DNA FINGERPRINTING. PlflRfMDIM. Kurt Weising Hilde Nybom Kirsten Wolff Wieland Meyer. CRC Press Boca Raton Ann Arbor London Tokyo

Transcription

1 V) DNA FINGERPRINTING in PlflRfMDIM Kurt Weising Hilde Nybom Kirsten Wolff Wieland Meyer CRC Press Boca Raton Ann Arbor London Tokyo

2 TABLE OF CONTENTS Chapter 1 Introduction 1 Chapter 2 Genetic Variation at the DNA Level 3 I. A Short Survey of Molecular Markers 3 A. Properties of Molecular Markers 3 B. Protein Markers 3 C. DNA Sequencing 4 D. Restriction Fragment Length Polymorphisms (RFLPs) 5 1. RFLPs in Nuclear DNA 5 2. RFLPs in Cytoplasmic DNA: cpdna and mtdna 7 E. DNA Fingerprinting Based on Hybridization 8 F. Molecular Markers Based on DNA Amplification 9 G. Miscellaneous Markers 10 II. Minisatellites and Simple Sequences: The Molecular Basis of Classical DNA Fingerprinting 10 A. The Nomenclature of Tandem-Repeated DNA Elements 10 B. DNA Fingerprinting with Tandem-Repetitive Probes Human Minisatellites Other Minisatellites Simple Sequence Probes Factors Affecting the Level of DNA Fingerprint Polymorphism 17 C. Chromosomal Localization of Minisatellites and Simple Sequences 17 D. Mechanisms Responsible for Minisatellite and Simple Sequence Polymorphism ; 20 E. Functional Significance of Minisatellites and Simple Sequences Telomeres Centromeres Transcription Transcriptional Regulation ; Recombination Replication General Considerations 23 III. Detection of DNA Polymorphism by PCR-Based Fingerprinting 24 A. Principle of the PCR..24 B. PCR with Minisatellite and Simple Sequence Primers 27 C. PCR with Arbitrary Primers 28

3 Chapter 3 Laboratory Equipment 37 Chapter 4 Methodology 43 I. Safety Precautions 43 II. DNA Isolation and Purification 44 A. Isolation of Plant DNA Collection and Preservation of Plant Tissue Isolation Protocols 51 a. CTAB Procedure 51 b. DNA Minipreparation 55 c. Potassium Acetate Procedure 55 d. DNA Preparation via Nuclei DNA Purification via CsCl Density Gradient Centrifugation 59 B. Isolation of Fungal DNA Isolation and Cultivation of Fungi from Plant Material, Soil, and Airborne Spores Isolation and Cultivation of Human and Animal Pathogenic Fungi Long-Term Maintenance of Fungal Cultures 63 a. Storage in Sterile Water 63 b. Storage in Culture Tubes (Slants) 63 c. Storage as Frozen Glycerol Stocks Induction of Sporulation in Fungal Cultures Large-Scale Accumulation of Fungal Material for DNA Isolation. 65 a. Culturing of Filamentous Fungi in Liquid Media. 66 b. Culturing of Yeast-Like Fungi in Liquid Media..; DNA Isolation Protocols 68 a. SDS Procedures 69 b. TNS/PAS Procedure,...72 c. DNA Preparation via Protoplasts 73 C. Quantitation of DNA Ethidium Bromide Staining Spectrophotometry 76 III. DNA Fingerprinting Based on Hybridization 77 A. Restriction of DNA Choice of the Enzyme Reaction Components and Conditions Setting Up Reactions 79 B. Agarose Gel Electrophoresis 79 C. Gel Drying 82

4 D. Southern Transfer 84 E. Generation of Probes Commercially Available Probes Chemical Synthesis of Oligonucleotides Isolation of Fingerprint Probes from Genomic Libraries Isolation and Processing of the M13 Probe 89 a. Preparation of Competent Cells 89 b. Transfection of Competent Cells with Ml3 Phage 90 c. Amplification of M13 Phage 91 d. M13 DNA Isolation 91 e. Isolation of a 780-bp M13 Fragment 92 F. Labeling of Probes End Labeling Nick Translation Random Priming Removal of Unincorporated dntps 98 a. Chromatography on Sephadex G b. Spin-Column Technique Nonradioactive Labeling Procedures 100 G. Hybridization Hybridization with Oligonucleotide Probes 101 a. Gel Hybridization with Radioactive Probes 101 b. Blot Hybridization with Nonradioactive Probes Hybridization with M13 and Other Minisatellite Probes 103 a. Standard Protocol 103 b. Two-Step Protocol Stripping of Hybridized Gels and Membranes 105 H. Signal Detection Autoradiography Phosphorimaging Nonradioactive Assays 108 IV. PCR-Based DNA Fingerprinting 112 A. PCR with Arbitrary Primers Standard RAPD Protocol Influence of Reaction Conditions and Components 114 a. Primers 114 b. Polymerase 115 c. Thermocycler 115 d. Temperature Profile 116 e. Template Concentration 117 f. Mg 2+ Concentration 118 g. Reproducibility and Quality of Banding Patterns 118 h. Modifications RAPD Fragments Used as Probes 121

5 4. Standard DAF Protocol Standard AP-PCR Protocol 125 B. PCR with Simple Sequence and Minisatellite Primers 126 C. Separation and Detection of PCR Products, Separation Procedures 129 a. Agarose Gel Electrophoresis 129 b. Polyacrylamide Gel Electrophoresis Detection Procedures 133 a. Ethidium Bromide Staining 133 b. Silver Staining 135 V. Evaluation of Fragment Patterns 136 A. Fragment Sizing and Matching 136 B. Statistical Procedures Introduction Band Sharing, Similarity Index, and Genetic Distance Population Genetic Analyses 143 a. Calculation of Mutation Rate 143 b. Population Size 143 c. Heterozygosity 144 d. Inbreeding Coefficient 144 e. Genetic Diversity and Population Subdivision 144 f. Paternity Testing and Estimation of Relatedness 146 g. Outcrossing Rates Phenetic and Phylogenetic Analysis 148 a. Ordination Techniques 149 b. Construction of Dendrograms 149 c. Assumptions and Limitations Linkage Analysis and Genetic Mapping 152 Chapter 5 Applications of DNA Fingerprinting in Plants and Fungi..157 I. Development of Methods and Initial Investigations 157 A. Minisatellite DNA Probes Detect Genetic Variation 157 B. Oligonucleotide Probes are Introduced 158 C. PCR-Based Methods Prompt Increased Research Activity ;. 158 II. Wild Plant Species 159 A. DNA Fingerprints Detect Genetic Diversity and Relatedness in Plant Populations Cross-Pollination vs. Selfing: The Influence of the Breeding System on Diversity Vegetative Propagation, Sexuality, and Apomixis: The Influence of the Reproductive System on Diversity Rare Species 165

6 4. Gymnosperms General Considerations 166 B. DNA Fingerprints Yield Insights into Plant Taxonomy Apomictic Microspecies Sexual Species Other Plant Groups General Considerations 170 III. Cultivated Plant Species 171 A. Cultivar Identification and Estimation of Genetic Relatedness Vegetatively Propagated Cultivars Seed-Propagated Cultivars Vegetative Sports and Other Mutants In ViYro-Propagated Plant Material and Somaclonal Variation General Considerations 186 B. Paternity Testing and the Identification of Hybrids Seed-Propagated Plants and Sexual Hybrids Somatic Hybrids General Considerations 191 IV. Inheritance of DNA Fingerprints, Linkage Analysis, and Genome Mapping in Plants 191 A. Minisatellite and Simple Sequence Hybridization 192 B. PCR-Based Methods 192 C. General Considerations 200 V. Fungi 201 A. Human Pathogenic Fungi: Diagnostics and Epidemiology Candida Cryptococcus Sporothrix.., Pneumocystis Aspergillus Histoplasma 208 B. Plant Pathogenic Fungi: Population Genetics, Epidemiology, and Pathotype Identification Ascochyta Colletotrichum Erysiphe Fusarium Leptosphaeria Magnaporthe Ophiostoma Phytophthora Sclerotinia 217

7 C. Insect-Pathogenic Fungi 218 D. Nonpathogenic Fungi 218 E. Commercially Important Fungi Yeasts Mushrooms 220 F. Taxonomy and Phylogeny 222 G. Inheritance of DNA Fingerprints, Linkage Analysis, and Genetic Mapping 224 H. General Considerations 225 VI. Conclusions 226 Chapter 6 Comparison of Methods for Detecting Genetic Variation 229 I. Morphological Characters and Allozymes vs. DNA Markers 229 II. Different Kinds of Molecular Markers 230 A. Discriminatory Power and Consistency 230 B. Costs 232 C. Conclusions 233 Chapter 7 Future Prospects 235 I. Improvement of Existing Methods 235 II. Extending the Spectrum of DNA Typing Strategies 237 A. Polymorphic Single-Locus Hybridization Probes 238 B. PCR-Amplified Microsatellites 239 C. Sequence Characterized Amplified Regions (SCARs) 241 D. Miscellaneous Techniques 242 E. Outlook 244 References....:..245 Appendices 287 Index 315