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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. EQgher quality 6 ” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Infonnation Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 MOLECULAR ASPECTS OF SOYBEAN TOLERANCE AND RESISTANCE TO PHYTOPHTHORA SOJAE DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University by NamikKaya B.Sc. (Hons), M.Sc. (Hons.) ***** The Ohio State University 1997 Dissertation Committee: Approved by Dr. T. T. VanToai, Adviser Dr. J. Streeter, Co-Adviser Adviser Dr. A. F. Schmitthenner Dr. M. Garraway Adviser Department of Horticulture and Crop Science UMI Number: 9813281 Copyright 1998 by Kaya, Namik All rights reserved. UMI Microform 9813281 Copyright 1998, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT Soybean Glycine max (L.) Merr., is one of the most important crops in the United States. One of the major disease of soybean is Phytophthora root rot (Prr). Causative agent of this disease is a fungus called Phytophthora sojae. The fungus causes severe losses on soybean production each year. One of the most effective way to control the disease is genetic control which is divided into tolerance and resistance. Tolerance is a quantitative trait and control by more than one gene. Unlike tolerance, resistance is a qualitative trait controlled by a single gene. In this study we studied molecular aspects of soybean tolerance and resistance against Phytophthora sojae. In the first part of our study, we choose amplified firagment length polymorphism (AFLP) technique to study disease tolerance. Our specific objective was to identify AFLP markers and QTL (quantitative trait loci) associated to the disease tolerance. Clark x Harosoy isoline mapping population was used for molecular marker analysis. Prr tolerance phenotype was assayed by the inoculum layer tests with three replicates. Total 13 AFLP markers were added to the previously established Clark x Harosoy soybean map. Of the 13 AFLP markers, only 10 were linked to the existing linkage groups. The map consists of 231 markers on 36 linkage groups and 40 unlinked markers. Six loci were also found significant at the probability level of 0.01 by one way analysis of variance. Two significant QTL (LOD score ii 3 or above) associated with the P. sojae tolerance were identified on linkage group three (R ^ 30.4%) and linkage group eleven (R ^ 62.0%) using MAPMAKER/QTL. This map including AFLP markers spans 2222.8 cM distance. In the second part of the study, our specific objective was to determine differential gene expression patterns and clone differentially expressed genes in response to inoculation o ff. sojae Race 1 in soyhosn-Phytophthora sojae model system and attempt to clone R psl-k gene. R psl-k gene provides host resistance to P. sojae Race 1. We used differential display technique to screen for genes expressed in response to inoculation of P. sojae Race 1 zoospores to the roots of soybean isolines Elgin (susceptible variety without R psl-k gene ) and Elgin 87(resistant variety with R psl-k gene). Total RNA’s of Elgin and Elgin 87 were isolated at 3 h, 6 h, 13 h, and 17 h post-inoculation with zoospores of Race 1 of Phytophthora sojae and water as controls. Seven different gene expression patterns have been identified and 49 differentially expressed bands have been displayed. Three sequences related to the general response of soybean to Phytophthora sojae infection have been cloned and their differential expression was confirmed by northern blot analysis. Ill Dedicated to pioneers; who enlightened our ways, to my wife, for her patience, help, kindness, and endless support to my parents, for their prayer, support and love to my family, for their cooperation and help IV ACKNOWLEDGMENTS I would like to express my gratitude to my advisor, Dr. T. T. VanToai for her guidance and thoughtful suggestions throughout the research. I am also very thankful to the other members of my committee, Dr. J. Streeter, Dr. A. F. Schmitthenner, Dr. M. Garraway for their suggestions and comments. Special thanks to Ginny, Drs. Getachew and Preisner for their help, discussions, suggestions, and also sincere thanks to my fellows Linda and Peng. I would like to thank to Yuzuncu Yil University in Turkey for their support throughout my studies. I also acknowledge to College of Food, Agricultural, and Environmental Sciences, OSU for their financial support in Autumn 1997. I express my sincere appreciation and gratitude to Dr. Alves for his guidance, suggestions, and comments. His frienship and suggestions were unforgetable. I also would like to express my sincere appreciation and special thanks to Mr. Yetisir for his friendship and suggestions. My last words for my wife, and my family. Without their support and prayers, this study cannot be accomplished. VITA August 1, 1966 ..................................Bom - Van, Turkiye 1983-1988 ........................................ B. S., Yuzuncu Yil University (Hons) 1988-1990 ........................................ M.Sc., Yuzuncu Yil University (Hons) Research Assistant Department of Horticulture Yuzuncu Yil University 1990-1991 ........................................ Ph.D., Yuzuncu Yil University Research Assistant Department of Horticulture Yuzuncu Yil University 1991-1996 .......................................... Ph.D., The Ohio State University High Education Council Fellowship 1997-present .....................................Research Assistant Agr., Biol., Food Eng. Department The Ohio State University PUBLICATIONS Research Publication 1. Kaya, N. 1991. Adaptation studies of several local melon varieties to Van Region.Y.Y.U. Art and Sci. Jour. 3: 10-16 2. Tekintas, E., Kaya, N., Beyhan, O., Karatas, E., Akca, O., 1991. Production of vi different vegetables under greenhouses and plastic tunnels in Van. EX Annual Productivity Symposium on the Improvements for Eastern Turkey, pp: 100-110 3. Kaya, N., VanToai, T. T., Specht, J., and Schmitthenner, A. F. 1996. Mapping QTL and identification of markers linked to Phytophthora root rot of soybean. International Symposium of Soybean Molecular Genetics. Ill, pp. 135 4. Kaya, N., Alves, J. D., VanToai, T. T., and Schmitthenner, A. F. 1997. Differential display of soybean root genes after inoculation of Phytophthora sojae Race I. ICABERG. pp. 120 5. Alves, J. D., Presiner, J. H., VanTaoi, T. T., and Kaya, N. 1997. Differentially expressed soybean root genes under anoxia. International Brazilian Plant Physiologist Symposium. FIELDS OF STUDY Major Field: Horticulture and Crop Science Plant Molecular Biology and Biotechnology Minor Field: Molecular Plant Pathology and Stress Physiology Molecular pathology of Soyhesn.-Fhytophthora sojae interaction and anaeorobic stress physiology, biochemistry, and genetic under the guidance of Dr. Tara T. VanTaoi. Vll TABLE OF CONTENTS Eâgs ABSTRACT.................................................................................................................ii DEDICATION.............................................................................................................iv ACKNOWLEDGMENTS........................................................................................... v VITA............................................................................................................................ vi LIST OF TABLES.......................................................................................................x LIST OF FIGURES.....................................................................................................xi GENERAL INTRODUCTION...................................................................................1 CHAPTER 1 Introduction ...................................................................................................16 Materials and Methods ................................................................................. 25 Results ...........................................................................................................31 Discussion ......................................................................................................50
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