PHYLOGENETIC RELATIONSHIPS IN AUSTRALIAN TURF GRASS CULTIVARS OF THE GENERA STENOTAPHRUM and CYNODON A thesis submitted for the degree of Master of Science by Moon Sun Kim School of Biotechnology and Biomolecular Science University of New South Wales February, 2005 TABLE OF CONTENTS TABLE OF CONTENTS ACKNOWLEDGEMENTS i ABBREVIATION ii SUMMARY iii CHAPTER ONE: INTRODUCTION 1.1. Turf grass 1 1.1.1 The turf grass family Poeceae 2 1.1.1.1 Stenotaphrum specie 5 1.1.1.1.1 Stenotaphrum secundatum (Walt) Kuntze 6 1.1.1.1.2 Buffalo grass varieties and cultivars 9 1.1.1.2 Cynodon species 10 1.1.1.2.1 Cynodon dactyoln (L.) Pers. 11 1.1.1.2.2 Cynodon dactylon var. dactylon 13 1.1.1.2.3 Cynodon transvaalensis (Burtt-Davy) 13 1.1.1.2.4 Cynodon variants and cultivars 14 1.2. Taxonomic analysis in plant 18 1.2.1 Plant identification and Plant Breeder Rights 18 1.2.1.1 DNA profiling and PBR in Australia 19 1.2.2 Methodologies for plant taxonomic analysis 20 1.2.2.1 Morphological characteristics 20 1.2.2.2 Biochemical characteristics 20 1.2.2.3 Molecular characteristics 21 1.2.2.3.1 Protein-based markers 21 1.2.2.3.2 DNA-based markers 22 1.2.2.3.3 Polymerase Chain Reaction (PCR) 23 1.2.2.3.4 PCR-based genetic markers 25 1.2.2.3.5 Simple sequence repeat (SSR) and inter simple 27 sequence repeat (ISSR) molecular markers 1.2.3 Molecular phylogenic study of turf grass 31 TABLE OF CONTENTS 1.3. Genetic data Analysis 34 1.3.1 Types of data 34 1.3.2 Data Analysis 35 1.3.2.1 Distance matrix method 35 1.3.2.2 Maximum parsimony and Maximum likelihood methods 36 1.3.2.3 Steps in the data analysis based on Distance matrix 37 1.3.2.4 Evaluation of the test 38 1.4. Project objectives 39 CHAPTER TWO: MATERIALS AND METHODS 2.1. Materials 40 2.1.1 Plant materials 40 2.1.2 Primers 40 2.1.3 PCR kit 41 2.1.4 Enzyme and markers 41 2.1.5 General reagents 41 2.2. Methods 42 2.2.1 Extraction of plant genomic DNA 42 2.2.2 Quantitation of genomic DNA 43 2.2.2.1 Dye Binding Fluorescence 43 2.2.2.2 UV absorption 43 2.2.2.3 Electrophoresis for quantitation of genomic DNA 43 2.2.3 Polymerase Chain Reaction (PCR) 44 2.2.3.1 Optimisation of PCR conditions 44 2.2.3.2 ISSR PCR amplification 44 2.2.4 Electrophoresis and visualisation of gels 45 2.2.4.1 Agarose gel electrophoresis and ethidium bromide staining 45 2.2.4.2 Non-denaturing PAGE and silver staining 45 2.2.4.3 Fluorescent labeling detection 46 2.2.5 Statistical Analysis 47 2.2.5.1 Data scoring and Distance matrix 47 2.2.5.2 Clustering analysis and construction of phylogenetic trees 47 TABLE OF CONTENTS CHAPTER THREE: RESULTS AND DISCUSSION 3.1. Optimization of ISSR-PCR amplification 48 3.2. Preliminary screening 48 3.2.1 Template 48 3.2.1.1 Time of storage of tissue 49 3.2.1.2 RNA inhibition 51 3.2.1.3 Amount of DNA 52 3.2.2 Concentration of ISSR primer 53 3.2.3 Concentration of Taq Polymerase 54 3.2.4 Concentration of MgCl2 55 3.2.5 Number of thermal cycles 57 3.2.6 Annealing temperature 58 3.2.7 Summary of PCR optimal condition 61 3.3. Reproducibility and repeatability in turf grass 62 3.4. Genetic identification of Buffalo grass cultivars using 63 ISSR-PCR analysis 3.4.1 Informative ISSR primers for cultivar identification 63 3.4.2 Polymorphism analysis of Buffalo grass cultivars 66 3.4.2.1 Polymorphism as seen on agarose gel electrophoresis 67 3.4.2.2 Polymorphism as seen on non-denaturing PAGE 70 3.4.2.3 Polymorphism as seen on non-denaturing PAGE with 72 fluorescent labeling detection 3.4.3 Genetic variation of Buffalo grass cultivars 74 3.4.3.1 Specific markers for closely related cultivars 79 3.4.4 Phylogenetic relationship of Buffalo grass cultivars 82 3.4.4.1 Data matrices of Buffalo grass cultivars 82 3.4.4.2 Distance matrices of Buffalo grass cultivars 84 3.4.4.3 Clustering analysis and Phylogenetic construction of Buffalo 86 grass cultivars 3.4.5 Comparison of detection methods 92 TABLE OF CONTENTS 3.5. Genetic identification of Couch grass cultivars using 95 ISSR-PCR analysis 3.5.1 Informative ISSR primers for cultivar identification 95 3.5.2 Polymorphism analysis of Couch grass cultivars 98 3.5.2.1 Polymorphism as seen on agarose gel electrophoresis 98 3.5.2.2 Polymorphism as seen on non-denaturing PAGE 102 3.5.3 Genetic variation of Couch grass cultivars 105 3.5.3.1 Specific markers for closely related cultivars 108 3.5.4 Phylogenetic relationship of Couch grass cultivars 115 3.5.4.1 Data matrices of Couch grass cultivars 115 3.5.4.2 Distance matrices of Couch grass cultivars 120 3.5.4.3 Clustering analysis and Phylogenetic construction of Couch grass 120 cultivars 3.5.5 Comparison of detection methods 127 CHAPTER FOUR: CONCLUSION 4.1. Cultivar identification of turf grass using ISSR-PCR 128 REFERENCES 131 APPENDICES vi ACKNOWELDGEMENTS ACKNOWLEDGEMENTS At first, I would appreciate my supervisor, Dr. Ian McFarlane for experimental recommendation, reviewing of the drafts of my thesis to advance of the thesis’s skill and thanks for encouragement of my study. I am also grateful to Dr. Wendy Glenn for helping of advice in general molecular biology technique and Dr. Alan Wilton for helping of teaching of computer analysis involved with the phylogenetic analysis. I would like to thank Dr. Don Loch for providing cultivars for my study and help with cultivar origins and names. I also thank my laboratory colleagues and Korean friends who are studying in BABS for technical discussions and individual encouragement during the studying. Finally, I thank my parents for their supporting and my husband, Mickey, for helping emotionally to study very hard all the time. i ABBREVIATIONS ABBREVIATIONS The abbreviation and symbols used throughout this thesis follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature. AP-PCR Arbitrary Primed PCR CTAB Hexadecyltrimethylammonuim bromide DAF DNA Amplification Fingerprinting dNTP Deoxynucleotide triphosphate EDTA Ethylendiaminetetra-acetic acid EtBr Ethidium bromide FISSR Fluorescent Inter-Simple Sequence Repeat ISSR Inter Simple Sequence Repeat NJ Neighbour-Joining algorithm PAGE Polyacrylamide gel electrophoresis RAPD Randomly Amplified Polymorphic DNA PAUP Phylogenetic Analysis Using Parsimony PBRs Plant Breeder Rights PCR Polymerase Chain Reaction RFLP Restriction Fragment Length Polymorphism SSR Simple Sequence Repeat TEMED N,N,N',N' - tetramethylenediamine UV Ultra Violet ii SUMMARY SUMMARY Turf grass has coexisted with human beings for a long time and now has become indispensable for human environment. Many people have been eager to have gorgeous and economical place for their purpose. Therefore, many new turf varieties have been created and turf suppliers have selected and created more appropriate grass cultivars. The turf grass industry is now a large world-wide market. There is also a large market in Australia. Many turf grasses that have been developed for Australian circumstances have been used for beautification, recreational facilities and functional facilities. For example, some of the Buffalo grass cultivars (Stenotaphrum species) and Couch grass cultivars (Cynodon species) have been used in many areas and new varieties are still being developed. Moreover, the Australian government has ratified the intellectual property law that offers plant breeder protection in the Plant Breeder Rights Act of 1994. The granting of Plant Breeder Rights (PBRs) is based on being able to establish the Distinctness, Uniformity and Stability of the new variety (Smith, 1997). With developments in the field, cultivar identification and classification of turf grass cultivars is very important. The identification of plants and their variation by using morphological traits has been used for a long time. One obvious reason for this is the ease of observing and recoding external features. However, morphological comparisons may have limitations including subjectivity in the analysis of physical characteristics because of continuous variation. This is particularly so among cultivars with highly similar pedigrees. Therefore, it has been suggested that a more sensitive diagnostic test would benefit the identification of cultivars. iii SUMMARY Inter Simple Sequence Repeats (ISSRs), first developed in 1994, were used to analyse genetic diversity of Buffalo and Couch grass cultivars in this study. ISSRs are semi- arbitrary markers amplified by the Polymerase Chain Reaction (PCR) in the presence of one primer complementary to target microsatellites. Ten Buffalo and 50 Couch grass cultivars were screened using 100 commercial ISSR primers. Eighteen ISSR primers produced reproducible and informative polymorphic bands from the Buffalo grass cultivars using agarose gel electrophoresis with ethidium bromide staining and non-denaturing polyacylamide gel electrophoresis with silver staining to separate bands. In addition a newer detection method termed Fluorescent labeling detection (FISSR-PCR) was tried. Two of the informative primers were labeled with the fluorescent tag 6-FAM and screened against the 10 Buffalo grass cultivars. (AG)n and (AC)n repeat sequences produced the most useful polymorphic bands, from agarose gel electrophoresis and non-denaturing PAGE, to differentiate each Buffalo grass cultivar. Phylogenetic trees were created from the data using the Neighbour- joining algorithm. Interestingly the cultivars separated into two main groups. One contained those cultivars granted PBRs in the United States of America, for example Palmetto.