MORPHOLOGICAL AND GENETIC VARIABILITY OF MALAYSIAN CHANNA SPP BASED ON MORPHOMETRIC AND RAPD TECHNIQUES NORAINY BINTI MOHD HUSIN UNIVERSITI SAINS MALAYSIA 2007 MORPHOLOGICAL AND GENETIC VARIABILITY OF MALAYSIAN CHANNA SPP BASED ON MORPHOMETRIC AND RAPD TECHNIQUES by NORAINY BINTI MOHD HUSIN Thesis submitted in fulfillment of the requirements for the degree of Masters of Science FEBRUARY 2007 ACKNOWLEDGEMENTS Alhamdulillah, syukur ke hadrat illahi kerana dengan izin Nya saya dapat menyiapkan tesis ini. My sincere gratitude and appreciation to my supervisor Prof. Madya Dr. Siti Azizah Mohd Nor. She has helped me a lot in my research. Through her guidance, patience and encouragement, I have come this far. Thank you Dr. I also would like to say thank you to K. Za and Abg. Amir, who has taught and guide me at the beginning of my research. My gratitude also goes to other lab mates and friends Fatimah, Emi, Mila, Zam, Azad, Zahir, Abg Amir and Zarul. Thank you also to lab member of Lab 408 (Dr. Sofiman’s lab). To other friends who has enriched my life with their friendship and kindness. My profound gratitude and love to both of my parents for their unconditional love and support. Thank you also to my auntie and uncles for their encouragement. Last but not least to my husband and my beautiful children; Damia and Adam, I am blessed having you in my life. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES vi LIST OF FIGURES ix LIST OF PLATES xii LIST OF ABBREVIATIONS xiii LIST OF APPENDICES xiv LIST OF PUBLICATIONS & SEMINARS xv ABSTRAK xvi ABSTRACT xviii CHAPTER ONE : INTRODUCTION 1.1 The Channa - snakehead 1 1.1.1 Taxonomy and Morphology 1 1.1.2 General biology of the snakeheads 5 1.1.3 Morphological taxonomy of the snakeheads 7 1.2 Introduction on morphometrics 11 1.2.1 Principal Component Analysis (PCA) 14 1.2.2 Discriminant Function Analysis (DFA) 15 1.2.3 Studies based on the truss network 16 1.3 Genetic in Fisheries and Aquaculture 18 1.3.1 Molecular techniques 20 1.3.2 Application of RAPD-PCR in fishes 29 34 CHAPTER TWO : MATERIALS AND METHODS 2.1 Sampling Method 34 iii 2.2 Morphometric-Truss network measurement 38 2.2.1 Data transformation 42 2.2.2 Analysis of variance (ANOVA) 42 2.2.3 Principal component analysis (PCA) 43 2.2.4 Discriminant function analysis (DFA) 43 2.3 RAPD Analysis 43 2.3.1 DNA extraction 44 2.3.2 Primer Screening 48 2.3.3 Optimization 48 2.3.4 DNA amplification 49 2.3.5 Data Analysis 50 52 CHAPTER THREE: RESULTS 3.1 Morphometric 52 3.1.1 Descriptive analysis 52 3.1.2 ANOVA 59 3.1.3 Principal Component Analysis (PCA) 63 3.1.4 Discriminant Function Analysis (DFA) 71 3.2 RAPD analysis 83 3.2.1 DNA Extraction 83 3.2.2 Primer screening 85 3.2.3 Optimization of RAPD-PCR parameters 88 3.2.4 Intraspecific genetic variability of six Channa species 96 studied. 3.2.5 Phylogenetic relationship among Channa spp of 109 Peninsular Malaysia. 3.2.6 Diagnostic markers 112 iv CHAPTER 4: DISCUSSION 114 4.1 Morphometric study 114 4.2 RAPD Analysis 119 4.3 Morphological and molecular congruence 129 CHAPTER 5: CONCLUSION 134 BIBLIOGRAPHY 136 APPENDICES Appendix 1: Genetic distance generated by FreeTree software 157 v LIST OF TABLES Page Table 1.1 Recommendations of catch and release practices for 4 anglers regarding four Channa species (Malaysian Fishing Net, 2006 with some modification). Table 2.1 Origin of Channa species used in this study sampled from 37 different locations. Table 2.2 Eighteen variables of side view truss-network. 40 Table 2.3 Eleven variables of dorsal view truss-network. 41 Table 2.4 Reagent for the master mix and its volume for each PCR 51 tube. Table 3.1 Descriptive analysis for side view measurement. 53 Table 3.2 Descriptive analysis for dorsal view morphometric data. 57 Table 3.3 Oneway ANOVA of 18 side view measurements for six 60 Channa species. Table 3.4 Oneway ANOVA of 11 dorsal view measurements for six 61 Channa species. Table 3.5 Eigen values, percentage of variance explained and 65 percentage of cumulative values for side view variables. Table 3.6 Rotated Component Matrix for three components of 18 66 side view variables. Table 3.7 Eigen values, percentage of variance, percentage of 67 cumulative value for dorsal view variables. Table 3.8 Rotated Component Matrix for three components of 11 67 dorsal view variables. Table 3.9 Eigen values, percentage of variance, cumulative 72 advantage and canonical correlation for 18 side view characters. Table 3.10 Eigen values, percentage of variance, cumulative 72 percentage and canonical correlation for 11 dorsal view characters. Table 3.11 Structure matrix showing pooled within-group correlations 74 between discriminating variables and standardized canonical discriminant functions for 18 side view measurements. vi Table 3.12 Structure matrix showing pooled within-groups correlations 74 between discriminating variables and standardized canonical discriminant functions for 11 dorsal view measurements. Table 3.13 Classification results of predicted group membership for 79 side view Table 3.14 Classification results of predicted group membership for 80 dorsal view Table 3.15 Pairwise group comparisons for side view measurements. 82 Table 3.16 Pairwise group comparisons for dorsal view 82 measurements. Table 3.17 Summary of the number and characteristics of 87 amplification products obtained from the 40 primers screened. Table 3.18 Total number of fragments, number of polymorphic 97 fragments, percentage of polymorphism, number of genotypes and size of bands generated by C. striata amplified by nine primers. Table 3.19 Genetic distance of C. striata individuals based on nine 97 chosen RAPD primers. Table 3.20 Total number of fragments, number of polymorphic 99 fragments, percentage of polymorphism, number of genotypes and size of bands generated by C. marulioides amplified by nine primers. Table 3.21 Genetic distance of C. marulioides individuals based on 99 nine chosen RAPD primers. Table 3.22 Total number of fragments, number of polymorphic 101 fragments, percentage of polymorphism, number of genotypes and size of bands generated by C. gachua amplified by nine primers. Table 3.23 Genetic distance of C. gachua individuals based on nine 101 chosen RAPD primers. Table 3.24 Total number of fragments, number of polymorphic 103 fragments, percentage of polymorphism, number of genotypes and size of bands generated by C. micropeltes amplified by nine primers. Table 3.25 Genetic distance of C. micropeltes individuals based on 103 nine chosen RAPD primers. vii Table 3.26 Total number of fragments, number of polymorphic 105 fragments, percentage of polymorphism, number of genotypes and size of bands generated by C. lucius amplified by nine primers. Table 3.27 Genetic distance of C. lucius individuals based on nine 105 chosen RAPD primers. Table 3.28 Total number of fragments, number of polymorphic 107 fragments, percentage of polymorphism, number of genotypes and size of bands generated by C. melasoma amplified by nine primers. Table 3.29 Genetic distance of C. melasoma individuals based on 107 nine chosen RAPD primers. Table 3.30 Summary of intraspecies variability among six Channa 108 species. Table 3.31 List of the diagnostic markers for species and genus 113 specific of six Channa species studied. viii LIST OF FIGURES Page Figure 2.1 Map of Peninsular Malaysia showing location of sampling 36 sites for six Channa species (Modified from Leong, 2003). Figure 2.2 Morphological landmarks were identified along the outline 39 of the fish and eighteen side view measurements were taken for each specimen (a) and (b). Figure 2.3 Morphological landmarks were identified along the outline 41 of the fish and eleven dorsal view measurements were taken for each specimen. Figure 3.1 The summary of SNK tests showing the five subsets for 62 both side and dorsal measurements. Figure 3.2 Scatter plot of individuals’ component score between 69 Component 1 and Component 2 for side view. Figure 3.3 Scatter plot of individuals’ component score between 70 component 1 and component 2 for dorsal view. Figure 3.4 Canonical discriminant functions for side view. 76 Figure 3.5 Canonical discriminant functions for dorsal view 77 Figure 3.6 Total genomic DNA extracted using Pure-Gene® Tissue 84 DNA Extraction Kit. Lane M : Lambda DNA Marker Hind III. Lanes 1-4: Representative DNA extract of Channa species. Figure 3.7 Screening of primer OPAB-1 to OPAB-20 from Life 86 Technologies. Highlighted number were the primers used in this study. M is 100 bp plus DNA ladder from Fermentas. Figure 3.8 Screening of primer OPF-1 to OPF-20 from Life 86 Technologies. Highlighted number were the primers used in this study. M is 100 bp plus DNA ladder from Fermentas. Figure 3.9 The optimization of genomic DNA quantity on RAPD 89 banding patterns generated by primer OPAB-2. M is 100 bp plus DNA ladder (Fermentas); DNA concentration of 10 ng (Lane 1); 25 ng (Lane 2); 40 ng (Lane 3); 60 ng (Lane 4); 75 ng (Lane 5) and 100 ng (Lane 6). ix Figure 3.10 The optimization of MgCl2 concentration on RAPD banding 89 patterns generated by primer OPAB-2. M is 100 bp plus DNA ladder (Fermentas) ; MgCl2 concentration of 2.0 mM (Lane 1); 2.5 mM (Lane 2); 3.0 mM (Lane 3); 3.5 mM (Lane 4); 4.0 mM (Lane 5); 4.5 mM (Lane 6); 5.0 mM (Lane 7); 5.5 mM (Lane 8); 6.0 mM (Lane 9) and 6.5 mM (Lane 10).