Genetic Diversity of False Gharial Tomistoma schlegelii based on Cytochrome b-Control Region (cyt b-CR) Gene Analysis Muhamad Farhan Bin Badri (34975) Bachelor of Science with Honours (Aquatic Resource Science and Management) 2015 UNIVERSITJ MALAYSIASARAWAK Grade A- Please tick <V) Final Year Project Report Masters PhD DECIA RATJON OF ORIGINAL WORK This declaration is made on the... .;l... day of.. .. ;Jr.,-.v!y" year .....;;;,;;J].C;S:­ Stude nt's Declaration: I .. _ ~~_~!l.~_~.I? .f!J~_~~ __ . ~?.: ___~~e!: .. .J..3_ f:{~~ _./. £~... ____ .....-.---- ..----- ....-------.----......--.--..---­ (PLEASE INDIC TE NAME. MATRIC NO. AND FACULTy) hereby declare that the work entitled. .~ft~ _ !l! ~~.'!.!J - f!~ - ~ b.':>!~.I-- !: --~~-~ ['JJ'.~! - .b.i.'.J--~:! ..~L~.":~B - ~ !.~.- ~ -~~?: is my original work. I have not copied from any other students' work or from any other sources with tbe exception where due reference or acknowledgement is made explicitly in th e text. nor has any part of the work been written for me by another person. r / 7 / :»')1 ~ MV~fI~AO F~IlH~'" p,Itv Il/!PlT G4'11S') Date submitted Name of the student (Mabic No.) Supervisor's Declaration: I,-- -OP,- - ~!!~~..~~!!.. !!~~_~ _ ...............__ .__ (SUPERVISOR'S NAME), herehy certify that the work entitled, q~~~-- ~.'".'!~~ - ~~~ - ~~~.~X: - ~~-~~i. ~}!lg-"'-- ~t_<;t ·~~jdTITLE ) was prepared by the aforementioned or above mentioned student, and was submitted to the "FACULTY' as a * partiaJJfuil fulfillment for the conferment of ._.¥h__J!~!.~..$.~~~_~L ~~A._ ~f!~~~- . - - .. --------­ (PLEASE INDICATE THE DEGREE TITLE), and the aforementioned work, to the best of my knowledge, is the said student's work Received for examination by: o~ M ·lIl ~A II ~H4'" Date: (Name of the supervisor) I declare this Project!Thesis is classified as (Please tick (fJ): o CONFIDENTIAL (Contains confidential information under the Official Secret Act 1972)* o RESTRICTED (Contains restricted information as specified by the organisation where research was done)* ,)3OPEN ACCESS I declare this Project/Thesis is to be submitted to the Centre for Academic Information Selvices (CAlS) and uploaded into UNIMAS Institutional Repository (UNIMAS IR) (Please tick (fJ): ~ES Po~~ VaJidation of Pl'ojecttrhesis I hereby duly affirmed with free consent and willingness declared that this said Project!Thesis shall be placed officially in the Centre for Academic Information Selvices with the abide interest and rights as follows: • This Project!Thesis is the sole legal property of Universiti Malaysia Sru'awak (UNIMAS), • The Centre for Academic Information Services has the lawful right to make copies of the Project!Thesis for academic and research purposes only and not for other purposes, • The Centre for Academic Information Services has the lawful right to digitize the content to be uploaded into Local COlltent Database. • The Centre for Academic Information Services has the lawful right to make copies of the Project!Thesis if required for use by other parties for academic purposes or by other Higher Learning Institutes, • No dispute or any claim shall arise from the student hinIself / herself neither a third party on this Project!Thesis once it becomes the sole property of UNlMAS. • This Project!I'hesis or any material, data and information related to it shall not be distributed, published or disclosed to any party by the student hinIselflherself without first obtaining approval from UNIMAS. Student's signature --,~,,---_=,,---,_____ _ Supervisor"s signature: -'--~--aFte=;)'fIH---- (Date) Notes: * If the Project!Thesis is CONFIDENTIAL or RES1'RICTED, please attach together as aIlllexure a letter from the orgaIIisation with the date of restriction indicated, and the reasons for the confidentiality and restriction. - [The instrument was prepared by The Centre for Academic Information Services] DECLARATION No portion of the work referred to in this dissertation has been submitted in support of an application for another degree qualification in this or any other university or institution of higher learning. Muhamad Farhan Bin Badri Aquatic Resource Science and Management Programme Faculty of Resource Science and Technology Universiti Malaysia Sarawak II Acknowledgement Alhamdulillah and my utmost gratitude to Allah SWT, for giving me strength, wisdom and knowledge to complete this dissertation and to keep me through many obstacles experienced prior the completion of this project. Hereby, I wish to take this opportunity to express my gratitude and appreciation to all those that have assisted, involved and guided me throughout the completion of this project. First of all, special thanks to Dr Ruhana Hassan, the Head of Department of Aquatic Science UNIMAS who is also my supervisor for trusting me to do this project and for giving me constructive advice, encouragement, guidance and assistance along the way until the completion of this project. Thank you very much for being so patient with me. Next, my appreciation goes to all postgraduate students in Aquatic Molecular Lab especially Miss Nursyuhaida Md Shahid and Mr Amirul Arib for their kind assistance in lab work and useful discussion that help me a lot in this project. Both of them had been actively and always been available to advise me and gave their countless help. Thank you very much. I would also like to extend my gratitude to my labmates and friends, especially to Miss Farhah Binti Dzarif for giving me continuous moral support. I could not finish this dissertation without their help. Last but not least, all my love and thanks goes to my family for always being there, giving me their support. Thank you. III Table of Contents Title and Front Cover I Declaration II Acknowledgement III Table of Contents IV List of Abbreviations VII List of Tables VIII List of Figures IX Abstract 1 1.0 Introduction 2 2.0 Literature Review 4 2.1 Taxonomy 4 2.2 Tomistoma schlegelii 4 2.2.1 Description 4 2.2.2 Classification based on Morphology 6 2.2.3 Previous Studies related to Tomistoma 7 2.2.4 Ecology and Habitat 8 2.2.4.1 Historical Distribution 8 2.2.4.2 Current Distribution 9 2.3 Mitochondrial Genes 10 2.3.1 Mitochondrial DNA 10 2.3.2 Cytochrome b Gene 11 IV 2.3.3 mtDNA Control Region 12 2.4 Molecular Study Approach 13 3.0 Materials and Methods 14 3.1 Sampling Site 14 3.2 Field Work 15 3.2.1 Blood Collection and Preservation Method 15 3.3 Laboratory Work 15 3.3.1 Preparation of buffer solution for modified CTAB method 15 (Doyle and Doyle, 1987) 3.3.2 DNA Extraction 17 3.3.3 Agarose Gel Electrophoresis 18 3.3.4 Gel Documentation of DNA Bands 19 3.3.5 Optical Density Reading 19 3.3.6 Polymerase Chain Reaction (PCR) Amplification 20 3.3.7 Sequencing 22 3.3.8 Data Analysis 22 4.0 Results and Discussion 23 4.1 Total Genomic DNA Extraction 23 4.1.1 DNA Extraction of T. schlegelii Blood Sample 23 4.1.1.1 Optical Density Reading 26 4.1.1.2 Comparison of Gel Electrophoresis and Optical Density 28 Reading Values 4.1.2 DNA Extraction of T. schlegelii Fresh Egg Sample 30 V 4.1.2.1 Optical Density Reading 31 4.2 Polymerase Chain Reaction (PCR) 32 4.2.1 Amplification of cyt b-CR gene for T. schlegelii Blood Sample 32 4.2.2 Amplification of cyt b-CR gene for T. schlegelii Fresh Egg Sample 35 4.3 Sequencing Analysis 37 4.4 Genetic Divergence Analysis 40 4.5 Phylogenetic Analysis 43 5.0 Conclusion and Recommendations 46 6.0 References 47 7.0 Appendices 52 VI List of Abbreviations Abbreviation Full Term bp Base pairs CIA Chloroform-Isoamyl Alcohol CITES Convention on International Trade in Endangered Species CTAB Cetyl-trimethyl Ammonium Bromide Cyt b Cytochrome b Cyt b-CR Cytochrome b-control region dNTPs Deoxynucleotide triphosphate DNA Deoxyribonucleic acid EDTA Ethylene diaminetetra-acetic acid EtOH Ethanol EtBr Ethidium bromide g Gram IUCN International Union for Conservation of Nature MgCl2 Magnesium Chloride min Minutes ml Milliliter mtDNA Mitochondrial DNA NaCl Sodium chloride rpm Revolutions per minute s Seconds VNTRs Variable number tandem repeats μl Microliter ºC Degree Celcius VII List of Tables No Title Page Table 3.1 2x CTAB buffer for 500 ml stock. 16 Table 4.1 OD reading of total genomic extraction products from blood sample 27 that involved in this study. Table 4.2 OD reading of total genomic extraction products from egg sample 31 that involved in this study Table 4.3 Summary of genetic distance value in percentage (%) for cyt b-CR 40 gene sequences of T. schlegelii. MW indicates samples from Matang Wildlife Center, MF indicates samples from Miri Crocodile Farm, TS01 indicates sample from Asajaya while CC indicates outgroup sample, C. porosus. VIII List of Figures No Title Page Figure 2.1 Taxonomy of Tomistoma schlegelii (adapted from www.iucnredlist.org). 4 Figure 2.2 The difference of snout between false gharial and Indian gharial. (a) 5 indicates the snout of false gharial while (b) indicates the snout of Indian gharial (both figures were adapted from www.arkive.org). Figure 2.3 Distribution of Tomistoma schlegelii (Bezuijen et al., 2010). 9 Figure 2.4 Mitochondrial DNA (adapted from www.bmb.leeds.ac.uk). 10 Figure 2.5 Location of cyt b gene on mtDNA (adapted from www.nature.com). 11 Figure 3.1 Map of Sarawak showing the the collection sites of Tomistoma samples. 14 (a) indicates Matang Wildlife Center in Kuching while (b) indicates Asajaya, Sarawak. Figure 3.2 Location of the blood sampling site – the supravertebral vein at post 15 occipital site (photo refer to C. porosus, adapted from Kaur et al., 2012). Figure 3.3 Cyt b-CR from the 3’ of cyt b to Domain II, before the B box.