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Evaluating DNA Extraction and Analysis of Ancient Seeds: a Study in Peach

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Evaluating DNA Extraction and Analysis of Ancient Seeds: a Study in Peach Lakehead University Knowledge Commons,http://knowledgecommons.lakeheadu.ca Electronic Theses and Dissertations Electronic Theses and Dissertations from 2009 2015 Evaluating DNA Extraction and Analysis of Ancient Seeds: A Study in Peach Joseph, Felicia http://knowledgecommons.lakeheadu.ca/handle/2453/804 Downloaded from Lakehead University, KnowledgeCommons Evaluating DNA Extraction and Analysis of Ancient Seeds: A Study in Peach A thesis presented to The Faculty of Graduate Studies of Lakehead University by Felicia Joseph In partial fulfillment of requirements for the degree of Master of Science in Biology July 2015 © Felicia Joseph, 2015 Declaration: I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person, except where due acknowledgment has been made in the text. Felicia Joseph II Abstract The peach (Prunus persica) is one of the most widely grown stone fruit in the world but its origin and domestication is still being debated. Genetic research has focused on identifying desirable genes for agricultural purposes, suitable genetic targets for species identification, the development of a genetic linkage map for the peach, and ultimately the publication of the whole genome by the International Peach Genome Initiative. However, there has been no genetic research applied to ancient peach stones. In the present study DNA was extracted from modern peach samples collected from the Zhejiang Province in China to assess the most effective methods to extract DNA from seeds. These methods were then applied to an archaeological peach stone collected from the Maoshan archaeological site in China dated to the Liangzhu Culture Period. A search for previously sequenced genetic targets from P. persica and its wild species P. ferganensis, P. mira, P. kansuensis, and P. davidiana was performed to search for suitable genes for genetic and phylogenetic analysis. Phylogenetic analysis was used to evaluate the genetic targets for species identification. A Type II chlorophyll a/b-binding protein gene (Lhcb2) was identified as an ideal marker for species identification for ancient peach specimens due to its small amplicon size and ability to amplify DNA from low quantities. Chloroplast DNA and microsatellite markers were assessed for their ability to distinguish between peach and its various wild species. Several chloroplast genes successfully amplified DNA in the modern peach samples (rbcL, psbM-trnD, clpP), but were not successful when applied to ancient samples. GC-MS analysis was performed on the ancient peach stones to determine whether miscoding lesions were present in the DNA sequence which may have led to amplification failure. Damage due to oxidation and hydrolysis are consistent with the location in which the stone was collected and suggest the possibility of exposure to water and burning. Both sources of damage may explain the lack of amplification of these archeological samples. III Lay Summary "Faculty and students in the Department of Biology are bound together by a common interest in explaining the diversity of life, the fit between form and function, and the distribution and abundance of organisms." The peach is one of the most widely grown fruit in the world, and it is of great importance agriculturally, economically, and socially. Specifics’ regarding where the domesticated peach originated and how it came to become part of everyday life is still being debated. An origin in China has been well documented but no DNA analysis has been performed on an archeological sample from this area. In this study we attempted to analyse DNA from two modern peach samples from the Zhejiang Province in China and one ancient peach sample collected from the Maoshan site in China. The purpose of this study was to assess the success of different DNA extraction and analysis methods and to attempt to provide an insight into the origins of the peach and its relationship to the fruit we see today. IV Acknowledgements Firstly, thank you to Dr. Carney Matheson for giving me the opportunity to continue my studies of ancient DNA at Lakehead University. I appreciate the guidance that you have given to me and encouraging me to never give up. Thank you to my committee, Dr. Kam Leung and Dr. Lada Malek , and to my external reader for your support and evaluation throughout my studies. Thank you to my friends and colleagues of CB3037 both past and present; especially Karen, Margaret, Christy, Heidi, and Jessie for all your support. The long days and nights were much more enjoyable with your presence. I cannot thank you enough for your words of encouragement and many laughs. To the staff of the Paleo DNA Laboratory, Steven and Renee Fratpietro and Karen Maa, you were an incredible sounding board and never stopped pushing me to continue. The opportunity and experience you provided me with contributed in my success. Mum, dad, and Sabrina; it has been a long road and I could not have made it through this without your love and support coming all the way from Hamilton and Ottawa. Sarah, Paulina, Paula and Erin, thank you for the long talks every visit home. To Nichola, you are simply the best, always the voice of reason and reminding me of my strength to keep going. Lastly, to all my Thunder Bay family, Sarah, Karen, Byron, and Steph, thank you for always pushing me to keep moving forward and being there for me through my endeavors. V Table of Contents Abstract ............................................................................................................................. III Lay Summary .................................................................................................................... IV Acknowledgements ............................................................................................................ V Table of Contents .............................................................................................................. VI List of Tables ................................................................................................................. VIII List of Figures ................................................................................................................... IX List of Abbreviations ........................................................................................................ XI 1.0 Introduction ............................................................................................................. 1 1.1 History and Structure of DNA .................................................................................. 1 1.2 Ancient DNA ............................................................................................................ 3 1.3 DNA Damage............................................................................................................ 4 1.3.1 Hydrolytic Damage ............................................................................................ 5 1.3.2 Oxidative Damage ............................................................................................. 6 1.4 DNA Preservation ..................................................................................................... 7 1.5 Peach Background and Domestication ..................................................................... 8 1.6 Genetic Studies of Peach ........................................................................................ 12 1.6.1 Characterisation of the Type II Chlorophyll a/b-binding Protein Gene (Lhcb2*Pp1) in Peach ............................................................................................... 21 2.0 Methodological Background ....................................................................................... 23 2.1 DNA Extraction ...................................................................................................... 23 2.2 DNA Purification .................................................................................................... 24 2.2.1 Silica Bead Purification ................................................................................... 24 2.2.2 Micro Bio Spin Column with Bio-Gel P30...................................................... 25 2.3 The Polymerase Chain Reaction ............................................................................. 26 2.4 Sequencing .............................................................................................................. 27 2.5 Phylogenetic Analysis of Sequences ...................................................................... 29 2.6 Gas Chromatography Mass Spectrometry to Detect DNA Damage ....................... 30 3.0 Methods and Procedures ............................................................................................. 32 3.1 Analysis of Genes Sequenced in Prunus persica and its Wild Species .................. 32 3.2 Sample Collection and Preparation ......................................................................... 32 3.2.1 Preparation of Modern Samples....................................................................... 35 3.2.2 Preparation of Ancient Samples....................................................................... 36 3.3 DNA Analysis ......................................................................................................... 37 3.3.1 DNA Extraction ............................................................................................... 37 3.3.2 Purification
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