Wildlife DNA Analysis

Adrian M. T. Linacre, Flinders University, Adelaide, Australia Shanan S. Tobe, University of Strathclyde, Glasgow, UK

Foreword ix Preface xi

About the Authors xiii

Acknowledgements xv

1 Introduction 1

1.1 Importance of wildlife forensic science investigations 1

1.2 Role of forensic science in wildlife crimes 3

1.3 Legislation covering wildlife crime 4

1.4 Role of non-human DNA in forensic science 8

1.5 Development of wildlife DNA testing 9

1.5.1 History and current state of wildlife DNA forensic science 10

1.5.2 Wildlife forensic science testing 11

1.5.3 Performing DNA typing in wildlife investigations 13

1.6 Accreditation and certification 14

1.7 Standardisation and validation 20

1.8 Collection of evidential material, continuity of evidence and transportation to the laboratory 24

1.9 Note taking and maintenance of a casefile 29

1.10 Case assessment and initial testing 30

1.11 Scope of book 32

Useful websites 32

References 33

2 DNA, Genomes and Genetic Variation 37

2.1 Introduction 37

2.2 The DNA molecule 37

2.3 Chromosomes and nuclear DNA 39

2.4 Genomes 41

2.4.1 Nuclear DNA 41

2.4.2 Mitochondrial and chloroplast DNA 44

2.5 DNA mutation and genetic variation 47

2.5.1 Genetic variation of repetitive DNA 48

2.5.2 Single base changes leading to genetic variation 48

2.5.3 Genetic loci used in species testing 50

2.6 DNA polymorphisms leading to speciation 53

2.6.1 Genetic isolation 54

2.6.2 Other processes leading to speciation 56

2.7 What is a species? 56

2.7.1 Subspecies 60

2.7.2 Genus to Kingdom 61

2.8 Summary 63

References 64

3 Methods in Wildlife Forensic DNA Analysis 69

3.1 Introduction 69

3.2 Protein polymorphisms 69

3.3 DNA isolation, purification and concentration 70

3.3.1 Generic aspects of DNA isolation 70

3.3.2 Lysis step 71

3.3.3 DNA purification: silica-based extraction 72

3.3.4 DNA purification: Chelex R  100 resin 73

3.3.5 DNA purification: organic extraction 74

3.3.6 Microconcentration 76

3.4 DNA quantification 76

3.5 Restriction fragment length polymorphisms (RFLP) 78

3.6 Methods based on the polymerase chain reaction 81

3.6.1 Factors affecting PCR efficiency and optimisation of PCR 84

3.6.2 PCR-based methods of DNA quantification 88

3.6.3 Random amplification of polymorphic DNA 91

3.6.4 Amplification of fragment length polymorphisms (AFLP) 93

3.7 PCR set-up 95

3.8 PCR clean-up 98

3.9 DNA sequencing 99

3.10 SNP typing 100

3.11 New generation of DNA sequence methods 102

Suggested reading 104

4 Species Testing 105

4.1 Introduction 105

4.2 Species 106

4.2.1 Genetic variation and correspondence with taxonomy 106

4.3 Attributes of a species testing locus 106

4.4 Application of a locus to a species 110

4.5 Tests available and how they are performed 110

4.5.1 Sequencing 111

4.5.2 Species-specific primers 124

4.6 Developing a species test 127

4.6.1 Use of data on GenBank and sequence alignment 128

4.6.2 Designing primers 135

4.6.3 Validation 156

4.7 Interpretation and reporting of results 159

4.7.1 Interpretation and reporting sequencing results 160

4.7.2 Interpretation and reporting species-specific testing results 169

4.8 Other limitations: hybrids and wild/captive bred 171

4.9 Future methodologies 173

References 173

5 Genetic Linkage 177

5.1 Introduction 177

5.2 Whole genome testing 177

5.3 Types of individualisation testing 178

5.3.1 Short Tandem Repeats 179

5.4 Identifying STR loci 182

5.4.1 DNA libraries 183

5.4.2 Locating novel microsatellite motifs using Next Generation Sequencing 184

5.5 Allele databases 190

5.5.1 Number of theoretical genotypes 192

5.5.2 Allelic ladders 192

5.6 Hardy–Weinberg equilibrium 193

5.7 Kinship factors and accounting for shared alleles 199

5.7.1 Rare or absent alleles on the database 202

5.8 Assessing the suitability of STR loci 203

5.8.1 The Genetic Data Analysis software (GDA) 205

5.8.2 The Excel Microsatellite Toolkit 214

5.8.3 Arlequin 220

5.8.4 API-Calc 228

5.8.5 Genepop 230

5.8.6 FSTAT 235

5.8.7 Structure 236

5.8.8 Summary 242

5.9 Genetic assignment: paternity testing 244

5.9.1 Genetic assignment: paternity testing if one parent is not available 249

5.9.2 Genetic assignment in paternity testing, incorporating kinship factor 251

5.10 Concluding comments 253

References 254

6 Interpretation, Evaluation and Reporting of Results 259

6.1 Introduction 259

6.2 Case assessment 260

6.3 Hierarchies of propositions 261

6.4 DNA evidence evaluation 262

6.4.1 The frequentist approach 263

6.4.2 Likelihood ratios 264

6.4.3 The Bayesian approach 266

6.4.4 Comparison of the three approaches 267

6.5 Evaluation of DNA evidence in wildlife cases 269

6.5.1 Case scenario 1 269

6.5.2 Case scenario 2 271

6.5.3 Case scenario 3 272

6.6 Role of the expert witness 273

6.7 Report writing 275

6.8 Summary and comments 277

Statement of witness 278

References 299

Measurements 303

Glossary 305

Appendix A Simulated Sample Populations 311

Appendix B Useful websites 323

Index 325