Genetic control of Apigenin di-C-glycoside biosynthesis in bread wheat grain and their role as yellow pigments of Asian alkaline noodles Submitted by Grace Yasmein Wijaya This thesis is submitted to Faculty of Sciences in fulfilment of the requirements for the degree Doctor of Philosophy Faculty of Science The University of Adelaide November 2012 This book is An Answer to Prayers of a Long list of Believers I have been very blessed and loved with all of your spiritual supports, for His guidance and protections, and sincerely will not have enough to thank you all..... May you all be blessed and loved, too As I have been always, yasmein Table of Content Table of Content i List of Tables x List of Figures xiii List of Supplemental Materials xxv Summary xxx Statement of Authorship xxxiv List of Publications xxxv Acknowledgement xxxvi List of Abbreviations xxxix Chapter I: General Introduction 1 1.1 Background 1 1.2 Knowledge Gap 2 1.3 Structure of thesis 2 Chapter II: Literature review 4 2.1 Asian noodles as one of the major end-products of Australian bread wheat 4 2.2 Yellow colour of YAN 5 2.2.1 Natural Compounds that contribute to the yellow colour of YAN 5 2.2.1.1 Types of natural compounds that contributes to the yellow colour of YAN and their roles in plants and human health 5 2.2.1.2 Contribution of xanthophylls and ACGs to the yellow colour of alkaline noodles 8 2.2.2 Factors influencing the measurement of the yellowness of noodles and the content of xanthophyll and ACG in wheat grain 10 i 2.2.3 The amount, tissue location and composition of xanthophylls and apigenin di-C- glycosides in wheat grain and recovery in flour following milling 11 2.3 Apigenin di-C-glycoside biosynthesis in plants 12 2.3.1 Flavonoid biosynthetic pathway 12 2.3.2 Pathway leading to apigenin di-C-glycoside biosynthesis in wheat grain 15 2.3.3 UDP-sugar recognition by the glycosyltransferases 20 2.4 Genetic regulation of flavone biosynthesis in cereal grain 21 2.4.1 Genetic loci involved in the control of compounds related to the yellow colour of alkaline noodles 21 2.4.2 Transcriptional regulation of flavonoid biosynthesis 23 2.4.3 Transcription factors controlling flavonoid biosynthesis in cereal grain 24 2.5 Wheat genomics for studying genetic regulation of flavone-di-C-glycoside biosynthesis 27 2.5.1 Organization of the wheat genome and wheat genomic information 27 2.5.2 Methods for studying regulation of gene expression in wheat 30 2.5.2.1 Reverse and forward genetic approaches for studying regulation of gene expression in wheat 30 2.5.2.2 Identification of transcription factor genes through genetic mapping and positional cloning 31 2.5.2.3 Availability of resources for synteny analysis to determine transcription factor gene sequences 33 2.5.2.4 Transcript analysis of wheat genes 36 2.6 Conclusion 37 2.7 Research questions 38 2.8 Research aims 38 ii 2.9 Significance/contribution to the discipline 39 Chapter III: General Materials and Methods 40 3.1 Introduction 40 3.2 Plant Materials 40 3.2.1 Genetic resources for experiment 1 (Chapter IV): Survey of genetic variation in ACG content and composition 40 3.2.2 Genetic resources for experiment 2 (Chapter V): Quantitative assessment of content and composition of the ACGs and their contribution to YAN colour in comparison to that of lutein 41 3.2.3 Population for experiment 3 (Chapter VI): Genetic control of the ACG content and composition in bread wheat grain 42 3.2.3.1 Parents of Australian doubled haploid populations 42 3.2.3.2 Sunco/Tasman doubled haploid population used in the QTL mapping 42 3.2.3.3 Other genetic stocks used in marker analysis 44 3.3 Location, time, and condition of planting of the genetic resources 45 3.4 Phenotyping 46 3.4.1 Data collection 46 3.4.1.1 ACG analysis 46 3.4.1.2 The Use of Internal and external standard solutions in ACG Analyses 47 3.4.1.2.1 The use of vanillin internal standard in ACG analyses 47 3.4.1.2.2 The used of rutin internal standard 48 3.4.1.2.3 The use of rutin external standard 48 3.4.1.3 Hundred grain weight 48 3.4.2 Data Analysis 49 3.4.2.1 Calculations of ACG traits 49 iii 3.4.2.2 Statistical Analysis 50 Chapter IV: Apigenin di-C-glycosides (ACG) content and composition in grains of bread wheat (Triticum aestivum L.) and related species 53 4.1 Introduction 53 4.2 Material and Methods 54 4.2.1 Plant materials 54 4.2.2 Apigenin di-C-glycoside analysis 56 4.2.3 Experimental design and data analysis 56 4.3 Results and discussion 57 4.3.1 Apigenin di-C-diglycoside analysis 57 4.3.2 Apigenin di-C-glycoside concentration and content per grain 58 4.3.2.1 ACG concentration 58 4.3.2.2 ACG content per grain 60 4.3.2.3 ACG content of reciprocal F1 and parental grain 64 4.3.2.4 ACG content of nullisomic-tetrasomic lines of Chinese Spring and Chinese Spring/Triticum spelta chromosome substitution lines 66 4.3.2.5 Relative effects of genotype and environment on ACG content 66 4.3.3 Apigenin di-C-glycoside composition, ACG1/ACG2 67 4.3.3.1 ACG composition and pedigree of Triticum aestivum L. cultivars 68 4.3.3.2 ACG1/ACG2 ratio in reciprocal F1 grains and their parents 69 4.3.3.3 ACG composition of nullisomic-tetrasomic lines of Chinese Spring 69 4.3.3.4 Correlation among ACG components 71 4.4 Conclusions 72 Chapter V: Contribution of Apigenin di-C-glycoside (ACG) to the colour of yellow alkaline noodles (YAN) in comparison to that of Lutein 77 iv 5.1 Introduction 77 5.2 Materials and Methods 79 5.2.1 Plant materials 79 5.2.2 Preparation of flour and noodles 80 5.2.2.1 Grain milling 80 5.2.2.2 Preparation of noodle sheets 81 5.2.3 Measurement of colour of YAN and WSN 82 5.2.4 Analyses of yellow pigment content in grain and flour 83 5.2.4.1 ACG analysis of grain and flour 83 5.2.4.2 Lutein analysis of grain and flour 84 5.2.4.2.1 Lutein extraction and analyses 84 5.2.4.2.2 The use of internal and external standard solutions in lutein analyses 85 The use of -apo-8’-carotenal as internal standard 85 The use of xanthophylls to construct the standard curves 86 5.2.5 Estimation of ACG contribution to yellowness of YAN 86 5.2.6 Data analysis 87 5.3 Results and Discussion 88 5.3.1 ACG and lutein content in wheat grain and flour 88 5.3.1.1 The effect of milling on the proportion of ACG and lutein in relation to their tissue location in the grains 87 5.3.1.2 Comparison of Bühler and Quadrumat Junior Mills 92 5.3.1.3 The influence of grain physical properties on milling 93 5.3.2 Yellowness of YAN and its correlation with lutein and ACG concentration 97 5.3.3 Yellow colour developed in the presence of alkaline salts 98 5.3.4 Prediction of the contribution of ACG to b* (YAN-WSN) 102 v 5.3.5 Contribution of ACG to b*(YAN –WSN) 103 5.4 Conclusions 105 Chapter VI: QTL associated with Apigenin di-C-glycoside (ACG) concentration and composition in bread wheat (Triticum aestivum L.) grain and identification of candidate genes 111 6.1 Introduction 111 6.2 Material and methods 113 6.2.1 Plant material 113 6.2.2 Validation of the QTL associated with variation in ACG composition, ACG concentration, and 100 grain weight in the Sunco/Tasman 114 6.2.2.1 Construction of new genetic maps for Sunco/Tasman incorporating additional markers 114 6.2.2.2 QTL analyses of ACG concentration, ACG composition and 100 grain weight. 115 6.2.3 Addition of markers to the major QTL for ACG concentration and composition located on 7BS 116 6.2.3.1 DNA Isolation 116 6.2.3.2 SSR marker analysis 117 Polymorphism test 117 Mapping polymorphic markers in the Sunco/Tasman doubled haploid population 118 6.2.3.3 Single nucleotide polymorphism (SNP) marker analysis 119 Source of SNPs 119 SNP marker analysis 119 6.2.3.4 Apigenin-C-diglycoside analysis 122 6.2.3.5 Linkage mapping and QTL analysis 122 vi Comparison of 7BS of Sunco/Tasman with other wheat maps 123 6.2.4 Synteny between wheat, rice and Brachypodium distachyon 123 6.2.4.1 Wheat 7BS and rice chromosomes 6 and 8 123 6.2.4.2 Wheat and Brachypodium distachyon 124 6.2.5 Multiple alignment and structural analysis of candidate sequences 124 6.3 Results and discussion 125 6.3.1 ACG content and composition of Sunco/Tasman parents and doubled haploid population 125 6.3.2 QTL for ACG content and composition in Sunco/Tasman 128 6.3.3 Fine mapping of ACG QTL on 7BS of Sunco/Tasman 132 6.3.3.1 Additional markers 132 6.3.3.2 The order of SSR markers within the QTL on 7BS of Sunco/Tasman compared with other populations and a consensus map 135 6.3.3.3 QTL for the ACG traits 135 6.3.4 Synteny between the wheat 7BS chromosome region harbouring the QTL associated with variation in ACG traits with the physical maps of rice and Brachypodium distachyon 141 6.3.4.1 Synteny with rice chromosomes 6 and 8 141 6.3.4.2 Synteny with Brachypodium distachyon chromosomes 1 and 3 147 6.3.5 Candidate genes related to variation in ACG biosynthesis in wheat 151 6.3.5.1 The glycosyltransferases 151 6.3.5.2 Other candidate genes for the major and minor QTL associated with ACG content and composition.