AN ABSTRACT OF THE DISSERTATION OF Veli Erdogan for the degree of Doctor of Philosophy in Horticulture presented on April 16, 1999. Title: Genetic Relationships among Hazelnut (Corvlus) Species. Abstract approved: Shawn A. Mehlenbacher Interspecific hybridization, pollen-stigma incompatibility, morphological and phenological characterization, and DNA sequence analysis were studied in hazelnut (Corylus) species. Interspecific crosses resulted in a wide range of cluster set from 0% to 77.8%. Reciprocal differences were common. In general, crosses involving C. avellana and C. heterophylla were more successful when used as pollen parents, but crosses involving C. americana were more successful when it was the female parent. C. cornuta, C. californica and C. sieboldiana intercrossed freely in both directions, as did C. colurna and C. chinensis. The Asian species C. sieboldiana, C. heteropyhlla, and C. chinensis were not cross-compatible with each other. Fluorescence microscopy showed that pollen-stigma incompatibility exists within and among wild hazelnut species, in addition to the cultivated European hazelnut C. avellana. Pollen-stigma incompatibility and embryo abortion (blank nuts) appear to be major blocks to interspecific gene flow. A phylogenetic analysis based on twenty three morphological and five phenological characters placed hazelnut species in three groups. The first two included all representatives of shrub species while the third group contained tree species. In addition, the chloroplast matK. gene and the Internal Transcribed Spacer (ITS) region of the nuclear ribosomal DNA (rDNA) were amplified and sequenced. The ITS region and wa/K were 666 and 1231 bp long, respectively. The matK. gene had extremely low divergence and resulted in ambiguity as the number of informative characters was only 10, thus was not very informative. The nuclear ribosomal ITS region contained relatively low levels of variation that only 22 characters were informative. However, several well supported clades and three distinct groups were formed based on the ITS sequences. First and second groups included the shrub species while the third group contained the tree species. In addition, one species was separated from the three groups. Corylus species were placed into four groups: 1- C. avellana, C. maxima, C. americana and C. heterophylla 2- C. colurna, C. chinensis, paperbark hazelnut and C. jacquemontii 3- C. cornuta, C. californica and C. sieboldiana 4- C. ferox. The strict consensus tree topology was congruent with the results of interspecific hybridization and morphological classification. Copyright by Veli Erdogan April 16, 1999 All Rights Reserved Genetic Relationships among Hazelnut {Corylus) Species by Veli Erdogan A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented April 16, 1999 Commencement June 1999 Doctor of Philosophy dissertation of Veli Erdogan presented on April 16, 1999 APPROVED: —1^7""" ^ "-*■ "^™L_" - •*- ^ -^ Major Professor, representing Horticulture - ^ - ■■— Chair of Department of Horticulture Dean of Graduate^1 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. Veli^rdogan,HCrd Author ACKNOWLEDGEMENTS I would like to express my gratitude to my major professor Dr. Shawn A. Mehlenbacher for his guidance, understanding and assistance. I also thank David C. Smith for his assistance and suggestions during my interspecific hybridization studies. I enjoyed working with them during my graduate research assistantship. I am thankful to Drs. Joseph Spatofora, Patrick Hayes, Tony Chen, and Jeffrey J. Jenkins for serving on my committee. I appreciate the technical advice and initial supply of ITS primers provided by Dr. Aaron Listen for my DNA studies. Many thanks go to Drs. Ruth Martin, Nahla Basil, David Martin and Johanne Brunei for technical advice, and help in lab studies and software use. The help of Randy Hopkins and the crew at the Smith Research farm of OSU for construction of pollination cages was greatly appreciated. I would like to acknowledge, the Oregon Hazelnut Commission for their support of my research, the USDA-ARS National Clonal Germplasm Repository for providing me with plant material, the Department of Horticulture for its open- door policy concerning the use of labs, the Central Services Lab of OSU for primer synthesis and DNA sequencing, and Ankara University, where I work, for letting me study abroad. Work in the Department of Horticulture would have not been much fun over the years without sharing feelings, ideas, support and suggestions with Glenn Creasy, Kevin Cook, Joel Davis, China Lunde, Qiang Yao, Rebecca Brown and many other graduate students. I am obliged to Nihat Guner for his invaluable friendship and assistance, and to Hamdi Ogut and Mustafa Toraman for their moral support. I also enjoyed the interactions with faculties especially with Dr Patrick Breen, and with office specialists. Finally, I owe a great debt and respect to my beloved family for their everlasting patience, support and encouragement. TABLE OF CONTENTS Page CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW 1 1.1 INTRODUCTION 1 1.2 LITERATURE REVIEW 2 1.2.1 Taxonomy, origin and distribution of hazelnuts 2 1.2.2 Methods and data used in determination of genetic relationships 4 1.2.2.1 Phenetics 5 1.2.2.2 Cladistics 6 1.2.2.3 Types of data 7 1.2.3 Interspecific hybridizations 10 1.2.4 Self-incompatibility 17 1.3 REFERENCES 22 CHAPTER 2. INTERSPECIFIC HYBRIDIZATION IN HAZELNUT (Corylus) 36 2.1 ABSTRACT 36 2.2 INTRODUCTION 37 2.3 MATERIALS AND METHODS 39 2.3.1 Genetic material 39 2.3.2 Crosses 44 2.3.3 Seed germination and growing seedlings 45 2.4 RESULTS ; 46 2.4.1 Interspecific crosses , 46 2.4.2 Seed germination and seedling growth 53 2.5 DISCUSSION 59 TABLE OF CONTENTS (continued) Page 2.6 REFERENCES 65 CHAPTER 3. SELF-INCOMPATIBILITY IN WILD Corylus SPECIES 69 3.1 ABSTRACT 69 3.2 INTRODUCTION 69 3.3 MATERIALS AND METHODS 71 3.4 RESULTS 73 3.5 DISCUSSION 78 3.6 REFERENCES 83 CHAPTER 4. CHARACTERIZATION OF Corylus SPECIES BASED ON MORPHOLOGY AND PHENOLOGY 86 4.1 ABSTRACT 86 4.2 INTRODUCTION 86 4.3 MATERIALS AND METHODS 95 4.3.1 Phenological data 95 4.3.2 Husk characters 96 4.3.3 Leaf characters 96 4.3.4 Nut characters 100 4.3.5 Other characters 100 4.3.6 Data analysis 101 4.4 RESULTS 101 4.5 DISCUSSION 106 TABLE OF CONTENTS (continued) Page 4.6 REFERENCES 110 CHAPTER 5. MOLECULAR ANALYSIS OF Corylus SPECIES BASED ON NUCLEAR rDNA ITS AND CHLOROPLAST matK GENE SEQUENCES 113 5.1 ABSTRACT 113 5.2 INTRODUCTION 114 5.3 MATERIALS AND METHODS 116 5.3.1 Plant material 116 5.3.2 Genomic DNA extraction 116 5.3.3 Polymerase Chain Reaction (PCR) amplifications 118 5.4 RESULTS 123 5.5 DISCUSSION 127 5.6 REFERENCES 133 CHAPTER 6. CONCLUSIONS 138 BIBLIOGRAPHY 141 APPENDIX 157 LIST OF FIGURES Figure Pages 3.1 Intraspecific pollen-stigma reactions in Corylus 75 4.1 Husk variation in C. avellana; (A) Italian geotypes (B) Turkish genotypes 88 4.2 Husk variation in (A) C. maxima and (B) a typical husk of C. americana 89 4.3 A typical husk of (A) C. heterophylla and (B) C. cornuta 90 4.4 A typical husk of (A) C. californica and (B) C. sieboldiana 91 4.5 Husk variation in (A) C. colurna and (B) C. jacquemontii 92 4.6 A typical husk of (A) C. chinensis and (B) C. ferox var. thibetica 93 4.7 Variation in nuts of hazelnut species 94 4.8 Strict consensus tree of 33 taxa based on botanical characters 105 5.1 Structure and relative position of the (A) Internal Transcribed Spacer (ITS) region of nuclear ribosomal DNA, and (B) chloroplast maiK gene, including relative positions of the PCR and sequencing primers used in the present study 120 5.2 Electrophoretic separation of PCR-amplified nuclear rDNA ITS and chloroplast matK. gene bands 122 5.3 Strict consensus tree of 30 genotype based on nuclear rDNA ITS region sequences 124 5.4 Strict consensus tree of 30 genotypes based on maiK. DNA sequences 126 LISTS OF TABLES Table page 2.1 Female parents ofCorylus species and genotypes used in 1995 40 2.2 Female parents ofCorylus species and genotypes used in 1996 41 2.3 Female parents ofCorylus species and genotypes used in 1997 42 2.4 Male genotypes (mix) used in Corylus crosses 43 2.5 Intra- and interspecific crossing results (% cluster set) of Corylus species in 1995 47 2.6 Intra- and interspecific crossing results (% cluster set) of Corylus species in 1996 48 2.7 Intra- and interspecific crossing results (% cluster set) of Corylus species in 1997 49 2.8 Mean intra- and interspecific crossing results (% cluster set) of Corylus species in three years 50 2.9 Percent blank formation in intra- and interspecific crosses of Corylus species (average of 1995, 1996 and 1997) 52 2.10 Percent seed germination results in intra- and interspecific crosses of Corylus species (average of 1995 and 1996) 54 2.11 Hybrid seedling survival results in intra- and interspecific crosses of Corylus species (average of 1995 and 1996) 56 2.12 Hybrid seedling vigor expressed as trunk diameter (in cm) of intra- and interspecific crosses ofCorylus species (average of 1995 and 1996) 58 3.1 Number of genotypes used in pollinations in Corylus species 72 3.2 Total number of compatible and incompatible pollinations within and between Corylus pecies 76 LIST OF TABLES (continued) Table Page 4.1 Cultivated and wild Corylus accessions used in evaluation of botanical characters 97 4.2 Character states for the 28 botanical characters investigated 98 4.3 Coding of the character states for Corylus accessions 102 5.1 Cultivated and wild Corylus accessions used in DNA studies 117 5.2 Primer sequences used for PCR amplifications and sequencing 121 LIST OF APPENDIX TABLES Table Pages Al.