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Application of High Resolution Melting Analysis for Haplotype Detection in Phylogeographic Research and Case Studies of Arenaria Ciliata, A

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Application of High Resolution Melting Analysis for Haplotype Detection in Phylogeographic Research and Case Studies of Arenaria Ciliata, A Application of High Resolution Melting analysis for haplotype detection in phylogeographic research and case studies of Arenaria ciliata, A. norvegica and Minuartia recurva (Caryophyllaceae) Xiaodong Dang In fulfilment of the requirement for the degree of Doctor of Philosophy National University of Ireland, Maynooth Department of Biology September 2012 Head of department: Prof. Paul Moynagh Supervisor: Dr. Conor Meade Table of contents Table of contents i Declaration vi Acknowledgement vii List of Figures ix List of Tables xi Abstract xiii 1. General introduction 1 1.1 Biogeographic questions on arctic/alpine carnation species in Ireland 2 1.1.1 Arctic/alpine carnation plants in Ireland 2 1.1.2 Principles and methods of phylogeographic research 9 1.2 High Resolution Melting analysis as a potential method of haplotype detection in phylogeographic research 13 1.3 Limitations of HRM analysis in haplotype detection and the scope for improvement 15 1.3.1 Inherent limitation of HRM analysis 16 1.3.2 HRM sensitivity and amplicon size 18 1.3.3 HRM sensitivity to SNPs: Discriminating between haplotypes with the smallest possible template variation 21 1.3.4 Evaluating HRM analysis with in silico simulation 22 1.4 Objectives 25 2. High Resolution Melting analysis: case studies in carnation species 27 2.1 Introduction 28 2.2 Materials 29 2.2.1 Arenaria ciliata and A. norvegica 29 i 2.2.2 Minuartia recurva 32 2.3 Methods 34 2.3.1 DNA extraction and initial assays 34 2.3.2 Locus selection and internal primer design for HRM analysis 35 2.3.3 High-Resolution Melting analysis: in vitro protocols 39 2.3.4 Interpretation and analysis of HRM curve profiles 43 2.3.5 Determination of haplotype identities based on melting profiles 46 2.3.6 In silico simulation of HRM analysis and correlation tests 48 2.4 Results 49 2.4.1 Haplotype detection for Arenaria species 49 2.4.1.1 Uniformity of the assay within a plate 49 2.4.1.2 Inter-batch repeatability of HRM analysis 52 2.4.1.3 Haplotype detection in the sampled populations with rps16 54 2.4.1.4 Haplotype detection in the sampled populations with trnT-trnL 63 2.4.1.5 Concatenated haplotypes 72 2.4.2 Results for Minuartia recurva 73 2.4.2.1 Haplotype detection with rps16 73 2.4.2.2 Haplotype detection with trnT-trnL 75 2.4.2.3 Concatenated haplotypes 81 2.5 Discussion 82 2.5.1 Discussion based on the HRM analysis with the Arenaria species 82 2.5.2 Discussion based on the HRM analysis with M. recurva 88 2.6 Conclusion 90 3. Phylogeography, population genetics and demographic history analysis of Arenaria ciliata and A. norvegica in Europe 92 3.1 Introduction 93 3.2 Methods 99 ii 3.2.1 Haplotype phylogeny in A. ciliata and A. norvegica 99 3.2.2 Population genetic diversity 102 3.2.3 Demographic history 105 3.3 Results 110 3.3.1 Haplotype phylogeny in A. ciliata and A. norvegica 110 3.3.2 Phylogeography of A. ciliata and A. norvegica 116 3.3.3 Population genetic diversity and structure 124 3.3.4 Demographic history 134 3.4 Discussion 141 3.4.1 Phylogeny and subspecies status of the Arenaria ciliata Complex 141 3.4.2 Phylogeography based on haplotypes in clade I and clade II/III 143 3.4.2.1 Clade I 145 3.4.2.2 Clade II and III 147 3.4.3 Co-occurring and co-migration of different clades 149 3.4.4 The structure of the populations 150 3.4.5 The origin of Irish populations and the possibility of Irish refugia 153 3.5 Conclusion 157 4. Phylogeography, population genetics and demographic history analysis of Minuartia recurva in Europe 158 4.1 Introduction 159 4.1.1 Background 159 4.1.2 Objectives 160 4.2 Materials and Methods 161 4.2.1 Haplotype phylogeny in M. recurva 161 4.2.2 Population genetic diversity 165 4.2.3 Demographic history 167 4.3 Results 169 iii 4.3.1 Haplotype phylogeny and phylogeography in M. recurva 169 4.3.2 Population genetic diversity and structure 175 4.3.3 Demographic history 181 4.4 Discussion 183 4.4.1 Phylogeography of M. recurva 183 4.4.2 The structure of the populations 186 4.4.3 The origination of the Irish populations 187 4.5 Conclusion 188 5. High Resolution Melting analysis: evaluation of its efficiency in phylogeographic research 189 5.1 Introduction 190 5.1.1 Background 190 5.1.2 Aims and objectives 191 5.2 Methods 198 5.2.1 Evaluation of HRM performance with different mutation classes 198 5.2.2 Evaluation of the effect of amplicon size on the occurrence of multiple melting domains 200 5.2.3 Evaluation of the effect of amplicon size on the sensitivity of HRM analysis 201 5.2.4 In silico HRM analysis of published data 203 5.3 Results 205 5.3.1 HRM performance with different mutation classes 205 5.3.2 The effect of amplicon size on occurrence of multiple melting domains 210 5.3.3 The effect of amplicon size on sensitivity of HRM analysis 212 5.3.4 HRM performance with published data 216 5.3.4.1 HRM analysis with mitochondrial loci on Hyla sarda 216 5.3.4.2 HRM analysis with a mitochondrial locus on Littorina sp. 223 iv 5.3.4.3 HRM analysis with chloroplast loci on Cedrela fissilis 228 5.3.4.4 HRM analysis with chloroplast loci on Palicourea padifolia 233 5.3.4.5 HRM analysis with chloroplast loci on Arenaria species 236 5.3.4.6 Overall evaluation on the in silico HRM analysis with published data 239 5.4 Discussion 241 5.4.1 HRM sensitivity to different mutation types 241 5.4.2 The effect of amplicon size on HRM efficacy 244 5.4.3 Post-hoc HRM performance with the published data 245 5.4.4 Reducing missed detection by the cross-check strategy 248 5.4.5 Missed detection rate: from theoretical assessment to realistic evaluation 250 5.5 Conclusion 252 6. General discussion 254 6.1 The prospect of applying HRM analysis in phylogeographic research 255 6.1.1 Empirical evaluation of HRM sensitivity 255 6.1.2 Quantitative evaluation of HRM sensitivity 257 6.1.3 Possible improvements of HRM sensitivity 260 6.2 Phylogeographic history of the studied species in Europe and in Ireland 262 7. Bibliography 265 Appendix Figure 1 277 Appendix Figure 2 278 v Declaration This thesis has not been submitted in whole, or in part, to this, or any other University for any other degree and is, except where otherwise stated, the original work of the author. Signed:__________________________________ Xiaodong Dang vi Acknowledgements First of all I would like to thank my supervisor, Dr. Conor Meade for giving me the opportunity to pursue my PhD study at NUI Maynooth, and for the advice he provided throughout my works, both in bench works and in methodology. During my study, my co-supervisor, Dr. Colin Kelleher and other researchers from the National Botanic Gardens of Ireland (Glasnevin) also provided advice and support for both laboratory and field works. Emma Howard-Williams has helped me a lot, especially in carrying me in the car looking for the plants everywhere in Ireland and Scotland. Undergraduate students Brian Collopy, Dale Oflaherty, Kevin Logan, Matthew Brocklebank, Ronan Morris and Ross Campbell, who each worked in the lab for months, also have contributed part of the DNA data. I owe my big thanks to them for their support. Here I also thank other collaborators including Adreas Tribsch (Salzburg), Kevin Walker (BSBI), Bogdan Jovanovic (Heidelberg), Pablo Vargas (Madrid), Patrick Kuss (Bern) and Zachary Dwight (Salt Lake City), who helped in software use, locating, collecting and identifying samples, and vii giving advice. Their helps are essential for the works in the present study. I owe my thanks to department and college staffs, as well as researchers from other labs in the department of biology, who helped me in laboratory teaching, using and maintaining the instruments, delivering reagents and devices, and in those paper works for administrative use. Their helps have made my life easier in Ireland. I am also grateful to the university who granted the John & Pat Hume Scholarship to support my study for the past four years. As part of the bigger project of a biogeographic research on Irish arctic/alpine plants, the study was funded by Science Foundation Ireland (Grant No.: SFI/08/RFP/EOB1545). The Roche LightCycler® 480 System real-time PCR instrument used in the study was also funded by Science Foundation Ireland (Grant No.: SFI/07/RFP/GEN/F571/ECO7). viii List of Figures Chapter 1 Figure 1.1 8 Figure 1.2 9 Figure 1.3 23 Chapter 2 Figure 2.1 39 Figure 2.2 44 Figure 2.3 50 Figure 2.4 51 Figure 2.5 51 Figure 2.6 60 Figure 2.7 66 Figure 2.8 71 Figure 2.9 74 Figure 2.10 77 Figure 2.11 78 Figure 2.12 79 Figure 2.13 81 Chapter 3 Figure 3.1 111 Figure 3.2 112 Figure 3.3 114 Figure 3.4 117 Figure 3.5 120 ix Figure 3.6 122 Figure 3.7 127 Figure 3.8 129 Figure 3.9 136 Figure 3.10 138 Figure 3.11 140 Chapter 4 Figure 4.1 170 Figure 4.2 171 Figure 4.3 173 Figure 4.4 176 Figure 4.5 182 Figure 4.6 184 Chapter 5 Figure 5.1 211 Figure 5.2 213 Figure 5.3 215 Figure 5.4 227 Figure 5.5 232 x List of Tables Chapter 2 Table 2.1 31 Table 2.2 33 Table 2.3 36 Table 2.4 36 Table 2.5 37 Table 2.6 41 Table 2.7 42 Table 2.8 50 Table 2.9 53 Table 2.10 55 Table 2.11 56 Table 2.12 57 Table 2.13 67 Table 2.14 68 Table 2.15 69 Table 2.16 72 Table 2.17 73 Table 2.18 74 Table 2.19 76 Table 2.20 79 Table 2.21 82 Table 2.22 85 85 Chapter 3 Table 3.1 96 xi Table 3.2 97 Table 3.3 125 Table 3.4 126 Table 3.5 128 Table 3.6 131 Table 3.7 133 Table 3.8 134 Chapter 4 Table 4.1 162 Table 4.2 162 Table 4.3 175 Table 4.4 178 Table 4.5 180 Table 4.6 181 Chapter 5 Table 5.1 207 Table 5.2 210 Table 5.3 214 Table 5.4 219 Table 5.5 221 Table 5.6 224 -225 Table 5.7 229 Table 5.8 230 Table 5.9 235 Table 5.10 240 xii Abstract The present study includes two aspects in the field of phylogeography.
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