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Molecular Marker Based Genetic Diversity in Symplocos Spp. from the Two Biodiversity Hotspots in India

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Molecular Marker Based Genetic Diversity in Symplocos Spp. from the Two Biodiversity Hotspots in India MOLECULAR MARKER BASED GENETIC DIVERSITY IN SYMPLOCOS SPP. FROM THE TWO BIODIVERSITY HOTSPOTS IN INDIA THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY BY SOFIA BANU RESEARCH GUIDE Dr. VIDYA S. GUPTA PLANT MOLECULAR BIOLOGY GROUP, DIVISION OF BIOCHEMICAL SCIENCES, NATIONAL CHEMICAL LABORATORY, PUNE – 411008, INDIA OCTOBER 2008 Molecular marker based genetic diversity in Symplocos spp. from the two biodiversity hotspots in India A thesis submitted to the University of Pune for the Degree of DOCTOR OF PHILOSOPHY in Biotechnology SOFIA BANU Plant Molecular Biology Group, Division of Biochemical Sciences, National Chemical Laboratory, Pune – 411008, INDIA OCTOBER 2008 CERTIFICATE This is to certify that the work incorporated in the thesis, “Molecular marker based genetic diversity in Symplocos spp. from the two biodiversity hotspots in India” submitted by Ms. Sofia Banu, for the Degree of Doctor of Philosophy, was carried out by the candidate under my supervision in the Division of Biochemical Sciences, National Chemical Laboratory, Pune - 411 008, India. Such material as has been obtained from other sources has been duly acknowledged in the thesis. Dr. Vidya S. Gupta (Research Supervisor) October 2008 Pune ii DECLARATION I hereby declare that the thesis “Molecular marker based genetic diversity in Symplocos spp. from the two biodiversity hotspots in India” submitted for the degree of Doctor of Philosophy to the University of Pune has not been submitted by me for a degree/diploma to any other University. Sofia Banu October 2008 Pune iii Dedicated to my beloved parents iv INDEX Page No. Title Acknowledgements vi - vii List of Abbreviations viii - ix List of Figures x - xi List of Tables xii - xiv Chapter 1: Introduction and review of literature 1 - 12 Chapter 2: Genetic diversity in S. laurina based on chloroplast 37 - 94 and mitochondrial DNA markers • Section A: Analysis of populations based on 37 - 77 chloroplast and mitochondrial universal markers using PCR- RFLP technique • Section B: Analysis of populations with chloroplast 78 - 94 microsatellite markers Chapter 3: Understanding the distribution of genetic diversity in 95 - 118 S. laurina based on chloroplast genome sequence to elucidate the phylogeographical structure Chapter 4: Assessing the phylogeographic pattern of genetic 119 - 144 structure in S. laurina using nuclear markers Chapter 5: Thesis summary and future directions 145 - 151 Bibliography 152 - 177 Biodata 178 - 180 v ACKNOWLEDGEMENT The journey in the path of research during last five years led me to associate with many people, their views, ideas, traits and criticism all that have influenced and contributed immensely in making of this thesis. At the outset, I wish to express my sincere gratitude to my research guide, Dr. Vidya Gupta, National Chemical Laboratory (NCL), Pune, for accepting me as a student; I am previleged to have worked under her supervision. I am grateful for her constant support, encouragement, teachings, guidance and her godmotherly care during my trying times during the course of this work. Her enthusiastic attitude, ideas and scientific temperament have inspired me profoundly and have silently contributed to my understanding of the subject. Her views have given new diamensions to my opininion and my overall growth as a researcher. The concepts developed in the thesis are outcome of long sessions of pondering, framing- reframing of statements/views and brainstroming stimulating discussions with her. I wholeheartly thank her for everything. My sincere thanks to Dr. Meena Lagu for always advicing me in planning the research work especially the collection tours. Her meticuolous ways have taught me a lot and has always inspired me to perform better. The constant help and encouragement rendered by her during my Ph.D. programme will always remain arched in my mind. I want to express my heartfelt gratitude to Dr. Narendra Kadoo, without whom this thesis would not have had the present form. I especially thank him for his innovativeness in carrying out the sequencing reactions and for rendering all possible help in generating the data. He always inspired me to learn new techniques, especially has taught me a lot about computers and helped me to improve my technical skills. His good wishes and company will always be cherished. I thank Dr. BG Kulkarni, Dr. PSN Rao, Dr. HS Suresh, Dr. Mao, Dr. A Baishya, Dr. PJ Handique for their generous help extended during field trips. I thank all the field personals from various forest offices who have helped me immensely during sample collection, whose names I might not recollect now but I am grateful to them. I would like to thank the Director, National Chemical Laboratory for making available all the facilities for my research work. I also acknowledge financial support from the Council of Scientific and Industrial Research, New Delhi in the form of Research Fellowships and Department of Biotechnology, New Delhi in the form of research grant during the tenure of my work. I am happy to thank sincerely to my collection team members Ram and Rasika, who stood behind all my decisions taken during field tours and had taken all the pains to make this project a success. I thank them for their critical views, stimulating ideas and mostly for supporting me unconditionally all these years. My sincere thanks to Dr. Mohini Sainani, Dr. S. P Joshi and Dr. Ashok Giri for their help and advice. vi I am grateful to Dr. Gauri Apte, Dr. Abhay, Dr. Savita Borse, Dr. Elangovan, Dr. Sirsha and Dr. Bhusan. They have immensely contributed to my intellectual growth during various phases of my research and have provided food for thought, encouraged me to analyze things in all possible ways and mostly inspired me to look beyond what is apparent. I thank Anand, Bornali, Shraeddha, Lalitha, Sanjeeb Roy, Sharmili, Sujoy and Subhash for their constant support and friendship. They always raised my spirits and showed me the brighter side of life and inspired me to look forward. I also wish to thank my seniors and more over my friends Ajay, Suhas, Ashwini, Rupali, Ramya, Neeta, Varsha, Sadhana, Suvarna, Rashmi, Trupti, Gayatri, Rohini, Charu, Gauri, Vaiju, Manasi, Radhika, Sagar, Tejas, Hemangi, Manasi M, Arun, Krishna, Shekhar, Vivek, Pavan, Prabhash, Yashwant, Rebecca, Abhijit, Medha, Pradeep, Manje, Richa, Aditi, Laxmi, Neha, Priya, Pankaj, Hemlata, Ajit, Jagtap ji, Nishant, Suman, Shweta, Poorva, Uma for their friendship and helping hands. Knowingly or unknowlingly all have contributed to my learning and growth, making this journey a memorable one. The conception and progress of this research project has been positively gained due to valuable and critical inputs from many people and I remain forever indepted for their kind suggestions and critical views. I acknowledge all friends and well-wishers, whom I might have left out due to oversight, but whose best wishes have always encouraged me. I would like to mention Carl Sagan, whose quote “There are many hypotheses in science which are wrong. That's perfectly all right; they're the aperture to finding out what's right” has influenced me immensely and kept me going all these years when I was struggling hard to find answers to many of the questions that boggled my mind. Lastly, I would like to mention my parents for whom it is more than a dream and who have been eagerly looking forward to this milestone. I would like to thank my brothers for always asking me when I will finish my Ph. D work. I thank them for being there always, with their endless encouragement, support and most importantly for keeping faith in me. Sofia vii LIST OF ABBREVIATIONS AFLP Amplified fragment length polymorphism AMOVA Analysis of molecular variance BHS Biodiversity hotspots bp base pair BP Before Present BSI Botanical Survey of India CAPS Cleaved amplified polymorphic sequence cpDNA Chloroplast DNA cpSSR Chloroplast simple sequence repeat CTAB Cetyl-trimethyl-ammonium bromide DNA Deoxy ribonucleic acid dNTPs Deoxy ribonucleotide triphosphate EDTA Ethylene diamine tetra acetic acid EG Eastern Ghats ESU Evolutionary significant unit g gram h hour ha hectar IAA Iso-amyl alcohol ISSR Inter simple sequence repeat kb kilobase pair kg kilogram km2 square kilometer LB Luria Bertonii LGM Last glacial maxima m meter M molar mg milligram min minute mL milliliter mm millimeter viii mM millimolar MoEF Ministry of Environment and Forest msl mean sea level MSN Minimum spanning network MST Minimum spanning tree mtDNA Mitochondrial DNA mya million years ago nDNA nuclear DNA NEI Northeast India ng nanogram °C Degree celsius PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction PEC Primer enzyme combination RAPD Randomly amplified polymorphic DNA RFLP Restriction fragment length polymorphism rpm revolution per minute s second SI South India SSD Sum of squared deviation TAE Tris-acetate-EDTA TBE Tris-borate-EDTA TE Tris-EDTA WG Western Ghats μg microgram μL microlitre μM micromolar ix LIST OF TABLES Chapter 1 Table 1.1 Comparison between the number of species in India and the world Table 1.2 The eight terrestrial biogeographic realms recognized all over the world Table 1.3 Mutation rates in plant nuclear and organellar genomes Chapter 2 - Section A Table 2.A.1 Sampling locations of 12 populations of S. laurina in India Table 2.A.2 List of chloroplast DNA and mitochondrial DNA PECs used for analysis of S. laurina population Table 2.A.3 Distribution of PCR-RFLP chlorotypes in 12 populations of S. laurina Table 2.A.4 Distribution of PCR-RFLP mitotypes in 12 populations of S. laurina Table 2.A.5 Genetic diversity indices for 12 populations of S. laurina in India Table 2.A.6 Genetic diversity indices for S. laurina for different geographical regions Table 2.A.7 Analysis of molecular variance (AMOVA) for the whole range of S.
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