AN ABSTRACT OF THE THESIS OF Rebecca Nelson Brown for the degree of Doctor of Philosophy in Horticulture presented on July 2. 2001. Title: Traditional and Molecular Approaches to Zucchini Yellow Mosaic Virus Resistance in Cucurbita Abstract approved: 7 James R. Myers 7 Zucchini yellow mosaic virus (ZYMV) is an important disease of Cucurbita worldwide. Resistant cultivars are the best means of control. ZYMV resistance is quantitative in the wild species C. ecuadorensis, and is controlled by at least three genes in tropical C moschata. Very little molecular work has been done in Cucurbita, and no markers are available to assist in selecting for ZYMV resistance genes. The objectives of this research were threefold: to develop molecular markers for use in breeding for ZYMV resistance, to develop a framework map of Cucurbita, and to transfer ZYMV resistance from C. ecuadorensis to C. maxima 'Golden Delicious'. The identification of a DNA marker linked to ZYMV resistance from C moschata TSIigerian Local' was attempted using random amplified polymorphic DNA (RAPD) analysis of resistant and susceptible bulks from the crosses C moschata 'Waltham Butternut' x CWaltham Butternut' x TSIigerian Local'). No marker consistently linked to ZYMV resistance was identified. Marker polymorphism was 14% between the parents. The cross C pepo A0449 x (A0449 x TSTigerian Local') was used as the mapping population to construct the framework map. The completed map consists of 153 loci in 29 linkage groups covering 1,981 cM. Approximately 75% of the Cucurbita genome is covered. Three morphological traits, two quantitative trait loci and a putative secondary ZYMV resistance gene have been placed on the map; an additional eight traits and nine RAPD markers remain unlinked. A study of the inheritance of ZYMV resistance was conducted as part of the efforts to mark and map resistance. Resistance appears to be controlled by at least three dominant genes in C. pepo. Environment and time since inoculation affect expression of resistance, and resistance should be treated as a quantitative trait. Development of ZYMV-resistant 'Golden Delicious' has progressed through five generations. In 2000, thirty-five plants were selected which were fiilly virus resistant and had reasonable processing quality. ZYMV resistance levels throughout the project were 31% for the F2 and 12% for the first backcross in 1998, 3% for the backcross progeny of the 1998 selections in 1999, and 15% for the selfed progeny of the 1999 selections in 2000. Traditional and Molecular Approaches to Zucchini Yellow Mosaic Virus Resistance in Cucurbita by Rebecca Nelson Brown A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presentedjuly2,2001 Commencement June 2002 Doctor of Philosophy thesis of Rebecca Nelson Brown presented on July 2. 2001. APPROVED: Major Professor, representing Herticulture Head of Departing of Horticulture KJ- Dean of71 tfaje 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. Rebecca Nelson Brown, Author Acknowledgement Many people helped to make this work possible. I would like to thank Augie Gabert and the other folks at Sunseeds for getting me started on this project, and for providing breeding lines, virus inoculum, and facilities. I would like to thank Jim Myers for the willingness to take on a crop that was new to him, and find the money to support this research. I would like to thank the members of my committee for their advice and support, and the folks of the OSU vegetable crops program for their support and assistance. Most of all I would like to thank my husband for supporting me both financially and emotionally, and for his willingness to move to Oregon. 11 TABLE OF CONTENTS Page INTRODUCTION 1 Economic significance of Cucurbita 1 The Impact of Viruses on Squash Production 3 Research Objectives 4 LITERATURE REVIEW 6 Zucchini Yellow Mosaic Virus 6 Resistance to Other Viruses in Cucurbita 8 Classical Cross Protection 10 Genetically Engineered Cross Protection 11 Genetics of Virus Resistance in Other Cucurbits 12 Cucumber 12 Melon 13 Watermelon 14 The Use and Development of Molecular Breeding Tools in Cucurbita 15 Molecular Genetics and Mapping — Cucumis and Citrullus 18 Cucumis 18 C. melo 18 C sativus 20 Citrullus 22 in TABLE OF CONTENTS (continued) Page Random Amplified Polymorphic DNA (RAPDs) as a Tool for the Identification of Trait-Linked Markers and Molecular Map Construction 23 Advantages of RAPD Markers 25 Potential Difficulties with RAPD Markers 27 The Inheritance of Morphological Traits in Cucurbita 29 The Genetics of Fruit Color in Cucurbita pepo 29 Base Colors 30 Green, Yellow or White Fruit 30 Precocious Yellow Fruit 34 Inhibition of mature fruit color: ImfOi I-mc. 35 Color modifiers 36 Extending the effects of B: Ep-1 and Ep-2 36 Pigment intensifiers: LI and L2 37 Striped Fruit 39 Other effects oi L2. 42 Dark Stems and Fruit 42 The color reducing gene r 43 Quiescent intense {qi) 44 Plain light fruit coloration {pi) 44 Turning orange at maturity: mo-1 and mo-2 45 Fruit Color Genes in Cucurbita moschata and Cucurbita maxima 46 Precocious yellow 46 Genes for Green and Motded Green Fruit in C. moschata 48 Genes for Blue, Green and Red Fruit in C maxima 48 IV TABLE OF CONTENTS (continued) Page Genes for other fruit traits 49 Warts 49 Rind Hardness (Lignification) 49 Fruit Flesh Color and Carotenoid Content 51 Fruit Shape 52 Genes Affecting Leaves and Stems 54 MotdeLeaf. 54 Genetic Yellowing in Leaves: Ses-B 55 Internode Length: Vine or Bush? 56 Powdery Mildew Resistance 59 A FRAMEWORK MAP OF CUCURBITA 60 Introducdon 60 Materials and Methods 60 Plant Materials 60 Morphological Traits 62 Disease Resistance 62 DNA Extraction 63 Polymerase Chain Reaction 64 Data Analysis 65 Results and Discussion 67 Morphological and Disease Resistance Traits 67 Days and nodes to first flower 67 Leaf mottle 70 Leaf shape and texture 74 Internode length and plant habit, tendrils 78 Fruit shape and set 79 Fruit rind color 83 TABLE OF CONTENTS (continued) Page Fruit Flesh Color 87 Fruit surface texture 88 Powdery mildew resistance 90 ZYMV resistance 92 RAPD Markers 94 The Map 95 References 105 A SEARCH FOR A RAPD MARKER LINKED TO ZYMV RESISTANCE FROM CUCURBITA MOSCHATA 'NIGERIAN LOCAL' USING BULKED SEGREGANT ANALYSIS 108 Introduction 108 Materials and Methods 109 Plant Material 109 Virus inoculation 109 DNA extraction 110 RAPD screening 110 AFLP screening Ill Results and Discussion 112 Segregation of ZYMV resistance 112 RAPD Analysis 113 AFLP Screening 113 Conclusion 113 References 115 Vl TABLE OF CONTENTS (continued) Page BREEDING 'GOLDEN DELICIOUS' TYPE CUCURBITA MAXIMA FOR RESISTANCE TO ZUCCHINI YELLOW MOSAIC VIRUS 117 Introduction 117 Materials and Methods 119 Plant Material 119 Greenhouse and Field Techniques 121 ZYMV Resistance Screening 122 Processing Quality 124 Results and Discussion 125 Conclusions 141 References 143 SUMMARY 145 BIBLIOGRAPHY 148 APPENDIX 166 vu LIST OF FIGURES Figure Page 2-1. Days to first flower for the BC, progeny of the cross A0449 x (A0449 x TSIigerian Local') 68 2-2. Nodes to first flower for the BC, progeny of the cross A0449 x (A0449 x 'Nigerian Local') 68 2-3. Days to first flower and nodes to first flower are correlated for the BC, progeny of the cross A0449 x (A0449 x 'Nigerian Local') (R=0.83) 69 2-4. The mottle leaf trait in Cucurbita 70 2-5. Pointed versus rounded leaf points 75 2-6. The distribution of indentation between the primary leaf veins in the progeny of the cross Cucurbita pepo A0449 x (A0449 x C moschata "Nigerian Local") 76 2-7. Leaf indentation in the parents and BC, of the mapping population 77 2-8. The distribution of intemode lengths among plants of the BC, of the cross Cucurbita pepo A0449 x (A0449 x C. moschata TMigerian Local') 79 2-9. Mature fruit of plants of the BC, of the cross Cucurbita pepo A0449 x (A0449 x C. moschata TSTigerian Local') showing the variation in fruit shape 81 2-10. The mature fruit of C pepo A0449 (left) and C. moschata TSfigerian Local'(right) 84 2-11. Powdery mildew resistance in the BC, of the cross Cucurbita pepo A0449 x (A0449 x C moschata 'Nigerian Local') 90 2-12. ELISA scores for the BC, of the cross Cucurbita pepo A0449 x (A0449 x C moschata TSTigerian Local') 94 2-13. Segregating RAPD bands from DNA of the BC, of the cross Cucurbita pepo A0449 x (A0449 x C moschata TSfigerian Local') amplified with Operon primer A20 95 vm LIST OF FIGURES (continued) Figure Page 2-14. Linkage map of the BC, of the cross A0449 x (A0449 x 'Nigerian LocalO 97 2-15. Graphical illustrations of marker distortion along each linkage group for the BC, of the cross A0449 x (A0449 x TSTigerian Local') 100 4-1. A mature 'Golden Delicious' fruit (left) and 'Golden Delicious' foliage (right) showing symptoms of ZYMV infection 118 4-2. Fruit of Cucurbita ecuadorensis. 120 4-3. Fruit weights for the selected virus resistant plants from the Cucurbita maxima breeding program in 2000 131 4-4. Equatorial flesh thickness of fruit from plants selected for virus resistance in the Cucurbita maxima breeding program in 2000 131 4-5. Puree thickness of the ZYMV resistant Cucurbita maxima selections for 2000, measured as percent total solids 132 4-6. Percent soluble solids in ZYMV resistant C. maxima selections for 2000 133 4-7. Some of the harvested fruit from 2000 135 4-8. Pedigrees of the 15 best C. maxima plants selected for ZYMV resistance and fruit quality in 2000 139 4-9.