Characterizing the host response and genetic control of resistance in 'Honeycrisp' to apple scab (Venturia inaequalis) A Dissertation SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Matthew Daniel Clark IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY James J. Luby & James M. Bradeen January, 2014 © Matthew Daniel Clark 2014 Acknowledgements I must express considerable gratitude to my advisors Dr. Jim Luby and Dr. Jim Bradeen for their expertise, guidance, and support throughout this project. The opportunities to travel to meetings, participate in a large research project, and conduct research in other laboratories around the world would not have been possible without their gracious support. Additionally, my committee members Drs. Hokanson, Stupar, and Blanchette provided editorial feedback and leadership throughout this process. David Bedford provided specific data sets and plant material, but more importantly provided his time and expertise on his successful approach to apple breeding. Dr. Vincent Bus was a gracious collaborator and host who provided the technical tools and the deep knowledge base to initiate and complete this project. The other researchers at Plant & Food Research Ltd., New Zealand, Susan Gardiner and Heather Bassett also provided data for Chapter 3. Dr. Stefanie Dukowic-Schulze was instrumental in microscopy work in the Chen lab. Josh Friell and Michael Nelson were great statistical resources and sounding boards for troubleshooting analyses in R. Computational resources provided through the Minnesota Super Computing Institute made much of this work possible. Much of the work in this thesis would not have been possible without the collaboration of Cari Schmitz, an excellent scientist and a good friend. The RosBREED project (USDA SCRI 2009-51181-05808) afforded collaboration in Amy Iezzoni’s lab where Dr. Umesh Rosyara became an important resource on numerous projects (including Chapter 1). Dr. Nahla Bassil, Elisabeth Alperin, and April Nyberg had a key role in DNA extraction when failures at UMN set us back. Other RosBREED experts Drs. Kate Evans, Susan Brown, Cameron Peace, Eric van de Weg, and Marco Bink developed many tools and provided invaluable insight and direction that could only be achieved within such a willing, collaborative environment. Many students in the RosBREED project also were instrumental including: Megan Mathey, and Drs. Sujeet Verma, Travis Stegmeir, and Yingzhu Guan. Many thanks go to the Luby lab group (Steven McKay, Cari Schmitz, Luke Haggerty, Soon Li Teh, Nick Howard, Megan Mathey, John Tillman, Allison Wang) and the support staff (Ken Mullin, Les Miroslawski, Tracy Sickler, Jim Elskamp, John Mazzarella) who I cannot even begin to list the numerous ways each has been helpful on this project. To Lynne Medgaarden, you were right; this was the right path for me! I was fortunate to receive a number of awards that provided financial support and recognition including: the University of Minnesota Doctoral Dissertation Fellowship, the Hueg Landscape Arboretum Research Fellowship, William H. Alderman Memorial Graduate Award, Daniels Fellowship in Horticulture, ASHS Travel Grants, MPGI Travel Grant, and APS Travel Grant. A final thank you to friends and family who supported this crazy endeavor! Carrie Eberle, Ben Clasen, Mohamed Yakub, Erin Trieber, Mandy Waters, Jessica Biever, Garret Beier and so many others at UMN made this process exciting and fulfilling for me personally. To Dan Caldwell, who has been by my side throughout this project and now knows more about plants than he probably ever intended. i Dedication I dedicate this dissertation to those who showed me how plants were important, interesting, and such a joy to explore. The list of those farmers and green thumbs is much too long to list here. So I dedicate this work to those who taught me to plant seeds, to graft trees, to divide rhizomes, to recognize and name different species, to put-up the bounty, and to enjoy cooking and eating the food that I produced. ii Abstract Two novel apple scab resistance loci have been identified in the apple cultivar Honeycrisp, an emerging cultivar in North America that is utilized in apple breeding programs worldwide. Greenhouse inoculation experiments with the apple scab fungal pathogen, Venturia inaequalis, identified a resistance defense response in ‘Honeycrisp’ and its ancestors ‘Keepsake’, ‘Frostbite’, and ‘Northern Spy’ seven days after inoculation. The defense response ranged from necrotic and chlorotic lesions to stellate necrosis and class 3b lesions (with sporulation). A hallmark of the resistance defense response is autofluorescence at the infection site in cleared leaf tissue. Several ‘Honeycrisp’ progeny populations were screened with monoconidial isolates of V. inaequalis and segregated 3:1 for resistance, suggesting two resistance genes inherited from ‘Honeycrisp’. A consensus ‘Honeycrisp’ linkage map with 1091 SNP markers was constructed for use in mapping. Two resistance loci were mapped using linkage mapping and quantitative trait loci mapping approaches. Marker haplotypes were constructed to trace the inheritance of resistance loci. Rvi19 mapped on linkage group 1 at ~50 cM. In the Rvi19 haplotype, the 138 bp allele for the Ch-Vf1 marker cosegregates with resistance, and is identical by state (IBS) with the Rvi17 resistance in ‘Antonovka’. Rvi19 is transmitted from ‘Frostbite’ to ‘Keepsake’ to ‘Honeycrisp’, and into the resistant progeny of ‘Honeycrisp’. The other locus, Rvi20, mapped onto linkage group 15, and is IBS with a marker haplotype found in the susceptible cultivar Golden Delicious. Rvi20 is transmitted to ‘Honeycrisp’ from an unknown parent. Molecular marker haplotypes were used to identify advanced selections in the University of Minnesota breeding program iii with pyramided scab resistance. Candidate genes were identified at each haplotype that can serve as starting points for identifying the functional genes conferring resistance. A collection of V. inaequalis isolates was assembled and curated from six locations in Minnesota for screening scab resistance. These 80+ isolates were examined for genetic diversity and population structure and provide a snapshot of the diversity of the pathogen present at this time. iv Table of Contents Page List of Tables ............................................................................................................x List of Figures ...........................................................................................................xiv Introduction ..............................................................................................................1 The Host: Apple .............................................................................................4 The Pathogen: V. inaequalis ..........................................................................6 Collection and Curation of Fungal Isolates ...................................................9 Inoculation and Disease Screening ................................................................10 Defense Response in Apple ...........................................................................12 Genetics and Mapping Approaches ...............................................................16 Known Resistance Genes ...............................................................................18 Quantitative and Polygenic Resistance ..........................................................22 Fruit Quality and the Cultivar Honeycrisp .....................................................23 Marker-Assisted Selection .............................................................................24 Molecular Tools .............................................................................................26 Candidate Genes ............................................................................................27 Transgenic Apple ...........................................................................................29 Venturia inaequalis Population Structure ......................................................30 Hypothesis and Objectives .............................................................................32 Summary ........................................................................................................34 Tables ............................................................................................................38 v Figures............................................................................................................39 Chapter 1 A consensus ‘Honeycrisp’ apple (Malus × domestica) genetic linkage map from three full-sib progeny populations ....................................................................42 Introduction ....................................................................................................43 Materials and Methods ...................................................................................47 Plant Materials ...................................................................................47 DNA Extraction Protocol ...................................................................48 Marker Data Generation and Analysis ...............................................49 Linkage Mapping ...............................................................................51 Results ............................................................................................................53 Parental Linkage Maps ......................................................................54