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Resistance in Bread Wheat (Triticum Aestivum L.) Accession Citr 2401

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Resistance in Bread Wheat (Triticum Aestivum L.) Accession Citr 2401 Genetics of Russian wheat aphid (Diuraphis noxia) resistance in bread wheat (Triticum aestivum L.) accession CItr 2401 By THANDEKA NOKUTHULA SIKHAKHANE Submitted in accordance with the requirements for the degree of MASTER OF SCIENCE in the subject LIFE SCIENCES at the UNIVERSITY OF SOUTH AFRICA SUPERVISOR : Prof T.J. Tsilo CO-SUPERVISOR : Dr V.L. Tolmay JANUARY 2017 DECLARATION Name: ______________________________________________________ Student number: ______________________________________________________ Degree: ______________________________________________________ Exact wording of the title of the dissertation or thesis as appearing on the copies submitted for examination: Genetics of Russian wheat aphid (Diuraphis noxia) resistance in bread wheat (Triticum aestivum L.) accession CItr 2401 I declare that the above dissertation/thesis is my own work and that all the sources that I have used or quoted have been indicated and acknowledged by means of complete references. ________________________ _____________________ SIGNATURE DATE STUDENT NUMBER: 57652538 i ACKNOWLEDGEMENTS I would like to extend my sincere appreciation and gratitude to: My supervisors, Prof Toi Tsilo and Dr Vicki Tolmay for always being there for me whenever I required assistance, for teaching and grooming me, The Agricultural Research Council and the National Research Foundation for funding, Dr Astrid Jankielsohn, for all the RWA information she shared with me and her images and figures she allowed me to use, Dr Scott Sydenham, for all the molecular study information, practical demonstrations and editing of my writings, Ms Emma Mollo, Ms Bongiwe Nhlapho and Mr Timmy Baloyi, for their technical assistance, Ms Juliette Kilian, for her assistance with acquiring literature, Ms Sandiswa Figlan and Mr Learnmore Mwadzingeni for all their invaluable inputs and support, Students at the ARC-Small Grain Institute, for all their assistance, My mentor and dear friend Dr Oluwatosin Ijabadeniyi, his wise advice, continuous encouragement and for always believing in me, My God-fearing parents, Siphiwe and Zanele Sikhakhane, for believing in me and for all their emotional support and prayers during this project, Above all, My Lord and Saviour Jesus Christ for giving me the strength, knowledge and understanding, for always being present and for constantly reminding me that through him, all things are possible. ii ABSTRACT The Russian wheat aphid (RWA) (Diuraphis noxia Kurdjumov) is one of the important insect pests of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and other grasses. To date, there are four RWA biotypes identified in South Africa. The virulent biotypes emerged, partly due to climate change and new genetic variations within populations of RWA; hence there is a need to improve host-plant resistance, as an effective control measure. Bread wheat (Triticum aestivum L.) accession Cereal Introduction (CItr) 2401 is known to be resistant to all RWA biotypes worldwide. The goal of this study was to use a backcrossed near-isogenic line (NIL) BC5F5 mapping population, developed from a cross between CItr 2401 and susceptible Kavkaz, to identify and validate single nucleotide polymorphism (SNP) markers linked to the resistance phenotype in CItr 2401. This was achieved by (i) conducting a preliminary study that evaluated the suitability of simple sequence repeat (SSR) markers previously reported in literature for discriminating stacked RWA resistance genes and, (ii) employing SNP markers for the first time in a RWA resistance study as a future alternative to the widely used SSR markers. None of the tested SSR markers showed potential use in marker-assisted selection (MAS). The mapping population was phenotypically evaluated for RWA resistance using the four South African biotypes, viz. RWASA1, RWASA2, RWASA3 and RWASA4. Analysis of variance (ANOVA) showed significant (P<0.001) differences of genotypes after confirming the normality of residuals and homogeneity of variance. The Illumina iSelect 9,000 wheat SNP platform was used to genotype the two crossing parents and a selection of 24 NIL genotypes from the mapping population. Eight SNP markers found to be linked to the phenotype were converted to breeder-friendly and high-throughput Kompetitive allele-specific polymerase chain reaction (KASP) markers. The designed KASP markers were validated on the two crossing parents, the 24 NIL sent for SNP genotyping, on the mapping population and on the preliminary study genotypes for their effectiveness. The KASP assays developed in this study will be useful for stacking the RWA resistance from CItr 2401 with other Dn genes effective against the RWA. Keywords: gene stacking, genotyping, KASP assay, linkage mapping, resistance, Russian wheat aphid, sequencing, simple sequence repeats, single nucleotide polymorphism, wheat iii TABLE OF CONTENTS DECLARATION ............................................................................................................................... i ACKNOWLEDGEMENTS ............................................................................................................. ii ABSTRACT ..................................................................................................................................... iii LIST OF FIGURES ........................................................................................................................ vi LIST OF TABLES ........................................................................................................................ viii LIST OF APPENDICES ............................................................................................................... viii LIST OF ABBREVIATIONS AND ACRONYMS....................................................................... ix CHAPTER 1 ..................................................................................................................................... 1 INTRODUCTION ............................................................................................................................ 1 1. Introduction ......................................................................................................................... 1 2. Motivation of the study ....................................................................................................... 4 3. Aim and Objectives of the study ........................................................................................ 5 4. Dissertation Outline ............................................................................................................ 5 CHAPTER 2 ..................................................................................................................................... 7 LITERATURE REVIEW ................................................................................................................ 7 2.1 Russian wheat aphid .............................................................................................................. 7 2.1.1 Brief background .............................................................................................................. 7 2.1.2 RWA feeding and symptoms ............................................................................................ 9 2.1.3 RWA distribution in different areas of the world ............................................................. 9 2.1.4 Types of host-plant resistance mechanisms .................................................................... 11 2.1.5 Resistance genes for RWA control ................................................................................. 15 2.2 Wheat .................................................................................................................................... 16 2.2.1 Brief background ............................................................................................................ 16 2.2.2 Uses, health benefits and side effects of wheat consumption ......................................... 17 2.2.3 Climatic preferences of wheat ........................................................................................ 18 2.2.4 Bread wheat (T. aestivum L.) production ....................................................................... 18 2.2.5 Genomics of T. aestivum L. ............................................................................................ 21 2.3 Sequencing ............................................................................................................................ 22 2.3.1 DNA sequencing ............................................................................................................ 22 2.3.2 Next-generation sequencing ........................................................................................... 23 2.3.3 Genotyping-by-Sequencing (GBS) ................................................................................. 25 iv 2.4 Molecular markers .............................................................................................................. 26 2.4.1 Brief background ............................................................................................................ 26 2.4.2 Microsatellite markers .................................................................................................... 27 2.4.3 SNP markers ................................................................................................................... 27 2.4.4 Genetic Mapping ...........................................................................................................
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