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Boselaphus Tragocamelus, Antilope Cervicapra) and Cervidae (Axis Axis, Axis Porcinus) in Pakistan

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Boselaphus Tragocamelus, Antilope Cervicapra) and Cervidae (Axis Axis, Axis Porcinus) in Pakistan MOLECULAR PHYLOGENY AND DIVERSITY ANALYSIS OF BOVIDAE (BOSELAPHUS TRAGOCAMELUS, ANTILOPE CERVICAPRA) AND CERVIDAE (AXIS AXIS, AXIS PORCINUS) IN PAKISTAN BY GHULAM ABBAS 2011-VA-748 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF VETERINARY & ANIMAL SCIENCES, LAHORE 2016 The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore. We, the supervisory committee, certify that the contents and form of the thesis, submitted by Mr. Ghulam Abbas, Regd. No. 2011-VA-748 have been found satisfactory and recommend that it be processed for the evaluation by the External Examiner(s) for the award of the degree. Supervisor: _______________________ Dr. Asif Nadeem Member: ___________________________ Prof. Dr. Masroor Ellahi Babar Member: _______________________ Dr. Muhammad Tayyab DEDICATED To My Dear Parents i ACKNOWLEDGEMENTS All praises to “ALLAH”, the Almighty, Most Gracious, the most Merciful and the Sustainer of the worlds, Who created the jinn and humankind only for His worship and raised among the inhabitants of Makkah the Holy Prophet “Muhammad” (peace be upon him.) from among themselves, who recited to them His revelations and purified them, and taught them the Book (Holy Quran) and the Wisdom. Foremost thanks to Almighty Allah Who blessed me with good health and valued teachers to accomplish my Ph. D studies. I feel great pleasure in expressing my heartiest gratitude and deep sense of obligation to my supervisor Dr. Asif Nadeemand to the distinguished member of my supervisory committeeProf. Dr. Masroor Ellahi Babar (Tamgha-e-Imtiaz) for their able guidance, keen interest, skilled advice, constructive criticism, constant encouragement, valuable suggestions and kind supervision throughout the course of my study and research work. In addition, I owe my thanks to my committee memberDr. Muhammad Tayyabfor his suggestions. I need to offer my heartiest thanks to my friends, research fellows and colleagues at the University especially to Dr. Mohammad Sajid Tahir, Dr. Tanveer Hussain, Dr. Mohammad Haseeb Saeed and Mr. Muhammad Aqeel for their support and fruitful suggestions duringmy research. I would like to offer my special thanks to Mr. Ghulam Abbas Pathan and all those who helped me during the sample collection In the end, I could not envisage the endeavors that my parents made for successful completion of my higher education. They sacrificed all available resources in their lives for making me able to get higher education that ultimately lead me to successful completion of the degree. No acknowledgement could ever adequately express my obligation to my affectionate parents for leading their children into intellectual pursuits. “May Allah rest their souls in piece” (Aa’meen). I also extend my heartiest compliments to my wife, kids and sister whose love, prayers and support made it possible to reach this position. Ghulam Abbas Haral ii TABLE OF CONTENTS DEDICATION…………………………………………………………i ACKNOWLEDGEMENTS……………………………………………ii TABLE OF CONTENTS………………………………………………iii LIST OF TABLES……………………………………………………..iv LIST OF FIGURES……………………………………………………v SR. NO. CHAPTERS PAGE NO. 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 8 3 MATERIALS AND METHODS 24 4 RESULTS 45 5 DISCUSSION 106 6 SUMMARY 115 7 LITERATURE CITED 116 iii LIST OF TABLES TABLE TITLE PAGE NO. NO. 3.1a Sampling details of Boselaphus tragocamelus 30 3.1b Sampling details of Antilope cervicapra 31 3.1c Sampling details of Axis axis 32 3.1d Sampling details of Axis porcinus 33 3.2 List of primer sequences 39 3.3 Recipe for each multiplex of primers 40 3.4 PCR reaction composition for sequencing products 42 3.5 PCR reaction conditions for sequencing products 42 4.1 Polymorphisms identified in cytochrome-b gene of Boselaphus 46 tragocamelus 4.2 Polymorphisms identified in cytochrome-c gene of Boselaphus 50 tragocamelus 4.3 Polymorphisms identified in d-loop region of Boselaphus tragocamelus 54 4.4 Cytochrome-b, cytochrome-c and d-loop region based evolutionary 59 analyses of Boselaphus tragocamelus 4.5 Polymorphisms identified in cytochrome-b gene of Antilope cervicapra 62 4.6 Polymorphisms identified in cytochrome-c gene of Antilope cervicapra 65 4.7 Polymorphisms identified in mitochondrial d-loop region of Antilope 68 cervicapra 4.8 Cytochrome-b, cytochrome-c and d-loop region (collectively) based 72 evolutionary analyses of Antilope cervicapra 4.9 Polymorphisms identified in cytochrome-b gene of Axis axis 74 4.10 Polymorphisms identified in cytochrome-c gene of Axis axis 77 4.11 Polymorphisms identified in mitochondrial d-loop of Axis axis 79 4.12 Cytochrome-b, cytochrome-c and d-loop region based evolutionary 83 analyses 4.13 Polymorphisms identified in cytochrome-b gene of Axis porcinus 84 4.14 Polymorphisms identified in cytochrome-c gene of Axis porcinus 87 4.15 Polymorphisms identified in mitochondrial d-loop region of Axis porcinus 90 4.16 Cytochrome-b, cytochrome-c and d-loop region (collectively) based 94 Evolutionary analyses of Axis porcinus iv LIST OF FIGURES FIG. TITLE PAGE NO. NO. 3.1 Map of Pakistan showing selected areas for sampling of Boselaphus tragocamelus 26 3.2 Map of Pakistan showing selected areas for sampling of Antilope cervicapra 27 3.3 Map of Pakistan showing selected areas for sampling of Axis axis 28 3.4 Map of Pakistan showing selected areas for sampling of Axis porcinus 29 Photograph of Boselapus tragocamelus Antilope cervicapra (Bovidae), Axis axis 3.5 34 and Axis porcinus (Cervidae) Multidimensional scaling plot for cytochrome -b gene of Boselaphus 4.1 47 tragocamelusof various regional origins Cytochrome-b gene based genetic variation plot of Boselaphus tragocamelus of 4.2 48 various regional origins Phylogenetic Neighbor Joining tree (circular) of cytochrome-b gene of Boselaphus 4.3 49 tragocamelus Multidimensional scaling plot of mitochondrial genomic gene, cytochrome-c for 4.4 51 Boselaphus tragocamelus 4.5 Cytochrome-c based genetic variation plot of Boselaphus tragocamelus 52 4.6 Phylogenetic tree (circular) of cytochrome-c gene of Boselaphus tragocamelus 53 Multidimensional scaling plot of mitochondrial genomic d-loop region for 4.7 Boselaphus tragocamelus 55 4.8 D-loop sequence based Genetic variation plot of Boselaphus tragocamelus 56 4.9 Phylogenetic tree (circular) of d-loop region of Boselaphus tragocamelus 57 Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.10 58 cytochrome-c and d-loop region for Boselaphus tragocamelus Cytochrome-b, cytochrome-c and d-loop region based genetic variation plot of 4.11 Boselaphus tragocamelus 59 Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region 4.12 61 of Boselaphus tragocamelus. 4.13 cytochrome-b based multidimensional scaling plot of Antilope cervicapra 63 4.14 Cytochrome-b based genetic variation plot of Antilope cervicapra 63 4.15 Phylogenetic tree (circular) of cytochrome-b gene of Antilope cervicapra. 64 4.16 Multidimensional scaling plot of cytochrome-c gene for Antilope cervicapra 66 4.17 Cytochrome-c based genetic variation plot of Antilope cervicapra 66 4.18 Phylogenetic tree (circular) of cytochrome-c gene of Antilope cervicapra 67 4.19 Multidimensional scaling plot of d-loop region for Antilope cervicapra 69 4.20 d-loop region based genetic variation plot of Antilope cervicapra 69 4.21 Phylogenetic tree (circular) of d-loop region of Antilope cervicapra 70 Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.22 cytochrome-c and d-loop region (collectively) for Antilope cervicapra 71 v Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic 71 4.23 variation plot of Antilope cervicapra Phylogenetic tree (rectangular) of Cytochrome-b, cytochrome-c and d-loop region 4.24 73 (collectively) of Antilope cervicapra 4.25 Multidimensional scaling plot of mitochondrial cytochrome-b gene for Axis axis. 75 4.26 Cytochrome-b based genetic variation plot of Axis axis 75 4.27 Phylogenetic tree (circular) of cytochrome-b region of Axis axis 76 4.28 Multidimensional scaling plot of cytochrome-c gene for Axis axis 77 4.29 Cytochrome-c based genetic variation plot of Axis axis 78 4.30 Phylogenetic tree (circular) of cytochrome-c region of Axis axis 78 4.31 Multidimensional scaling plot of mitochondrial d-loop region for Axis axis 80 4.32 D-loop region based genetic variation plot of Axis axis 80 4.33 Phylogenetic tree (circular) of d-loop region of Axis axis 81 Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.34 82 cytochrome-c and d-loop region (collectively) for Axis axis Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic 4.35 variation plot of Axis axis 82 Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region 4.36 (collectively) of Axis axis 83 4.37 Multidimensional scaling plot of mitochondrial cytochrome-b for Axis porcinus 85 4.38 Cytochrome-b based genetic variation plot of Axis porcinus 85 4.39 Phylogenetic tree (circular) of cytochrome-b region of Axis porcinus 86 4.40 Multidimensional scaling plot for Axis porcinus based on cytochrome-c 88 4.41 Cytochrome-c based genetic variation plot of Axis porcinus 88 4.42 Phylogenetic tree (circular) of cytochrome-c region of Axis porcinus 89 4.43 Multidimensional scaling plot of d-loop region for Axis porcinus 91 4.44 D-loop region based genetic variation plot of Axis porcinus 91 4.45 Phylogenetic tree (circular) of d-loop region of Axis porcinus 92 Multidimensional scaling plot of mitochondrial genomic cytochrome-b, 4.46 93 cytochrome-c and d-loop region for Axis porcinus Cytochrome-b, cytochrome-c and d-loop region (collectively) based genetic 4.47 93 variation plot of Axis porcinus Phylogenetic tree (rectangular) of cytochrome-b, cytochrome-c and d-loop region 4.48 95 (collectively) of Axis porcinus Phylogenetic tree (radiation) of cytochrome-b gene of selected four species 4.49 96 (Boselaphus tragocamelus, Antilope cervicapra, Axis axis and Axis porcinus).
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