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Enid Perez Lara Mapping of genomic regions associated with agronomic traits and resistance to diseases in Canadian spring wheat by Enid Perez Lara A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science Department of Agricultural, Food and Nutritional Science University of Alberta © Enid Perez Lara, 2017 Abstract Wheat breeders, in addition to phenotypic selection, employ molecular markers in their programs for different purposes, including parental selection, quality control, analysis of advanced lines (cultivars), on genetic purity and identity, and for markers assisted selection. In the first study of this thesis we evaluated 158 recombinant inbred lines (RILs) population for flowering, maturity, plant height and grain yield under field conditions. With a subset of 1809 single nucleotide polymorphisms (SNPs) and 2 functional markers (Ppd-D1 and Rht-D1) we identified a total of 19 quantitative trait loci (QTLs) associated with flowering time under greenhouse (5) and field (6) conditions, maturity (5), grain yield (2) and plant height (1). These QTLs explained between 6.3 and 37.8% of the phenotypic variation. Only the QTLs on both 2D chromosome (adjacent to Ppd-D1) and 4D chromosome (adjacent Rht-D1) had major effects and, respectively reduced flowering and maturity time up to 5 days with a yield penalty of 436 kg ha-1 and reduced plant height by 13 cm, but increased maturity by 33 degree days. In the second study, we used genome-wide association analysis (GWAS) to identify markers associated with the wheat diseases leaf rust, stripe rust, tan spot, common bunt and three host selective toxins (HST) from Pyrenophora tritici-repentis (Ptr ToxA, B and C). We were able to identify 94 markers associated with all traits except Ptr ToxC sensitivity. Two major effect genomic regions on 5B and 1A were associated with Ptr ToxA sensitivity, of which the former coincided with the Tsn1 gene. For Ptr ToxB, two other major effect regions on chromosomes 2B and 5B. The genomic regions associated with common bunt mapped on chromosomes 2B, 4B and 7A, while those associated with leaf rust mapped at two positions on 2B. A single marker-trait was associated each to tan spot on 7B and for yellow rust on 2A. Finally, we investigated the phenotypic effect of 50 markers associated with 16 genes for resistance to rust and tan spot, and Ptr toxin reaction ii in a subset of 70 cultivars. We first report the marker makeup of the 70 cultivars to aid spring wheat breeders in parental choice for future crossing programs. We also identified 6-8 markers for yellow rust, 4-6 markers for leaf rust, 5-9 markers for tan spot resistance and 6-11 markers for Ptr ToxA insensitivity as the best predictors of the phenotypic variation observed across the 70 cultivars. iii Preface A version of Chapter 2 of this dissertation has been published as: Perez-Lara E, Semagn K, Chen H, Iqbal M, N’Diaye A, Atif Kamran, Alireza Navabi, Curtis Pozniak, Dean Spaner (2016) QTLs Associated with Agronomic Traits in the ‘Cutler’ × ‘AC Barrie’ Spring Wheat Mapping Population Using Single Nucleotide Polymorphic Markers. PLoS ONE 11(8): e0160623. doi: 10.1371/journal.pone.0160623. The population ‘Cutler’ x ‘AC Barrie’ presented in Chapter 2 was developed by Dr. Muhammad Iqbal and was phenotyped by Dr. Muhammad Iqbal, Dr. Atif Kamran, Dr. Alireza Navabi and Dean Spaner under field and greenhouse conditions from 2008 to 2011. I joined the project in 2012. I was partially responsible for collecting data in the field during seasons 2012 – 2013. I also participated in the extraction and shipping of the DNA samples to University of Saskatchewan, where they were genotyped using iSelect Illumina SNP Array. I carried out the genotyping of vernalization (Vrn-A1, Vrn-B1 and Vrn-D1), photoperiod (Ppd-A1, Ppd-B1 and Ppd-D1) and semi-dwarf genes (Rht-B1 and Rht-D1) genes in the population and carried out the statistical analysis and the QTL mapping in conjunction with Dr. Kassa Semang and Dr. Hua Chen. I wrote a manuscript that was edited significantly by Dr. Kassa Semang as well as by the co-authors and editors. A version of Chapter 3 has been submitted as a manuscript for publication to Crop Sciences as Enid Perez-Lara, Kassa Semagn, Van Anh Tran, Izabela Ciechanowska, Hua Chen, Muhammed Iqbal, Amidou N’Diaye, Curtis Pozniak, Stephen E. Strelkov, Pierre J. Hucl, Robert J. Graf, Harpinder Randhawa, D. Spaner. Population structure and genome-wide association analysis of resistance to wheat diseases and insensitivity to Ptr toxins in Canadian spring wheat using 90K SNP array. iv The wheat collection called Variety Composite used in Chapter 3 and 4 was originally put together by Dr. Curtis Pozniak and Pierre Hucl at the University of Saskatchewan. For chapter 3, I extracted the DNA samples and shipped them to the University of Saskatchewan where they were genotyped using an eSelect Illumina SNP Array. The disease scores for leaf rust, tan spot and common bunt in the diseases nursery in 2011 to 2015 were carried out by the wheat breeding group of the University of Alberta at the Edmonton Research Center, University of Alberta. Stripe rust scoring was done in Lethbridge and Creston and recorded by Dr. Harpinder Singh Randhawa and Dr. Dean Spaner. The infiltration and scoring of the Ptr ToxA, B and C was carried out by MSc. student Anh Van Tran in Dr. Stephen Strelkov’s Lab. I was responsible for genotyping the genetic polymorphism for the rust markers (Lr21, 34, 37, 46, 67 and 68; Yr10 and Sr2) as well as the tan spot markers tsn1 and tsc2. In collaboration with Dr. Kassa Semang and Dr. Hua Chen we carried out the data analysis and the GWAS analysis. I wrote the manuscript that was edited by Dr. Kassa Semang according to feedback from the co-authors and editors. For the experiment presented in Chapter 4, I carried out the genotyping of the population using 16 PCR-based markers. I did the DNA extraction, genotyping of the population, scoring of the results and wrote the manuscript. The data were analyzed in collaboration with Dr. Hua Chen and Dr. Kassa Semang who also participated in the manuscript edition for publication. I was responsible for data analysis, interpretation and presentation of the results in the manuscripts of all the studies of presented in this thesis. Dr. Dean Spaner, Dr. Kassa Semang， Dr. Hua Chen, and Dr. Muhammad Iqbal provided considerable editorial and assistance over the duration of writing the thesis. All chapters and papers submitted and all other parts of the thesis have been edited extensively by Dr. Kassa Semang and Dr. Dean Spaner. v Acknowledgments I would like to thank a huge amount of wonderful people but I will start for the most relevant for this PhD Thesis: my supervisor Dr. Dean Spaner his guidance, teaching and financial support of my studies throughout my entire PhD program. I specially thank Dr. Spaner in the first place for the opportunity to join his research team and the chance to work for him during more than five years. Thanks for his wise advice and teachings about wheat breeding, work ethics, English communication and many other topics, as well as allowing me the freedom to develop my ideas and explore new methods. I am heartedly grateful to my supervisory committee member Dr. Stephen Strelkov for his support and valuable advice to my research work and also for accepting to be a part of my committee from the very beginning of my program and for his support during my entire program. I would also like to thank my committee members: Dr. Harpinder Randhawa, Dr. Andreas Hamann and my external examiner Dr. Aaron Beattie for accepting to participate in my exam. I am also much obliged to the past committee members: Dr. Habibur Rahman, Dr. Allen Good and Dr. Michael Deyholos who challenged me and encourage me to learn new topics. I want to thank and I’m in debt to Dr Hua Chen and Dr Kassa Semagn who have been unconditional supporters as well as invaluable source of knowledge for me. Many thanks to Dr. Harpinder Randhawa (Agriculture and Agri-Food Canada, Lethbridge) and Dr. Curtis Pozniak, Dr. Amidou N’Diaye, Dr. Pierre J. Hucl (University of Saskatchewan), Dr. Muhammad Iqbal, Dr. Atif Karman (Seed Centre, The University of Punjab in Pakistan), Dr. Alireza Navabi (University of Guelph) for their contributions to my thesis. vi Many thanks for technical assistance and support throughout my research project to my supervisor’s team: Hiroshi Kubota, Klaus Strenzke, Izabela Ciechanowska, Fabiana Dias, Joe Moss, Russell Puk, Darcy Bemister, Nguyen Khan (Kan) and all the other members who had been in the wheat breeding team during different periods of my program: Joe Back, Dr. Alexander Pawari, MSc. Neshad Pazuki, Dr. Glen Hawkins, Aggar Frias Luna, Tom Keady, Ivan Adamyk and several summer students. Many thanks to Dr. Stephen Tsoi, Dr. Harleen Kaur, Dr. Juan Jovel, Dr. Jordan Patterson, Dr. Paul Stoddard for the technical advice during some part of my projects. Also thanks to Dr. Urmila Basu, Dr. Kelley Dunfield, Dr. Michael Vinsky, Dr. Francois Paradis, Dr. Iliana Strelkov, Dr. Reem Aboukhaddour, Dr. Muhammad Rahman, Dr. Rudolph Fredua- Agyeman, Dr. Berisso Quevede, Anh Van Tran, Homa Azkarian, Alireza Akhavan, for more than 5 years of technical support throughout the different steps of my projects, for lending me or sharing with me not just their knowledge but their equipment and resources. Special thanks to Swati Megha, Aarohi Suasnabas, Azam Nikzad,Aidin Foroutan Naddafi, Azadeh Yasari my partners in the lab, as well as Reza Salehi and Seyed Abolghasem Fatemi for their advice and for all the emotional support.
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