Ariffin, Nur Asma

Ideris, Aini

Ismail, Patimah

Kassim Shaari, Norazila

Universiti Putra Malaysia Institutional Repository

National Topdown Biotechnology ProjectsDevelopment of DNA Markers for Identification of Colour Varieties and Sex in Tiger BarbsPatimah Ismail. Norazila Kasslm 5haari. Nur Asma Ariffin and Aini IderisFacultyof MedicineandHealthSciencesUniversitiPutraMalaysia43400 UPM,Serdang,SelangorMalaysiaE-mail olCorrespondingAuthor:patimah@putra.upm.edu.myKey words: mtDNA, RAPD marker, tiger barb.IntroductionThe beautiful and exotic colours ofornamental fish have led an interest tobreed and study many species of or-namental fish. The use of moleculartechniques has becoming very popularin the study of stock identificationwhich is very useful to aquaculture andfishery management. Ornamental fishindustry plays an important role in thefisheries sector in Malaysia and manylocal species such as Harlequin fish(Rasbora heteromorpha), Arowana(Sc/eropages formosus) and Tiger barb(Puntius tetrazonai has potential forcommercial value. Phylogenetic analy-sis and genetic variability of the fishhad been widely used in fisheries re-search, as it is technically simple, fastand economical for determining thelevels of polymorphism within stocksand between stocks.The objectives of the projects are todetermine the genetic markers of tigerbarbs using RAPD and MtDNA analy-sis, as these will minimise the incorrectidentification and stock contaminationfor commercial purposes.Materials and MethodsGenetic variability between and withinthree varieties of domesticated Tigerbarb was studied using random ampli-fied polymorphic DNA (RAPD) mark-ers and MtDNA analysis. Fifty randomindividuals of each variety (normal,green and yellow) and twelve of 9-10mer random primers with 50-77.7% ofG+C content were used to generate theRAPD markers separable by agarosegel electrophoresis. Isolation of ge-nomic DNA and analysis ofmtDNAwere conducted using muscle tissuefollowing the procedures by Pure-GeneTissue Kit II (BST TechLab). Twoportion of the mtDNA gene were am-plified. Primer l6SL (5'-CGC CTGTTT ATC AAA AAC AT-3') and16SH (5'-CCG GTC TGA ACT CAGATC ACG T-3') were used to amplifya partial region of l6S rDNA (about620 bp). The second portion was ob-tained from the 12s ribosomal RNAand tRNA-Val gene (about 730 bp)was amplified using the followingprimer pair: OMTl6SF ('5 - TGCCAG CCA CCG CGG ITA TAC CT-3') and tRNA02 (5' - GGA TGT CITCTC GGT GTA AG- 3') (Saiki et al.,1988). The temperature regimen for 30cycles was I min at 95°C, I min at55°C and 2 min at 70°C. The ampli-fied products were purified and con-centrated by enzyme kit (PCR productPre- Sequencing Kit, Pharmacia Bio-tech); the PCR product was mix withthe mixture of SAP, EXON I, deion-ised water and incubated at 37°C for 15minutes followed by 80°C for 15 min-utes. The sequence amplification wasperformed with the Big Dye Termina-tor cycler sequencing kit (AB!, Phar-macia Biotech); the purified productswere mixed with 1 III reaction mix and0.5 III primer (for each reaction theduplicate for forward and reverseprimer was prepared). The reactionmixture was performed with the pa-rameter of 25 cycle at 96°C for 30second, IS second at 50°C and 4 min at60°C. Purification of sequence reac-tion of PCR product was done with themixture of 3M sodium acetate and 95%ethanol (2:30). Then the mixture wasmixed with the PCR product and incu-bated at room temperature before itwas centrifuged at 15,000 rpm for 20minutes and mixed the pellet with 70%ethanol. Formamide was added intothe sample and it was then analyzedusing automated sequencer of AB!prism 310 genetic analyzer (Perkin-Elmer).The analysis of the multiple alignmentsof sequences was done using theClustel V program with default settings(Higgins and Sharp, 1988). Computergenerated alignments were comparedto secondary-structure models de-scribed by Neefs et al., (1991) and Ortiet al., (1996). The PHYLIP ver. 3.572package and PAUP* 4.0 were used forthe phylogenetic analyses. Geneticdistance was calculated using the Jukeset al.-Cantor model (Jukes et al. andCantor, 1969), Kimura's et al.twoparameter (K2) model (Kimura et al.,1980) and a neighbor joining trees(Saitou et al.and Nei, 1987) was con-structed.The RAPD markers were analysedusing Nei and Li's Similarity Index(SI) in which bands were scored aspresent or absent for each variety andvariation among varieties was quanti-fied using the index of dissimilarity.The same variety from different stateswas grouped in the same cluster.Results and DiscussionA total of 73 RAPD markers werefound in the normal variety, 79 in thegreen variety and 84 in the yellow vari-ety with an average of 6-7 markers perprimer. Each variety of fish was foundpolymorphic with the normal varietyidentified as the most polymorphic(50.57%), followed by the yellow(45.95%) and the green was the leastpolymorphic (40.68%). Total DNAfrom three varieties of tiger barb wasamplified at 2 region of mtDNA geneusing two set of primer (16SUI6SHand OMTl6SFI tRNA02). Primer16SL and 16SH amplified at 620 bpwhile primer OMT16SF and tRNA02amplified at 750 bp and the consensussequence for a partial region of the 16SrDNA gene and l2s ribosomal RNAand tRNA- Val gene in Tiger barb was500 bp and 539 bp respectively.ConclusionsThe RAPD analysis suggests that thelow polymorphisms among individualsof the same variety indicate a highlevel of inbreeding among the fishpopulations. There is a close geneticrelationship between the normal andthe green variety compared to the377UPM Research Report 1997-2000, Section 2-Extended Abstractsyellow variety. The MtDNA study is aspecific study breeding program. can ~eformulated to increase the genetic van-ability within the varieties and popula-tions using varieties with high SI andlower SI (green and yellow varieties).Benefits from the studyThe availability of genetic markers toidentify fish stocks as well as individ-ual fishes will enable commercialbreeders to have propriety tags of theircultural strains. Examination ofmtDNA has indicated that certain areasof the genome may be more variablethan others and maximum amount ofinformation can be gained by se-quencing the mtDNA fragment. .Though at present, this is an expensiveand time consuming procedure, but thisgives more information than RAPDanalysis. It will allow fish seed to bemonitored the levels of genetic varia-tion in the broodstocks to avoid delete-rious effects of inbreeding.Literature cited in the textFelsenstein, J. 1998. PHYLIP (PhylogenyInference Package) ver. 3.572. Universityof Washington.Jukes. T.H. and Cantor. C.H. 1969.Evolution of protein molecules In"Mammalian Protein Metabolism" Ed byHN Munro, Academic Press. New York,pp 21-132.Kimura. M. 1980. A simple method forestimating evolutionary rates of basesubstitution through comparative studiesof nucleotide sequences. J. Mol Evo116:111-120.Neefs. J.M .• Peer. Y.V.D .•Rijk, PD .•Goris.A. and Wachter. R.D. 1991. Compilationof small ribosomal subunit RNAsequences. Nucleic Acids Res 19: 1987-2015.Orti, G.• Petry. P.• Porto. J.I.R .• Jegu, M.and Meyer. A. 1996. Patterns ofnucleotide change in mitochondrialribosomal RNA genes and the phylogenyof piranhas. J. Mol. Evol. 42: 169-182.Saiki. R.K .• Gelfand. D.H .• Stoffel. S .•Schart, S .•Higuchi. R.. Horn. R.• Mullis.K.B. and Frlich. H.A. 1988. Primer-directed enzymatic amplification of DNAwith thermostable DNA-polymerase.Science 239:487-491.Saitou, N. and Nei. M. 1987. Theneighbour-joining method: a new methodfor reconstructing phylogenetic tree. MolBioi Evol. 4: 406-425.Seatle.Higgins, D.G and Sharp. P.M. 1988.CLUST AL: a package for performing378multiple sequence alignment on amicrocomputer. Gene 73: 237-244.Project Publications inRefereed JournalsNone.Project Publications inConference ProceedingsAndi Parenrengi, Lokman Shamsudin,Patimah Ismail and Nakisah Mat Amin.2000. Preliminary Study on DNA LevelMarker of Grouper at Different BufferPreservation and DNA ExtractionMethod. Proceeding of the 4thNationalCongress on Genetics. 26-28 September2000. Genting Highlands. Malaysia.Andi Parenrengi, Lokman Shamsudin.Patimah Ismail and Nakisah Mat Amin.2000. RAPD Fingerprinting of AreolatedGrouper. Epinephelus Areolatus.Presented at National FisheriesSymposium. 31 October to 2 November2000. Grand Blue Wave Hotel. JohorBahru, Malaysia.Lokman Sharnsudin, Andi Parenrengi,Patimah Ismail and Nakisah Mat Amin.2000. The Development of GeneticMarker of Grouper by Optimizing PCR-RAPD Analysis. Proceeding of the 4thNational Congress on Genetics. 26-28September 2000. Genting Highlands.Malaysia.Lokman Shamsudin, Andi Parenrengi,Patimah Ismail and Nakisah Mat Amin.2000. Fingerprinting of a commercializedmarine species of grouper by PCR-RAPDanalysis. Proceeding 12'hNationalBiotechnology Seminar. 12-15 November2000. Damai Laut Country Resort.Lurnut, Perak Darul Ridzuan, Malaysia.Lokman Shamsudin, Andi Parenrengi,Patimah Ismail. and Nakisah Mat Amin.2000. A study on the fingerprinting ofEpinephelus areolatus population forbreeding purpose in aquaculture. Paperpresented at Biotechnology Symposium.Benefits and Concerns for Agriculture.Industry and the Environment. 12-13February 2001. Monash UniversityMalaysia. Bandar Sunway, Petaling Jaya,Malaysia.Norazila. K.S .• Patimah Ismail andRidzwan, B.H 2000. Variasi Genetik BagiPopulasi Timur Laut Di Malaysia.Seminar Penyelidikan Fakulti SainsKesihatan Bersekutu UniversitiKebangsaan Malaysia. Oktober 24. 2000.Patirnah Ismail. Asma, A.N. & Shahreza,M.S. 2000. Development of DNAMarkers Using Random AmplifiedPolymorphic DNA (RAPD) for TheIdentification Of Tiger Barbs (PuntiusTetrazons). Proceedings 12thNationalBiotechnology Seminar "Biotechnologyas the Catalyst for Economic Growth"From Genes to Protein. Damai Laut,Perak pplO-12.Patirnah, I..Asma, A.N. and Shahreza, M.S.1998. Genetic Differences of Tiger barbs(Puntius tetrazona) in Perak PopulationUsing Random Amplified PolymorphicDNA (RAPD) Markers. Proceedings ofloth National Biotechnology Seminar"Biotechnology Towards the NextMillennium". 27th- 28thOctober 1998.Sirim, Selangor.Patimah, I..Norazila, K.S. and Ridwan, H.Genetic Polymorphism of Sea Cucumber(Hurothuroidae). Paper sent forPenyelidikan & Pembangunan SainsTeknology. Universiti KebangsaanMalaysia.Patimah, I..Norazila, K.S. and Ridzwan,B.H.1999. Polimorfisme Genetik TimunLaun (Holothuroidae). Paper presented atIRPA-UKM Seminar 1999. AwanaResort. Genting Highlands. 30thJuly - 1"August 1999.Patimah, I.. Shahreza, M.S. and Asrna A.N.1998. Genetic Differences of Tiger Barbs(Puntius tetrazona) in Perak PopulationUsing Random Amplified PolymorphicDNA (RAPF) markers. Proceedings oflOthNational Biotechnology Seminar"Biotechnology towards the nextmillennium" 27 & 28 October 1998.Sirirn, Selangor.Patimah.L, Shahreza, M.S. and Asrna A.N.1998. Phenotypic Expression fromCrosses Between Three Varieties of TigerBarbs (Puntius tetrazona). Proceedings oflOthNational Biotechnology Seminar"Biotechnology towards the nextmillennium" 27 & 28 October 1998.Sirim, Selangor.Patimah. I.. Shahreza. M.S. and Asma, A.N.1998. Phenotypic Expression fromCrosses Between Three Varieties of TigerBarbs (Puntius tetrazona). Proceedings of10'"National Biotechnology Seminar"Biotechnology Towards the NextMillennium". rr - 28'" October 1998.Sirim, Selangor.Patirnah, I.. Tam. B.B .• Ambok, B.A.M .•Saad, C. and Cheah, W.L. 1999.Preliminary trials on astaxanthin to eggproduction of Koi angel fish(Pterophyllum). Paper presented at theWorld Conference on Ornamental FishAquaculture. World Trade Centre.Singapore. 3'd - 6thJune 1999.Ruzainah, A.• Siti Azizah M.N. and PatimahIsmail. 2000. The Optimization OfRAPD Technique For The Study OfGenetic Variation In Pangio. Proceeding12thNational Biotechnology Seminar. 12-15 November 2000. Darnai Laut CountryResort. Lumut, Perak.National Topdown Biotechnology ProjectsGraduate ResearchAndi Parenrengi. 200 l. Biotechnology/Population Genetics [M.Sc.]. UniversitiPutra Malaysia.Norazila Kassim Shari. 2001.BiotechnologylPopulation Genetics[M.Sc.]. Universiti Putra Malaysia.Nor Asma Ariffin. 2000. Biotechnology[M.Sc.]. Universiti Putra Malaysia.Shahreza Md. Shariff. 2000.Breeding/Biotechnology [M.Sc.].Universiti Putra Malaysia.379

Development of DNA markers for identification of colour varieties and sex in tiger barbs

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Development of DNA markers for identification of colour varieties and sex in tiger barbs

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