i QTL MAPPING AND NIRS ESTIMATION OF CYANOGENIC GLUCOSIDES IN FLAXSEED BY ADAM SCOTT CHIN-FATT A Thesis Submitted to the Faculty of Graduate Studies of the University of Manitoba in Partial Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE Department of Plant Science University of Manitoba Winnipeg, Manitoba, Canada Copyright © 2014 ii THE UNIVERSITY OF MANITOBA FACULTY OF GRADUATE STUDIES COPYRIGHT PERMISSION QTL MAPPING AND NIRS ESTIMATION OF CYANOGENIC GLUCOSIDES IN FLAXSEED BY ADAM SCOTT CHIN-FATT A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfilment of the requirements of the degree of MASTER OF SCIENCE Copyright © 2014 The authority of the copyright has granted permission to The Library of the University of Manitoba to lend or sell copies of this thesis/practicum, to the National Library of Canada to microfilm this thesis and to lend or sell copies of the film, and to University Microfilms Inc. to publish an abstract of this thesis/practicum. The reproduction or copy of this thesis has been made available by the authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as by copyright laws or with express written authorization from the copyright owner. iii ACKNOWLEDGEMENTS To my family, for keeping me grounded and somewhat sane throughout this experience and for instilling in me a love of science and a curiosity for the unknown. To an all-star team of lab technicians and post-docs. In particular, thanks to Debbie and Elsa, my surrogate work mothers, who were akin to walking manuals on ‘how to be a good scientist.’ To Andy, for his steady guidance and refreshingly dark humor. To Santosh and Raja, for being accommodating in their impressive wealth of experience and knowledge. To the Chemistry experts of the Canadian Grain Commission. In particular, thanks to Ray, and his amazing attention to detail and his vast insight into the complexities of analysis and application. To Bert, whose experience has proved invaluable in navigating the infernal NIRS apparatus. To my supervisor Sylvie, a bastion of efficiency, for keeping me on track with this project and inspiring me to be a better researcher. Finding a more capable mentor is unimaginable. Without her, this project submission would no doubt have been procrastinated for another couple of months. To the aforementioned and all the other people I’ve met on this journey that have made this project possible. I wish them all the best and it has been an honor to collaborate with people of such great esteem and proficiency. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... iii TABLE OF CONTENTS ................................................................................................... iv LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii LIST OF ABBREVIATIONS ............................................................................................ ix LIST OF APPENDICES ................................................................................................... xii ABSTRACT ..................................................................................................................... xiii FOREWORD .................................................................................................................... xv 1.0 GENERAL INTRODUCTION ..................................................................................... 1 1.1 Background ............................................................................................................... 1 1.2 Research Objectives .................................................................................................. 3 1.3 Basic Strategy ............................................................................................................ 3 2.0 LITERATURE REVIEW ............................................................................................. 7 2.1 The Utility of Flax ..................................................................................................... 7 2.2 Anti-Nutrients in Flaxseed ........................................................................................ 9 2.3 Processing Methods to Remove Cyanogenic Glucosides ....................................... 13 2.4 Accumulation of Cyanogenic Glucosides ............................................................... 14 2.5 The Effect of Abiotic Factors on Cyanogenic Glucoside Accumulation ................ 15 2.6 Functions of Cyanogenic Glucosides ...................................................................... 17 2.7 Evolution of Cyanogenic Glucoside Biosynthesis Pathway ................................... 21 2.8 Biosynthesis of Cyanogenic Glucosides ................................................................. 22 2.9 Degradation of Cyanogenic Glucosides .................................................................. 26 2.10 Detoxification of Hydrocyanic Acid ..................................................................... 27 2.11 Regulation of Cyanogenic Glucoside Activity ...................................................... 27 2.12 Cyanogenic Glucoside Biosynthetic Gene Organization ...................................... 28 2.13 Genetic Engineering of Cyanogenic Glucosides ................................................... 31 2.14 Analytical Methods of Cyanogenic Glucosides .................................................... 32 2.15 Extraction .............................................................................................................. 33 2.16 Acid Hydrolysis ..................................................................................................... 34 2.17 Enzymatic Hydrolysis ........................................................................................... 35 v 2.18 Measurement of Cyanide ...................................................................................... 36 2.19 König method ........................................................................................................ 36 2.20 Guignard method ................................................................................................... 37 2.21 Feigl Anger method ............................................................................................... 38 2.22 Reaction with Nitrobenzaldehyde ......................................................................... 38 2.23 Titration ................................................................................................................. 39 2.24 Chromatography .................................................................................................... 40 2.25 Direct Measurement of Intact Cyanogenic Glucosides by Gas Chromatography ....................................................................................................................................... 40 2.26 Direct Measurement of Intact Cyanogenic Glucosides by High Performance Liquid Chromatography ................................................................................................ 41 2.27 Direct Measurement of Intact Cyanogenic Glucosides by High Performance Thin-Layer Chromatography (HPTLC) ........................................................................ 42 3.0 IDENTIFICATION OF QTL ASSOCIATED WITH THE ACCUMULATION OF LINUSTATIN AND NEOLINUSTATIN IN THE MATURE FLAX SEED ............ 43 3.1 Abstract ................................................................................................................... 43 3.2 Introduction ............................................................................................................. 45 3.3 Materials and Methods ............................................................................................ 47 Plant Materials and DNA isolation ............................................................................ 47 Linkage Map .............................................................................................................. 48 Measurement of Cyanogenic Glucosides .................................................................. 49 QTL Analysis ............................................................................................................ 50 Anchoring to the physical map and fine-mapping ..................................................... 51 Candidate gene identification .................................................................................... 52 3.4 Results ..................................................................................................................... 53 Linkage Map .............................................................................................................. 53 Phenotypic Analysis .................................................................................................. 56 QTL Analysis ............................................................................................................ 61 Fine Mapping and Physical mapping of the CG QTL ............................................... 64 3.5 Discussion ............................................................................................................... 68 Genetic Linkage Map ...............................................................................................