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Phylogenetic Analysis of Two Fish Genus: Osteochilus and Cyclocheilichthys

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Phylogenetic Analysis of Two Fish Genus: Osteochilus and Cyclocheilichthys PHYLOGENETIC ANALYSIS OF TWO FISH GENUS: OSTEOCHILUS AND CYCLOCHEILICHTHYS Nadia Syahirah binti Radzali (35034) Bachelor of Science with Honours (Aquatic Resource Science and Management) 2015 UNlVERSfT I MALAYSIA SARAWAK Grade: _ _ ___ Ple ase tic k (..J) Final Year Project RepOtt [2] Maste!'s D PhD D DECLARATION OF ORIGINAL WORK This decla ration is made on the . .....day ot: ... .... .o.. .. year , S t.uuellt:" ~ D ~ cl R rH. (iou . I ......N . ~.I.~..\~. ~ . ~:tl~ .~ .~~...~!.~ .f. ~ .. ~.~. ~.~~. l L . f . F~!.~,...f(l..0:!!CJT.dl.. ~-..~~J~ _~_ ~ (f. _ .x0~ re Iec.", YlO "':;J r<rLEASE INDICAT~ NAME, MATRIg NO. tf.p. FACULTy) ~f}' y declare that t e work entitled, .. · ~l!I.f;.g~ ·""~t ._ A!':-C?f-Jl-tl!-.lr~~.fJ} m.....'1.~L : . b\Ie. f.! 1S my original work I have not copi ed"from any ~e r s t~DtB work or from any other sources with the exception where due reference or acknowledgement " made explicitly in the text, nor has any part of the work been written for me by another person. ¥~~\ .:........' ­ Date sub mitted Name of the student (Matric No.) N~'O\A ~AH'I~AH t,Itv[{ t1.A 07./1 t I "?t;ll3 'f Supprvisor's Declaration: I,_..__ .___ ..fJl1j'l'l_Ql__U _ . _~. :?..Lz. ____ _.___ .__ ...____ .. (SUPERVISORS NAME), hereby certify that the work entitled, r.l~J.A3;g~j1,b .~ _ ... A~~~!~U.Li::f . l~~ _ fi1J_ ._ k£JJ.N .I. ( TITLE) was prepared by the a t' or ementJ. onea-Ul.lkI",.a.~S ,lb oA,-«ve- m e~.Loflt lOned{ bo··stuere."'Ol · <4...~nt, amr, .j rwas. su b'mltt e d to t' h e "FACUh LT' Y" as a * partiaVfull fulfillment for the conferment of .fPC' _ ![~IIU ' _i. .i'. 5_ !-_{ _~ _ 1!\>-:""-J _ . ~!:t _ •• _.~nlt,"'_ "_ J_ ~'j.1I\H ,<, ",I:'f. ", C.e ItA < .... (I? q. fVl t; ~ ' fe Mr (pLEASE INDICATE THE DEGREE TITLE). and the aforementioned work, to the est uf rny knowledge, is the said studenfs work - Received fo r examination by: . Date ~ {i, (~tr (Name ofthe supervisor) I declare this ProjecUThesis is classified as (please tick (,/J): D CONFIDENTIAL (Contains co nfidential information under the Official Secret Act 1972);' D RESTRICTED (Contains restricted information as specified by the organisation where re~ e arc h was done)':' [LlOPEN ACCESS I declare this Project/Thesis is to be submitted to the Centre for Academic Information Services (CAlS) and uploaded into UNIMAS Institutional Repository (UNlMAS IR) (Please tick (,/J): D 'ES c;3NO Validation o f ProjectfI'besis I hereby duly affirmed with free consent and willingness declared that this said Projecttrhesis shall be placed officially in the Centre for Academic Information Services with tbe abide interest and rights as follows: • This ProJect/Thesis is the sole legal property of Universiti Malavsia Sarawak (UNlMAS). • The Centre for Academic Information Services has the lawful right Lo make copies of the ProjectlThesis for academic and research purposes only and not for other purposes. • The Centre for Academic Information Services bas the lawful right to digitize the content to be uploaded into Local Co ntent Database. • The Centre for Academic Information Services has the lawful right to make copies of the Pro]ecttI'hesls If required ror use by other parties for academIc purposes or by other Higher Learning Institutes. • No dispute or any claim shall arise from the studellt himself / herself neither a third party on this Projecttrhesis once it becomes the sole propelty of UNIMAS. • This Project/Thesis or any material, data and information related to it sha ll not be di..s.tributed, pllolish8d or diB-doBeo. to aT1Y party by tbp 'St\lr:l ent him<:l.plfi'hp.rself withol11 first obtaining approval from UNIMAS. I;'A • ~ k>.L" Student's signature - -- - -'L"J'-4----:--:.7'C Supervisor's signature (Date) '2-6/G/I) Current Address' Lot l\} 1} ! Ie M c,v-. S: Al~1i !N Il ~ I Notes: 0); If the Projer:t/Thesis is CONF IDENTIAL Or RESTRI C'r ED please attach together as annexure a letter from the organisation with the date of restriction indicated, and the reasons for the - confldentiality and restrktioD. [The instrument was prepared by The Centre for AcademIC Information Services] PHYLOGENETIC ANALYSIS OF TWO FISH GENUS: OSTEOCHILUS AND CYCLOCHEILICHTHYS Nadia Syahirah binti Radzali (35034) This project report is submitted in partial fulfillment of the requirements for the Degree of Bachelor of Science with Honours (Aquatic Resource Science and Management) Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARAWAK 2015 Declaration I hereby declare that no portion of the work referred in this project has been submitted in support of an application for another degree qualification of this or any other university or institution of higher learning. NADIA SYAHIRAH BINTI RADZALI Aquactic Resource Science and Management Programme Faculty of Resource Science and Technology Universiti Malaysia Sarawak 3 Acknowledgement I would like to express my gratitude to Dr Fazimah Aziz, my supervisor for guiding me during the completion of this final year project. Thank you also to Dr Khairul Adha A. Rahim as my co-supervisor. Their knowledge and guidance was tremendous help in conducting this project. Next, I would also like to thank the Mr. Zaidi Ibrahim, Ms. Lucy and Mr. Zulkifli Ahmad for their assistance for their help during laboratory work and field work. My gratitude goes to all my helpful coursemates for their support and guidance. My appreciation also goes to my family especially my parents for supporting me emotionally and financially. Lastly, I thank everyone that directly or indirectly contributed to the completion of this final year project. 4 Table of Content Declaration i Acknowledgement ii Table of Content iii-iv List of Abbreviations v List of Tables vi List of Figures vii Abstract viii 1.0 Introduction 1 - 2 2.0 Objectives 2 3.0 Literature Review 3 3.1 Cyprinidae Family 3 3.2 Osteochilus sp. 3-4 3.3 Cyclocheilichthys sp. 5 3.4 Molecular Study 5-6 3.5 Mitochondrial DNA (mtDNA) 6 4.0 Materials and Methods 7 4.1 Study Site 7 4.2 Sample Collection 8 4.3 Laboratory Work 8 4.3.1 Muscle Tissue Extraction 8 4.3.2 Genomic DNA Extraction 9 4.3.3 Gel Electrophoresis 10 4.3.4 Polymerase Chain Reaction (PCR) 10-11 4.35 DNA Purification 11-12 4.4 Mitochondrial DNA Data Analysis 12 5.0 Results 13 5.1 Genomic DNA Extraction 13 5.2 Polymerase Chain Reaction (PCR) 14 5.3 Sequence Analysis 15-17 5.4 Genetic Divergence 18-19 5.5 Phylogenetic Analysis 20-23 6.0 Discussion 24-25 7.0 Conclusion 26 8.0 References 27-28 9.0 Appendix 30 5 List of Abbreviations COI Cytochrome C Oxidase Subunit I PCR Polymerase Chain Reaction NJ Neighbor-Joining ML Maximum-Likelihood MP Maximum-Parsimony EDTA Ethylenediaminetetraacetic acid TBE Tris-borate-EDTA T Thymine G Guanine A Adenine C Cytosine 6 List of Tables Page Table 4.1 : PCR cocktails for the amplification of COI gene of Cyclocheilichthys spp and 10 Osteochilus sp Table 5.1: Summary of BLAST results of Cytochelichthys sp. and Osteochilus sp. COI gene 16 sequence. Table 5.2: Percentage of nucleotide composition of Cyclocheilichthys sp. 16 Table 5.3: Percentage of nucleotide composition of Osteochilus sp. 16 Table 5.4: Percentage of nucleotide composition of Cyclocheilichthys sp. and Osteochilus sp. 17 Table 5.5 : Estimates of Evolutionary Divergence between Sequences 19 7 List of Figures Page Figure 3.1: Picture of Osteochilus sp. 4 Figure 3.2: Picture of Cychlocheilichthys sp. 5 Figure 4.1: Map of Batang Kerang, Balai Ringin, Sarawak 7 Figure 4.2: Polymerase chain reaction profile 11 Figure 5.1: Agarose gel photograph showing genomic DNA extraction product of 13 Cyclocheilichthys sp. and Osteochilus sp. using Lane 1-1kb ladder (Promega), Lane 2- Cyclochelichthys sp., Lane 3-Cyclocheilichthys sp., Lane 4- Cyclochelichthys sp., Lane 5- Osteochilus sp. , Lane 6- Osteochilus sp. Figure 5.2: Agarose gel photograph showing polymerase chain product for tissue samples of 14 Cyclocheilichthys sp. and Osteochilus sp.. Lane 1-1kb ladder, Lane 2- Cyclocheilichthys sp., Lane 3- Cychlocheilichthys sp., Lane 4- Cyclocheilichthys sp., Lane 5- Osteochilus sp., Lane 6- Osteohilus sp. Figure 5.3: Neighbor-Joining (NJ) tree showing the relationships among cytochrome c oxidase I 21 (COI) gene of Osteochilus sp. and Cyclocheilichthys sp. rooted with Channa Striata. The number on the branhes indicate bootstrap value of Neighbor-Joining based on 500 replications. Figure 5.4: Maximum-Likelihood tree to show relationships among the COI gene of 22 Cyclocheilichthys sp. and Osteochilus sp. rooted with Channa striata. the values on the branches are percentages of bootstrap value based on 500 replications. Figure 5.5: Maximum-Parsimony tree to show the relationships among COI gene of 23 Cyclocheilichthys sp. and Osteochilus sp. rooted with Channa striata. The numbers on the branches shows the bootstrap values based on 500 replications. 8 Phylogenetic Analysis of Two Fish Genus: Osteochilus and Cyclocheilichthys NADIA SYAHIRAH BINTI RADZALI (35034) Aquatic Resource Science and Management Faculty of Resource Science and Technology Universiti Malaysia Sarawak Abstract Osteochilus spp and Cyclocheilichthys spp are part of the Cyprinidae family. The aim of this study was to construct the phylogenetic tree of Osteochilus spp and Cyclocheilichthys spp using Cytochrome C Oxidase Subunit I (COI) gene and to study the genetic divergence of the Osteochilus spp and Cyclocheilichthys spp using COI gene. Samples were collected from Batang Kerang, Balai Ringin, Serian using three-layered gill net method.
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