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Development and Use of Molecular Tools in Fragaria

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Development and Use of Molecular Tools in Fragaria AN ABSTRACT OF THE DISSERTATION OF Wambui Njuguna for the degree of Doctor of Philosophy in Horticulture presented on March 1, 2010. Title: Development and Use of Molecular Tools in Fragaria Abstract approved: Nahla V. Bassil This dissertation describes the application and development of molecular tools that have great potential for use in studying variation in strawberry germplasm. The first study evaluated 91 microsatellite (simple sequence repeat, SSR) markers for transferability in 22 Fragaria species and their utility in fingerprinting octoploid strawberries. Out of the transferable markers, a reduced set of four SSRs was developed based on polymorphism, multiplexing ability, reproducibility and ease of scoring. Unique SSR fingerprints were obtained for over 175 Fragaria samples representing 22 Fragaria species used in the study. Testing of two molecular markers linked to the red stele and anthracnose resistances identified potential sources of resistance in previously untested genotypes. Further characterization of these accessions is warranted to validate resistance and usefulness in breeding. In the second study, 20 SSRs polymorphic in wild Asian diploids, F. iinumae and F. nipponica, from Hokkaido, Japan were selected for genetic analysis of 137 accessions from 22 locations. Principal coordinate analysis followed by non-parametric modal clustering grouped accessions into two groups representing the two species. Further clustering within the groups resulted in 10 groups (7-F. iinumae, 3-F. nipponica) that suggest lineages representative of the diversity in Hokkaido, Japan. The third study tested plant DNA barcodes, the nuclear ribosomal Internal Transcribed and the chloroplast PsbA-trnH spacers, for Fragaria species identification. The ‘barcoding gap’, between within species and between species variation, was absent and prevented identification of Fragaria species. The fourth study evaluated the genetic diversity of 94 accessions representing 22 Fragaria species using four universal chloroplast SSR loci. Genetic diversity was moderate (0.54) despite the homoplasy observed. Species-specific haplotypes for F. nipponica, F. orientalis, F. iinumae and F. nilgerrensis were identified. Sequencing whole chloroplast genomes using Illumina in a final study revealed a close maternal genome relationship between F. vesca ssp. bracteata and the octoploid species supporting a North American origin of the octoploids and the polyphyly of F. vesca. Calculation of divergence time of Fragaria revealed young evolutionary age of the genus at 2.7 million years and of the octoploids at 450,000 years. ©Copyright by Wambui Njuguna March 1, 2010 All Rights Reserved Development and Use of Molecular Tools in Fragaria by Wambui Njuguna A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented March 1, 2010 Commencement June 2010 Doctor of Philosophy dissertation of Wambui Njuguna presented on March 1, 2010. APPROVED: Major Professor representing Horticulture Head of the Department of Horticulture Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Wambui Njuguna, Author ACKNOWLEDGEMENTS I would like to acknowledge the tremendous contribution of my major advisor, Dr. Nahla V. Bassil, during my studies at Oregon State University. Her constant support, patience, encouragement and guidance has been invaluable. Her enthusiasm for research and openness to new technology facilitated interesting projects carried out during my PhD research. I would like to thank my committee members, Drs. Chad Finn, Aaron Liston, Shawn Mehlenbacher, and Jennifer Parke for their advice, guidance and useful suggestions. I also acknowledge Dr. Kim E. Hummer’s contribution to my research. Her availability for discussions on strawberry species and interest in my PhD research were a great support! I would like to sincerely thank Dr. Aaron Liston for his constant availability for consultation, his insightful direction and patience. I acknowledge the useful advice and contribution offered by Dr. Christopher Richards and the analytical assistance by Patrick Reeves. I thank Dr. Sushma Naithani for her technical assistance. I also thank Dr. Richard Cronn for his technical guidance and advice during the chloroplast genome sequencing project. Special thanks goes to the staff at NCGR, Corvallis for their continuous support and encouragement. Many thanks to Ted Bunch, Missy Fix, Barb Gilmore, April Nyberg, Jim Oliphant, Jack Peters, and Deb Tyson for their invaluable technical support. I thank present and former horticulture graduate students, Nina Castillo, Danny Dalton, Michael Dossett, Kahraman Gurcan, Vidyasagar, Sathuvalli, Esther Uchendu, and Sugae Wada for their friendship and technical help when needed. I am also very thankful for the technical assistance offered by Brian Knaus, Stephen Meyers, Matt Parks, and Sarah Sundholm. I would like to acknowledge the support and encouragement offered by friends from Westside community church and the Kenyan community in Corvallis. I also thank Daphne Kagume for her friendship that has been a great support. I would like to especially thank Brendan Young and his family for their timely support and encouragement! CONTRIBUTION OF AUTHORS Dr. Nahla V. Bassil assisted in the experimental design, analysis and writing of all chapters. Drs. Kim Hummer and Thomas Davis collected plant material used for chapter three, and assisted in the experimental design and writing of the chapter. Dr. Christopher Richards helped with writing and data analysis of chapter three. Drs. Aaron Liston and Richard Cronn assisted with analysis and experimental design of chapter six and Dr. Aaron Liston also assisted in writing the chapter. TABLE OF CONTENTS Page CHAPTER 1: Development and Use of Molecular Tools in Fragaria L. ……… 1 Wild strawberry species ……….……………….……….……….……… 2 Strawberry origin and breeding ……….……….……….……….………. 11 Origin ...……….……….……….……….……….……….……… 11 Breeding progress ……….……….……….……….……….……. 11 Breeding gene pool ……….……….……….……….…………… 13 Strawberry phylogeny ……….……….……….……….……….………… 14 Characterization of strawberry germplasm……….……….……….…… 17 Morphological identification of strawberries ……….……….….. 17 Isozymes ……….……….……….……….……….……….…….. 18 DNA-based PCR markers ……….……….……….……….……. 19 Linkage mapping in strawberry ……….……….……….……….……… 33 DNA barcoding ……….……….……….……….……….……….……. 36 Next generation sequencing ……….……….……….……….…………. 39 Overall objectives and justification of research……….……….……….. 42 References ……….……….……….……….……….……….……….…. 46 CHAPTER 2: A Reduced Molecular Characterization Set for Fragaria L. (Strawberry) ……….……….……….……….……….……….… 66 Abstract ……….……….……….……….……….……….……….…….. 67 Introduction ……….……….……….……….……….……….………… 69 Material and methods ……….……….……….……….……….……….. 75 Results ……….……….……….……….……….……….……….……… 80 Discussion ……….……….……….……….……….……….……….…. 83 References ……….……….……….……….……….……….……….… 89 CHAPTER 3: Genetic Diversity in Japanese Strawberry Species ……….……... 128 Abstract ……….……….……….……….……….……….……….…….. 129 Introduction……….……….……….……….……….……….…………. 130 TABLE OF CONTENTS (Continued) Page Material and methods ……….……….……….……….……….………. 134 Results ……….……….……….……….……….……….…………….. 138 Discussion ……….……….……….……….……….……….………… 139 References ……….……….……….……….……….……….………... 143 CHAPTER 4: DNA Barcodes for Species Identification in Fragaria L. (Strawberry) ……...……….……….……….……….………… 155 Abstract……….……….……….……….……….……….…………… 156 Introduction ……….……….……….……….……….……….……… 157 Material and methods ……….……….……….……….……….…….. 160 Results ……….……….……….……….……….……….…………….. 162 Discussion. ……….……….……….……….……….……….………… 164 References ……….……….……….……….……….……….………… 167 CHAPTER 5: DNA Chloroplast SSR Diversity in Fragaria L. species……… 181 Abstract ……….……….……….……….……….……….…………… 182 Introduction ……….……….……….……….……….……….………. 183 Material and methods ……….……….……….……….……….…….. 186 Results ……….……….……….……….……….……….……………... 188 Discussion. ……….……….……….……….……….……….………... 190 References ……….……….……….……….……….……….………… 195 CHAPTER 6: Whole Chloroplast Genome Sequencing of Wild Fragaria Species ……….……….……….……….……….……….……… 215 Abstract ……….……….……….……….……….……….……………. 216 Introduction ……….……….……….……….……….……….………... 217 Material and methods ……….……….……….……….……….……… 221 Results ……….……….……….……….……….……….………………. 225 Discussion. ……….……….……….……….……….……….………….. 228 References ……….……….……….……….……….……….………….. 236 TABLE OF CONTENTS (Continued) Page CHAPTER 7: Concluding Remarks ……….……….……….……….…………. 255 Conclusion ……….……….……….……….……….……….………….. 256 References ……….……….……….……….……….……….………….. 259 BIBLIOGRAPHY ……….……….……….……….……….……….………….. 260 APPENDICES ……….……….……….……….……….……….………………. 286 LIST OF APPENDICES Appendix Page A High Throughput DNA Extraction Protocol ….……….………….………. 287 B High Throughput Medium Scale DNA Extraction Protocol .….……….…. 290 C Isolation of Pure Chloroplast DNA (cpDNA)………………….………… 294 D Equimolar Pooling of PCR Fragments ………………...……….…………. 300 E Summary of Number of Reads Obtained from PCR Illumina Preparations …………………...……….……………………………… 307 F Representation of Four Indels Mapped on the Phylogenetic Tree of Fragaria …….……………………………………………....... 312 G Graphical Display of the Chloroplast Genome Coverage in Fragaria…....... 315 H Ninety-one SSRs Tested for Cross Transferability in Fragaria .…………….
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