DEVELOPMENT OF SPECIES-SPECIFIC MICROSATELLITE MARKERS OF TERMITE Globitermes sulphurues Haviland (BLATTODEA: TERMITIDAE) FOR GENETIC POPULATION ANALYSIS BY USING NEXT-GENERATION SEQUENCING (NGS) APPROACH NUR AIZATUL NATHASHA BINTI KHIZAM UNIVERSITI SAINS MALAYSIA 2019 DEVELOPMENT OF SPECIES-SPECIFIC MICROSATELLITE MARKERS OF TERMITE Globitermes sulphurues Haviland (BLATTODEA: TERMITIDAE) FOR GENETIC POPULATION ANALYSIS BY USING NEXT-GENERATION SEQUENCING (NGS) APPROACH by NUR AIZATUL NATHASHA BINTI KHIZAM Thesis submitted in fulfilment of the requirements for the degree of Master Science June 2019 ACKNOWLEDGEMENT First and above all, I am grateful to the Almighty God for granting me the strength and capability to complete my Master journey. Alhamdulillah, thank you God for blessing me much more than I deserve. Also, I would like to express my very great appreciation to my supervisor, Associate Professor Dr. Abdul Hafiz Bin Ab Majid for his valuable and constructive suggestions during the planning and development of this research work. His willingness to give his time so generously has been very much appreciated. I wish to thank various people for their contribution to this master research project in term of suggestions, guidance, and times. This research work would not have been completed without them. Special thanks to my beloved parents, Mr. Khizam Hashim and Mrs. Maizatul Akmal for their countless support, understanding and encouragement throughout my entire life. Praise to God, along with their patience and consecutive prayers, I managed to complete my Master studies with ease. I always knew that they believed in me and wanted the best for me. I also really appreciate how all of my family members including my cousins, Muhammad Abid Rosmadi, Muhammad Adib Rosmadi, Aina Khaireena Rozman and Aida Khairunisa Rozman who have been involved in my research studies process. Also, I am really grateful for having a supportive and thoughtful fiancee, Mr. Alif Amin Bin Abdullah who kept me accompanied during my sampling days just to obtain my samples collection. I thank you for your part through thick and thin in my life journey. I would like to take a moment to thanks a group of people, Nurul Akmar, Siti Nor Ain, Fadhlina Hazwani, Faezah Syukriah Sabtu, Husna Noordin and Nor Izzaty ii Abd Manan who I feel simply haven’t been thanked enough, who helped me in giving ideas and helpful assistance in solving my problems through the process of lab work completion. Thanks for the valuable friendship and experience they bring into my days spent in USM. Next, I have furthermore to extend my sincere gratitude to Universiti Sains Malaysia (USM) for giving me a great opportunity to be one of the postgraduate students studying in their prestige university. Finally, assistance provided by all staff School of Biological Science, either directly or indirectly was greatly appreciated. iii TABLE OF CONTENTS ACKNOWLEDGEMENT ii TABLE OF CONTENTS iv LIST OF TABLES viii LIST OF FIGURES x LIST OF SYMBOLS AND ABBREVIATIONS xi LIST OF APPENDICES xii ABSTRAK xiii ABSTRACT xv CHAPTER 1 : INTRODUCTION 1 1.1 History of Study 1.2 Problem statement 3 1.3 Significance of study 5 1.4 Hypothesis 6 1.5 Objectives 6 CHAPTER 2 : LITERATURE REVIEW 2.1 Higher Group Termites: History, Caste system, Colony dynamics 7 2.2 Foraging mechanism of subterranean termites 9 2.3 Biology of Globitermes sulphureus Haviland, 1898 10 2.3.1 Taxonomy of Globitermes sulphureus 10 2.3.2 Morphological characteristics of Globitermes sulphureus 10 2.3.3 Nesting and Foraging territory of Globitermes sulphureus 11 2.3.4 Defensive system of Globitermes sulphureus 11 iv 2.3.5 Research studies of Globitermes sulphureus 12 2.4 Transcriptome 13 2.5 Recognition of Next Generation Sequencing (NGS) 15 2.5.1 Potential uses and Practises of NGS 17 2.6 Molecular or Genetic marker 19 2.7 Microsatellite marker 21 2.7.1 Types and Classifications of microsatellite 23 2.7.2 Advantages of microsatellite marker 25 2.7.3 Applications of microsatellite marker 26 2.8 Primer Design 28 2.9 Polymorphism study of microsatellite marker 29 2.10 Population Genetic Study on Higher Group Termite 30 CHAPTER 3: DEVELOPMENT OF SPECIES-SPECIFIC MICROSATELLITE MARKERS FROM TRANSCRIPTOME DATASET OF Globitermes sulphureus Haviland (BLATTODEA: TERMITIDAE) 3.1 Introduction 33 3.2 Materials and Procedures 36 3.2.1 Assembly of transcripts, microsatellite repeat motif detection and locus- 36 specific primer design 3.2.2 Primer design 38 3.2.3 Dissolving and dilution of developed primer pairs 40 3.3 Results 41 3.3.1 Repeat motif detection 41 3.3.2 Development of species-specific microsatellite primers 44 v 3.4 Discussion 48 3.5 Conclusion 50 CHAPTER 4 : CHARACTERIZATION OF SPECIES-SPECIFIC MICROSATELLITE POLYMORPHISM IN Globitermes sulphureus (Haviland) 4.1 Introduction 51 4.2 Materials and Procedures 54 4.2.1 Field sampling of Globitermes sulphureus 54 4.2.2 Genomic DNA extraction for polymorphism studies 59 4.2.3 PCR reaction and estimation of polymorphic information content 63 4.3 Results 68 4.4 Discussion 72 4.5 Conclusion 78 vi CHAPTER 5 : POPULATION GENETIC STRUCTURE AND BREEDING PATTERN OF Globiteremes sulphurues FROM SELECTED LOCATIONS IN NORTHERN OF PENINSULAR MALAYSIA 5.1 Introduction 79 5.2 Materials and Procedures 81 5.2.1 Globitermes sulphureus termites sample collection 81 5.2.2 Genomic DNA isolation for population studies 86 5.2.3 Microsatellite genotyping 87 5.2.4 Population genetic data analysis 90 5.3 Results 94 5.4 Discussion 108 5.5 Conclusion 119 CHAPTER 6 : GENERAL SUMMARY AND SUGGESTIONS 120 REFERENCES 124 APPENDICES vii LIST OF TABLES Page Table 3.1 Core criteria for designing primers 39 Table 3.2 Different types of di-, tri-, tetra-, penta- and hexanucleotide repeat 42 motifs Table 3.3 Numbers of microsatellites with di-, tri-, and tetranucleotides repeat 43 motifs in different repeat unit classes, the total number of microsatellites with each motif and the number of di-, tri-, and tetranucleotides microsatellites with the number of repeat units equal to or greater than 4, 5, and 6, respectively in the transcriptome dataset of G. sulphureus Table 3.4a Information on the molecular structure of 15 dinucleotide 45 microsatellite developed from G. sulphureus showing number of repeats, type of repeats and sequences Table 3.4b Information on the molecular structure of 10 trinucleotide 46 microsatellite developed from G. sulphurues showing number of repeats, type of repeats and sequences. Table 3.4c Information on the molecular structure of 5 tetranucleotide 47 microsatellite developed from G. sulphurues showing number of repeats, type of repeats and sequences. Table 4.1 Detailed sampling information of G. sulphureus individuals of each 56 regions Table 4.2 Summary for PCR touchdown protocol 64 Table 4.3 List of reagents used in PCR reaction 64 Table 4.4a Primer sequences and descriptions of 15 dinucleotide microsatellite 69 markers developed in G. sulphureus Table 4.4b Primer sequences and descriptions of 10 trinucleotide 70 microsatellite markers developed in G. sulphureus Table 4.4c Primer sequences and descriptions of 5 tetranucleotide 71 microsatellite markers developed in G. sulphureus Table 5.1 Detailed 8 collection points of G. sulphurues worker individuals 83 Table 5.2 Detailed information for 7 chosen loci used in microsatellite 87 genotyping viii Table 5.3 Variability of 7 polymorphic species-specific microsatellite loci in 94 Peninsular Malaysia (Kedah and Penang) for natural and metropolitan populations Table 5.4 Breeding structure of natural and metropolitan populations of the 95 G. sulphureus subterranean termite Table 5.5 Summary of Wright's F-statistics and relatedness coefficient (r) for 98 workers of G. sulphureus from simple, mixed family colonies and all populations focused in Northen part of Peninsular Malaysia Table 5.6 Summary of Wright's F-statistics for workers of G. sulphureus from 98 natural and metropolitan populations focused in Northen part of Peninsular Malaysia Table 5.7 Relatedness coefficient (r) between nestmate reproductive and 99 inbreeding (FIS) reproductive in simple family colonies of G. sulphureus Table 5.8 Gene diversity measures for workers of G. sulphureus from both 100 natural and metropolitan populations Table 5.9 Summary AMOVA analysis 102 Table5.10a Hardy-Weinberg Equilibrium (HWE) exact test in the subterranean 104 termite G. sulphureus populations based on populations Table5.10b Hardy-Weinberg Equilibrium (HWE) exact test in the subterranean 105 termite G. sulphureus populations based on loci Table 5.11 Bottleneck test analysis that showed P-value within 8 populations 106 of G. sulphurues in Peninsular Malaysia Table 5.12 Summary of probability value for mutation-drift equilibrium from 107 Wilcoxon test. ix LIST OF FIGURES Page Figure 2.1 Schematic diagram representing the arrival of DNA 17 sequencing technologies Figure 2.2 Classification of genomic DNA mutation phenomena 20 Figure 2.3 Types and classifications of microsatellite marker 23 Figure 3.1 A screenshot of the MSATCOMMANDER application 37 running on Window 10 Figure 4.1 Sampling study sites of G. sulphureus termites for the selected 55 areas of Northen part of Malaysia (Kedah and Penang) Figure 4.2 Research sites for G. sulphurues collections 57 Figure 4.3 Research sites for G. sulphurues collections 58 Figure 4.4 Flowchart for DNA extraction of G. sulphureus workers 61 protocol Figure 4.5 Laboratory workflow procedure 62 Figure 4.6 Fragment Analyzer TM Automated CE System Machine used 66 and preparation to load samples into 96 well plate Figure 4.7 PROsize v3.0 software package main interface 66 Figure 4.8 Calibration curve interface 67 Figure 4.9 CERVUS v3.0 software interface 67 Figure 5.1 Eight sampling location of G. sulphurues termite species 84 between natural and metropolitan regions Figure 5.2 Sample collection activities on the research sites.