to apple scab (Venturia inaequalis)

249 Pages·2014·2.6 MB·English

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.
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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.

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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
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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.
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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
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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
Consensus Linkage Map ....................................................................54
Comparison of Genetic Positions to Physical Map ............................55
Segregation Distortion .......................................................................55
Discussion ..........................................................................................56
Tables .................................................................................................64
Figures................................................................................................67
Chapter 2 Characterization of the defense response to Venturia inaequalis in
‘Honeycrisp’ apple, its ancestors, and progeny .........................................................81
Introduction ....................................................................................................82
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Materials and Methods .......................................................................87
Experiment 1 ..........................................................................87
Experiment 2 ..........................................................................89
Results ............................................................................................................90
Experiment 1 ......................................................................................90
Experiment 2 ......................................................................................93
Discussion ......................................................................................................93
Tables ............................................................................................................101
Figures............................................................................................................103
Chapter 3 Identification of unique sources of apple scab resistance (Rvi19 and Rvi20) in
the apple (Malus × domestica) cultivar Honeycrisp ..................................................109
Introduction ....................................................................................................110
Materials and Methods ...................................................................................115
Plant Materials and V. inaequalis Isolates .........................................115
Inoculation and Screening ..................................................................116
DNA Extraction .................................................................................117
Molecular Marker Analysis and R Gene Mapping ............................117
SSR Marker and SNP Mapping .........................................................119
Validation of the Resistance in a New Zealand Population ...............121
Candidate Genes ................................................................................121
Gene Pyramiding in Disease Resistant Selections .............................122
Results ............................................................................................................123
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Discussion ......................................................................................................127
Tables ............................................................................................................134
Figures............................................................................................................144
Chapter 4 Population genetic structure of Minnesota isolates of Venturia inaequalis
collected from diverse Malus species and cultivars ...................................................156
Introduction ....................................................................................................157
Materials and Methods ...................................................................................162
Monoconidial Isolate Collection ........................................................162
DNA Extraction and SSR Genotyping ..............................................164
Data Analysis .....................................................................................166
Results ............................................................................................................168
Discussion ......................................................................................................171
Tables ............................................................................................................178
Figures............................................................................................................188
Conclusions ............................................................................................................192
Literature Cited .......................................................................................................198
Appendix Field screening of four ‘Honeycrisp’ full-sib families identifies QTL for apple
scab resistance using a pedigree based mapping approach ........................................218
Materials and Methods ...................................................................................218
Plant Material .....................................................................................218
Inoculum ............................................................................................218
Scab Assessment ................................................................................219
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