Faculty of Resource Science and Technology Population Genetics of Tor douronensis in Sarawak Muhammad Amirul Arib bin Md Adzhar (31344) Bachelor of Science with Honours Aquatic Resource Science and Management 2014 Population Genetic of Tor douronensis in Sarawak Muhammad Amirul Arib bin Md Adzhar (31344) A report submitted in partial fulfilment of the Final Year Project STF 3012 Supervisor Dr Ruhana binti Hassan Co-supervisor Prof. Dr. Lee Nyanti Aquatic Resource Science and Management Department of Aquatic Science Faculty of Resource Science and Technology Universiti Malaysia Sarawak 2014 i DECLARATION No portion of the work referred in this dissertation has been submitted in support of an application for another degree qualification of this or any other university or institution of higher learning. _________________________________ Muhammad Amirul Arib bin Md Adzahar Aquatic Resource Science and Management Department of Aquatic Science Faculty of Resource Science and Technology Universiti Malaysia Sarawak I Acknowledgement In the name of Allah The Most Gracious and The Most Merciful. Alhamdulillah, I would like to express my outmost gratitude to Allah SWT, for giving me the strength to complete my Final Year Project. Hereby, I wish to take this opportunity to express my gratitude and appreciation to all those have assisted and guided me throughout the completion of this Final Year Project. First of all, special thanks to Dr Ruhana Hassan as Head of Aquatic Department and my supervisor for trusting me do this project and for giving me constant advice, guidance, encouragement and moral support along the way until the completion of this project. I would like to express my co-supervisor Prof Dr Lee Nyanti for his advice and useful discussions and also Mr Zaidi and Mr Nazri for all kind help during sample collection. My sincere appreciation also goes to all postgraduates of molecular aquatic lab especially Mohd Khairulazman Sulaiman and Kak Nurhartini Kamalia Yahya for their guidance and advice during molecular works. Deepest thanks also to Kak Nursyuhaida Md Shahid for her valuable advice and guidance on some difficulties during data analysis. I also would like to thank all my beloved lab mates for their kind assistance, companionship and support that helped me throughout this project. I would like to thank all my course mates and friends especially for their encouragement and support. Finally, all my love and greatest appreciation goes to my family member especially my parents, Mr Md Adzhar Shamsudin and Mrs. Rosnah Mohammad Idris for their support and encouragement. Thank you. II Table of Contents Declaration i Acknowledgement ii Table of Content iii List of Abbreviations v List of Tables and Figures vi Abstract ix 1.0 Introduction 1 2.0 Literature Review 3 2.1 The Cyprinids 3 2.2 The Southeast Cyprinids 3 2.3 The Genus Tor 4 2.4 The T. douronensis Valenciennes 5 2.5 Molecular Studies on Cyprinidae 6 2.6 Mitochondrial DNA 8 3.0 Materials and Methods 10 3.1 Collection of samples 10 3.2 Total Genomic DNA extraction 11 3.2.1 Preparation of buffer solution for modified CTAB method (Doyle and Doyle, 1987) 11 3.2.2 Preparation of tissue for total genomic DNA extraction 12 3.2.3 Total genomic DNA extraction using modified CTAB method (Doyle & Doyle, 1987) 12 3.3 Agarose gel electrophoresis 13 3.4 Optical density (OD) reading 14 3.5 Primer dilution 15 3.6 Polymerase chain reaction 16 III 3.7 Data analysis 17 4.0 Result and Discussion 20 4.1 Total Genomic DNA Extraction of Tor douronensis 20 4.1.1 Agarose Gel Electrophoresis 20 4.1.2 Optical Density Reading 22 4.2 Amplification of CO1 gene 25 4.3 Sequencing Analysis 27 4.4 Genetic Divergence Analysis 28 4.5 Population Genetics Analysis 30 4.5.1 Nucleotide Divergence 30 4.5.2 Phylogenetic Analysis 33 4.5.3 Network Analysis 36 4.5.4 Analysis of Molecular Variance 39 5.0 Conclusion and Recommendation 43 6.0 References 45 7.0 Appendix 50 IV List of Abbreviations Abbreviations Description CO1 Cytochrome oxidase 1 DNA Deoxyribonucleic Acid mtDNA Mitochondrial DNA % Percentage EDTA Ethylene diaminetetra-acetic acid CTAB Cetyl-trimethylammonium bromide CIA Chloroform Isomyl Alcohol ml Mililiter g Gram sdH2O Sterilized distilled water µl Micro liter rpm Rotation per minute oC Degree celcius UV Ultraviolet PCR Polymerase Chain Reaction µM Micro molar EtBr Ethidium Bromide V List of Figure Pages Figure 7.1 Location of T. douronensis populations involved in this study. 9 Figure 4.1 Agarose gel electrophoresis photograph showing total genomic DNA 20 extraction product Figure 4.2 Agarose gel electrophoresis photograph showing Polymerase Chain 25 Reaction product Figure 4.3 A maximum parsimony 50% majority rule consensus tree constructed 34 of CO1 gene sequence of T. douronensis from Sarawak with H. dispar and N. stracheyi as outgroup. Figure 4.4 Bayesin inference of the 50% majority rule consensus tree of CO1 gene 35 sequences of T. douronensis Figure 4.5 The minimum-spanning network (MSN) generated by Network 4.5.0.2 36 (Bandelt et al. 1999) illustrating the relationships of T. douronensis in Sarawak Figure 4.6 Mismatch distribution for Sarawak T. douronensis at each locality. 37 Figure 7.2 DNA Extraction Protocol 50 Figure 7.3 PCR profile of amplification using oligoreductase primer for CO1 genes 51 VI List of Tables Pages Table 4.1 Optical density reading for T. douronensis DNA extraction product. 22 Table 4.2 Summary of BLAST result of T. douronensis CO1 gene sequence obtain 27 in this study Table 4.3 Summary of genetic distance in percentage (%) for CO1 gene sequences 29 of T. douronensis Table 4.4 Samples of T. douronensis analyzed for DNA (CO1) gene sequence 31 variation with locality, GPS reading, field voucher and identified haplotype. Table 4.5 Measures of haplotypes and nucleotide diversity within populations of T. 32 douronensis analyzed by location Table 4.6 Measures of nucleotide diversity (π) and net nucleotide divergence 32 among populations of T. douronensis analyzed by location Table 4.7 Summary statistics of cytochrome oxidase subunit 1 (COI) mtDNA 38 sequence variation in 4 populations of T. douronensis in Sarawak Table 4.8 Measures of geographical population differentiation in T. douronensis 40 based on an analysis of molecular variance approach with cytochrome c oxidase subunit 1 data VII Table 4.9 Genetic differentiation matrix of populations calculated by ΦST. P 41 values are shown in parenthesis (below the diagonal) Table 4.10 Measures of nucleotide subdivision (Nst), population subdivision (FST), 41 and gene flow (number of migrants, Nm) among 4 populations of T. douronensis Table 7.1 2X CTAB buffer recipe for 500ml stock 50 Table 7.2 PCR master mixture 51 VIII Population Genetics of Tor douronensis in Sarawak Muhammad Amirul Arib bin Md Adzhar Aquatic Resource Science and Management Department of Aquatic Science Faculty of Resource Science and Technology Universiti Malaysia Sarawak ABSTRACT Tor douronensis, known locally as Semah, is one of the valuable and potential resources in Sarawak due to its high food value and demand as game fish. Several molecular studies related to T. douronensis had been carried out but limited in populations. No study had involved T. douronensis from Bakun. Therefore, this study was designed to infer population subdivision, the genetic structure, evolutionary neutrality, and population expansion of T. douronensis among 4 populations from Sarawak including Bakun using partial DNA sequencing of the Cytochrome c oxidase I (COI) mtDNA gene. A total 465 bp of CO1 gene of T. douronensis had been successfully amplified, and based on phylogenetic tree, there are 3 distinct geographical population subdivision observed (Central, Southern, and Northern population); 1st clade (haplogroup I) from Bakun, 2nd clade (haplogroup II) from Layar and 3rd clade (haplogroup III) from Ba Kelalan and Ulu Limbang. Overall, there were 13 haplotypes and none is shared by any population. Low level gene flow has been observed. Small number of migrants per generation (Nm<1.0) among the population indicated the small population or separated populations with large geographical and topological barrier. Population expansion was undergone for the species for the whole populations except for northern population as shown by small and non-significant values of the sum of the standard deviation of the observed (SSD<0.5) and expected mismatch distributions (unimodal) and Harpending raggedness index (r <1.2). Furthermore a large negative value and significant test of Fu’ Fs in Bakun population suggested recent expansion. The result also suggested that all the populations do not deviate with evolutionary neutrality supported with no significant in Tajima’s neutrality test (p<0.05). Key words: Tor douronensis, COI gene, Population expansion, Population subdivision, evolutionary neutrality. ABSTRAK Tor douronensis dikenali sebagai Semah oleh penduduk tempatan merupakan salah satu sumber bernilai dan berpotensi di Sarawak kerana nilai makanan dan permintaan dalam sukan pancing. Beberapa kajian molecular telah dilakukan berkaitan spesis ini namun hanya melibatkan beberapa populasi. Tiada kajian lagi dilakukan untuk populasi Bakun. Justeru, kajian ini bertujuan untuk melihat pecahan populasi, struktur genetik, kebiasaan genetik dan pengembangan populasi Semah di Antara empat populasi Semah temasuk Bakun di Sarawak dengan menggunakan rantaian CO1 gene. Sebanyak 465 bp rantaian CO1 gene berjaya dianalisa, dan berdasarkan peta phylogenetic, terdapat 3 pecahan kumpulkan populasi dikenal pasti iaitu, Kumpulan I daripada Bakun, Kumpulan II daripada Layar dan Kumpulan III daripada Ulu Limbang dan Ba Kelalan. Terdapat 13 haplotype secara keseluruhan tanpa ada percampuran haplotype dalam setiap populasi.Tahap pengaliran gen yang rendah dapat diperhatikan dengan jumlah migrasi rendah (Nm <1.0) menunjukkan populasi yang kecil atau populasi tersebit terpisah dengan keadaan geogafi yang besar. Pengembangan populasi berlaku untuk semua populasi kecuali untuk populasi Ba Kelalan- Limbang dengan kadar kecil dan tidak signifikan kadar taburan kaitan (SSD<0.5), jangkaan mismatch (unimodal) dan nilai pengembangan Harpending (r<1.2).