Improvement of Australian Wheat Grain Functionality for Breadmaking by Introgression of Novel High-Molecular Weight Glutenin Subunits Into Australian Cultivars
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Improvement of Australian wheat grain functionality for breadmaking by introgression of novel high-molecular weight glutenin subunits into Australian cultivars NANDITA ROY A thesis submitted to Murdoch University in fulfilment of the requirements for the degree of Doctor of Philosophy Australia-China Joint Centre for Wheat Improvement School of Veterinary and Life Sciences Murdoch University Perth, Western Australia 2019 Declaration I am Nandita Roy, certify that: This work contains no material previously submitted for a degree or diploma in any University or other tertiary institution and, to the best of my knowledge and belief, no material which has been published or written by any other person except where due reference is made in the text. i Table of Contents Declaration i Table of Contents ii Acknowledgments x Abstract xii Abbreviations xiv Publications and conferences xvi List of Tables xvii List of Figures xviii Chapter 1 1 General Introduction 1 1.1 Aims of the project 1 1.2 Background 1 1.3 Thesis outline 3 1.4 References 4 Chapter 2 6 Literature review 6 2.1 Wheat 6 2.1.1 Evolution of bread wheat 6 2.1.2 Australian Wheat 7 2.1.3 Grain composition of wheat 7 ii 2.1.4 Storage protein and baking quality 8 2.2 Wheat grain protein composition and classification 10 2.2.1 Albumins 11 2.2.2 Globulins 11 2.2.3 Gluten 11 2.2.4 Gliadins 12 2.2.5 Glutenins 12 2.3 The genetics of gluten (gliadins and glutenins) 14 2.3.1 x and y-type HMW-GSs 15 2.3.2 Expression pattern of HMW glutenins genes in different types of wheat 16 2.4 The role of HMW-GS 17 2.4.1 Allelic variation of HMW-GSs in Australian wheat cultivars 17 2.4.2 Interaction effects of HMW-GSs on baking quality 18 2.4.3 Modifying HMW-GS composition to improve baking quality 20 2.4.4 Novel HMW-GS to improve grain quality 20 2.5 Conventional solution for introducing novel protein subunits 21 2.6 The relationship between grain yield and protein content: a wheat breeding challenge 21 2.7 Genetic and environmental effects on the expression of HMW glutenin content 21 2.8 Tools for the identification of target HMW-GS for wheat breeding 22 2.9 Protein, dough, and breadmaking quality testing 23 2.10 Summary 25 2.11 References 26 iii Chapter 3 37 Agronomic and grain quality characterisation of 1Ay HMW-GS reciprocal introgression near-isogenic lines (NILs) in commercial wheat backgrounds 37 3.1 Abstract 37 3.2 Introduction 37 3.3 Materials and Methods 38 3.3.1 Plant material and breeding procedure 38 3.2.2 Target protein identification 40 3.3.3 Ay21* subunit identification and quantification 41 3.3.4 Statistical analysis 43 3.4 Results 43 3.4.1 Gluten protein composition comparison between the introgressed Ay21* reciprocal NILs 43 3.4.2 Grain and protein quality of the NILs 44 3.4.3 Plant morphological characteristics 44 3.5 Discussion 44 3.6 References 55 Chapter 4 58 Expression of the 1Ay HMW glutenin subunit in Australian wheat cultivars has a positive effect on wheat grain quality 58 4.1 Abstract 58 4.2 Introduction 58 4.3 Materials and Methods 60 4.3.1 Plant materials 60 iv 4.3.2 SDS-PAGE 60 4.3.3 MALDI-TOF-MS 60 4.3.4 SE-HPLC 61 4.3.5 RP-HPLC 61 4.3.6 Quality attributes 61 4.3.7 Statistical analysis 62 4.4 Results 62 4.4.1 Identification and characterization of Ay21* HMW-GSs 62 4.4.2 Alteration in overall protein composition 62 4.4.3 Influence of the Ay glutenin subunit on agronomic performance and dough quality 63 4.5 Discussion 64 4.6 References 72 Chapter 5 76 Introgression the of 1Ay21* gene into Australian wheat cultivar Lincoln resulted in increased protein content and baking quality without yield reduction 76 5.1 Abstract 76 5.2 Introduction 76 5.3 Materials and Methods 78 5.3.1 Introgression of the active Ay allele into Australian cultivar Lincoln 78 5.3.2 Characteristics of the wheat seed and flour physical properties 78 5.3.3 Characterisation of flour protein 79 5.3.4 Characterising dough properties 80 5.3.5 Baking and bread quality assessment 80 v 5.3.6 Statistical analysis 81 5.4 Results 81 5.4.1 Grain protein composition 81 5.4.2 Agronomical and grain quality characteristics 81 5.4.3 Flour and dough quality 82 5.4.4 Bread quality 83 5.5 Discussion 83 5.5.1 Grain yield and grain protein content relationship in NILs 83 5.5.2 Influence of the expressed Ay21* allele on dough rheological properties and bread quality 84 5.6 Conclusion 85 5.7 References 93 Chapter 6 96 Evaluation of breadmaking performance of introgression NILs carrying the expressed HMW-GS Ay subunit alleles Ay21* and AyT1 in Australian wheat cultivar 96 6.1 Abstract 96 6.2 Introduction 96 6.3 Materials and Methods 98 6.3.1 Plant material 98 6.3.2 Field trials 98 6.3.3 Characteristics of the wheat seed and flour physical properties 98 6.3.4 Identification of Ay HMW-GSs 98 6.3.5 Characterisation of dough properties 99 vi 6.3.6 Baking and bread quality assessment 99 6.3.7 Statistical analysis 99 6.4 Results 99 6.4.1 HMW-GS composition of Ay21* and AyT1 introgression NILs 99 6.4.2 Protein attributes of Ay21* and AyT1 introgression NILs 100 6.4.3 Morphological and physical characteristics of wheat grain and flour in the Ay introgression lines 100 6.4.4 Rheological properties of flour and dough of NILs 101 6.4.5 Effects of introgressed expressed Ay alleles on baking quality 101 6.5 Discussion 102 6.5.1 Differential influence on protein content and composition 102 6.5.2 Differential effects of two different, expressed Ay subunits on dough rheological properties 103 6.5.3 Differential influence of the expressed HMW-GS alleles Ay21* and AyT1 on baking quality 104 6.5.4 Influence of expressed HMW-GS Ay alleles on agronomic and grain physical properties 104 6.6 References 111 Chapter 7 114 Cloning and characterisation of the expressed 1Ay HMW glutenin subunit allele AyT2 from durum wheat and its introgression into Australian wheat cultivars 114 7.1 Abstract 114 7.2 Introduction 114 7.3 Materials and Methods 115 7.3.1 Characterization of the complete ORFs of 1AyT2 from tetraploid wheat line TTD201 115 vii 7.3.2. Phylogenetic tree construction 116 7.3.3 MALDI-TOF MS analysis 116 7.3.5 Grain and protein quality measurements 117 7.3.6 Statistical Analysis 117 7.4 Results 118 7.4.1 Identification of the AyT2 subunit 118 7.4.2 Molecular characterization of AyT2 gene 118 7.4.3 Introgression of the AyT2 subunit into Australian wheat cultivars 119 7.4.4 Protein quality and agronomical characteristics of NILs carrying the AyT2 HMW-GS 119 7.5 Discussion 119 7.5.1 Identification of a novel expressed 1Ay HMW-GS allele has great potential to improve protein quality of bread wheat 119 7.5.2 Gene and protein characterisation confirmed that the AyT2 subunit is different from the other two alleles studied in this thesis and hence novel 120 7.5.3 This study demonstrated that the AyT2 subunit is transferrable to bread wheat without causing adverse effects and that is has potential to improve protein quality in the grain 121 7.6 References 140 Chapter 8 142 General discussion 142 8.1 Introduction 142 8.2 Major outcomes of this thesis 142 8.3 General discussion 143 8.3.1 The Ay21* and AyT1 HMW-GS have the potential to improve protein and dough quality 143 viii 8.3.2 The NILs possessing expressed Ay HMW-GS can be utilised to improve quality of Australian wheat for the bakery industry 144 8.3.3 In cv. Lincoln background, the expression of the 1Ay21* allele increased the protein content without a yield penalty 145 8.3.4 Cloning and characterisation of a novel AyT2 HMW-GS allele 145 8.4 Future research directions 146 8.4.1 Field trials and quality testing of Ay21* introgression wheat lines 146 8.4.2 Development of specific molecular markers for the AyT2 subunit 146 8.5 Novelty of this thesis and potential implications for future wheat grain quality research 147 8.6 Conclusion 147 8.6 References 150 ix Acknowledgments I would like to express my sincerest gratitude to my supervisors Professor Wujun Ma and Dr. Shahidul Islam who are the mentors of my research career. I sincerely appreciate your suggestions, motivation, cordial approach and enormous support that has made this journey successful. I must express special thanks to my principal supervisor, Professor Wujun Ma, for his priceless suggestions, valuable input and support to my project. I owe him lots of gratitude for showing me a new area of research, his dynamic view of research and his provision of resources. I must thank him for giving me the freedom to manage this research while providing guidance when needed, and a research assistantship that has helped me immensely. I would like to acknowledge Dr. Shahidul Islam for unwavering support and coaxing to get the level of scientific rigour in this thesis. I appreciate all his contributions of time, ideas, productive discussions, constructive comments and guidance in the preparation of this thesis.