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Genetic Mapping and Molecular Characterisation of Russian Wheat Aphid Resistance Loci in Wheat

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Genetic mapping and molecular characterisation of Russian wheat aphid resistance loci in wheat By Surendran Selladurai B.Sc in Agriculture (1st Class); PG. Dip. (Biotech. and Mol. Biol.) The thesis is presented in fulfilment of the requirements for the degree of Doctor of Philosophy School of Veterinary and Life Sciences, Murdoch University, Western Australia April 2016 This thesis was supported by Murdoch University (Western Australia) providing infrastructure at the State Agricultural Biotechnology Centre (SABC) and funds through Murdoch University Research Scholarship. A co-contribution was provided by a Grains Industry Research and Development Corporation. Declaration I declare that this thesis is my own account of my research and contains as its main content work which has not been previously submitted for a degree at any tertiary education institute. Surendran Selladurai Date: ii Abstract The Russian wheat aphid (RWA, Diuraphis noxia Kurdmojov) is considered as one of the most destructive pest of wheat around the world, causing significant yield loss in wheat cultivation. A continuous process of searching for novel resistance loci (Dn) to combat evolving new RWA biotypes has been successful in providing RWA resistance to breeding programs. Australia was declared as a RWA free country but Infestation of RWA was first time reported in Tarlee, South Australia in April, 2016. A novel resistance source, PI94365 with expressing resistance to several biotypes found in other countries was selected to incorporate its resistance into the Australian cultivar EGA Gregory. A double haploid (DH) population developed through the microspore technique was phenotyped in South Africa, Turkey and Morocco with respective biotypes. A genetic linkage map was constructed with 4053 molecular markers including simple sequence repeats (SSR), genome by sequencing (GBS) and Diversity array technology (DArT) molecular markers. Major QTLs to RWA resistance were mapped on 1DS, 7DS and 7BL and minor QTLs were mapped on 3BL, 4AS and 4DL. POPSEQ genetic map distances for the QTLs identified on chromosomes 1DS and 7DS were determined by comparative genomics studies with published consensus and POPSEQ maps. A large number of molecular markers have been identified in the region of RWA resistance loci for the marker assisted plant breeding. Proteomics studies in the absence of live aphids (due to quarantine restriction in Australia) were carried out in order to reveal the resistance mechanism driven by constitutive genes. Ten proteins were significantly differentially expressed between resistance and susceptible lines selected from the double haploid population that was mapped in detail through haplotype analysis. These proteins were annotated using the current wheat genome assembly and functional annotation in relation to RWA resistance. iii Studies identified several induced proteins with RWA infestations. Differentially expressed genes identified in these studies annotated to the wheat genome together with their genetic map location assigned some of the genes to major RWA resistance QTLs and thus this study provided some new insights into RWA resistance. Over all, the work carried out in this study delivered RWA resistant wheat lines for breeding resistance cultivars that are well characterized by a broad range of molecular markers in the regions of the RWA resistance loci. The high density of new molecular markers provides for the efficient tracking of RWA resistance loci in the pipe-line of cultivar development within the framework of quarantine restrictions. iv Publications and presentations Publications in preparation Selladurai, S., Appels, R., Diepeveen, D., Tolmay, V. and EI Bouhssini, M. Genetic mapping of Russian wheat aphid resistance loci in a doubled haploid mapping population. Selladurai, S., Appels, R. and Diepeveen, D. A proteomic approach to unravel host plant resistance mechanism to Russian wheat aphid in double haploid lines derived from double haploid mapping populations. Presentations Oral Presentation: Royal Society of Western Australia – Postgraduate Symposium 2015 Title: Molecular interaction between Russian wheat aphid and wheat and its control via host plant resistance. Poster Presentation: Selladurai, S., Appels, R., Cakir, M., Webster, H., Tolmay, V., Turanli, V., EI Bouhssini, M., Berryman, D. and O’Hara, G. Genetic mapping of Russian wheat aphid resistance genes in a Double haploid population derived from Australian wheat cultivar. International Wheat conference Sydney Australia (IWC2015), 20-25 September 2015, pp142. Selladurai, S., Appels, R., Cakir, M., Tolmay, V., Turanli, V. and EI Bouhssini, M. Genetic mapping of Russian wheat aphid resistance genes in wheat. Veterinary and Life Sciences poster day, Murdoch University, November 2014. v Acknowledgements The goals achieved by this thesis were made possible by collaboration and contribution from many individuals. First and foremost I would like to thank my supervisors Professors Rudi Appels and Dean Diepeveen for their support and encouragement. My sincere gratitude goes to my principal supervisor Professor Rudi Appels for sharing his research wisdom and providing valuable advice, tireless effort and constant encouragement. I am also grateful to Rudi for adopting me (he always says) as his PhD student showing me the respect as a research colleague rather PhD student and also sharing his office and nibbles. It is unforgettable Rudi, your constant guidance, support and encouragement that you have shown me even in the difficult times you faced. There are not enough words to express my appreciation. I would like to thank Dean who joined with me as a team member in the latter part of my PhD career. Your help on statistical analysis, thesis correction and providing moral support and strength were valuable. I would like to thank Dr. Mehmet Cakir, and Professor Michael G. K. Jones for initially giving me the opportunity to change my carrier from private research to PhD research. Special thanks to Mehmet for providing seed materials and valuable advice and helping me to conduct experiments with overseas collaborators. Dr. Hollie Webster’s contributions at the early stages of my thesis production are gratefully acknowledged. Valuable contributions from Dr. Vicki Tolmay (ARC-Small Grain Institute, South Africa), Professor Ferit Turanli (Ege University, Istanbul, Turkey) and Dr. Mustapha EL-Bouhssini (International Centre for Agricultural Research in the Dry Areas (ICARDA) to conduct Russian aphid screening experiments in overseas were greatly appreciated. vi Thanks to A/P Graham O’Hara and Dr. David Berryman for their advice towards my PhD project. The financial support provided by Murdoch University is kindly acknowledged. I would like to thank Dr. Reetinder Gill and Mirza Nazim Ud Dowla for their help to conduct field experiments here in Australia. My sincere thanks go to Ms Bee Lay Addis (SABC administrative officer) for processing my purchase orders and Ms Frankie DeRousie (Academic support & Streaming Coordinator, College of Veterinary Medicine) for facilitating seminar room each week. I also thank SABC staff members Professor Michael G. K. Jones (SABC Director), Dr. David Berryman (SABC Manager) and Ms Frances Brigg (SABC Officer) for providing excellent facilities to carry out my research at the State Agricultural Biotechnology Centre (SABC), Murdoch University. To my friends at the State Agricultural Biotechnology Wujun Ma, Steve Wylie, John Fosu-Nyarko, Mike Frankie, Shahid Islam, Rongchang Yang, Chris Florides, Rowan Maddern, Karl Benton, Mirza Nazim Ud Dowla, Samier Dilipkhot, Jamie Ong, Shu Hui, Sadia Iqbal, Sharon Wescott, Xiaolong Wang, I say thank you all for having helpful discussion and social gatherings. Lastly my family, I wouldn’t hold this without you. To my wife, Vathani your love, constant encouragements and tireless support during this bumpy long journey with hurdles are unforgettable and I am forever indebted. My beautiful daughters Asmitha and Lakshana, your dedication and sacrifice in your childhood life and vii staying with me patiently while I was working was marvellous. I offer big thank you and heartfelt kiss. You have made this whole journey fruitful. viii List of abbreviation Abbreviations Description AAMPs Aphid-associated molecular pattern(s) ABA Abscisic acid ABS Australian Bureau of Statistics ADP Adenosine diphosphate AFLP Amplified Fragment Length Polymorphism ANT Adenine nucleotide translocator ARC Agricultural Research Council ATP Adenosine triphosphate AUD Australian dollar BMV Brome mosaic virus BSA Bovine serum albumin BYDV Barley yellow dwarf virus CC-NB LRR Coiled-coil nucleotide-binding domain leucine-rich repeat CIMMYT International Maize and Wheat Improvement Centre cM Centimorgan cMap Comparative map CPO Corproporphyrinogen DAFWA Department of Agriculture and Food Western Australia DArT Diversity Array Technology ddRAD double digest Restriction-Site Associated DNA sequencing DH Double haploid Dn Diuraphis noxia DNA Deoxyribonucleic acid EDU Education EPG Electrical penetration graph ET Ethylene ETI Effector-triggered immunity FAO Food and Agriculture Organisation of the United Nations FDR False discovery rate GA Gibberellic acid GBS Genotyping-by-sequencing GDC Glycine decarboxylase dehydrogenase complex GMOD Generic model organism database GST Glutathione transferases GWAS Genome wide association study HR Hypersensitive response HR-CD Hypersensitive response cell death HSP Heat shock protein ix IAA Indole-3-acetic acid ICIM Inclusive
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