AN ABSTRACT OF THE THESIS OF Peter Boches for the degree of Master of Science in Horticulture presented on March 30. 2005. Title: Microsatellite Marker Development and Molecular Characterization in Highbush Blueberry (Vaccinium corymbosum L.) and Vaccinium Species Abstract approved: Nahla V. Bassil Pre-existing Expressed Sequence Tag (EST) libraries (1,305 sequences) and a newly constructed microsatellite-enriched genomic library (136 clones) from the V. corymbosum cultivar 'Bluecrop' were screened for unique Simple Sequence Repeat (SSR)-containing sequences. Our goal was to develop a set of robust SSR markers for use in managing the National Clonal Germplasm Repository blueberry collection. Of 153 microsatellite loci detected in the DNA sequences, 94 primer pairs were tested for amplification and polymorphism in a panel of 12 Vaccinium germplasm accessions. A total of 30 loci (20 EST and 10 genomic) repeatedly amplified a well conserved and highly polymorphic locus of the expected size. The average cross-amplification in 12 Vaccinium species of 44 V. corymbosum-derived SSR that amplified a single locus on agarose gels ranged from 59% in V. macrocarpon Ait. to 95% in diploid V. corymbosum (95% confidence interval for mean 73- 82%). The genetic similarity among 114 accessions of North and South American species of Vaccinium L. were investigated using 5 SSR loci that exhibited high allelic diversity. The genetic similarity measure reproduced many well-accepted taxonomic relationships between species in the genus Vaccinium and show great promise for future use in phylogenetic studies. Within section Cyanococcus, our results supported the recognition of V. elliottii Chapm. as a genetically distinct diploid highbush species and the theory of widespread hybridization among polyploid taxa: A set of 71 accessions representing wild and domesticated highbush blueberry germplasm were genotyped using 28 SSR loci. A total of 627 alleles were detected and produced unique fingerprints for all accessions. The duplication of two accessions being maintained as separate inventories was verified. The highest numbers of taxon-specific alleles were detected in wild V. corymbosum and some cultivars. Genetic similarity measures grouped wild and domesticated blueberries separately. Domesticated blueberries of the southern highbush type formed a group distinct from the northern highbush types. Northern highbush accessions grouped among ancestral clones that have been extensively used in blueberry breeding such as 'Rubel' and 'Stanley'. These SSR markers show excellent promise for future use in germplasm identification, studying wild Vaccinium populations, and linkage mapping. Microsatellite Marker Development and Molecular Characterization in Highbush Blueberry (Vaccinium corymbosum L.) and Vaccinium Species by Peter Boches A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented March 30, 2005 Commencement June 2005 Master of Science thesis of Peter Boches presented on March 30, 2005. APPROVED: l^T Major Professor, representing Horticulture ^ * • -' » i' V Head of the Department of Horticulture C^ ^^ Dean of t^/Gfadthe Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University Libraries. My signature below authorizes release of my thesis to any reader upon request. Peter Boches, Author ACKNOWLEDGEMENTS The author wishes to express sincere appreciation to numerous groups and individuals. This research was funded by USDA-CRIS 5358-21000-033-00D and the Northwest Center for Small Fruits Research. Thanks are due foremost to Nahla Bassil who provided technical guidance in all aspects of this research and exhibited exceptional kindness and patience in her role as Major Advisor; and to Kim Hummer who also provided technical guidance and whose dedication to agricultural research has made this project possible. The committee overseeing the production of this thesis included Chad Finn, Glenn Howe, Steve Strauss, and Steve Knapp. Chad Finn provided advice about blueberry breeding and access to blueberry germplasm, Glenn Howe generously offered advice about genetics and molecular markers, Steve Strauss provided administrative oversight and advice about molecular markers, and Steve Knapp also offered comments on genetics and molecular markers. Jeannie Rowland of the U.S. Department of Agriculture (USDA) provided extensive collaboration in the development of the DNA markers used in this research. Lori Winton helped with the enrichment protocol (chapter 2). Shawn Mehlenbacher graciously allowed the use of his lab during the initial phases of this research. Dave Brazelton and Adam Wagner (Fall Creek Farm and Nursery) provided extensive advice and access to blueberry germplasm. Mark Ehlenfeldt (USDA) provided blueberry pedigree information and advice about tetraploid inbreeding coefficients. Blueberry breeders Paul Lyrene and Jim Hancock graciously gave us permission to use their patented cultivars in this study. I am grateful to the American taxpayers and the staff of the National Clonal Germplasm Repository, for making crop germplasm publicly available; and to the staff of the Oregon State University Horticulture Department for enabling horticultural research endeavors. Megan Kitner and Jeanine DeNoma volunteered at the NCGR and helped directly with much of the lab work. Isabela Mackey and Barb Gilmore of the NCGR also assisted directly with lab work. Special thanks are due to my wife, family, and friends for their affection and help throughout my life. CONTRffiTJTION OF AUTHORS Dr. Nahla Bassil assisted with the design, data collection, and writing of all parts. Dr. L.J. Rowland provided access to the EST library used in chapter 2, advice on DNA extraction in blueberry, DNA of the plants 'W-20', 'W-23', and 'NJ88' used in chapter 3, and comments on chapter 4. Mark Ehlenfeldt provided the tetraploid coefficients of coancestry used in Chapter 4. TABLE OF CONTENTS Page CHAPTER 1. INTRODUCTION 1 The Genus Vaccinium 2 Molecular Markers 5 Microsatellite Markers 6 Research Objectives 10 References 11 CHAPTER 2. 'BLUECROP' GENOMIC AND EST-SSRS FOR USE IN MANAGING THE VACCINIUM COLLECTION 18 Abstract and Introduction 19 Materials and Methods 23 Results 34 Discussion 46 References 50 CHAPTER 3. EVALUATION OF 'BLUECROP'-DERIVED SSRS FOR STUDYING SPECIES RELATIONSHIPS IN VACCINIUM 56 Abstract and Introduction 57 Materials and Methods 64 Results 71 Discussion 79 References 84 TABLE OF CONTENTS (continued) Page CHAPTER 4. GENETIC DIVERSITY IN THE HIGHBUSH BLUEBERRY ANALYZED WITH SSR MARKERS 87 Abstract and Introduction 88 Materials and methods 94 Results 103 Discussion 114 References 121 CHAPTER 5. CONCLUDING REMARKS 128 Conclusion 129 References 131 Bibliography 133 Appendices 147 LIST OF FIGURES Figure Page 2.1 Distribution of SSR motifs in EST and genomic enriched libraries 35 2.2 SSR length in ESTs as opposed to SSR-enriched genomic library 36 2.3 BLAST similarities of SSR containing sequences 38 2.4 Cross-species amplification of 44 SSR loci in 9 sections of Vaccinium 41 3.1 Neighbor joining (NJ) tree of diploid species taxa, based on the proportion of shared alleles distance for five SSR loci 73 3.2 NJ dendrogram of diploid and polyploid taxa, compressed to show major clades 74 3.3 NJ dendrogram of diploids and polyploids, lower portion compressed 75 3.4 NJ dendrogram of diploid and polyploid taxa, upper portion compressed 76 4.1 Taxon-specific alleles 106 4.2 Neighbor joining dendrogram of Vaccinium cultivars based on proportion of shared alleles distance for 28 SSR loci 113 LIST OF TABLES Table Page 2.1 Germplasm used for primer screening 26 2.2 Forty nine SSR loci of the expected size 27 2.3 DNA sequence information of 49 SSRs 30 2.4 Germplasm used for cross-species amplification and polymorphism 32 2.5 Marker yields from the EST andgenomic libraries 34 2.6 Six classes of predicted SSR location: 5' UTR, 3' UTR, intron, post 5', and not applicable (NA) 29 2.7 SSR usefulness in 12 Vaccinium species 43 3.1 Species described in Vaccinium §Cyanococcus by Camp (1945), Vander Kloet (1988) and Galleta and Ballington (1996) 61 3.2 Domesticated blueberries used in the phylogenetic analysis 66 3.3 Vaccinium species accessions used in the phylogenetic analysis 68 3.4 Allelic diversity statistics for five SSR loci in Vaccinium species 71 4.1 The 71 blueberry accessions analyzed with 28 SSR loci 98 4.2 Twenty eight SSR loci used to analyze relationships among blueberries..:.... 101 4.3 Allelic diversity among northern highbush blueberries for 28 SSR loci 107 4.4 Allelic diversity for single copy SSRs with predicted locations 109 4.5 Alleles associated with low chilling in southern highbush cultivars 110 LIST OF APPENDICES Appendix Page A. DNA extraction for Vaccinium 148 B. High throughput DNA extraction protocol 159 C. A rapid SDS-based DNA extraction protocol 162 D. Additional SSR loci and candidate genes for traits of interest 166 E. DNA fingerprints for 69 blueberry cultivars using 28 SSR loci 182 LIST OF APPENDIX FIGURES Figure Page A.l Ten (il of blueberry genomic DNA visualized after separation on a 1.5% agarose gel 153 A.2 Agarose gel electrophoresis photograph of 6 jxl of genomic DNA from 3 extraction methods 155 A.3 Agarose gel image of 2 (xl of genomic DNA extracted using 4 combinations of LB and PPS combinations from the PUREGENE kit (Gentra) or the method of Accotto et al. (2000) 156 A.4 Simultaneous 95% confidence intervals (Tukey