QTL Mapping, Gene Identification and Genetic Manipulation of Glucosinolates in Brassica rapa L. BY Arvindkumar H. Hirani A Thesis Submitted to the Faculty of Graduate Studies at the University of Manitoba in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Plant Science University of Manitoba Winnipeg, Canada R3T 2N2 ©Copyright 2010 by Arvindkumar H. Hirani July 2011 ii ACKNOWLEDGEMENTS I would like to express my very sincere thanks to my supervisor, Dr. Genyi Li, Department of Plant Science, University of Manitoba, for providing excellent guidance and valuable advice throughout my PhD. program. I am also grateful for his encouragement and assistance in the course of my research. I would also like to thank my advisory committee members, Dr. Peter B. E. McVetty, Department of Plant Science, University of Manitoba and Dr. Anne C. Worley, Department of Biological Sciences, University of Manitoba for their valuable suggestions, helpful discussions and guidance for my research. I would like to thank Dr. Fouad Daayf for giving me access to the HPLC equipment facility in his Lab. I would like to extend my thanks to Mr. Lorne Adam and Dr. Abdelbasset El Hadrami for their assistance in HPLC for glucosinolate analysis. I would like to thanks Dr. Tom Ward for valuable help at the LC-MS facility in the Manitoba Chemical Analytical Laboratory, Department of Chemistry, University of Manitoba. My sincere thanks go to Dr. Carla Zelmer, Dr. JianFeng Geng, Dr. Gao Feng, Dr. Santosh Kumar, Ravneet Behla and Roger Watts for their technical support and helpful discussions. I appreciate all the past and present members of Dr. Li’s Lab for their advice and help in molecular techniques. My thanks also go to the greenhouse staff, Ian Brown and Cathy Bay. I would like to thanks all the members of Department of Plant Science. I am heartily grateful to the Heavenly Father, God, who gifted me this life and being always my source of strength, energy and hopes for the best. I praise and thank you my almighty Creator. I am deeply and forever indebted to my parents, Hasmukhray and Kantaben for their love, support and encouragement throughout my entire life. I am also very grateful to my wife, Ashmita. Without her love, encouragement, understanding and support, it would not have been possible to finish this thesis. I am also thankful to my only sister iii Sangita, and my brothers Vipul and Dilip. Special thanks to my friends for their help and support. Finally, I acknowledge that this research would not have been possible without the financial assistance of Genome Canada, Genome Alberta, NSERC, the University of Manitoba Graduate Studies and the Department of Plant Science at the University of Manitoba. I express my gratitude to all of them for financial support throughout my PhD. program. iv TABLE OF CONTENTS ACKNOWLEDGEMENT ................................................................................................ ii TABLE OF CONTENTS ................................................................................................ iv LIST OF TABLES ........................................................................................................... vi LIST OF FIGURES ........................................................................................................ vii ABBREVIATIONS .......................................................................................................... ix ABSTRACT ...................................................................................................................... xi FOREWORD .................................................................................................................. xiv 1. GENERAL INTRODUCTION ................................................................................. 1 2. LITERATURE REVIEW .......................................................................................... 6 2.1. Historical background of Brassica species ........................................................... 6 2.1.1. Genomic relationships in Brassica species .................................................... 7 2.1.2. Low glucosinolate rapeseed and canola ......................................................... 9 2.2. Economic importance of Brassica species .......................................................... 10 2.3. Plant secondary metabolites and their functions ................................................. 11 2.3.1. Glucosinolates as secondary metabolites ..................................................... 12 2.3.2. Chemical structures of glucosinolates ......................................................... 14 2.3.3. Glucosinolates and their biological functions in agriculture and nature ...... 16 2.3.3.1. Aliphatic glucosinolates ....................................................................... 16 2.3.3.2. Indole glucosinolates ............................................................................ 18 2.3.3.3. Aromatic glucosinolates ....................................................................... 20 2.3.4. Biosynthesis pathway of aliphatic glucosinolates ........................................ 21 2.3.4.1. Parental amino acid biosynthesis and condensation.............................22 2.3.4.2. Glucosinolate core skeleton formation ................................................. 24 2.3.4.3. Side chain modification in aliphatic glucosinolates ............................. 26 2.3.5. Diversity of glucosinolates in Brassicaceae ................................................ 29 2.4. Quantitative trait loci for glucosinolates in major Brassica species ................... 30 2.5. Glucosinolate identification and quantification approaches ............................... 33 2.6. Molecular markers and their applications for different traits in Brassica rapa .. 35 2.7. Homoeology between the A and C genomes.......................................................38 v 2.8. Inter or intra specific breeding for trait improvements in Brassica .................... 39 3. IDENTIFICATION OF QTL AND CANDIDATE GENES FOR LEAF AND SEED ALIPHATIC GLUCOSINOLATES IN Brassica rapa L. ............................. 43 3.1. Abstract ............................................................................................................... 44 3.2. Introduction ......................................................................................................... 43 3.3. Materials and Methods ........................................................................................ 47 3.4. Results ................................................................................................................. 52 3.5. Discussion ........................................................................................................... 57 4. IDENTIFICATION OF QTL FOR LEAF AND SEED GLUCOSINOLATES IN Brassica rapa L. RECOMBINANT INBRED LINES ............................................... 70 4.1. Abstract ............................................................................................................... 71 4.2. Introduction ......................................................................................................... 72 4.3. Materials and Methods ........................................................................................ 74 4.4. Results ................................................................................................................. 82 4.5. Discussion ........................................................................................................... 87 5. HOMOEOLOGOUS GENES REPLACEMENT AND INTROGRESSION FOR CHANGING ALIPHATIC GLUCOSINOLATES IN Brassica rapa L. BY MARKER ASSISTED BACKCROSS ..................................................................... 105 5.1. Abstract ............................................................................................................. 106 5.2. Introduction ....................................................................................................... 107 5.3. Materials and Methods ...................................................................................... 110 5.4. Results ............................................................................................................... 114 5.5. Discussion ......................................................................................................... 118 6. GENERAL DISCUSSION ..................................................................................... 128 7. CONCLUSIONS ..................................................................................................... 138 8. LITERATURE CITED .......................................................................................... 142 9. APPENDICES ......................................................................................................... 162 vi LIST OF TABLES Table 2.1 Trivial name and chemical formula of R side-chains of glucosinolates identified in Brassica species ... .......................................................................14 Table 2.2 Some of the important traits mapped and molecular markers developed in B. rapa for marker assisted selection in breeding .. .............................................37 Table 3.1 SCAR and SNP markers integrated on different linkage groups for glucosinolate biosynthesis loci ... .....................................................................62 Table 3.2 Genotype of BrMAM1-1 marker and 5C aliphatic glucosinolate