Microsatellite-based phylogenetic tree analysis of Australian Prunus fruit varieties Cheryl Chan (MScForSci) Centre for Forensic Science University of Western Australia This thesis is presented in partial fulfilment of the requirements for the Master of Forensic Science 2010 I declare that the research presented in this thesis, for the Master of Forensic Science at the University of Western Australia, is my own work. The results of the work have not been submitted for assessment, in full or part, within any other tertiary institute, except where due acknowledgement has been made in the text. Cheryl Chan i ACKNOWLEDGEMENTS First, I would like to thank my supervisors A/P Guan Tay and Prof. Ian Dadour. Guan for providing guidance, support and knowledge for the duration of this project. Ian for providing the facilities and support for the completion of this project. To the members of the lab who provided their friendship, support and reviews of my written work. Stephen Iaschi who helped me on a daily basis by dispensing much of his knowledge relating to DNA based research and statistic analysis. Catherine Rinaldi and Haifa Khoory for their knowledge in DNA based research. The other members of Guan’s research group Aishah Kadher, Habiba Al Safar, and James Meagher who provided knowledge, support and friendship. I would like to thank Alexandra Knight and the other members of staff at the Centre for Forensic Science who keep the Masters course running smoothly. Special thanks to my parents and Jonathan, who may be miles away but I would not have made it to the end of this project without their constant support, understanding, and love. Last but not least, He who made everything possible. ii PREFACE The main objective of the research undertaken here was to make use of DNA and chemical profiling to differentiate a newly patent premium hybrid of stone fruit, Nadia™ (Prunus salicina), from the rest of the Prunus family members. This thesis contains 3 chapters. The first chapter reviews the current literature associated with fraudulence in the food and beverage industry and its forensic significance. It also introduces some common forensic DNA markers and their applications, including microsatellite analysis. Chapter 2 describes the DNA profiling of Nadia™ and a range of different Prunus varieties based on 3 microsatellite markers. PCR and Gene Scan Analysis (ABI) was based on using genetic distances of the combined microsatellites for differentiation. The phylogenetic tree that was constructed effectively showed a distinction between Nadia and the rest of the 13 other Prunus varieties tested. Chapter 3 describes the chemical assessment of Nadia™ and other closely related stone fruits. It is the first attempt to report the chemical assessment of Nadia™, revealing its premium quality in terms of physical appearance, desirable post-harvest traits and high nutritional values. The chapter also attempts to introduce β-carotene as one of the potential chemical profiles used to effectively differentiate Nadia™ from the rest of the stone fruits. Chapter 2 is presented as a manuscript submitted to Journal of Agricultural and Food Chemistry, and is written in the format required by the journal. The entire thesis is tied together with the obligatory abstract and bibliography section. iii TABLE OF CONTENTS TABLE OF CONTENTS……………………….………………………………….……………....iv ABSTRACT………………………………………………..………………….……...ix CHAPTER 1 .................................................................................................................... 1 LITERATURE REVIEW .............................................................................................. 1 1.1 FRAUDS IN FOOD AND BEVERAGE INDUSTRY ............................................... 2 1.2 PROTECTING NEW VARIETIES WITH PLANT BREEDER’S RIGHTS (PBR) UNDER THE UPOV 1991 ACT .................................................................................. 4 1.3 METHODS OF APPLYING FOR PLANT PATENT UNDER THE RULES AND REGULATION OF IP AUSTRALIA ......................................................................... 9 1.4 CASE STUDY ON DICKSONIA ANTARTICA ....................................................... 10 1.5 THE PRUNUS FAMILY AND THE MOTIVATION BEHIND BREEDING NEW VARIETIES ................................................................................................................ 11 1.6 MARKET OF PRUNUS SPECIES IN AUSTRALIA/WORLDWIDE ................. 12 1.7 CASE STUDY: PINK LADY™ APPLES AND ITS SUCCESS ........................... 17 1.8 METHODS OF TESTING AUTHENTICITY OF FRUITS .................................. 18 1.8.1 TAXONOMIC CLASSIFICATION ..................................................................... 19 1.8.2 CHEMICAL TESTING ......................................................................................... 20 1.8.3 BIOLOGICAL TESTING ..................................................................................... 22 1.9 DNA IN FORENSIC ANALYSIS ............................................................................. 22 1.10 WHAT IS DNA? ......................................................................................................... 24 1.11 FORENSIC GENETICS ............................................................................................ 26 1.12 POLYMERASE CHAIN REACTION (PCR) IN FORENSIC ANALYSIS ......... 29 1.13 FORENSIC DNA MARKERS FOR PLANT GENOME ANALYSIS .................. 31 1.13.1 RESTRICTION FRAGMENT LENGTH POLYMOPHISM (RFLP) .............. 33 1.13.2 RANDOMLY- AMPLIFIED POLYMORPHIC DNA MARKERS (RAPD) .... 33 1.13.3 AMPLIFIED FRAGMENT LENGTH POLYMORPHSIM (AFLP) ................ 34 1.13.4 MICROSATELLITES ........................................................................................... 34 1.14 CURRENT PLANT/ FRUIT DATABASES ............................................................ 35 1.15 AIMS OF STUDY ...................................................................................................... 38 CHAPTER 2 (PART 1) ................................................................................................ 41 MATERIALS AND METHODS ................................................................................. 41 2.1.1 LEAF SAMPLE COLLECTION .............................................................................. 42 2.1.2 DNA EXTRACTION FROM PRUNUS LEAVES .................................................. 43 2.1.3 DNA PURIFICATION ............................................................................................... 43 2.1.4 GENOMIC PCR AMPLIFICATION OF MICROSATELLITE MARKERS ..... 44 2.1.5 VISUALISATION OF PCR PRODUCTS BY GEL ELECTROPHORESIS ....... 45 2.1.6 DETERMINATION OF MICROSATELLITE ALLELE SIZE BY GENE SCAN ............................................................................................................... 45 2.1.7 PHYLOGENETIC TREE ANALYSIS .................................................................... 45 CHAPTER 2 (PART 2) ................................................................................................ 47 RESULTS AND DISCUSSION ................................................................................... 47 2.2.1 AMPLIFICATION OF MICROSATELLITE REGIONS BY PCR ..................... 48 2.2.2 POLYMORPHISM IN DNA BAND PROFILES .................................................... 54 iv 2.2.3 GENE SCANNING .................................................................................................... 56 2.2.4 POLYMORPHISM INFORMATION CONTENT (PIC) ...................................... 64 2.2.5 PHYLOGENETIC TREE ANALYSIS .................................................................... 65 2.2.6 CONCLUSION ........................................................................................................... 69 CHAPTER 3 .................................................................................................................. 71 Abstract .............................................................................................................................. 74 Introduction ............................................................................................................................. 75 Results .............................................................................................................................. 82 Discussion and Conclusion ..................................................................................................... 84 Safety .............................................................................................................................. 87 Acknowledgement ................................................................................................................... 87 Literature Cited ....................................................................................................................... 87 BIBLIOGRAPHY ......................................................................................................... 98 v LIST OF FIGURES Figure 1: Development of Plant Variety Protection from 1973 to 2003 showing the increasing number of titles being enforced each year since 1981. (Adapted from: Jordens, 2005) ...................................................................................... 7 Figure 2: Production of various stonefruits in millions of megatonnes (MT) by the respective top 6 producing countries from year 2000 to 2007 showing increasing trend in the total number of the respective Prunus fruit production. (Data obtained from FAOSTAT, 2007)