AN ABSTRACT OF THE THESIS OF Venkata Krishna Kishore for the degree of Doctor of Philosophy in Crop Science presented on March 21, 2002. Title: Mapping Quantitative Trait Loci Underlying Genome-Wide Recombination Rate and Mating System Differences in Meadowfoam Redacted for Privacy Abstract approved: Steven J. Knapp Meadowfoam (Limnanthes a/ba Bentham; Order: Brassicales; Family: Limnanthaceae) is a self-compatible, predominantly allogamous, insect pollinated species. Meadowfoam oil is a source of novel unsaturated very-long-chain (VLC) seed oils(C20andC22)with low concentrations of saturated fatty acids (typically less than 2%) and outstanding oxidative stability. Here we report the development of 389 SSR markers for meadowfoam. All the 389 SSRs were screened on 14 meadowfoam germplasm accessions to assess their utility and efficiency. Ninety-six percent of the SSR markers (373 out of 389) were polymorphic among the 14-germplasm accessions (from nine taxa) with a mean heterozygosity of 0.63. We also report that the physical size of the meadowfoam genome was estimated to be 5.52 pg using flow cytometry; thus, the meadowfoam genome is ca. 16 times larger than the Arahidopsis genome. Karyotype analyses revealed that the meadowfoam genome is made up of two metacentric and three submetacentric chromosomes. Meadowfoam has two pairs of chromosomes with subterminal nucleolar organizing regions (NOR's). A genetic map comprised of 84 SSR loci dispersed among five linkage groups with 11 to 22 SSR loci per linkage (6 SSR loci segregated independently) was constructed. The map was 988.7 cM long with a mean density of 11 .8 cM and minimal clustering of loci. A total of 20 quantitative trait loci (QTL) were identified for five mating system characters in meadowfoam, using the SSR linkage map of meadowfoam. Individual QTL for mating system traits peta1 area (pa), seeds per plant (spp) and seeds per flower (spI)I account for up to 20% of the backcross phenotypic variance, with most traits showing QTL effects of 5-15%. The QTL for protandry and chiasma frequency were adjacent to the QTL forSJ)andspfThis study has provided evidence that the correlation between the chiasma frequency and the type of mating system is not a direct developmental relationship between these factors, but is due to a selective advantage of the combination of the characters found. The speculation that the genetic factors underlying chiasma frequency and autonomous seed set have co-evolved during evolution negates the self-fertilization as an 'evolutionary dead end". Mapping Quantitative Trait Loci Underlying Genome-Wide Recombination Rate and Mating System Differences in Meadowfoam by Venkata Krishna Kishore A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented March 21, 2002 Commencement June 2002 Doctor of Philosophy thesis of Venkata Krishna Kishore presented on March 21, 2002 APPROVED: Redacted for Privacy Major Professor, representing Crop Science Redacted for Privacy Chair of the Department of Crop and Soil Science Redacted for Privacy Dean of Gthduate 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. Redacted for Privacy Venkata Krishna Kishore, Author ACKNOWLEDGMENTS I am completely indebted to Dr. Steven J. Knapp, my research guide and major professor, for his uncountable practical suggestions during the conduct of research work, timely insights throughout the writing of this text, and critical review of each and every line of the text. I am also grateful to him for his encouragement and enthusiasm, which inspired me to work better. I also thank him for the financial support he provided throughout my doctoral program. I am thankful to members of my graduate committee, Dr. Patrick M. Hayes. Dr. James C Peterson, Dr. Tony Chen and Dr. Gregory Thompson for their time and guidance. Special thanks and gratitude are extended to Jimmie Crane"The walking and talking encyclopedia of meadowfoam" for sharing the information about cultivation of meadowfoam and also helping with the greenhouse work. I am thankful to Dr. Mary B. Slabaugh, who taught me all aspects of molecular techniques and was always generous with her time. Her encouragement, support, concern, and warm heart will be forever appreciated. Thanks are due to the individuals in the lab: Sonali, Shunxue, Pablo, Judy. Ju-Kyung, Felix, Cate and Adam for their warm smile, kindness and friendship throughout my study period. I am also grateful to Dr. Pablo Velasco-Pazos for his unreserved help, and technical suggestions especially during the preparation of the manuscript. The faculty and staff of the Department of Crop Science who directly or indirectly contributed to the success of this work are gratefully acknowledged. I wish to thank Caprice Rosato who assisted in automated genotyping of SSR markers using GeneScan and Genotyper software. Help from Nanci Adair, and Joan Rowe for sequencing DNA is appreciated. Appreciation is extended to Beth Parsons, Elias Sabry, Corwin Willard and Julie Oughton for their help with flow cytometry analysis. I am also thankful to Al Soeldner for his help with the electron microscopy analysis of the buds and flowers of meadowfoam. I owe a debt of gratitude to Dr. Oscar Riera-Lizarazu and Dr. Isabel Vales for allowing me to use their microscope facilities and help in getting started with the karyotype analysis. The funding and support from USDA grant #99-34407-7509to Dr. S.J. Knapp is also acknowledged. Though far from here, my work owes completely to my parents, sister, brother and my in-laws, for whom my success is their success. Special thanks are due to my brother Shreenivasarao, who took over and fulfilled my responsibilities towards our family during the course of my doctoral program. Last but not the least, I thank my better half of the life 'Sonali D. Gandhi", for her love and constant encouragement at home as well as at work. She has been an excellent team worker with me right from my bachelor's degree. I wouldn't have achieved whatever little I have so far without her patience, understanding and solid support. CONTRIBUTION OF AUTHORS Dr. Steven J. Knapp initiated, advised, and supervised all aspects of the projects. He substantially assisted in formulating hypotheses and revising manuscripts. Sonali D. Gandhi helped in acquiring data for karyotype and chaismata frequency analysis. She also helped in phenotyping the mating system traits. Jimmie Crane helped with all the technical aspects of growing meadowfoam in the greenhouse. He developed meadowfoam lines OMF64, OMF4O- 11 and OMF 109-3 in association with Dr.Steven J. Knapp. Dr. Pablo Velasco helped in SSR screening for the SSR project. Dr. Mary B. Slabaugh contributed and helped in all aspects of molecular techniques and analyses. TABLE OF CONTENTS Page CHAPTER 1 INTRODUCTION........................................................................................................ CHAPTER 2 THE DEVELOPMENT AND UTILITY OF SIMPLE SEQUENCE REPEAT MARKERS FOR DNA FINGERPRINTING AND HIGH-THROUGHPUT GENOTYPING IN MEADOWFOAM .........................................................................5 ABSTRACT .............................................................................................6 INTRODUCTION...................................................................................7 MATERIALS AND METHODS...........................................................11 RESULTS.............................................................................................. 20 DISCUSSION........................................................................................ 59 REFERENCES.......................................................................................70 CHAPTER 3 THE MEADOWFOAM GENOME AND SIMPLE SEQUENCE REPEAT GENOMEMAP .........................................................................................................77 ABSTRACT...........................................................................................78 INTRODUCTION................................................................................. 79 MATERIALS AND METHODS ...........................................................82 RESULTS.............................................................................................. 91 DISCUSSION...................................................................................... 104 REFERENCES.....................................................................................114 CHAPTER 4 MAPPING THE QUANTITATIVE TRAIT LOC1 UNDERLYING THE SHIFTS IN THE MATING SYSTEM OF MEADOWFOAM FROM ALLOGAMY TO ALJTOGAMY.............................................................................122 TABLE OF CONTENTS (CONTINUED) Page ABSTRACT .123 INTRODUCTION...............................................................................124 MATERIALS AND METHODS .........................................................128 RESULTS............................................................................................ 135 DISCUSSION......................................................................................152 REFERENCES.....................................................................................162 CHAPTER 5 CONCLUSIONS.......................................................................................................167 BIBLIOGRAPHY.....................................................................................................172