Population Genetic Analysis of Red Foxes (Vulpes vulpes ) in Hedmark Country, Norway - A Pilot Study Aarathi Manivannan Master Degree in Applied & Commercial Biotechnology HEDMARK UNIVERSITY COLLEGE Department of Natural Sciences and Technology 2013 Acknowledgement Coming to the end of my journey through the master program in Applied and Commercial Biotechnology, I would like to express my gratitude to all those who have encouraged me through it. A special thanks to my parents, S. Manivannan and M. Revathi who trusted me and gave me this opportunity to study in a privileged university, Hedmark University College. Thank you Appa for giving me a priceless gift “my studies” no matter whatever obstacles I came across. Thank you Amma for taking care of me no matter the distance between us. This achievement could not be possible without the guidance of my supervisor Robert C. Wilson. I am grateful for your never-ending support, excellent and enthusiastic supervision during this project. I would also like to thank my Associate Professor, Arne Linløkken for guiding me through the statistical part of my project. I dedicate this thesis to my fiancé, Aravindh Suryamoorthy. Thank you for your support, assistance and motivation in completing this thesis. Love you! Last but not the least, special thanks to all my friends in Norway and India who proved that “wherever you are, friends will never let you down”. Thank you for being with me at every possible situation! Hamar, May 31 st 2013 Aarathi Manivannan 2 Table of Contents Abstract ...................................................................................................................................... 5 1. Introduction ........................................................................................................................ 6 1.1. Fox ................................................................................................................................. 6 1.1.1. Physical characteristics of Foxes ...................................................................................................... 6 1.1.2. Distribution of Foxes ........................................................................................................................ 7 1.1.3. Diet of the Foxes .............................................................................................................................. 7 1.2. Vulpes Species ................................................................................................................ 7 1.2.1. Red Fox and its Distribution ............................................................................................................. 8 1.2.2. Physical Characteristics of Red Foxes ............................................................................................... 9 1.2.3. Diet of Red foxes ............................................................................................................................ 10 1.2.4. Reproduction ................................................................................................................................. 10 1.2.5. Predation ........................................................................................................................................ 11 1.3. DNA markers and Genotyping ....................................................................................... 12 1.3.1. Application of Microsatellite markers ............................................................................................ 15 1.3.2. Sex Determination ......................................................................................................................... 16 1.4. Advantages of microsatellites ....................................................................................... 19 1.5. Microsatellite marker/ primer design ............................................................................ 19 1.6. Common techniques used for genotyping...................................................................... 20 1.6.1. Applications of Multiplex PCR ........................................................................................................ 22 1.7. PCR Inhibitors ............................................................................................................... 22 1.7.1. Methods to overcome Inhibition ................................................................................................... 22 1.8. 16-capillary 3130 xl Genetic Analyzer (Applied Biosystems) ............................................ 23 2. Aim of Study ..................................................................................................................... 26 3. Materials and Methods .................................................................................................... 27 3.1. Scheme of the study ..................................................................................................... 27 3.2. Extraction of DNA ......................................................................................................... 27 3.2.1. From tissue ..................................................................................................................................... 27 3.2.2. From Hair ....................................................................................................................................... 28 3.2.3. From Scat ....................................................................................................................................... 28 3.3. Multiplex Primer Design ............................................................................................... 29 3.4. Multiplex PCR ............................................................................................................... 30 3.5. Agarose Gel Electrophoresis .......................................................................................... 30 3.6. Sequencing of PCR Amplicons ....................................................................................... 30 3.7. Genotyping .................................................................................................................. 31 3.8. Bioinformatics Analysis ................................................................................................. 32 4. Results............................................................................................................................... 33 4.1. Isolation of DNA ........................................................................................................... 33 4.2. Development of new primers for the established markers ............................................. 34 4.2.1. Marker choice ................................................................................................................................ 34 4.2.2. Primer design ................................................................................................................................. 34 4.2.3. Primer Testing ................................................................................................................................ 36 4.2.4. Multiplexes ..................................................................................................................................... 38 4.3. Genotyping .................................................................................................................. 40 4.3.1. Sex Differentiation ......................................................................................................................... 45 4.4. Sequencing Analysis ..................................................................................................... 47 4.5. Bioinformatics Analysis ................................................................................................. 49 4.5.1. LOSITAN Analysis ............................................................................................................................ 50 4.5.2. MICRO-CHECKER Analysis .............................................................................................................. 51 4.5.3. Population Diversity Analysis on the genotype data ..................................................................... 52 4.6. Relationship Analysis .................................................................................................... 62 5. Discussion......................................................................................................................... 66 5.1. Isolation of DNA ........................................................................................................... 66 5.2. Microsatellite Primers .................................................................................................. 66 5.3. Genotype Analysis ........................................................................................................ 67 5.4. Sex Differentiation ....................................................................................................... 68 5.5. Sequencing Analysis ..................................................................................................... 68 5.6. Population Genetics ..................................................................................................... 69 5.6.1. Selection of Markers ...................................................................................................................... 69 5.6.2. Identification of Genotyping