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ABSTRACT BIAN, YANG. Genetic Diversity And ABSTRACT BIAN, YANG. Genetic Diversity and Population Structure of Cultivated Blueberries (Vaccinium section Cyanococcus spp.). (Under the direction of Dr. Allan Brown). Blueberry (Vaccinium section Cyanococcus spp.) is an important small fruit crop native to North America with an incredible amount of genetic diversity that has yet to be efficiently characterized. Through broad natural and directed hybridization, the primary and secondary genepools currently utilized includes several distinct species and species hybrids in the section Cyanococcus. To date, only a limited number of cultivated blueberries have been assessed for genetic diversity in individual taxonomic groups using a limited number of molecular markers. A source of genomic SSRs is currently available through the generation and assembly of a draft genomic sequence of diploid V. corymbosum (‘W8520’). This genomic resource allows for a genome-wide survey of SSRs and the large scale development of molecular markers for blueberry genetic diversity studies and beyond. Of ~ 358 Mb genomic sequence surveyed, a total number of 43,594 SSRs were identified in 7,609 SSR-containing scaffolds (~ 122 counts per Mb). Dinucleotide repeats appeared the most abundant repeat types in all genomic regions except the predicted gene coding sequences (CDS). SSRs were most frequent and longest in 5’ untranslated region (5’ UTR), followed by 3’ UTR, while CDS contained the least frequent and shortest SSRs on average. AG/CT and AAG/CTT motifs were most frequent while CG/CG and CCG/CGG motifs were the least frequent for dinucleotide and trinucleotide motifs, respectively, in transcribed DNA. AAT/ATT motif was the most frequent trinucleotide motif in the nontranscribed DNA. In this study, 150 blueberry accessions of 8 cultivar types were evaluated for genetic diversity, population structure and genetic relationships using 42 genomic and EST-SSR markers. An average of 14.2 alleles and 56.0 allele phenotypes per locus were detected. The level of genetic diversity in rabbiteye was higher than in southern highbush, and southern highbush was higher than northern highbush accessions. Hexaploid V. virgatum was remarkably distinct from the rest of cultivar types. The 150 blueberry accessions clustered by species, ploidy levels and cultivar types in the neighbor-joining tree. Three groups were detected among highbush accessions: a group of the descendents of cultivar ‘Weymouth’, a group of primarily northern highbush accessions, and a group of primarily southern highbush accessions. A trend toward decreasing genetic distance was found among rabbiteye cultivars over cycles of recurrent selection. Genomic SSR markers produced greater mean pairwise distance than EST-SSR markers did among rabbiteye accessions, which indicated that EST-SSR markers are likely to be less polymorphic relative to genomic SSR markers in genetic diversity studies. © Copyright 2012 by Yang Bian All Rights Reserved Genetic Diversity and Population Structure of Cultivated Blueberries (Vaccinium section Cyanococcus spp.) by Yang Bian A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Horticultural Science Raleigh, North Carolina 2012 APPROVED BY: _______________________________ ______________________________ Allan Brown, Committee Chair James Ballington ________________________________ ______________________________ Jason Osborne Bryon Sosinski ________________________________ Todd Wehner BIOGRAPHY Yang Bian was born on August 3rd, 1986 in Nanjing, China. He graduated from Huazhong Agricultural University with Bachelor of Science in Horticulture. He came to the U.S. at 2009 as a M.S. graduate student at North Carolina State University Department of Horticultural Science. ii ACKNOWLEDGMENTS Thanks are due foremost to Dr. Allan Brown who provided directional guidance in all aspects of this research and exhibited exceptional kindness and encouragement in his role as Major Advisor. I would like to thank other committee members, Drs. James Ballington, Bryon Sosinski, Jason Osborne and Todd Wehner, for their advice, support and comment on my research and thesis. This thesis would not have been possible without the bioinformatic support from Archana N. Raja and Phuc (Peter) Pham. Drs. Gad Yousef and Ivette Guzman provided technical instruction, supervised the lab and field work and managed the instrument and reagents. I would also like to thank other laboratory members for all their help with the experiment in the field and lab. I owe many thanks to the staff of Piedmont and Sandhills Research Stations, for planting blueberry and field management; and to the staff of Plants for Human Health Institute and Department of Horticultural Science (NCSU), for providing the excellent facility, service and environment. Drs. Nahla Bassil and Jeannie Rowland of the U.S. Department of Agriculture (USDA), Dr. James Ballington of North Carolina State University (NCSU) and Dr. James Olmstead of the University of Florida (UFL) graciously provided plant and DNA materials used in this research. Dr. Mark Ehlenfeldt (USDA) kindly provided the coefficients of coancestry for some highbush and rabbiteye blueberries. I am grateful to Dr. Penelope Perkins-Veazie for being my extension mentor. I would additionally like to thank all of the excellent instructors of my M.S. courses at NCSU. It is their passion for education that has led me to reach further in my own pursuit of knowledge. Special thanks are due to my parents, family and friends for their affection throughout my life. This research was funded by UNC GA Fund “Generation and Validation of a Draft Genomic Sequence of Blueberry”. iii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. VI LIST OF FIGURES........................................................................................................... VII CHAPTER ONE LITERATURE REVIEW...........................................................................1 Economic importance and health benefits ..........................................................................1 Breeding history................................................................................................................3 Genetic diversity and relationships among blueberries.......................................................7 Comparison of EST and genomic SSR markers ...............................................................20 CHAPTER TWO - CHARACTERIZATION OF GENOMIC MICROSATELLITES AND MARKER DEVELOPMENT..............................................................................................24 Introduction.....................................................................................................................24 Materials and methods.....................................................................................................25 Survey of genomic SSRs .............................................................................................25 Primer design for genomic SSRs..................................................................................26 EST-SSR markers from EST libraries and transcriptome sequencing ...........................26 PCR and genotyping....................................................................................................26 Results.............................................................................................................................28 SSR frequency and distribution in blueberry genome...................................................28 Preferential motifs .......................................................................................................29 Marker validation and polymorphism ..........................................................................31 Discussion.......................................................................................................................32 The most abundant SSR types......................................................................................32 Frequencies of SSRs in the blueberry genome..............................................................33 Motif preference in genomic fractions .........................................................................34 Estimate of genetic diversity in blueberry ....................................................................35 The potential use of these markers ...............................................................................35 CHAPTER THREE - GENETIC RELATIONSHIPS AND POPULATION STRUCTURE OF CULTIVATED BLUEBERRY (VACCINIUM SECTION CYANOCOCCUS SPP.) ....36 Introduction.....................................................................................................................36 Materials and methods.....................................................................................................38 Plant materials .............................................................................................................38 Genetic diversity..........................................................................................................39 Genetic relationship analysis........................................................................................40 Population structure analysis........................................................................................41 Comparison of the two types of markers in constructing
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