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Note to Users NOTE TO USERS The original manuscript received by UMI contains broken or light print. All efforts were made to acquire the highest quality manuscript from the author or school. Page@)were microfilmed as received. This reproduction is the best copy available PEA AND POWDERY MILDEW: GENETICS OF HOST-PATHOGEN INTERACTION AND IDENTIFICATION OF MOLECULAR MARKERS FOR HOST RESISTANCE A Thesis Submitted to the Faculty of Graduate Studies, The University of Manitoba, by Khusi Ram Tiwari In partial fulfillment of the requirernents for the degree of Doctor of Philosophy Graduate Genetics Program (c) June 1998 National Library 8ibliothèque nationafe du Canada Acquisitions and Acquisitions et Bibliogaphic SeMces seMoes bibiiographiques 395 WelI'ington Street 395. rue Wellington OLtawaOPl K1AW Oüawâ ON KIA ON4 canada camda The author has granted a non- L'auteur a accorde une licence non exclusive licence dowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distriiute or sell reproduire, prêter, distri'buer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/iih, de reproduction sur papier ou sur format électronique. The author retahs ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. FACL'LTY OF GRmUATE STUDiES ***** COPYRIGHT PERhIISSION PAGE PXA BRD POUDERY MILDEU: GEHETICS OF HOST-PATEOGEN ~RACTIO~AND KDENTIFICATIOH OF MOLECUUR MARKEES FOE HOST RESISTAHCE A ThesidPracticum subrnitted to the Faculty of Graduate Studies of The University of Manitoba in partiaI fulfhent of the requirements of the degree of DOCTOR OF PHILOSOPHY Rhusi Barn Tivari *G1998 Permission has been granted to the Library of The University of Manitoba to Iead or sen copies of this thesisfpracticum, to the Xational Library of Canada to microfilm this thesis and to lend or sel1 copies of the film, and to Dissertations Abstracts International to publish an abstract of this thesis/practicum. The author reserves other publication rights, and neither this thesis/practicum nor extensive extracts from it may be printed or othenvise reproduced without the au thor's written permission. GENERAL ABSTRACT Tiwari. Khusi Ram Ph.D.. The University of Manitoba. June 1998. Pea and Powdery Mildew: Genetics of Host-Pathogen Interaction and Identification of Molecular Markers for Host Resistance. Pea (Pisum sativurn L.) suffers significant yield and quality losses because of infection by the parasitic fungus Eyiphe pisi Syd., the causal agent of powdery mildew. Resistant cultivars and lines were intercrossed and crossed with susceptible lines to determine the genetic basis of resistance. A high level of resistance in most of the resistant lines. including field pea cultivars grown in Canada (Highlight, AC Tamor and Tara). was conferred by er-1: resistance in JI 2480 was conferred by er-2. Variability in virulence was examined in naturally occurring populations of E. pisi in western Canada and NW USA. Thirty-one single colony isolates were tested on a set of 14 different pea lines, using a detached leaf assay. A low level of variability arnong the isolates was evident. Ten of the 14 pea lines were evaluated for powdery mildew reaction in Canada NE USA. SW USA, NW USA. UK and Nepal. Reaction in Nepal differed from that obsewed in other locations for three of the ten lines. The cultivarsllines Highlight, .JI 2480: JI 1559. Ji 2 10, JI 82. Radley and JI 1758 were suggested for use as differential lines for future studies. In a study of winter survival strategies of E. pisi in Manitoba, cleistothecia from infected leaves and stems were examined microscopically on a periodic bais throughout the winter of 1996/97. Most ascospores were degraded by spring under field conditions. In a seed- transmission study, where seeds fiom severely infected plants were sown in a greenhouse in 1996 and 1997, none of the 4200 plants examined was infiected with powdery mildew. Powdery mildew inoculua fiom other plant species found in the vicinity of pea fields did not infect pea. As molecular markers are useful in gene pyramiding and marker-assisted selection, three random amplified polyrnorphic DNA (RAPD) markers. OP048,OPE-16 and OPL-6 were identified as linked to er-l by screening progenies of the cross Highlightl Radley (susceptible cultivar), using bulked segregant analysis. Five amplified fragment length polymorphism (AFLP) markers Iinked to er-2 were identified by screening progenies of the cross JI 2480Radley using bulked segregant analysis. ACKNOWLEDGMENTS I would like to take this opportunity to thank my advisor Dr. Greg A. Penner (Cereal Research Centre. Winnipeg) for his guidance. encouragement, valuable time and for accommodating me in his busy laboratory. 1 sincerely thank Dr. T. D. Warkentin (Morden Research Centre. Morden) for offering me this interesting project and his support throughout this course of study. I gratefully acknowledge my Ph.D. advisory cornmittee members Dr. B. Fristensky (Plant Science) and Dr. G.R. Klassen (Microbiology) for their constructive suggestions and for their time to read this thesis. 1 wish my special thanks to Dr. J.G. Menzies and Dr. K.Y. Rashid for their assistance in pathological studies. 1 sincerely thank Dr. A. E. Slinkard for serving as my extemal examiner. My sincere appreciation goes to Dr. M. Arnbrose (John imes Institute, UK), Dr. N. F. Weeden (Corne11 University, USA), Dr. F. Muehlbauer (Washington State University. USA), Ms. S. Joshi (Nepal Agriculture Research Council. Nepal) for testing Our experimental materials and providing valuable information. 1 sincerely appreciate the help of C. Vera (Melfort Research Centre. SK.), D. D. OIT (Lacombe Research Centre. AB .) for providing inoculua of E. pisi. I sincerely acknowledge G. Casey, L. Bezte and S. J. Lee for their help and advise in the lab expenments. 1 am thankful to dl lab members uicluding Chris Anderson. Marcy Johnson. Allison Zacharias, Elsa Reimer, YdenZhang, Erica Reidel. Debbi Ugali. Zlatko Popovic and Nancy Fineberg. The help of A. Sloan, I. Wolfe, E. Muller. Frank Matheson. B. Gillis, and Blaine Recksiedler is greatly appreciated in field and iii greenhouse studies. 1 thank R. Sims for his photographie expertise and Mike Malyk and Marsha Bowyer for library services. 1 would like to thank my wife Arnbika Tiwari for her cooperation. understanding. and help during this course of study and in the difficult times. This thesis would not have been possible without her patience. This program of graduate study was too long for our children Bibek Tiwari and Binita Tiwxi. 1 missed them very much while working extra houn. 1 am indebted to my parents Ntr. Nanda Prasad Tiwari and Mrs. Shanta Devi Tiwari for their inspiration and patience. I appreciate the encouragement of my brother Bama Dev Tiwari. sisters Thuli Ghimire, Muna Bard, Sita Dhungana and Rama Dhitai and their families. To al1 our fiends: Ann Peters, Godfiey Chongo, Sangamesh Angadi, Vashantharajan Janakirarnan and the local Nepalese community including ChitrafManika Pradhan. AndyRlrmila Kuzyrnko, Mahesh/Hema Ghimire, LaxmardSulochana Tnpathy . DanyKamaIa Galay. ruid Maiya Uprety, who provided a great inspiration and fun Company during the course of this study, 1 Say "thank you very much. Financial support, provided by Western Grains Research Foundation (WGRF). is gntefully acknowledged. DEDICATION This thesis is dedicated to my parents Mr. Nanda Prasad Tiwari and Mrs. Shanta Devi Tiwari FOREWORD This thesis is written in a paper format. The results are presented in the form of 5 papers. The first paper (Chapter 3) was published in the Canadian Journal of Plant Science. 77: 307-3 10 (1997). The second paper (Chapter 4) was published in the Canadian Journal of Plant Pathology, 19: 267-271 (1997). The third paper (Chapter 6) is in press in Genome (1 998). The fourth paper (Chapter 5) is submitted to the Canadian Journal of Plant Pathology. The fifth paper (Chapter 7) will be submitted to Molecular Breeding. A general introduction and literature review precede the papers. This is followed by a general discussion and conclusion, literature cited and an appendix. TABLE OF CONTENTS GENERAL ABSTRACT ..................................................................................................... i ... ACKNO WLEDGEMENTS ............................................................................................... .iri DEDICATION ..............................................................................................................v FOREWORD...................................................................................................................... vi .. TABLE OF CONTENTS....................................................................................... ... ......... vri LIST OF TABLES ............................................................................................................ .xi .. LIST OF FIGURES ........................................................................................................... XII LIST OF PLBBREVIATIONS............................................................................................ xv 1. GENERAL INTRODUCTION ..........................................
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