL RNAase (20 mg/mL solution) and left at room temperature (25-28"C) for 15-30 minutes.
The samole was treated with 500 mL solvent mixture phenol: chloroform:isoamyl alcohol (25:24:1) and then gently vortexed to homogenize it.The sample was left at room temperature (25-28"C) for 10 minutes before centrifugation at 13,000 rpm (11,000 x g) for four minutes.The top aqueous layerwas removed to a new 1.5-mL microcentrifuge tube.The step of adding the solvent mixture was repeated twice.The sample was treated with one volume of chloroform : isoamyl alcohol (24:1) and then centrifuged at 13,000 rpm (11 ,000 x g) for four minutes.Two volumes of cold absolute ethanolwere mixed to the top aqueous layer by inversion of the tubes severaltimes.Precipitated DNA was collected at the bottom of the tubes as a white pellet after centrifugation at 6,000 rpm (4,000 x g) for 30 minutes, The pellet was washed with one milliliter ethanol 70o/o and then centrifuged at 6,000 rpm (4,000 x g) for 15 minutes.The genomic DNA was allowed to dry at room temperature (25-28"C) and then resuspended with TE (Tris-EDTA) buffer.
The genomic DNA was electrophoresed in a 0.8% horizontal agarose gel at 55 volts for 1-2 hours in 1 x TBE (Tris-Borate-EDTA) buffer.The gelwas stained with 0.5 pglmL ethidium bromide for 20-30 minutes and then washed with distilled waterfor 5-10 minutes prior to photographing.

DNA Purity and QuantitY
Ten microliters of genomic DNA were diluted in 490 mL distilled water (dilution factor of 50) in a 0.5- mL cuvetle tube prior to measuring the DNA purity and quantity.Genomic DNA and purity of DNA were estimated using a UVIKON Spectrophotometer (Kontron Instrument) at wavelengths of 260 nm and 280 nm.The reading of absorbency at 260 nm (OD,u) allows calculation of DNA concentration of the sample.
The quantity of DNA was quantitatively determined byfollowingtheformula of Linacero ef a/. (1998).Pu- rity of DNA was quantitatively estimated from the ra- tio between the reading of absorbency at 260 nm and 280 nm (OD26o/OD2so) and also qualitatively observed through the appearance of the single band formed on the agarose gel.

DNA Amplification
A total of 20 arbitrary primers (OPA01-20) were screened for a randomly single individual in orderto find the suitable primer for DNA amplification of grouper.Among them, six primers (based on the clarity and sharpness of the fragments) were selected to use in further analysis (fable 1).DNA amplification reac- tions consisted of 1 x reaction buffer; 3.5 mM MgCl,;  OPA=Operon Technologies Primer Set A A=Adenine, C=Cytosine, G=Guanine, T=Thymine 0.4 mM dNTPs mixture; 0.4 mM primer; 2.0 units taq polymerase; 50 ng template DNA and distilled water in a final volume of 25 mL.The amplification was per- formed using a programmable temperature cycler of GeneAmp PCR system 2400 from Perkin Elmer.The amplification was programmed at 45 cycles for 30 seconds ofdenaturation at 94"C, 30 seconds of an- nealing at 36"C, 1 minute of extension at72'C, and2 minutes of finalextension at72'C.
A mixture of 10 mL PCR product and 2.5 mL loading dye was electrophoresed on a 2.0o/o horizontal agarose gel at 55 volts for2-3 hours and then stained with 0.5 mg/mL of ethidium bromide for 20-30 minutes.The gel was washed with distilled water for 5- 10 minules priorto documentation with lmage Master VDS (Pha rmacia-Technology).

Data Analysis
The molecular weight of fragments was estimated based on the standard DNA fragment pattern from GeneRuler DNA ladder marker.The fragments were valued as polymorphicwhen they are absent in some samples but changes in banding intensity are not considered as polymorphic.Clear fragments were manually scored as present (1) or absent (0) at a par- ticular position ordistance migrated on the gel.A data matrix of 1's and 0's was entered into the data analysis package.Data analysis was performed by using lndonestan Flsherles Research Journal Vol. 8 No.1 the program MPDistance Package Software Version 1.04 (Amstrong et a|.,1994) and NumericatTaxonomy and Multivariate Analysis System (NTSYS) Version 1.80 (Rohlf, 1994).The similarity index of grouperwas calculated across all possible pairwise comparisons of individuals using the formula: S*u = 2n*u/n" + n" (Nei and Li, 1979).Where n,u is the nurhber of fragments shared by individualx and y; n, and nu are the number of fragmentsscored foreach individual.2).Linacero ef a/. (1998)  noted that the determination of DNA purity and quantity is an important step in performing a genetic analysis such as RAPD.The purity of genomic DNA should be in the range of 1 .8-2.0 for PCR requirement of DNA amplification.However the resuli of this study indi- cated that the DNA purity of slightly lowerthan 1.8   and higherthan2.0showed good RAPD banding pat- terns.lt is necessary to determine the quantity of genomic DNA in order to find out the DNA concentration for optimization of PCR-RAPD analysis The high purity level and the clear bands formed on agarose gel of genomic DNA obtained in this study indicated that Phenol-Chloroform is a very powerful technique for DNA extraction in fish especially E.
areolatus.Using the Phenol-Chloroform technique for routine total DNA isolation, Taggart et al. (1992) ob- tained a consistently good quality of high molecular weight DNA extracted from 12 species of the genus Salmo, Sa/velinus, Oncorhynchus, Coregonus, and Thymalus.Shima (1999) also reported that DNA ex- traction using the Phenol-Chloroform technique on the mudskipper, Peiophthalmus schosserl was more re- liable than the DNA extraction of wizard genomic DNA purification kit.The Phenol-Chloroform technique for DNA level extraction was also employed in studies on DNA marker of rivercatfish, Mystus numerus (Krm,   1998) and tiger barb, Puntius tetrazona (Asma, 1999).

RAPD ProfiIes
The RAPD banding patterns were examined for the presence or absence of bands associated with a certain primer used.A set of each 10-mer arbitrary primers of different oligonucleotide sequences was found to generate different RAPD profiles within indi- viduals.Using the six primers, 46 random markers (loci) were generated from DNA amplification of E.
areolatuis.The RAPD banding patterns from repre- sentative samples of E. areolatus generated by each primerareshown in Fig. 1.
The number and size of fragments amplified by different primers varied from twoto eight and 350-3000 bp, respectively (Table 3).The OPA-10 produced the highesi number and size of fragments.Asma (1999)  also reported the different number and size of fragments revealed by different primers in a study on ge- netic differences of tiger barb, P tetrazona.The number and size of amplification products ranged from one to nine fragments and 2"lO-255 bp respectively.A range of the molecularweight fragments amplified by the four different primers (OPA-14, OPA-17, OPA-18, and OPA-1 9) on genomic DNA of Malaysian river catfish, Mystus numerus was 200-2000 bp (Lim, 1998).
Different RAPD profiles between two species of groupers have been reported by Bakar &Azizah (1999).They found that a fragment of 700bp produced from a primer OPA-05 was present in E. tauvina but not in E. bleekeri.Similar findings were also reported by Williams et al. (1990) and Dinesh et al. (1993).Their results showed that DNA amplification in the fingerprinting technique can be tailored to produce patterns of varying complexity by changingthe primersequence length.

Polymorphism
Levels of variability were estimated according to the proportion of polymorphic bands within primers ).Among the primers, the OpA-10 was most vari- able, having an average of polymorphic bands of 63%.There was considerable variation in the level of polymorphism seen in different primers, ranging from as high as 63% in OPA-1 0 to 44a/o in OpA-06.The high polymorphism scored with RApD markers is prob- ably due to preferential amplification of non-coding repetitive regions of the genome.Since primers are conslructed at random, both coding and non-coding regions may be targets of PCR amplification.Lynch and Milligan (1994) noted that the RApD technique was expected to scan the genomic more randomly than conventional methods.Genotypes obtained in each primer varied from three to five in percentage ranging from 5.9% to 58.8%.The highest genotype numberwas revealed by primer OPA-10 and the low- est by primer OPA-16.The high genotype observed indicated the great genetic variability of this species.
A high level of diversity ranging from 33.33% to 50.00% was detected in the freshwater shrimp, Macrobrachium borellii with the RAPD marker (D'Amato and Corach, 1996).Different species of tilapine fishes showed different polymorphic bands (Mwanja ef a/., 1996).The resutts of their investiga_ tion on five species of tilapia showed that the number of pof ymo rph ic bands was 49% to 81 o/o ( O re och ro mi s n i I otic u s), 33% to 7 8% (O.escu/enf us), 640/o to 7 8o/o (O.le o c o st i ct u s), 7 0% (S a rot h e ro d o n g a t i I ae u s), and 55% to 75o/o (Tilapia zilli).

Similarity Index and Genetic Distance Level
The similarity index among individuals of F. areo latu s r anged from O.41 37 -1.0000 with an average of 0.60156 t 0.1045 (Iable 5).The simitarity index of 1.000 indicated that individuals showed the genetic identicaf such as individual between 1 and 4: and 2 and 7.The similarity index was found to be lowest (0.4137) between individual 10 and 12.The simitarity index of tiger barb, P. tetrazona varied from 0.6277g-0.73131in Perak population and 0.33333-0.83222in Johor population (Asma, 1999).viduals of E. areolafus constructed according to the genetic distance from Nei and Li's indices.The ge- netic distance level obtained ranged from 0.119 to 0.462.A high level of genetic distance of this species indicated a low level of inbreeding among individuals.
It is also supported by the fact that the samples were collected from a wild population.On the other hand, the low genetic distance, indicating the high inbreeding activity level, has been reported forseveralcultivable fishes such as tiger barb, tilapia, discus and hilsa shad.Asma (1999) The indexof similarity was used to calculate the genetic distance values and to construct the dendrogram.The dendrogram was constructed using the Unweighted Pair- Group Method of Aritmethic (UPGMA) employing Sequential, Agglomerative, Hierarchical and Nested Clustering (SAHN) from NTSYS-pc program (Rohtf, 1 994).RESULTS AND DISCUSSIONDNA ExtractionGenomic DNA of E. areolatus was successfully extracted by the Phenol-Chloroform technique.The DNA purity and quantity of E. areolatus obtained from this technique was 2.0049 + 0.1436 and 67.03 t 15.52 ngiml respectively(Table

Figure 2
Figure 2 shows the dendrogram of seventeen indi- Fig.2

Table 1 .
The sequence, nucleotide length, and G+C content of primers used in

Table 2 .
The purity and quantity of genomic DNA of E. areolatus