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Random Amplified Polymorphic DNA (RAPD) View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by CMFRI Digital Repository Aquaculture Research, 2004, 35, 10 06 ^ 10 09 d o i: 10.1111/ j.1365 -2109.20 04.01112. x SHORT COMMUNICATION Random amplified polymorphic DNA (RAPD) fingerprinting resolves species ambiguity of domesticated clown fish (genus: Amphiprion, family: Pomacentridae) from India Pallipuram Jayasankar Central Marine Fisheries Research Institute,Tatapuram PO, Cochin, Kerala, India Correspondence: P Jayasankar, Central Marine Fisheries Research Institute, PB No1603, Ernakulam North PO, Cochin 682 018, Kerala, India. E-mail: [email protected] Clown ¢shes of the genus Amphiprion are a valuable di¡erent groups of marine ¢shes such as striped bass resource from the point of view of the aquarium (Bielawski & Pumo 1997), large mouth bass (Wil- trade. There are about 27 species distributed widely liams, Kazianis & Walter 1998), goat¢shes (Mamuris, in the Indo West Paci¢c region, extending from the Stamatis, Bani & Triantaphyllidis1999) and groupers eastern Indian Ocean to the Indonesian and Philip- (Govindaraju & Jayasankar 2004). pine archipelagos and from the coast of northwest- The domesticated species of clown ¢sh at Vizhin- ern Australia to the Ryukyu Islands of Japan (Allen jam Marine Aquarium on the southwest coast of 1980; Fautin & Allen 1992). Allen (1991) used mor- India was identi¢ed as Amphiprion chrysogaster phological similarities to designate six taxonomic (Gopakumar, George & Jasmine 1999). However, the complexes of closely related species. Apart from col- species, which was domesticated at Mandapam Ma- our variation, tooth shape, scalation on the head rine Aquarium on the southeast coast of India, was and body proportions are used to identify the species identi¢ed as A. sebae (Ignatius, Rathore, Jagdis, Kan- of clown ¢sh (Fautin & Allen1992).The occurrence of dasami & Victor 2001). Since the stock of the latter multiple colour variants in most of the species, be- was drawn from the former, the present study was sides variations in colour patterns between juveniles carried out to ratify the species status of this import- and adults of the same species, makes unambiguous ant marine ornamental ¢sh. identi¢cation in clown ¢shes di⁄cult. Caudal ¢n clippings were taken from eight in Molecular techniques have become a major tool dividual clown ¢sh each from Mandapam Marine for systematic ichthyologists and may also be useful Aquarium (65^117mm total length) and Vizhinjam to ¢shery biologists for rati¢cation of taxonomic prob- Marine Aquarium (38^108 mm total length). Liver lems at species and population levels (Chow, Clarke tissues of 8 individuals of A. chrysogaster, collected & Walsh 1993). Random ampli¢ed polymorphic DNA from Mauritius waters, were obtained from Dr Joel El- (RAPD) technique allows detection of DNA poly- liot (Biology Department, University of Puget Sound, morphisms by randomly amplifying multiple regions Tacoma, WA, USA). Morphological identi¢cation of the genome by PCR using single arbitrary primers of the clown ¢sh was done based on the description designed independent of target DNA sequence of Fautin and Allen (1992). Original photographs (Welsh & McClelland 1990; Williams, Kubelik, Livak, and type specimens of domesticated clown ¢sh Rafalski & Tingey1990; Hardys, Balick & Schierwater species fromVizhinjam and Mandapam aquaria were 1992). Random ampli¢ed polymorphic DNA markers sent to Dr Gerald Allen for con¢rmation of species have been used to investigate the taxonomic status of identity. 10 06 r 2004 Blackwell Publishing Ltd Aquaculture Research, 2004, 35, 1006^1009 RAPD ¢ngerprinting of domesticated clown ¢sh from India P Jayasankar Extraction of genomic DNA and RAPD ampli¢ca- ter analysis. For this, the unweighted pair-group tion were done as per Govindaraju and Jayasankar method with arithmetic (UPGMA) (Sneath & Sokal (2004). Initially, a total of 15 primers from kits A and 1973) contained in the NEIGHBOR program of PHYLIP F of Operon Technologies (Alameda, CA, USA) were ver 3.57c (Department of Genome Science, University used to amplify the DNA. Based on repeatability and of Washington, Seattle, WA, USA), based on Nei’s robustness of the loci generated by them, the ampli¢- (1978) genetic distance values calculated for all pri- cation results of four primers, OPA 08 (50 GTGACG- mers, was used. Data resampling (1000 replicates) TAGG 30), OPA 10 (50 GTGATCGCAG 30), OPF 02 (50 and matrix calculations for bootstrap analysis were GAGGATCCCT 30) and OPF 03 (50 CCTGATCACC 30) performed using WinBoot, an UPGMA-based pro- were the only ones analysed. The size of RAPD bands gram (Yap & Nelson1996). was determined by comparison with a l DNA diges- In order to con¢rm the repeatability of the results, ted with EcoRI/HindIII molecular weight marker. For each tissue was ampli¢ed with each of the primers at all the primers, presence (1) or absence (0), of a frag- least three times and considered the loci for further ment was scored and RAPD patterns of individuals analysis only if they occurred in all the three ampli¢- were compared within and between species. Biopro¢l cations. The four primers generated three to nine (Bio-1D,Vilber Lourmet, Cedex1, France) was used to stable and clear loci in the size range of 100^ calculate the fragment sizes of the RAPD bands with 3500 bp. Figure1shows the RAPD pro¢les of di¡erent reference to molecular size markers.The‘species-spe- individuals of domesticated species from India and ci¢c diagnostic’ markers are de¢ned in the present Mauritius generated by two of the selected primers. study as those RAPD bands which are exclusive to a The average GD (genetic distance) and SI (similarity species for a given primer. index) values were calculated for all the four primers The similarity index between all possible pairwise together. The SI value derived between Mandapam comparisons of individuals was calculated (Nei1978) and Vizhinjam samples was 0.936, as expected for and the phylogenetic relationships between indi- the same species, while that between them and viduals and populations were constructed using clus- Mauritius species was very low,0.340. Figure 1 RAPD ¢ngerprints of clown ¢sh generated by Primer OPA 08 (a) and OPF 03 (b). Lanes 1^8, eight individuals of Amphiprion sebae from Mandapam marine aquarium; lanes 9^16, 8 individuals of A. sebae from Marine Aquarium of Vizhinjam; lanes17^24, A. chrysogaster from Mauritius; lane M, DNA size marker, l HindIII/EcoRI. r 2004 Blackwell Publishing Ltd, Aquaculture Research, 35, 1006^1009 10 07 RAPD ¢ngerprinting of domesticated clown ¢sh from India P Jayasankar Aquaculture Research, 2004, 35, 1006^1009 Table 1 Species diagnostic RAPD loci (in bp) in two species of clown ¢shes Species OPA 08 OPA 10 OPF 02 OPF 03 Amphiprion sebae 1650 and 2027 947 4973 500 and 4268 A. chrysogaster 1584 500 1375, 1000 1000 and 900 RAPD, random ampli¢ed polymorphic DNA. in Indian waters but commonly occur in Mauritius waters (pers. comm.). Morphological (Fautin & Allen 1992) and RAPD ¢ngerprinting (present study) have rati¢ed the species status of the domesticated clown ¢sh in the marine aquariums of Mandapam and Vizhinjam as A. sebae. Acknowledgments I am thankful to Dr Mohan Joseph Modayil, Director, CMFRI and Dr R. Paul Raj, Head, Division of Physiol- ogy, Nutrition and Pathology for facilities and useful suggestions. Thanks are due to Dr Gerald Allen for identifying clown ¢sh specimens and to Dr Joel Elliot, Dr G. Gopakumar and Boby Ignatius for kindly pro- viding tissue samples of clown ¢sh from Mauritius, Vizhinjam and Mandapam respectively. I am also Figure 2 UPGMA dendrogram showing relationships grateful to the Indian Council of Agricultural Re- among 24 individuals of two species of Amphiprion.Based search for ¢nancial help. on genetic distance values calculated by Nei (1978) from data for all primers. Bootstrap estimates (as percentage) are indicated above the branches. MM, Mandapam; VIZ, References Vizhinjam; MR, Mauritius. Allen G.R. (1980) Anemone Fishes of theWorld: Species, Care The similarity index between all possible pair- and Breeding. Aquarium Systems, Mentor, OH, USA. A l le n G. R. (1991) Damsel Fishes of theWorld. Mergus Publish- wise comparisons of individuals from all primers was ers, Melle, Germany. calculated and phylogenetic relationship between in- Bielawski J.P. & Pumo D.E. (1997) Random ampli¢ed poly- dividuals of Mandapam,Vizhinjam and Mauritius sam- morphic DNA (RAPD) analysis of Atlantic coast stripped ples was constructed using cluster analysis (UPGMA bass. Heredity 73,32^40. contained in PHYLIP 3.57c package). The results Chow S., Clarke M.E. & Walsh P.J. (1993) PCR-RFLP analysis (Fig. 2) showed clustering of Mandapam and Vizhin- on 13 western Atlantic snapper (Subfamily Lutjanidae): a jam samples together, clearly separated from Mauri- single method for species and stock identi¢cation. United tius samples. Thus all the individuals of each species States National Marine Fisheries Service Fishery Bulletin formed monophyletic species clusters. Some RAPD 91,619^627. fragments have shown ¢xed frequencies and hence Fautin D.G. & Allen G.R. (1992) Anemone Fishes And Their they can be used as species diagnostic markers in A. Hosts Sea Anemones: A Guide forAquarists and Divers.West- ern Australian Museum, Perth,WA, Australia. sebae and A. chrysogaster (Table 1). Both the species Gopakumar G., George R.M. & Jasmine S. (1999) Breeding had at least one diagnostic marker from each primer, and larval rearing of the
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