CAPABILITY OF MITOCHONDRIA DNA D . LOOP MARKERS FOR SHARK SPECIES IDENTIFICATION

ldentification of dry-fin shark was conducted by mitochondria DNA (mtDNA) D-loop markers. Eighteen of thiity samples have been successfully amplified the mtDNA D-loop region. The average total length of mtDNA D-loop was approximately 1790 bp. The differences among samples were clearfy identified using polymorpishm of seven restriction endonucleases, A/ul, Hintl, Haelll, Hindlll, Mbol, Rsal, and lagl. KEWI|ORDS: dry-finshark, identification, mtDNA, endonucleasis In this paper, the capability of mitochondria DNA D-loop marker was examined to identify shark species based on their fin as a prelirninary activity to find an easy and applicable marker for the field activity of study on the genetic structure of shark, to support their future management and conservation. MATERIALSAND METHODS

In this paper, the capability of mitochondria DNA D-loop marker was examined to identify shark species based on their fin as a prelirninary activity to find an easy and applicable marker for the field activity of study on the genetic structure of shark, to support their future management and conservation.

Mitochondria D-loop analysis
The mtDNA D-loop region and parts of cytochrome b gene and l65rRNA genes were amplified using PCR as described by Martin et al. (1992).The mtDNA D- loop region was amplified in volumes of 50 pL each consisting of 100 ng of template DNA, 1x reaction buffer(10 pM Tris-HCl pH 8.3, 50 pM KCI 1 pM MgCl2), 5 trtL of 2.5 pM dNTP mixture, 2.5 pL of each 10 pM oligonucleotide primer and 0.5 unit of lag polymerase.Amplification cycles consisted of 30 cycles for one minute at 94'C, one minute at 45"C, ancl one minute at72'Q, followed by one cycle for seven minutes at 72'C.Primer sequences are as follows: L-15,560 (23 bases from 15,5601h base of light strand, 5'a 3' CAT ATT AAA CCC GAA TGA TAT TT) and H1067 (25 bases from 1,067th base of heavy strand, 5'a3'ATA ATA GGG TAT CTA ATC CTA GTT T).The mtDNA D. loop regions were digested using the fourand fivebase recognition endonucleases, i.e.Alul, Hinfl, Haelll, H i n dlll, Mbol, Rsal, and T aql.The frag ments were separated onto 2o/a agarose gel in 1x TBE buffer, stained with ethidium bromide and photographed.Fragment patterns generated by each of restriction endonuclease were compiled for each individualas a haplotype.For shark species identification, the haplotypes of "blind" samples were compared to those of reference species.A total of three composite haplotypes, one haplotype consists of 7 letters representing the fragment patterns generated by each of the restriction endonuclease, were observed among samples.According to the species reference, fourof the 18 amplified samples were included to haplotype I as A. vulpinus species, while 14 others can be suggested as P. g/auca which can be further differentiated into six samples from lonian Sea (haplotype ll) and eight samples from South Adriatic (haplotype lll) (Table 1 ).

DISCUSSION
About sixty percent of dry-fin samples have been amplified the mtDNA D-loop region.The reasons for un-amplified mtDNA D-loop region in severalsamples might be due to: i) sample was denatured and/or ii) un-matched with printers used.The flrst reason may influence the quality of whole DNA taken, therefore the decreasing of DNA purity was occured.The same case was noted for three spines stickle back from Hokaido and Fukushin"ra, Japan.Consequently, other DNAextraction methnds should be used ordeveloped.The second reason is possibly the samples were other shark species.However, this possibility is not strong, as several primers have been used to amplify the IFR JoumalVol.T No. 1 Tahun 2a01 mtDNA D-loop regions forthese samples.lf this rea- son is really happened, it may be suggested that the totalmtDNAof this species should be observed firstly.
The length of mtDNA D-loop fragments of shark fin was comparable with greateramberjack (Nugroho ef a/., 2000), red sea bream (Nugroho, 2001), and grouper (Nugroho & Taniguchi, unpublished) that used also the same primer for mtDNA DJoop amplification.Iagl revealed more fragment patterns than other endonucleases.As well in the previous study, this enzyme restricted three types of fragment, while two types were observed by otherendonucleases.lt means that allof the amplified samples are of shark from species A. vulpinus and P. glauca.According to the haplo- type of species reference used, it is estimated that four from the total of 1 8 amplified samples (r.e. samples no 4, 1 3, 21 , and 22) are classified as the species A.
Even other technique such as AFLP (Amplified Fragment Length Polymorphism) is also useful for species identification as shown in previous experiment (Taniguchi & Nugroho, 1999).However higher skill and costare relatively needed.Therefore, forfield application it is unfavorable.lt suggests that sequence of shark's mtDNA should be found out as much as possible in order to develop the mtDNA primer.Once the accurate primer is available, amplification of mtDNA D-loop is not difficult, and this can be widely used in the field.coNclUsroNs Mitochondria DNA D-loop marker is available for 'blind'fin fish identification to an optimum degree of accuracy.The differences between shark species (A.vulpinusand P. glauca) detected by polymorphism of mtDNA D-loop sequences were generated using all of seven endonucleases, while differences between locations in P. glauca is possible to be found out by restriction endonuclease, Iag l.REFERENCES Martin, A.P., R. Humphreys, and S.R. Palumbi, 1992.Population geneiic structure of the armorhead, Pseudpentaceras wheeleru, in the North Pacific Ocean: Application of the polymerase chain reaction to fisheries problems.Canada Journal of Fisheries Aquatic Science 49: 2389-2391.o o t- of thirty samples have been amplified the mtDNA D-loop region.The average total length of mtDNA D-loopwas approximately 1790 bp.Polymor- phic restriction fragment was observed among samples for all seven endonucleases.An example of fragment patterns is shown in Fig. 1.Digestion of the mtDNA D-loop region with lagl revealed three fragment pat- terns, while the other endonucleases obtained two patterns.Restriction site of three patterns by endonuclease laq I were 640, 390, 300,230, and 230 bp (type A); 630, 480, 300, and 250bp (type B); and 640, 540, 300, and 250 bp (type C).

Figure 1 .
Figure 1.Restriction patterns of 'blind'shark samples (written as number)generated by Taqlendonuclease, with three'reference' samples at left side (AV4, Pg-4, and Pg-14), and a 50 bp marker ladder size at right size