Pyramiding Approaches for Potato Disease Resistance Breeding

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Kristen M. Brown-Donovan [email protected]

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Recommended Citation Brown-Donovan, Kristen M., "Pyramiding Approaches for Potato Disease Resistance Breeding" (2020). Electronic Theses and Dissertations. 3286. https://digitalcommons.library.umaine.edu/etd/3286

This Open-Access Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected]. PYRAMIDING APPROACHES FOR POTATO DISEASE RESISTANCE BREEDING

Kristen Michelle Brown-Donovan

B.A. Susquehanna University, 2004

B.S. Susquehanna University, 2011

M.S. University of Maine, 2013

A DISSERTATION

Submitted in Partial Fulfillment of the

Requirements for the Degree of Science

Doctor of Philosophy

(in Plant Science)

The Graduate School

The University of Maine

December 2020

Advisory Committee:

Gregory A. Porter, Professor of Crop Ecology and Management, Co-Advisor

Ek Han Tan, Assistant Professor of Plant Genetics, Co-Advisor

Michael E. Day, Associate Research Professor of Physiology and Ecophysiology (ret.)

Jianjun Hao, Associate Professor of Plant Pathology

Kathleen G. Haynes, Potato - Research Plant Geneticist, USDA (ret.) PYRAMIDING APPROACHES FOR POTATO DISEASE RESISTANCE BREEDING

By Kristen Michelle Brown-Donovan

Dissertation Advisors: Dr. Gregory A. Porter & Dr. Ek Han Tan

An Abstract of the Dissertation Presented In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (in Plant Science) December 2020

Potato is the fourth most important staple crop worldwide, with both high nutritional and economic values. Breeders seek improvements for many traits related to yield, dry matter, and surface and internal defects. Resistances to several diseases are also desired traits that breeders try to incorporate into their programs. The advent of DNA-based genetic technologies help breeding programs facilitate faster selection, including the use of marker-assisted selection. When specific alleles or linked markers may be present, breeders can test offspring early in the process and eliminate unwanted plant material that does not possess the trait of interest. However, these programs only work when breeders know what genes are in their program. Resistance to late blight, one of the most devastating potato diseases, is graded on a spectrum and facilitated by dozens of resistance genes throughout the Solanum genera. Genes from S. demissum, S. bulbocastanum, S. phureja and others have been introgressed into S. tuberosum, generating late blight resistant varieties like Defender, Yukon Gold, Tollocan, and Missaukee, yet their genotypes are largely unknown. Gene stacking is generally the preferred approach to incorporating gene resistance and is being pursued through trans- and cisgenic biotechnologies. But gene stacking is difficult to achieve through traditional breeding techniques without knowing what genes are present within the resistant cultivars. This study establishes the start of late blight genetic profiles for varieties and clones used as potato breeding material across the United States, as well as offering time-saving methodologies through a multiplex protocol. Additionally, a Mendelian use of marker-assisted selection is employed to estimate allele dosage in breeding clone NY121. This protocol solves a long-standing problem in potato breeding and can be easily utilized for any gene in potato to give an estimate for allele number. TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………………...v

LIST OF FIGURES………………………………………………………………………………vi

CHAPTER 1 PYRAMIDING LATE BLIGHT R GENES IN POTATO: A REVIEW…………..1

1.1. Introduction/Disease Overview……………………………………………………………1

1.2. Late Blight Resistance Genes in Potato…………………………………………………...3

1.3. Late Blight Pathology and Host Pathways of Resistance…………………………………9

1.4. Resistance Theory and Pyramiding LB R Genes………………………………………...11

1.5. Examples of R Gene Pyramiding in Potato LB Resistance………………………………12

1.6. Ways to Pyramid…………………………………………………………………………15

1.7. Potential Gene Interactions for Resistance and Susceptibility…………………………...19

1.8. Genotype Access Through Databases……………………………………………………21

1.9. Conclusion………………………………………………………………………………..23

CHAPTER 2 LATE BLIGHT RESISTANCE PROFILES OF ELITE POTATO

GERMPLASM IN THE UNITED STATES…………………………………………………….24

2.1. Abstract………………………………………………………………………………….24

2.2. Introduction……………………………………………………………………………...25

2.3. Methods………………………………………………………………………………….27

2.3.1. Plant Material……………………………………………………………………...27

2.3.2. Markers……………………………………………………………………………28

2.3.3. Polymerase Chain Reaction (PCR)………………………………………………..28

2.3.4. Phenotyping……………………………………………………………………….32

2.3.5. Field Data………………………………………………………………………….34

ii 2.3.6. Statistical Analysis………………………………………………………………...34

2.4. Results…………………………………………………………………………………...34

2.4.1. Marker Results…………………………………………………………………….34

2.4.2. Pyramided Varieties and Clones…………………………………………………..37

2.4.3. Detached Leaf Assay Results……………………………………………………...37

2.4.4. Field Data………………………………………………………………………….40

2.5. Discussion……………………………………………………………………………….43

2.5.1. Start of Late Blight Genetic Profile for Resistant Cultivars………………………49

2.6. Conclusion………………………………………………………………………………50

CHAPTER 3 ESTIMATION OF ALLELE DOSAGE OF TWO RESISTANCE GENES

IN POTATO CLONE NY121…………………………………………………………………...52

3.1. Abstract………………………………………………………………………………….52

3.2. Introduction……………………………………………………………………………...53

3.3. Methods………………………………………………………………………………….56

3.3.1. Plant Material……………………………………………………………………...56

3.3.2. DNA Isolation……………………………………………………………………..56

3.3.3. PCR Assay………………………………………………………………………...57

3.3.4. Statistical Analyses………………………………………………………………..57

3.4. Results…………………………………………………………………………………...58

3.5. Discussion……………………………………………………………………………….62

3.6. Conclusion………………………………………………………………………………65

REFERENCES…………………………………………………………………………………..67

APPENDIX A DETACHED LEAF ASSAY RESULTS AGAINST US-23 …………………...93

iii APPENDIX B LATE BLIGHT FIELD RATINGS ……………………………………………..97

APPENDIX C DISCARDED ASSAYS ……………………………………………………….104

BIOGRAPHY OF THE AUTHOR……………………………………………………………..107

iv LIST OF TABLES

Table 1.1. Cloned R genes that are functionally homologous and the Avr gene(s) they recognize…………………………………………………………………………………………..5

Table 2.1. Potato clones and varieties sampled in this study……………………………………..29

Table 2.2. Sequences of PCR primers used in the study…………………………………………..32

Table 2.3. Genetic profiles of late blight resistance genes in potato cultivars and varieties used in this study …………...... 35

Table 2.4. Combined ratings of potato clones’ or varieties’ susceptibility or resistance against US-23 from detached leaf assays.………………………………………………………..38

Table 2.5. Compilation of late blight field ratings of potato varieties and clones in three states (University of Maine, Aroostook Research Farm; The Pennsylvania State

University, State College, PA; Michigan State University, Clarksville Research Center) from 2011-2019………………………………………………………………………………….41

Table 2.6. The average monthly average temperatures of the three field testing sites during the growing season……………………………………………………………………….47

Table 3.1. Markers and primer pairs used for screening progeny of NY121……………………57

Table 3.2. Observed frequencies of markers in each family of NY121 progeny………………..58

Table 3.3. The p-values of the chi-square results comparing observed frequencies with expected frequencies if NY121 has one, two, or three or more copies of the respective alleles…………………………………………………………………………………………….60

TABLE B.1. Late blight field ratings of potato varieties and clones in three states from

2011-2019………………………………………………………………………………………..97

v LIST OF FIGURES

Figure 2.1. Potato responses to Phytophthora infestans US-23 analyzed using a detached leaf assay…………………………………………………………………………………………39

Figure 3.1. Detection of the TG689, RYSC3 and M6 markers and the control SUS3 marker from 8 individuals of family AF7505…………………………………………………….59

Figure A.1. Results of detached leaf assays against US-23 for each cultivar and clone in each trial…………………………………………………………………………………93

Figure C.1. The mean resistance of each variety and clone in a whole plant assay against late blight race US-23…………………………………………………………………...105

CHAPTER 1

PYRAMIDING LATE BLIGHT R GENES IN POTATO: A REVIEW

1.1. Introduction/Disease Overview

Late blight is one of the most devastating diseases of potato and tomato and is caused by the oomycete Phytophthora infestans (Agrios, 2005). When conditions are favorable, late blight devastates susceptible potato crops in a matter of days without intervention (Fry, 2008).

Worldwide 6 to 16% of the potato crop is lost to late blight (Haverkort et al., 2009; Savary et al.,

2019). Control of late blight is largely managed through chemical applications and cultural practices (Nowicki et al., 2012), including planting certified and treated seed, and eliminating infected cull piles and volunteers (S. B. Johnson, 2015). Metalaxyl and its stereoisomer mefenoxam proved to be suitable fungicides until the late 20th century (Goodwin et al., 1996), but were not effective deterrents again until 2009 (Saville et al., 2015). Forecasting models like

BLITECAST, SIM-CAST, NoBlight, and others were developed to aid farmers in determining when best to apply fungicides (Fry et al., 1983; Hansen, 2016; S. B. Johnson, 2015; Krause et al.,

1975), provided one is available in their region and incorporates local conditions and varietal information (Hijmans et al., 2000). These models are based on weather events and their expected effects on pathogen development, and more recent models may include additional factors like plant susceptibility, and pathogen virulence and aggressiveness (Mizubuti & Fry, 2006). New developments in spectroscopy and remote sensing are resulting in much earlier field detection of late blight and may allow chemical-based management to be deployed days before symptoms would have been detected in the past (Gold et al., 2020). Even so, if chemicals are not applied at the right time (D. A. Johnson et al., 2015) or if weather conditions are exceptionally favorable, late blight disease will still occur and can cause significant crop losses (Nowicki et al., 2012).

Host resistance is seen as an important component of integrated management of late blight disease in potato (Ahn & Park, 2013, per Enciso-Rodriguez et al., 2018; Fry, 2007;

Nowicki et al., 2012; Schepers & Bouma, 1999). Generally speaking, the primary breeding objective in potato continues to be incorporating disease resistance with excellent market traits

(Ortiz, 2020). The approach to obtain marketable and disease-resistant varieties has long been to rely primarily on phenotype (Bethke et al., 2017; Hirsch et al., 2013). However, breeding for late blight resistance is complicated by the variability seen in phenotypic response to the disease.

Resistance in the leaves and tubers appears to be managed by different genes (Rasmussen et al., 1998; Stewart et al., 1992), for example Rpi-blb1 and Rpi-bt1 conferred resistance in leaves but not in tubers (S. Chen et al., 2017). Furthermore, disease resistance may change over the course of life, displaying reduced resistance during senescence in hosts designated as resistant (Rodewald & Trognitz, 2013). Variability remains even when the same genes are present in different clones (Bisognin et al., 2002), indicating that the host genome may affect efficacy (Kramer et al., 2009). By far, the most difficult problem with late blight resistance, though, is lack of durability, as P. infestans is able to overcome host resistance and render previously resistant varieties susceptible (Black et al., 1953; Fry, 2008; Leach et al., 2001;

Vleeshouwers et al., 2011). The P. infestans genome is able to evolve quickly due to an unusually large genome size for its genus, with regions rich in mobile elements, and is able to quickly establish new avirulence (Avr) proteins (Martynov & Chizhik, 2020). The problem facing potato breeders seeking to include late blight resistance, then, is that applicable late blight resistance has been largely insufficient to this point.

One area of late blight resistance that has shown promise is gene pyramiding. Pedersen and Leath (1988) offered Nelson's (1978, 1979) thorough definition of pyramiding as “the

accumulation of genes into a single line or cultivar…[which] could be constructed with major genes, minor genes, defeated genes, effective genes, ineffective genes, race-specific genes, nonrace-specific genes, or any other type of host gene that confers resistance” against a disease.

Pyramiding genes for late blight has also been referred to in potato literature as “stacking,” especially in European literature (Haverkort et al., 2016), although stacking is more often used to describe multiple genes combined for resistances to more than one pathogen or effects (Halpin,

2005; Que et al., 2010; Taverniers et al., 2008). This review of pyramiding genes for late blight resistance in potato highlights the success thus far and relevant considerations for future pyramiding to remain successful.

1.2. Late Blight Resistance Genes in Potato

Resistance to late blight is classified into one of two categories. One type is vertical resistance, with resistance (R) genes causing cell death as a defense to infection. This is known as the hypersensitive response (HR) (Vleeshouwers et al., 2000). Potato R genes with narrow late blight resistance, or resistance to just one or a few late blight races, fall into this category. These

R genes respond to specific avirulence (Avr) proteins produced by P. infestans and are also referred to as ‘race-specific.’ The second type is horizontal or broad-spectrum resistance conferred by quantitative trait loci (QTL) (Vossen et al., 2014) and presents as delayed and/or ineffective infection, and reduced lesion growth, sporulation rates, and/or infectious periods

(Colon & Budding, 1988; Colon, Budding, et al., 1995; Dorrance et al., 2001). Broad-spectrum late blight R genes in potato respond to multiple late blight races. This latter form of resistance was initially thought to be polygenic (Ross, 1986; Turkensteen, 1993), but broad-spectrum resistance conferred by a single gene has been shown to be possible (Haverkort et al., 2016; J.

Song et al., 2003).

R genes make up 1 to 3% of the potato genome, in line with other plant species

(Rodewald & Trognitz, 2013). Dozens of late blight R genes have been identified to date (Tiwari et al., 2013), with searches continuing for novel R genes that confer late blight resistance (X.

Chen et al., 2018; Meade et al., 2020; Srivastava et al., 2018). The first late blight R genes described were R1 to R11, although not all of them have been cloned yet (Table 1.1.). They are derived from S. demissum and are the basis of the Mastenbroeck and Black differential sets, each a group of eleven potato clones differing in the presence of these R genes (Black et al., 1953;

Malcolmson & Black, 1966; Poppel et al., 2009). Since then, new nomenclature has been instituted and R genes have been given names that present some contextualization; for example,

Rpi-blb1 is a resistance gene against P. infestans found in S. bulbocastanum, because it was the first R gene cloned from S. bulbocastanum. However, this particular R gene was initially named

RB in the literature (J. Song et al., 2003). Similarly, Avr genes and proteins are now also preceded by the initials of the pathogen. Incidentally, the initial eleven R genes from S. demissum have been expanded to twelve with the discoveries of the linkage between genes R3a and R3b

(Huang et al., 2004). An extended differential set was established upon the discovery of these genes (Zhu et al., 2015). Some R genes from S. demissum have also been determined to be broad-spectrum, including R8 and R9/R9a (Haverkort et al., 2016). Haverkort et al. (2016) also classify R2 and R genes from other species as having ‘intermediate’ resistance, which apparently indicates genes that confer resistance to between 10% and 80% of P. infestans isolates in the

Netherlands.

Table 1.1. Cloned R genes that are functionally homologous and the Avr gene(s) they recognize.

Functionally Avr homologous R counterpart, if genes Spectrum known References R1 Narrow Avr1 Ballvora et al. 2002 R2 Both narrow Avr2 & Avr2 Park et al., 2005 R2-like and broad homologs Lokossou et al., 2009 Rpi-abpt Champouret, 2010 Rpi-blb3 Aguila-Galvez et al., 2018 Rpi-edn1 Kapos et al., 2019 Rpi-mcq1 Rakosy-Tican et al., 2020 Rpi-hjt1-1, -2, & -3 Rpi-snk1-1 & -2 R3a Narrow Avr3a Huang et al., 2005; Rpi-sto2 Champouret, 2010; Jo, 2013; Kapos et al., 2019 R3b Narrow Avr3b Li et al., 2011; Rietman, 2011 R8 Broad Avr8 Oosumi et al., 2009; Haverkort et al., 2016; Vossen et Rpi-bt1 al., 2016; Rpi-Smira2 R9a Broad AvrBLB2 Vossen et al., 2005; Rpi-blb2 Oh et al., 2009; Jo, 2013 Rpi-edn2 Song et al., 2003; Vossen et al., Rpi-blb1/RB Broad AvrBLB1 2003; Rpi-pta1 Vleeshouwers et al., 2008; Wang et al., 2008; Champouret, Rpi-sto1 2010; Vleeshouwers et al., 2011; Kapos et al., 2019 Rpi-mcd1 Narrow Lokossou, 2010 Śliwka et al., 2006; Foster et al., Rpi-phu1 Broad AvrVnt1 2009 Rpi-vnt1 Pel et al., 2009; Pel, 2010 Rpi-Smira1 Narrow AvrSmira1 Rietman et al., 2012 Rpi-Smira2 Broad AvrSmira2 Rietman et al., 2012

For the late blight R genes that have been characterized, all of the encoded proteins fall into the largest class of plant resistance (Gururani et al., 2012; Lokossou et al., 2009; Tiwari et al., 2013), having a cytoplasmic nucleotide-binding site (NBS) flanked by a leucine-rich repeat region (LRR) at the C-terminal end (NLR protein) (Jones & Dangl, 2006; Jupe et al., 2013;

Lokossou et al., 2009). The NBS domain contains conserved regions and is responsible for signaling (Collier et al., 2011; Kapos et al., 2019; Marone et al., 2013). The LRR domain is thought to be the region which detects the specific pathogen (Qi et al., 2012), owing to its relatively higher sequence variability across proteins (Collier & Moffett, 2009). In wheat, the

LRR region was found to be the domain which recognized the pathogen in powdery mildew

(Stirnweis et al., 2014). This domain also prevents autoactivation of downstream signaling

(Kapos et al., 2019). The NBS and LRR domains of each NLR appear to have co-evolved, as

LRR swapping yields constitutive activation (Marone et al., 2013). The N terminal region of many of these NLRs are coiled-coil (CC) (Rodewald & Trognitz, 2013; Witek et al., 2016), which sometimes have a leucine zipper (LZ) region (Lokossou et al., 2009). This domain interacts with other proteins and appears to also recognize infection (Collier et al., 2011; Collier

& Moffett, 2009), as well as signal for defense mechanisms (Kapos et al., 2019; Stirnweis et al.,

2014). In an R gene conferring resistance to Potato Virus X, Rx, the CC domain is reported as the recognition mechanism which creates a conformational change in the NBS domain, triggering a signal leading to the HR (Rairdan et al., 2008). The CC domain produced by late blight R gene

Rpi-blb1/RB also directly interacts with IPI-O (also known as AvrBLB1), specifically dependent on the amino acid at position 129 (Y. Chen et al., 2012), although not all variants of IPI-O are recognized by Rpi-blb1/RB (Y. Chen & Halterman, 2017). R3a has shown eight single amino acid substitutions across the length of the protein, indicating rapid response to P. infestans

changes (Segretin et al., 2014), and is localized to endosomes only in the presence of recognized effectors (Engelhardt et al., 2012). However, the interaction that stimulates movement is unknown, although a direct interaction between the R3a protein and the effectors is not expected

(Kapos et al., 2019). Resistance activation by R2 is mediated by BSU-LIKE PROTEIN1 (BSL1), requiring it to recognize Avr2 (Saunders et al., 2012).

Breeders and scientists look to wild relatives for crop improvement in potato (Jansky &

Spooner, 2018; Ross, 1986), in part because the progeny of cultivated germplasm can segregate for the valuable traits of the parent, leaving the progeny with fewer desirable attributes than the parents (Jansky & Spooner, 2018). However, linkage drag from the wild species can require at least three backcrossing events to eliminate the undesired wild traits (Black, 1949; Lauer, 1959;

Rudorf, 1958, per Bethke et al., 2017). Nevertheless, they have successfully introgressed resistant genes into cultivars from resistant tuber-bearing Solanum cousins such as S. demissum,

S. acaule, S. bulbocastanum, and S. phureja through conventional breeding. Germplasm from S. demissum was introduced to cultivated S. tuberosum in the US in the early 1900s (Jansky &

Spooner, 2018; Ross, 1986). Introgression has proven to be effective, but it is a time-consuming process. The first cultivar with S. demissum in its pedigree was released in 1945 (Ross, 1986), but other cultivars have taken longer. The following example is representative of introgression of resistance from wild sources: the late blight resistant varieties Bionica and Toluca took nearly 50 years to develop. These varieties utilize the R gene known as Rpi-abpt through a complex hybrid scheme: the triploid progeny of diploid S. bulbocastanum and tetraploid S. acaule were crossed with diploid S. phureja, and three subsequent backcrosses with tetraploid S. tuberosum yielded the cultivars (Haverkort et al., 2016). Despite major efforts like these to produce cultivars with late blight resistance, resistance to late blight in cultivars with race-specific R genes was lost

after a few seasons (Black et al., 1953, per Haverkort et al., 2016). For the most part, the sources of late blight resistance in modern cultivars are often unknown, in part because original introgression records are unavailable (Bethke et al., 2017), although at one point S. demissum was estimated to be integrated into half of the world’s potato varieties (Ross, 1986).

The few broad-spectrum R genes found give hope that more are out there, and the search continues in wild Solanum species (Bethke et al., 2017; Machida-Hirano, 2015; Witek et al.,

2016; Yang et al., 2017; J. Zheng et al., 2020). As discussed earlier, RB or Rpi-blb1 was one of the first broad-spectrum R genes cloned from S. bulbocastanum (J. Song et al., 2003). Homologs for R2/Rpi-blb3, R8, R9/Rpi-vnt1, and Rpi-blb2 were recently discovered in S. alandiae

(Muratova (Fadina) et al., 2019). Dozens of late blight resistant genes have been cloned (Table

1.1.) and more have been mapped (Champouret, 2010; Rodewald & Trognitz, 2013;

Vleeshouwers et al., 2008). One of the features of NLR genes is that they tend to cluster together

(Michaelmore & Myers 1998, and Young 2000, per Linden et al., 2004; Marone et al., 2013), and this remains true for potato (Enciso-Rodriguez et al., 2018; Gebhardt & Valkonen, 2001; Jo,

2013; Jupe et al., 2013; Lokossou et al., 2009; Nowicki et al., 2012). Comparative studies coupled with techniques such as resistance gene enrichment sequencing (RenSeq), in which NLR genes can be rapidly screened from long-read libraries, can help fast track discovery of novel sources of late blight resistance (Jupe et al., 2013). Similarly, RNA-Seq (Mosquera et al., 2016), diagnostic RenSeq, allele mining and other ‘omic’ tools (Van Weymers et al., 2016) have been shown to identify novel R genes. Van Weymers et al. (2016) also rightly pointed out that while wild species hold promise, additional novel sources of resistance appear to already exist in cultivated germplasm, so genebank accessions should also be investigated.

1.3. Late Blight Pathology and Host Pathways of Resistance

Late blight is a well-studied disease and the molecular mechanisms of infection and resistance are being actively investigated. Late blight disease manifests when P. infestans parasitizes live cells above ground and/or in the tubers and spreads through the plant, which, despite the reduced chances of sprouting, make infected seedpieces and volunteers particularly insidious (Andrivon, 1995; Lambert et al., 1998; Mizubuti & Fry, 2006; Zwankhuizen et al.,

1998). P. infestans can reproduce sexually and asexually. In the latter, sporangia penetrate the cells with a germ tube, and mycelium overtake the surrounding cells, creating necrotic lesions. In the leaves, sporangiophores grow through the stomata and are almost visible to the unaided eye.

A white fuzz on the underside of a leaflet in the morning with an accompanying asymmetrical water-soaked lesion is the primary diagnostic sign. The sporangiophores release more zoospores and sporangia, which can be transported via air and water. Zoospore production in P. infestans is expected to occur as described in P. cinnamomi (Fry, 2008); briefly, sporangia cytoplasmic pH rises and calcium ions increase in response to dropping temperatures, and the cytoplasm subdivides into the zoospore units, flagella are assembled, and the organelles form, polarize, and begin functioning (Hardham, 2005). Aerially, sporangia can travel for miles (Fry, 2007), remaining viable for hours in cloudy conditions (Mizubuti & Fry, 2006). Water is a necessary component for infection, as either zoospores propel themselves or sporangia germinate germ tubes that grow to compromised areas of the leaf epidermis (Fry, 2020).

As the hyphae of the pathogen grows intercellularly, finger-like organs called haustoria develop, each dissolving the cell wall and pressing into the cell membrane (Akino et al., 2014;

Whisson et al., 2016). A poorly delineated area of plant-pathogen interface called the extrahaustorial matrix is formed, and a series of proteins are secreted from the haustorium to

access the plant cytoplasm (Akino et al., 2014). The pathogen apparently co-opts host cellular machinery as some mechanisms of microbial macroautophagy are redirected by effectors to the haustoria (Dagdas et al., 2018). Importantly, effectors are believed to be discharged through the haustoria, but the precise pathways of effectors are still being elucidated (Hu et al., 2020; Liu et al., 2014; Wang et al., 2017; Whisson et al., 2007, 2016; Zheng et al., 2014). Wang et al. (2017) found that the apoplastic effector EPIC1 was delivered to the host nucleus through the ER-to-

Golgi apparatus pathway, but they could not find a delivery method for the cytoplasmic effector

Pi04314, which also located to the host nucleus. The effectors are Avr proteins and are collectively found in the RXLR class (Vleeshouwers et al., 2011), which is defined by the conserved Arg-Xaa-Leu-Arg sequence in the N-terminus following a secretion signal peptide and a diverse array of C-termini. Kapos et al. (2019) provides an excellent list of NLRs and their corresponding pathogen effectors. Effectors are not strictly inhibitory, however; some also upregulate genes to enhance infection, such as in the nuclear interactions of effector Pi04089 with host gene StKRBP1 (X. Wang et al., 2015). Generally, however, most Avr proteins’ functions are not known (Kapos et al., 2019; S. Wang et al., 2019).

NLR proteins signal that an infection has taken place in the host and are thought to induce a cascade of defense mechanisms, including b-1,3-glucanase (Agrios, 2005). It has been suggested, in the same effector-NLR setting, that upon formation of the haustorial matrix, endosomal modifications occur (Lu et al., 2012). For instance, R3a relocalizes to the endosome upon infection as a signal for defense, although its autoactive variant did not require relocalization to initiate defense (Engelhardt et al., 2012). For the R1-Avr1 interaction, R1 did not recognize Avr1 except in the host’s nucleus (Du et al., 2015). On the whole, NLRs respond to effectors that evolve quickly, with the flexibility of their domains the source of their nimbleness

and ability to respond. Because of this flexibility, NLRs have developed wide detection capacity that requires specific investigation of each (Kapos et al., 2019; Marone et al., 2013), including potential investigation within different species, as Nicotiana benthamiana was suggested to have different effector recognition mechanisms from potato (Akino et al., 2014). The specific mechanism for each NLR differs, and the activation of most NLRs remains unknown (Tamborski

& Krasileva, 2020), although a few interactions between NLRs and Avrs in Solanum species have been elucidated (Martynov & Chizhik, 2020), as were previously mentioned.

1.4. Resistance Theory and Pyramiding LB R Genes

There are several models proposed to describe host resistance to pathogen infection. The gene-for-gene theory operates on the premise that the host has a gene which encodes a protein that recognizes a protein from the pathogen (Flor, 1971). In potato, the R genes R1-R11 have been identified from S. demissum which recognize effectors from P. infestans, such that resistance gene R1 protein recognizes the protein from avirulence gene 1 (Avr1), R2 recognizes

Avr2, and so on. The pathway may be more complicated, however; for example, a recent study has shown that Avr2 has evolved at two loci such that it is recognized by two functionally homologous but otherwise unrelated R genes, R2 and Rpi-mcq1 (Aguilera-Galvez et al., 2018). A number of sources outline the relationships between P. infestans Avr genes and Solanum R genes, including Vleeshouwers et al. (2011), Jo (2013), and Martynov and Chizhik (2020).

The guard/decoy theory is an alternative to the gene-for-gene theory, where an intermediary component interacts with the Avr protein, and the results of that interaction are recognized by the NLR (Agrios, 2005). Differentiating the helper protein between a guardee and decoy is semantic from the perspective of host defense; the difference lies in whether the compound in question also fulfills another role which was established, evolutionarily speaking,

before acting as a signal eventually leading to the NLR (Kapos et al., 2019). A third theory, the bait model, works similarly to the guard/decoy model but the so-called bait directly interacts with the NLR (Jo, 2013). In some cases the bait is another NLR (Castel et al., 2019; Jones et al.,

2016; Tran et al., 2017). These theories could be combined into a single ‘helper model.’ The helper protein can then be delineated within its own context as a guard, decoy, bait, or anything else that may more accurately represent its function(s).

P. infestans has been shown to rapidly mutate and adapt to infect hosts (Nowicki et al.,

2012). This plasticity presented by P. infestans can affect the durability of R genes in potato clones, including displaying partial virulence (Vleeshouwers et al., 2011). However, if multiple

R genes are present, scientists have hypothesized that multiple mutations would be required to overcome the host, allowing late blight resistance to be maintained in the host for longer before breaking (Haesaert et al., 2015; Jo et al., 2016; Tiwari et al., 2013). Gene pyramiding is a natural extension of the gene-for-gene theory, and its modern understanding was described by Flor

(1971) after Person (1959) applied his theoretical model to Flor’s earlier (1954) flax-rust data.

The applicable solution to provide additional late blight resistance is to introduce lines that carry multiple late blight R gene loci through gene pyramiding.

1.5. Examples of R Gene Pyramiding in Potato LB Resistance

There are several lines of evidence that pyramiding R genes for late blight resistance have an additive effect as well enhancing resistance durability (Colon, Jansen et al., 1995; Colon,

Turkensteen, et al., 1995). Early supposition of pyramiding suggested that partial resistance, or

“residual resistance”, was conferred by “ineffective resistance genes” and considered homozygosity in pyramiding “essential” (Pedersen & Leath, 1988). Studies in other diseases in potato have shown that pyramiding provides additive effects. One example is from the

S pyramiding of GpaIV adg and Gpa5, two genes that provide resistance against pale cyst nematode

S (Dalton et al., 2013). On their own, GpaIV adg and Gpa5 confer partial resistance; clones with both show a greater reduction in cyst development.

For late blight resistance in potato, resistant clones and varieties have been measured for general combining ability (GCA), which is an indication of additive effects (Stewart et al., 1992, and Bradshaw et al., 1995, per Jansky, 2000). Hybrids produced from crosses between S. tuberosum and wild cousins showed good GCA (L. T. Colon, Jansen, et al., 1995). By the end of the 20th century, breeders were looking for parents that were able to reliably pass on the resistance trait. Stewart et al., 1992) tried to identify which parent varieties were best at conferring resistance to offspring in their study, and ultimately determined Stirling was the best parent for both foliar and tuber resistance.

Evidence to support the additive theory for durability has been tested by identifying genetic combinations already present in late blight resistant material, or by introducing novel combinations. For example, close inspection of one of the Mastenbroeck differentials, MaR9, revealed that its resistance was conferred by R1, R3a, R3b, R4, R8, Rpi-abpt1, and a new allele

R9a (Jo et al., 2015; Kim et al., 2012). Another Mastenbroeck differential, MaR8, was found to contain R3a, R3b, R4, and R8 (Kim et al., 2012). Both MaR8 and MaR9 appeared to exhibit durability to late blight resistance. Furthermore, potato cultivars, clones, and wild Solanum with excellent phenotypic resistance ratings have been found to have late blight pyramiding incorporated through conventional breeding and selection processes. The European variety

Fortuna has Rpi-blb1 and Rpi-blb2 (Dixelius et al., 2012). Sarpo Mira is well-known for its resistance durability, and it was found to contain five genes: R3a, R3b, R4, and two newly discovered genes Rpi-Smira1 and Rpi-Smira2, the latter of which confers quantitative resistance

(Rietman et al., 2012). Rietman et al. (2012) concluded that it was the combination of vertical and horizontal resistance that gave this cultivar its durability. In other notable studies, a cross between S. tuberosum spp. andigena and S. demissum yielded natural pyramiding of three R genes (E. C. Verzaux, 2010). Recurrent maternal half-sib selection from hybrid crosses between

S. phureja and S. stenotomum, yielded offspring with enhanced late blight resistance suggesting that gene pyramiding may have occurred (Haynes et al., 2014). Similarly, diploid populations with the Rpi-mcd1 gene from S. microdontum and the Rpi-ber gene from S. berthaultii were bred with diploid S. tuberosum, with offspring bearing both R genes performing better against late blight than offspring with either gene alone (Tan et al., 2010). Other recent diploids produced with R genes from S. avilesii (Rpi-avl1), S. tarijense (Rpi-tar1), S. chacoense (Rpi-chc1), and S. venturii (Rpi-vnt1) also showed greater resistance when two R genes were present than any one gene alone (Su et al., 2020).

While it is encouraging that combining resistance genes can lead to very good resistance against late blight, not all combinations work in an additive fashion. Subtractive resistance was found between some allelic powdery mildews NLRs in wheat, which the authors noted were independent of host background (Stirnweis et al., 2014). The authors also proposed this as a possible mechanism for phenotypic variation in resistance. In a potato study, when five S. tuberosum dihaploids underwent protoplast fusion with S. phureja IVP 101, the resulting progeny varied in tuber resistance strength relative to their disease-resistant parents, while only one individual displayed similar foliar resistance to parents; all other showed lower resistance

(Rasmussen et al., 1998). Rasmussen and colleagues (1998) pointed out several instances in which somatic hybrids were inconsistent in conferring additive effects, suggesting that the dilution effect proposed by De Maine (1978) (per Rasmussen et al., 1998) may be at work. In

these cases, the non-resistance genes of one parent are affecting the efficacy of the resistance genes from the other (Rasmussen et al., 1998).

While pyramiding is proposed as the best approach for late blight resistance durability, there is compelling evidence that pyramiding is an unsustainable solution. Pentland Dell, with its three narrow-spectrum R genes, was overcome in four seasons (Bethke et al., 2017). This strategy has been suggested to be a short-term solution with long-term implications of hypervirulence (Kapos et al., 2019). Defeated R genes still provide some resistance in the field

(Stewart et al., 2003), so they do not necessarily become irrelevant over time. Advocates for genetic engineering (GE) for late blight resistance in potato cite GE as a tool for varying genes in the field and across seasons as a method of preventing P. infestans from overcoming resistance

(Śliwka et al., 2019).

1.6. Ways to Pyramid

There are a number of approaches that can be taken by breeders to enable late blight R gene pyramiding strategies in their breeding programs. Marker-assisted selection (MAS) is already being utilized. MAS utilizes the presence of certain polymorphic sequences of markers tightly linked to R genes, or in some cases the sequences of the genes themselves, to determine whether germplasm has a gene of interest. Efficient MAS schemes are based on a selection index which combines genotype with phenotype scores using both sources of information for immediate decision-making in recurrent breeding programs (Hospital et al., 2000; Lande &

Thompson, 1990; Moreau et al., 1998; Servin et al., 2004). Such indices may already be maintained by the programs. A pyramiding theory has been suggested for diploids (Servin et al.,

2004) and may be useful for programs interested in both tetraploid and diploid breeding schemes.

Somatic fusion has been tried since the 1990s to quickly integrate late blight resistance into S. tuberosum from wild sources (Jansky, 2000). Rasmussen et al. (1998) were not able to detect any additive effects from their hybrids. Rakosy-Tican et al. (2015, 2020) were able to derive a hybrid with two broad-spectrum R genes, RB and Rpi-blb3, and two race-specific R genes, R3a and R3b. After backcrossing with late blight resistant Quarta and Baltica, eight progeny were selected that maintained these four R genes. These underwent field trials for late blight resistance and agronomic performance. While the hybrids with the broad-spectrum R genes survived a late blight outbreak and yielded well, some of the quality attributes (e.g. tuber morphology) were not quite ideal. The authors suggested that some of these lines have value as pre-breeding material.

In the last decade, Genetic Engineering (GE) has been exploited to develop pyramided R gene combinations that confer excellent late blight resistance in well-known varieties. In this approach, a vector delivers one or several genes to the selected host, or otherwise provides a means of altering genetic code. In potato for late blight resistance, Agrobacterium tumefaciens typically delivers gene(s) of interest. Rpi-vnt1.1 and Rpi-sto1 were successfully transferred to susceptible cultivars Atlantic, Bintje, and Potae9 (Jo et al., 2014). The two selected R genes were used because they are broad-spectrum, and the varieties are widely grown in the US, The

Netherlands, and DPR Korea, respectively. Potae9 already has R2 or a homolog in its background, and pyramiding it with either of Rpi-vnt1.1 and Rpi-sto1 gave it greater resistance compared to Atlantic or Bintje with only one gene. Eight successful transformation events from across the three cultivars were found, with resistance confirmed by detached leaf assays (DLA), including an ‘upgrade’ of all three varieties to broad-spectrum resistance.

The cultivar Desiree was transformed with Rpi-vnt1.1, Rpi-sto1, or an additional third broad-spectrum gene, Rpi-blb3 singly and in combination (Haesaert et al., 2015). Desiree is a susceptible cultivar widely grown in Europe. When transformed with only Rpi-vnt1.1 or Rpi- sto1, late blight disease resistance was increased by varying levels according to the transformation. When transformed with all three R genes, the GE Desiree remained unaffected by disease in the field until the very end of the growing seasons, outperforming untransformed late blight resistant varieties like Bionica, Toluca, and Nicola. None of these varieties have Rpi- vnt1.1, Rpi-sto1, or Rpi-blb3 in their background (Haesaert et al., 2015).

Another field study pyramided RB, Rpi-blb2, and Rpi-vnt1.1 into Desiree and Victoria with the intention of growing the crop without fungicide in an effort to save small-scale farmers money (Ghislain et al., 2019). The authors declared these GE varieties “completely resistant,” as they showed a three- to four-fold increase in yield over the national average of Uganda. Because the incidence of late blight was reduced in the GE Victoria, yields increased and farmers in sub-

Saharan Africa saw income increase by 40% (Ghislain & Barekye, 2019).

Genetic engineering offers an opportunity for late blight pyramiding, though it is not without drawbacks. While many of the studies already cited showed additive effects, there were difficulties. In using the Agrobacterium method of transformation, longer tDNA constructs (i.e. more genes) led to longer Agrobacterium regeneration times, increased variation which reduced true-to-type individuals (Haverkort et al., 2016), and reduced transformation frequency (Jo et al.,

2014). In order to ensure the success of GE methods, extensive knowledge of the late blight R genes would be required (Nowicki et al., 2012). In fact, the practical first step in any pyramiding approach is to “inventory” the necessary genes to keep, discard, or to combine (Vossen et al.,

2014).

Moreover, the results of GE technology are not always predictable. As part of the

Durable Resistance in potato against Phytophthora (DuRPh) project, a combination of Rpi-sto1 +

Rpi-vnt1.1 + Rpi-chc1 was transformed into Desiree but none of the individuals with the full tDNA construct fully expressed all three genes. In contrast, a three-gene construct of Rpi-vnt1.1

+ Rpi-blb3 + Rpi-sto1 selected in the same project did express all three genes in a majority of clones (Haverkort et al., 2016; Zhu et al., 2013). Likely, position affected the expression of the former combination (Stief et al., 1989). This example highlights the complications that occur with GE methods. However, despite these difficulties, broad-spectrum and durable late blight resistance still has a better chance of being deployed through GE than through conventional breeding and selection. These problems can be surmounted more quickly than can the length of time it will take to conventionally breed a similar marketable, three-gene-pyramided variety with broad-spectrum and durable late blight resistance.

Late blight resistance is ultimately about success in the field, and to that end a specific type of polyculture has been suggested as a method of durable resistance. While pyramiding multiple R genes into one variety has been effective as shown by studies already cited, a more dynamic tactic has been submitted as a way to prevent hypervirulence. Genetic polyculture is a type of intercropping in which the only variation in a planted crop is the resistance genes and alleles. In some ways, genetic polyculture is simply updated jargon for the multiline proposal

(Jensen, 1952) of the mid-twentieth century. Theoretically speaking, this is an improvement over planting susceptible varieties; practically speaking, the disease severity would be greater than planting a crop in which all plants were equally resistant because a neighbor’s disease severity will affect a plant’s ability to defend against disease (Connolly et al 1995, per Jansky, 2000). By the same token, disease severity is reduced by the presence of late blight resistant clones,

providing some late blight disease mitigation (Connolly et al., 1995). This idea has greater merit still; while this intercropping approach was not utilized in the DuRPh project, the underlying principle of multilines having little to no variation other than resistance reappeared in their “true- to-type” parameter, the modified variety had to be otherwise identical (Haverkort et al., 2016).

Zhu et al. (2015) (per Haverkort et al., 2016) have called this an “isogenic differential set”, and propose that “isogenic clones…grown in the future will vary in time and space according to the prevailing pathotypes of P. infestans” (Haverkort et al., 2016). When narrow-spectrum R genes become “non-functional,” they can be replaced (Kushalappa et al., 2016).

These studies underscore and support the contention made by Haverkort et al. (2016) that pathogen adaptation is hampered by a combination of genes with a range of effector specificity, thereby giving hosts durable resistance. Plans within the DuRPh project were based on this theory, and resulted in an ‘improved’ late blight resistant Desiree, a European variety which is typically late blight susceptible (Haesaert et al., 2015). In this study, transformed cultivars with pyramided R genes did not become infected at field sites while their transformed counterparts with single R genes did show infection, albeit delayed from untransformed Desiree (Haverkort et al., 2016). With some of their transformed cultivars, they achieved a 1:1 resistant:susceptible segregation ratio, indicating simplex inheritance.

1.7. Potential Gene Interactions for Resistance and Susceptibility

With the mechanisms of R genes needing to be unpacked on a case-by-case basis, and with so much yet to be discovered in plant defense generally, late blight pyramiding must be understood as a complex process that will likely require persistent correction. Gene interactions both helpful and obstructive seem to have an effect on resistance. To begin with, NLRs have been shown to self-associate (Y. Chen et al., 2012), oligomerizing as dimers (Kapos et al., 2019)

and pentamers (J. Wang et al., 2019) to initiate plant defense; the study of the latter form revealed that the CC domains joined to form a funnel integral to resistance initiation and cell death. This finding aligns with the hypothesis proposed for Rpi-blb1/RB by Y. Chen et al.

(2012). For dimers, while homodimerization is typical, at least one example of distinct NLRs combining has been found (Tran et al., 2017), although these NLRs had a Toll-like, Interleukin-1

Receptor (TIR) domain at their N-termini and were combining at their TIR domains. Even so, given that the specific mechanisms of so many late blight R genes are as yet unknown, including how additive resistance might be induced, oligomerization of R gene proteins is worth exploring in pyramided clones. It is possible that partial resistance is conferred because of a lack of oligomerization. Sarpo Mira may be an ideal candidate to begin such a study because there are so many R genes present in its germplasm.

The influence of host background on phenotypic expressivity, or the genetic context of the R gene in question, has been proposed as evidence for oligomerization of R genes (Gebhardt

& Valkonen, 2001) as well as epistasis (Colon, Jansen, et al., 1995; Gebhardt & Valkonen,

2001). In modeling late blight resistance for genomic selection in breeding, Enciso-Rodriguez et al. (2018) offered four models to assess genotypic effects; the general model they proposed showed more accuracy than their linear additive model in explaining variance in late blight resistance because the general model included factors of dominance and epistasis. Jansky (2000) highlighted examples of studies where the phenotypic segregation ratios in progeny diverged from expected resistant:susceptible ratios, and indicated that all authors supposed suppressor genes were at play in the host. In one case, the authors proposed that homozygosity of minor R genes provided the epistatic effect (L. T. Colon, Jansen, et al., 1995). Epistasis and allele dosage of R genes therefore remains an area of interest. MAS techniques may now be able to settle the

allele dosage question, but that is merely one use of the tool. MAS could play an important role in identifying potential epistatic genes as well as assisting with mechanism discovery. By using

MAS in conjunction with phenotype in progeny, individuals with a gene or genes of interest and varying degrees of resistance may be selected for comparative studies to interrogate mechanisms and pathways.

The role of susceptibility genes (S genes) must also be integrated into genetic studies of R genes (Langner et al., 2018). Susceptibility genes are those which produce proteins that are targets for pathogen effectors that either fail to trigger the immune response or promote pathogen growth. In potato, ethylene response factor 3 (ERF3) has been identified as an S gene, which when silenced allows for late blight resistance, and when overexpressed showed greater late blight growth (Tian et al., 2015). S genes offer another possible explanation for the divergent resistance ratios previously mentioned, as well as an explanation for variation in resistance among full-sib progeny. In their study of somatic fusion for late blight pyramiding, Rasmussen et al. (1998) suggested, among other things, that “non-resistance” genes from the susceptible parent diminished the effect of the R genes in a dilution effect. Such an effect may or may not be S genes, but their role in the late blight resistance pathway has nevertheless been implicated.

1.8. Genotype Access Through Databases

Vleeshouwers et al. (2011) described late blight resistance breeding this way: “Late blight resistance genes have been identified, bred, and deployed in agriculture in a ‘blind’ fashion without detailed knowledge of the effectors they are recognizing.” In fairness, genotyping is a recent capability. But as clones and cultivars are investigated for the genetic sources of their superior traits, it will be necessary to link genotype and phenotype data to each, especially if that clone is catalogued in a genebank (Bethke et al., 2017). Databases already hold

agronomic information or are in development for potato germplasm. Wageningen University hosts the European Potato Pedigree Database

(https://www.plantbreeding.wur.nl/PotatoPedigree/index.html), and lists late blight phenotype according to citation; for example, Abnaki is listed as having both susceptibility and medium resistance in leaf and tuber based on three different contributors. This conflict arises in part because the resistance phenotype varies in response to the pathogens in the field; in effect, this information is telling a partial story. For breeders looking to enhance late blight resistance, a cultivar like Abnaki may be overlooked as a potential parent because it is listed as ‘susceptible’ in a trial when it may be a good parent and contributor to pyramided progeny for the R genes it possesses. A potato database was at one point in progress through the SOL Genomics Network

(Bombarely et al., 2011 per Bethke et al., 2017), as was standardization of language and data

(Shrestha et al., 2010 and Guberma et al., 2011, respectively, per Bethke et al., 2017). Single nucleotide polymorphisms (SNPs) and corresponding high-resolution melting (HRM) point can be included as “functional allele activity (FAA) markers” (Yuan et al., 2009), as there is a push for genebanks to characterize holdings by allele frequencies (McCouch et al., 2012 per Bethke et al., 2017). Meade et al. (2020) suggested a “‘variome’ database” of important cultivars for the purpose of focusing marker diagnostics, as well as proposed kompetitive allele-specific PCR

(KASP) as a unifying, simple assay for gene detection and allele dosage. Perfect haplotype characterization is likely an impossibility owing the high rate of SNP in potato and the revelations that will likely come from the field of epigenetics (Spooner et al., 2014). Even so, these calls to centralize knowledge will, at the very least, help breeders interested in pyramiding late blight genes choose parents with different R genes. As our understanding of R genes matures, such databases will be instrumental for late blight resistance research.

1.9. Conclusion

Pyramiding provides the best opportunity to date for effective late blight resistance in the field, but late blight resistance remains a tricky problem with a likely complex and nuanced solution. Broad-spectrum R genes, especially in combination, appear to confer the greatest resistance and are likely to be more durable in the field. Novel R genes must continue to be sought so breeders can draw from as large a reserve as possible. As R genes are discovered, they need to be studied within the context of their genome, and together with S genes, their interactions will hopefully reveal the molecular mechanisms that confer resistance, especially additive resistance. These applications will expand the toolkit for potato breeders looking to strengthen crop resistance to late blight disease.

CHAPTER 2

LATE BLIGHT RESISTANCE PROFILES OF ELITE POTATO GERMPLASM IN THE

UNITED STATES

2.1. Abstract

Pyramiding resistance genes (R genes) in potato generally provides improved field resistance against late blight. Potato breeders interested in prioritizing late blight resistance in their programs may want to implement pyramiding so genetic profiles from elite varieties can enable targeted breeding schemes toward this goal. The purpose of this study was to begin genotyping late blight resistant elite germplasm and to phenotype the elite germplasm against contemporary late blight races. Forty varieties and clones were genotyped using markers for R genes R1, R2,

R3b, R8, Rpi-blb1/RB, and Rpi-phu1. For phenotyping, twenty varieties and clones underwent a detached leaf assay against late blight race US-23, and field data were obtained from three locations from 2011-2019. Fourteen varieties and clones were assessed across all three experiments. One late blight resistant cultivar, Abnaki, was found to be susceptible to US-23 in laboratory and field settings and genotyped with the R1 gene. The clone J117 was found to have

Rpi-blb1/RB and showed high resistance against US-23 in the laboratory and field. Several varieties were positive for R3b and these cultivars showed at least moderate resistance in the detached leaf assays. While two of the varieties that were pyramided with R1 also showed good field resistance, the detached leaf assay indicates that the combination is likely non-additive.

These findings can be readily applied by potato breeders looking to broaden late blight resistance by leveraging novel R gene combinations.

2.2. Introduction

Late blight is caused by the biotrophic oomycete Phytophthora infestans and remains one of the most devastating diseases in potato (Solanum tuberosum L.) worldwide (Savary et al.,

2019). Under conducive environmental conditions, late blight can destroy a field in less than a week (Fry, 2008). This is of particular concern for the potato, which is the third most important staple crop consumed in the world and provides carbohydrates and nutrients, including vitamin

C, folic acid, ascorbic acid, dietary fiber and potassium (Camire et al., 2009; King & Slavin,

2013). Late blight can be managed through fungicide applications, planting certified clean and treated seed, and removing volunteers and cull piles (S. B. Johnson, 2015; Nowicki et al., 2012).

Moreover, utilizing host resistance can help reduce chemical applications, which can cost more than 10% of the crop’s total value (Fry, 2007).

Efforts for developing host resistance against potato diseases have been centered on introgression from wild relatives (Jansky & Spooner, 2018), and a solution to the late blight problem began in the same way more than a century ago with crosses to S. demissum (Ross,

1986). Host resistance to late blight has proven to be complex. Phenotypic responses across the plant kingdom have been categorized as either qualitative or quantitative. Qualitative resistance, also known as vertical, narrow or race-specific, show cell death at points of inoculation and in the immediate area, known as the hypersensitive response. Resistance genes (R genes) which confer qualitative resistance respond to a specific effector, a protein associated with pathogenicity from P. infestans. Qualitative R genes include several discovered in S. demissum, such as R1 (Leonards-Schippers et al., 1992), R3a and R3b (Huang et al., 2004), R6 and R7 (El-

Kharbotly et al., 1996), and R10 (Xu et al., 2013). However, qualitative R genes which have been deployed to the field, either alone or in combination, can be overcome within a few seasons

(Bethke et al., 2017; Fry, 2008). Quantitative resistance is also described as horizontal, broad- spectrum and durable, and R genes that confer this type of resistance are able to respond to different P. infestans races. While quantitative resistance has previously been thought to be polygenic (Ross, 1986; Turkensteen, 1993), broad-spectrum resistance has been associated with single R genes including Rpi-blb1/RB (J. Song et al., 2003), Rpi-blb2 (Vossen et al., 2005), Rpi- blb3 (Lokossou et al., 2009), R8 (Vossen et al., 2016), Rpi-phu1 (Śliwka et al., 2006), Rpi-sto1

(Vleeshouwers et al., 2008), and Rpi-vnt1 (Pel et al., 2009).

Resistance gene pyramiding, the selective breeding of multiple R genes against a disease into one variety or clone, is expected to increase resistance in the field and also reduce selection pressure on the pathogen (Dangl & Jones, 2001; McDonald & Linde, 2002; Mundt, 2002; Xin et al., 2012). Pyramiding has sometimes been effective against potato disease (e.g. for pale cyst nematode (Dalton et al., 2013)), but not always (e.g. for root knot nematode (Tan et al., 2009)).

For late blight, pyramiding strictly qualitative R genes has been abandoned because of the durability issue. There is, however, growing evidence that pyramided quantitative R genes and pyramided qualitative and quantitative R genes in potato cultivars provide an additive resistance effect. The highly resistant Sarpo Mira was found to have five putative R genes conferring resistance, and the authors contended that durability is enhanced because of the combination of qualitative and quantitative traits (Rietman et al., 2012; Tomczyńska et al., 2014). The

Mastenbroeck differential MaR9 was found to harbor R2, R8, and R9a (Jo et al., 2015; Kim et al., 2012). Potae9, grown in DPR Korea, already has R2 or a homolog, and was found to have increased resistance when it was transformed with Rpi-vnt1.1 or Rpi-sto1 (Jo et al., 2014). The susceptible Desiree was transformed with three broad-spectrum R genes (Rpi-vnt1.1, Rpi-sto1, and Rpi-blb3) and found to be unaffected by late blight in the field, but showed variable

resistance when only one gene was present (Haesaert et al., 2015). A different combination of R genes was transformed into Desiree and Victoria (Rpi-blb1/RB, Rpi-blb2, and Rpi-vnt1.1), and these genetically engineered (GE) varieties, grown in Uganda without fungicides, outperformed the national yield averages by three- and four-fold (Ghislain et al., 2019; Ghislain & Barekye,

2019).

Based on the evidence that late blight R gene pyramiding may confer durable resistance, conventional breeders can potentially utilize molecular methods to pyramid known late blight genes to help select for blight resistant varieties. A current limitation is that the late blight R gene makeup of many elite parental clones or varieties is not known. This study utilized a marker- based approach to identify the presence or absence of six R genes (R1, R2, R3b, R8, Rpi-blb1/RB, and Rpi-phu1) in varieties and clones that conventional potato breeders find valuable as parents when late blight resistance is a priority in their program. The information gathered here can be used in decision-making for late blight genetic pyramiding. Additionally, the objective is to distinguish cultivars which may already have multiple R genes and observe their phenotypes for additive resistance. The findings may be utilized immediately by breeders to select parents with differing genotypes to produce offspring with multiple R genes for late blight resistance. These findings may also serve as an introduction of late blight genetic profiles that accompany the varieties’ and clones’ physiological and agronomic data.

2.3. Methods

2.3.1. Plant Material

The plant material collected for this study includes several named varieties popular in public breeding programs for their known late blight resistance, as well as unreleased clones with good late blight resistance. Tubers were supplied by Michigan State University, University of

Maine, and USDA-ARS Beltsville. Some advanced material from the University of Maine’s

Potato Breeding and Variety Enhancement Program were also selected because of their pedigree.

A list of varieties and clones used in the research and their pedigrees is provided in Table 2.1.

Five replicates of each clone or variety were grown from whole or partial tubers in a growth chamber. In growth chamber conditions, tubers were planted in four-inch pots in potting soil and watered as needed and fertilized weekly with a 15-15-15 NPK solution of between 50 and 100 ppm. The temperature and relative humidity remained constant at 21°C and 60%, respectively, and the plants received 16 hours of moderate light daily.

2.3.2. Markers

Markers associated with genes R1, R2, R3b, and R8 from S. demissum were selected based on being either allele specific or having SCAR-based protocols. Similarly, a marker associated with the Rpi-blb1/RB gene from S. bulbocastanum, and one with Rpi-phu1 for S. phureja were included in this study. As a check for DNA presence, a marker for starch synthase

(SUS3) was applied. The sequences, annealing temperatures, and the studies from which the procedures were derived can be found in Table 2.2.

2.3.3. Polymerase Chain Reaction (PCR)

DNA from greenhouse-grown plants was extracted using a modified protocol by Edwards et al. (1991). Fresh leaf tissue (1-2 g) was ground with metal beads in a buffer consisting of 200 mM Tris HCl (pH 8.0), 250 mM NaCl, 25 nM EDTA (pH 8.0), 0.5% SDS, and 200 µg/mL

RNAse A. After the DNA was washed with isopropanol and 70% ethanol, the DNA was eluted in a buffer of 10 mM Tris HCl (pH 8.0) and 0.5 mM EDTA (pH 8.0). The concentrations were low enough that no reduction was necessary.

Table 2.1. Potato clones and varieties sampled in this study. Pedigree where identified: acl Solanum acaule, adg S. andigena, blb S. bulbocastanum, dms S. demissum, mcd S. microdontum, phu S. phureja, vrn S. verneii; Database: WUR Wageningen UR Plant Breeding (https://www.plantbreeding.wur.nl/), NAI North American Potato Inventory (2016) (https://www.potatoassociation.org/publications-2/north-american-potato-variety-inventory/), ECP European Cultivated Potato (https://europotato.org/); S susceptible, MR moderately resistant, R resistant.

Against Resistance Originating US-8, per Clone Pedigree source, if program and/or Database Douches name known reference et al., (2004) USDA 1276-185 x Akeley et al., NAI, Abnaki -- USDA B 4116-2 1971 ECP MSG274-3Y x University of AF2376-5 -- EB8109-1 Maine University of AF3360-1 AF1896-5 x SA8211-6 -- Maine University of AF4081-2 Stirling x A95083-56 dms mcd -- Maine A95053-61 x AC93026- University of AF4303-1 -- 9 Maine A00487-22LB x University of AF4329-4 -- A92030-5 Maine AF4386- University of NY120 x CF77154-10 -- 16 Maine EB8109-1 x Jacqueline University of AF4561-1 -- Lee Maine University of AF4624-1 Nevsky x COA00329-3 -- Maine A97044-107LB x University of AF4626-3 -- A99034-2E Maine A98025-48LB x University of AF4631-3 -- A99031-1TE Maine University of AF4696-1 IND1072 x A00082-6 -- Maine AF5039- AF2376-5 x Harley University of -- 17 Blackwell Maine AF5382- University of AF84-4 x AF2376-5 -- 12 Maine

Table 2.1. Continued

AF5400-2 AF2376-5 x NY140 University of Maine -- AF5403-3 AF2376-5 x St. Johns University of Maine -- Dakota Pearl x AF5426-1 University of Maine -- AF2376-5 AC NAI, Belleisle x ND457 De Jong et al., 1995 -- Chaleur ECP Dakota A89163-3LS x A8914-4 Farnsworth et al., 2010 NAI -- Trailblazer adg KSA195-90 x Ranger NAI, Defender dms? Novy et al., 2006 R Russet ECP phu WUR, Dorita PH 66 x Libertas dms Holland, 1961 S ECP Elba D29-10 x NY27 adg Thurston et al., 1985 NAI R S. bulbocastanum PI 243510 + S. tuberosum Davis et al., 2012; J117 blb -- PI 203900 (somatic Helgeson et al., 1998 fusion) Jacqueline Tollocan x Chaleur Douches et al., 2001 NAI R Lee International Potato I-931 x AVRDC- Center late blight LBR-9 R 1287.19 differential, Douches et al., 2004 R (Kirk et Missaukee Tollocan x NY88 Douches et al., 2010 NAI al., 2010) Michigan State MSM171- dms University, R (Kirk et Stirling x MSE221-1 A mcd (Mambetova et al., al., 2010) 2018) MR (Kirk MSM182- dms Michigan State Stirling x NY121 et al., 1 mcd University 2010) Michigan State MSQ176-1 MSI152-A x Missaukee -- University Michigan State MSQ086-3 Onaway x Missaukee -- University Saginaw Michigan State Pike x NY121 -- Chipper University

Table 2.1. Continued

NY121 N43-288 x E74-7 Cornell University R MPI 185/14 x MPI WUR, Pirola phu Germany (BRD), 1976 -- 175/28 ECP WUR, USDA 96-44 x USDA Pungo USDA, 1950 NAI, -- 528-170 ECP S. bulbocastanum Acc. 243510 + Katahdin Butler, 2015; Helgeson Sbu 8.5 (protoplast fusion), blb -- et al., 1998 backcrossed with Ivory Crisp Scottish Crop Research dms WUR, R (Kirk et Stirling 8318(6) x 8204A(4) Institute, 1991; mcd ECP al., 2010) Bradshaw, 2009 USDA 96-56 x NAI, Superior Rieman, 1962 S Minn. 59-44 ECP WUR, Tollocan Junita x 58-ER-1 Mexico, 1980 R ECP acl Scottish Crop Research dms Torridon 8372A(17) x G 5833(5) Institute, 1988; ECP R mcd Bradshaw, 2009 vrn MR, dms (Whitworth Yukon Gem Brodick x Yukon Gold Whitworth et al., 2010 phu et al., 2010)

Table 2.2. Sequences of PCR primers used in the study.

Annealing Gene Primer Sequence Temp (℃) Reference R1 76-2Sf2 cac tcg tga cat atc ctc act a 55 Sokolova et al., 2011 gta gta cct atc tta ttt ctg caa gaa t R2 R2SP-S7F tac taa cct ttt cct aga tg 55 Ohbayashi et al., 2010 per R2SP-A9R aga act ttc tca cag ctt tt Mori et al., 2011 R3b R3bF4 gtc gat gaa tgc tat gtt tct cga ga 55 Kim et al., 2012 R3bR5 acc agt ttc ttg caa ttc cag att g R8 R8T1F.1 tcg act tct tct tac gag gtc tat a 66 Endelman Lab, U. of WI R8T1R.1 tcc ggg tta cag ttg cct tca aat t (personal comm.) RB RB1 cac gag tgc cct ttt ctg ac 50 Butler, 2015; 1' aca att gaa ttt tta gac tt Colton et al., 2006 Rpi-phu1 GP94F atg tat cac aat cac att ctt gct c 56 Śliwka et al., 2006 GP94R tgt aaa acc aac aag tag tgt tgc

DNA amplification occurred in a solution of GoTaqÒ Green Master Mix (8 µL; Promega

M712), upstream and downstream primers (Table 2.2., 4 nmol, 2 µL), nuclease free water (4 µL), and 1 µL DNA. The PCR environments were set to those prescribed by the initial study, except for SUS3, which ran in the same environment as the corresponding late blight marker for which it was a check. Markers were revealed using electrophoresis in 0.8% agarose gels for all markers except for GP94, which was run on 2% agarose gel. All gels contained GelGreen and were read with a blue light transilluminator (Dark Reader DR89X Transilluminator, Clare Chemical

Research).

2.3.4. Phenotyping

Phytophthora infestans race US-23 was used as inoculant in detached leaf assays (DLA).

Cultures were obtained from the University of Wisconsin’s and Cornell University’s plant pathology holdings. Inocula were made on Rye A media at 18°C with no light. Inoculation and

assay methods were modified slightly from Karki et al. (2020). To inoculate, sporangia were collected from Rye A media after 10 to 14 days of growth. The culture received 10 mL of deionized water, was mixed, and incubated at 4°C for 2 hours to release zoospores. Zoospore concentrations were measured by hemocytometer and adjusted to 15,000-20,000 zoospores/mL.

Additional races US-8, -11, and -24 were solicited from Cornell University, and additional US-8 isolates were provided from Oregon State University. DLA tests with these races are explained in Appendix C.

For the DLA, twenty varieties and clones were selected for phenotyping based on commercial or breeding importance for late blight resistance. The third to the sixth fully developed leaves were collected from growth-chamber-grown plants between two and five months after planting. Compound leaves of three or four leaflets were placed in Petri dishes lined with wet paper towels. The abaxial side of each leaflet was inoculated with 2 to 10 droplets of 10

µL zoospore solution. The inoculated leaflets were incubated in the dark at 18°C in a growth chamber, and rated 5 dpi for susceptibility, partial resistance, or resistance on a scale similar to

Malcolmson (1976), as described by Colton et al. (2006) and Karki et al. (2020). Essentially, a disease rating scale of 1 to 9 was used, where 1 was rated most susceptible (substantial sporangia present) and 9 was most resistant (no observable lesion). Four or five replicates of each variety or clone were used in each assay, and the assay was repeated twice. An average rating of each variety or clone was calculated in each assay, with corresponding conclusions of susceptible, partially resistant, or resistant, and a final assessment was made at the conclusion of the trials.

Katahdin, Superior, and Russet Burbank were included as susceptible checks in each assay.

Detailed results from these DLA screening trials are presented in Appendix A.

2.3.5. Field Data

Field data from late blight susceptibility screening trials were obtained from Michigan

State University, The Pennsylvania State University, and the University of Maine. Data from

Michigan State can be referenced in Enciso-Rodriguez et al. (2018). When known, the prominent late blight race found in the field each season is listed. In Michigan, disease severity was rated as the relative area under the disease progress curve (RAUDPC) (Enciso-Rodriguez et al., 2018). In

Pennsylvania, disease severity was measured as the percentage of the leaf area affected by late blight and by the area under the disease progress curve (AUDPC). In Maine, the average

AUDPC was presented, and its rating was offered with mean separation relative to Defender,

Kennebec, and Superior within the same year. Detailed results from these field screening trials are presented in Appendix B.

2.3.6. Statistical Analysis

The data were analyzed in Excel using the AVERAGE function. Box-and-whisker plots were also constructed in the program from the raw DLA data.

2.4. Results

2.4.1. Marker Results

The results of the marker trials are presented in Table 2.3. The primer sequences 76-

2sf2/76-2SR are described by Ballvora et al. (2002) as an allele-specific presence/absence marker for R1 with a length of 1.4 kb. Eight varieties and clones tested positive for the R1 gene, and one additional clone, AF4081-2, showed an inconclusively faint band (Table 2.3.), which could be attributed either to contamination or low DNA concentration. The marker for R2 utilizes sequences R2SP-S7/R2SP-A9 to identify a present/absent band 800 bp in length

(Obayashi et al., 2010, per Mori et al., 2011). Only Saginaw Chipper tested positive for R2

(Table 2.3.). R3b is one of several alleles for the R3 gene (Huang, 2005) and taken with the R3a allele may be considered part of the R3 complex (G. Li et al., 2011). It is an allele-specific present/absent marker 378 bp long and identified by the sequences R3bF4/R3bR5 (Rietman et al., 2011, per Kim et al., 2012).

Table 2.3. Genetic profiles of late blight resistance genes in potato cultivars and varieties used in this study. Genes or associated markers that showed positive are marked with “X;” those with unclear results are marked with “?”.

Clone/variety R1 R2 R3b RB R8 Rpi-phu1 Present Genes

AF4081-2 ? X X R3b, R8 Stirling X X X R1, R3b, R8

Torridon X X X R1, R3b, R8

Abnaki X X R1, R8 AF4303-1 X X R1, R8 AF5039-17 X X R1, R8 AF5382-12 X X R1, R8 AF5403-3 X X R1, R8 Pungo X X R1, R8 Saginaw X X R2, R8 Chipper MSQ086-3 X ? R3b Dorita X X R3b, R8 MSM171-A X X R3b, R8 MSM182-1 X X R3b, R8 Tollocan X X R3b, R8 Yukon Gem X X R3b, R8 J117 X X RB, R8 Sbu8.5 X X RB, R8 AF4626-3 ? LBR-9 ? Pirola X X R8, Rpi-phu1

AF2376-5 X R8 AF3360-1 X R8 AF4329-4 X R8

Table 2.3. Continued

AF4386-16 X R8 AF4561-1 X R8 AF4624-1 X R8 AF4631-3 X R8 AF4696-1 X R8 AF5400-2 X R8 AF5426-1 X R8 Chaleur X R8 Dakota X R8 Trailblazer Defender X R8 Elba X R8 Jac Lee X R8 Missaukee X R8 MSQ076-1 X R8 NY121 X R8 Superior [none]

Nine clones and varieties showed positive R3b results (Table 2.3.). The sequences

R8T1F.1/R8T1R.1 are used as a present/absent marker for the R8 gene, with a length of 500 bp

(J. Endelman, personal comm.). Tollocan and Missaukee are known to be positive for this marker and were used as positive controls. The R8 marker band appeared in every variety and clone except for the negative control Superior, although three bands were light enough to be inconclusive (Table 2.3.). The Rpi-blb1/RB gene is tested using the RB-629/RB628 sequences, with an allelic-specific present/absent band 629 bp in length (Colton et al., 2006). The clone

Sbu8.5, which derived the Rpi-blb1/RB gene through protoplast fusion followed by backcrossing several times with S. tuberosum Ivory Crisp (Butler, 2015), was used a positive control. Both

Sbu8.5 and J117 tested positive for this marker (Table 2.3.). The primer sequences GP94 are

described by Śliwka et al. (2006) as a pseudo-SCAR marker for Rpi-phu1 with a length of 300 bp. Only Pirola tested positive for Rpi-phu1 (Table 2.3.).

2.4.2. Pyramided Varieties and Clones

Half of the varieties and clones sampled in this study showed positive bands for two or more markers. Eight clones and varieties were positive for R1 and R8, and eight were positive for

R3b and R8. Both clones that were positive for Rpi-blb1/RB, Sbu8.5 and J117, also were positive for R8. Pirola tested positive for Rpi-phu1 and R8, and Saginaw Chipper tested positive for R2 and R8. Two varieties, Stirling and Torridon, were the only two sampled which tested positive for three markers; they were both positive for R1, R3b, and R8.

2.4.3. Detached Leaf Assay Results

Twenty samples representing all the R gene combinations discovered were selected for

DLA based on the genotyping results. Named varieties were given preference, as were elite breeding lines well-known to breeders for their late blight resistance. Seventeen named varieties and J117 and NY121 were ultimately selected (Table 2.4.), as well as clone Sbu 8.5, which was developed at the University of Maine and selected because of its usefulness to the University of

Maine Potato Breeding Program. The DLA overall responses to US-23 from all three trials of clones and varieties are summarized in Table 2.4.; while Appendix A provides the three experimental replicates from which these conclusions were drawn. Yukon Gem (containing R3b) and J117 (Rpi-blb1/RB) were found to be resistant from the DLA experiments (Figure 2.1.). Nine varieties and clones were found to be partially resistant, including Defender, Dorita (R3b),

Jacqueline Lee, NY121, Pirola (Rpi-phu1), Sbu 8.5 (Rpi-blb1/RB), Stirling (R1, R3b), Tollocan

(R3b), and Torridon (R1, R3b).

Table 2.4. Combined ratings of potato clones’ or varieties’ susceptibility or resistance against US-23 from detached leaf assays.

Name Against US-23 Abnaki susceptible AC Chaleur susceptible Dakota Trailblazer susceptible Defender partially resistant Dorita partially resistant Elba susceptible Jacqueline Lee partially resistant J117 resistant Katahdin susceptible Missaukee susceptible NY121 partially resistant Pirola partially resistant Pungo susceptible R. Burbank susceptible Sbu 8.5 partially resistant Stirling partially resistant Superior susceptible Tollocan partially resistant Torridon partially resistant Yukon Gem resistant

Figure 2.1. Potato responses to Phytophthora infestans US-23 analyzed using a detached leaf assay. Twenty potato clones subject to DLA are listed on the x-axis. A DLA scale of 1 to 9 was used, where 1 was rated most susceptible (substantial sporangia present) and 9 was most resistant (no observable lesion). A rating between 5 and 6.99 is considered partially resistant. The box and whisker plot reflect the mean (x) rating of each variety and clone. The R genes that are present in these clones identified in this study are labeled: * R1, ° R3b, + Rpi-blb1/RB, ‡ Rpi-phu1.

The remaining varieties and clones, including known susceptible clones Katahdin, Russet

Burbank, and Superior, were found to be susceptible, having an average rating of 4 or lower. In the DLA experiments, susceptible clones and varieties included: Abnaki (R1), AC Chaleur,

Dakota Trailblazer, Elba, Missaukee, and Pungo (R1) (Figure 2.1. and Table 2.4).

2.4.4. Field Data

The late blight ratings of 36 varieties and clones were assessed mostly against US-23, though a few lines were grown in years when race US-8 and US-22 were prevalent (Table 2.5.).

Average ratings for each line per year from the three programs may be found in Appendix B.

What is immediately obvious is that there are a few lines that showed consistency across ratings, including the expected susceptible Atlantic and Superior, but there are some lines that show a wider range of ratings even against one late blight race, such as MSQ086-3 (US-23) and

AF5400-2 (US-23) (Table 2.5.). Abnaki, Atlantic, and Superior were susceptible to US-23 in the field, as was clone AF4386-16 (Table 2.5.). Clones and varieties that showed consistent resistance in the field against US-22 include: NY121, Saginaw Chipper, Stirling, and Torridon

(Table 2.5.). Clones and varieties that showed consistent field resistance against US-23 include:

J117, NY121, Sbu 8.5, Jacqueline Lee, Stirling, and Torridon (Table 2.5.). Consistent partial field resistance was found in LBR9, MSM182-1, and Dakota Trailblazer against US-22, while consistent partial field resistance against US-23 was found in AF2376-5, Defender and Saginaw

Chipper (Table 2.5.). Jacqueline Lee and Missaukee gave variable results that ranged from susceptible to resistant against US-22 (Table 2.5.).

Table 2.5. Compilation of late blight field ratings of potato varieties and clones in three states (University of Maine, Aroostook Research Farm; The Pennsylvania State University, State College, PA; Michigan State University, Clarksville Research Center) from 2011-2019. The rating assessments were provided by the researchers overseeing the trials, except for those from Michigan. Those ratings were estimated in this study based on average RAUDPC, where calculations less than 10.00 were determined to be resistant, from 10.01 to 19.99 to be moderately resistant, and greater than 20.00 to be susceptible. More information about these data may be found in a supplemental table in Appendix B. The ratings key is as follows: VS = very susceptible; S = susceptible; MS = moderately susceptible; MS/MR = moderately susceptible to moderately resistant; MR/R = moderately resistant to resistant; R = resistant; VR = very resistant. If a line only has one season’s assessment, it is designated with ‘(1)’.

Ratings against Ratings Line Years Locations Range US- US-8 US-23 22 Abnaki VS-S 2011-2017 ME, PA S (1) None VS-S AF2376-5 MS-MR/R 2012-2019 ME, PA None None MS/MR-MR/R AF4303-1 MR-R 2010-2011 ME, PA MR/R-R None None AF4329-4 MR-MR/R 2010-2011 ME, PA MR-MR/R None None AF4329-7 MS 2012 ME None None None AF4386- VS-S 2013-2014 ME, PA None None VS-S 16 AF4561-1 MS-R 2011-2012 ME, PA MR/R (1) None MR/R (1) AF4624-1 MR-MR/R 2011 ME, PA MR (1) None None MS/MR- AF4626-3 2011 ME, PA MS/MR (1) None None MR AF4631-3 MR/R 2011 ME, PA MR/R (1) None None AF4696-1 MS-VR 2011-2016 ME, PA R (1) None MS-VR AF5039- S-R 2013-2016 ME, PA None None S-R 17 AF5382- S-MS/MR 2014, 2016 ME, PA None None S-MS/MR 12 AF5400-2 S-R 2014-2016 ME, PA None None S-R AF5403-3 MS-R 2014-2016 ME, PA None None MS-R AF5426-1 S-MR 2014-2016 ME, PA None None S-MR 2010, Atlantic S MI, PA None S (1) S 2016-2017

Table 2.5. Continued

Dakota MR-R 2010, 2012 MI None MR-R None Trailblazer Defender MR-R 2011-2019 ME, PA R (1) None MR-R Dorita MR 2016-2017 PA None None MR

Elba MR 2016-2017 PA None None MR

2011- J117 R-VR 2014, ME, PA R (1) None R-VR 2016-2019 2010- Jacqueline 2012, S-R MI, PA None S-R R Lee 2014, 2016-2017 LBR9 MR-R 2010, 2012 MI None MR-R None M171-A S-MR 2010-2012 MI None S-MR None 2010- M182-1 MR-R MI None MR-R R (1) 2011, 2013 ME, MI, MS/MR- Missaukee S-VR 2010-2019 None S-R PA VR 2010- NY121 R 2011, MI None R R 2013-2015

Q086-3 S-R 2010-2014 MI None S-MR S-R

Russet 2013- S-MR PA, ME None None S-MR Burbank 2016, 2019 2016- Sbu 8.5 MR-VR PA None None MR-VR 2017, 2019 2010- Saginaw MR-R 2012, MI None R MR-R Chipper 2014-2015 2010- 2012, Stirling R-VR MI, PA None R R-VR 2014, 2017-2019 ME, MI, Superior VS-S 2011-2019 S (1) None VS-S PA

Table 2.5. Continued

2010, Torridon R 2012, MI, PA None R R 2016-2017 Yukon MR-VR 2014-2019 ME, PA None None MR-VR Gem

2.5. Discussion

Gene pyramiding has been shown to be generally effective for improving late blight resistance in potato (Haesaert et al., 2015; Haynes et al., 2014; Jo et al., 2014; Kim et al., 2012;

Tan et al., 2010; Zhu et al., 2012) and is also present in wild potato germplasm (E. Verzaux et al., 2010). This study was undertaken with an objective of assessing which R genes are present in elite breeding germplasm in the US, and further determining whether late blight resistant varieties with multiple R genes show increased resistance against US-23. Analyzing phenotypic data in the context of the marker results gives nuanced understanding of the late blight resistance system and can inform future late blight resistance breeding. This study provided initial late blight profiles for more than a dozen varieties and clones, and indicated a non-additive combination of R genes.

Despite its being cloned and studies showing efficacy with some primer combinations, the R8 gene is a difficult gene to identify based on the markers that were tested in this study.

Several primer combinations were used in the course of this study, and the combination presented here was the first to separate resistant from the susceptible clone Superior. However, the primer pair used was apparently unable to show separation within resistant clones, owing to lack of specificity across resistant germplasm (D. Douches, personal comm.). An alternative though unlikely possibility is that this study may have found that the R8 gene is overrepresented

in favored late blight resistant parent material, echoing findings by Vossen et al. (2016). Based on previous experiments (e.g. Douches et al., 2004), some varieties are known to be resistant to

US-8, and therefore presumed to have R8, including Defender, Elba, Jacqueline Lee, NY121,

Missaukee, Stirling, Tollocan, Torridon, and Yukon Gem (MR) (Table 2.1.). The source of resistance in NY121 is novel (Mayton et al., 2010), so R8 is not a factor for this clone against this race. However, most of these other varieties indicate partial resistance against US-23 (Figure

2.1.). Given that the R8 gene has shown resistance to US-23 in other studies (Christensen et al.,

2018), it is not unexpected that the gene is present in the germplasm in this study. Based on the

DLA and field data, Defender, Dorita, Elba, Jacqueline Lee, Pirola, Tollocan, and Torridon are candidates for the R8 gene. Given that analysis of the R8 gene is speculative, it will not be considered in the remaining discussion.

Fourteen varieties and clones were assessed in the field and by DLA and were genotyped with the six markers. Of these, clones J117, Sbu 8.5, Abnaki, Dorita, Stirling, Torridon, and

Yukon Gem tested positive for markers associated with one or more of the following genes: R1,

R2, R3b, or Rpi-blb1/RB (Table 2.3.). Abnaki stands out as a late blight resistant variety which is susceptible to US-23 (Tables 2.4. & 2.5.). This variety was found to be positive for R1 (Table

2.3.), which is race-specific (Leonards-Schippers et al., 1992) and has been overcome in the field

(Ross, 1986). This genotype-to-phenotype correlation is therefore unsurprising and suggests that at least one avirulence protein produced by US-23 is unrecognized by the R1 product.

The clone J117 is notably resistant against US-23. This clone is the product of protoplast fusion of S. bulbocastanum and S. tuberosum (Helgeson et al., 1998) and was previously confirmed to have Rpi-blb1/RB (Davis et al., 2012). The clone Sbu 8.5 was the only other clone in this study to have Rpi-blb1/RB (Table 2.3.), and that clone was found to be partially resistant

in the DLA (Table 2.4.) and very resistant in the field (Table 2.5.) against US-23. The Rpi- blb1/RB gene is known to be broad-spectrum and confers partial resistance against most races

(Chen & Halterman, 2017; Chen & Halterman, 2011; Karki et al., 2020; Mambetova et al., 2018;

Song et al., 2003). That J117 performed better than Sbu 8.5 in the DLA indicates that there could be an additional component affecting the resistance response. As a somatic hybrid, J117 retains more genetic similarity with S. bulbocastanum than Sbu 8.5, as the latter is a product of somatic fusion followed by several rounds of backcrossing with S. tuberosum. Because S. bulbocastanum harbors additional resistance in Rpi-blb2 and Rpi-blb3, it is possible that J117 also contains either or both of these. Additional study with these markers would bear this out.

Yukon Gem also showed resistance against US-23, although it was found to have partial resistance in the field during one season at one location (Appendix B). This cultivar was found to be positive for the R3b gene, as were Stirling and Torridon (Table 2.3.). Stirling and Torridon were the only pyramided varieties subjected that were positive for three R genes, and both were found to have partial resistance in the DLA (Table 2.4.) and resistance in the field (Table 2.5.).

All three varieties share the R3b gene, while Stirling and Torridon also have R1 (Table 2.3.). R3b is also race specific (Huang et al., 2004). A fourth variety, Dorita, has R3b (Table 2.3.), and was found to have moderate resistance against US-23 in DLAs and in the field (Tables 2.4. & 2.5.).

Taken together, the data indicate that R3b may be valuable in contributing resistance against US-

23, but there does not appear to be additive resistance when R1 is combined with R3b. Relative to the other varieties and clones without Rpi-blbl1/RB in these trials, Stirling and Torridon do appear to have greater resistance in the field, to which pyramiding with an additional R gene is a possible explanation. To that end, Yukon Gem may also have another R gene present since it has good field resistance and has S. phureja is in its background. To date, only one gene has been

identified in the literature from S. phureja (Rpi-phu1, Śliwka et al., 2006) and Yukon Gem tested negative for this marker. However, there is a good possibility that more R genes will be found in

S. phureja in future studies. In that event, it will be worth testing Yukon Gem for these additional

R genes from S. phureja.

While combining DLA results with field data provides a wider view in understanding the relationship between genotype and phenotype, stepping back to look only at marker and DLA data shows that Tollocan also has R3b (Table 2.3. & 2.4.) and was found to have partial resistance to US-23, which tracks with Dorita and Torridon, and could provide further evidence that Yukon Gem may be pyramided with an additional R gene that has not yet been identified.

Pungo, genotyped with R1 (Table 2.3.) and found susceptible in the DLA against US-23 (Table

2.4.), thus far shares the same profile as Abnaki and one would predict Pungo would likely be susceptible in the field against US-23. Pirola is noted here specifically because it is the only variety genotyped as positive for Rpi-phu1 (Table 2.3.). Its phenotyped rating of partial resistance against US-23 in the DLA (Table 2.4.) is consistent with the broad-spectrum resistance of the gene (Śliwka et al., 2006). At least partial resistance would be expected in the field. Including Pirola, Pungo, and Tollocan in field tests against US-23 in the future would validate their profiles.

The results of the DLA in this study generally trend more susceptible than the field data.

These results may be attributed to the laboratory environment. Field conditions can vary and favor either the host or the pathogen. In these DLAs, the environment was skewed to favor the pathogen. As is typical of DLA assays, they were conducted under constantly high relative humidity levels, which is not typical of field conditions. UV light is known to have a negative effect on P. infestans (Mizubuti & Fry, 2006). The DLA’s were conducted in the dark, so the late

blight inoculant was not inhibited by this factor; moreover, the leaflets likely experienced stress by reduced photosynthesis, which could have compounded the effects of disease. Additionally, cooler temperatures are favorable for late blight growth, although different races have different ideal temperatures (Mizubuti & Fry, 2006). A temperature of 18°C is generally lower than the average monthly temperatures in two of the three locations where the field testing took place

(Table 2.6.).

Table 2.6. The average monthly average temperatures of the three field testing sites during the growing season. The closest weather station to each location is listed in parentheses.

Average monthly average temperatures (°C)1 Location May June July August September Presque Isle, ME (Caribou, ME) 10.8 15.9 18.7 17.5 12.8 Clarksville, MI (Grand Rapids, MI) 14.8 20.2 22.5 21.6 17.1 State College, PA (Williamsport, PA) 15.4 20.3 22.6 21.7 17.4

1. Data collected from NOAA.gov

Daily maximum highs, especially during a stretch of warm weather, will reduce disease growth in the field. Temperature was therefore also not a constraint for P. infestans in the DLA where it can be in the field.

While field results provide the best assessment of late blight phenotype (Fry, 1978), a

DLA can provide proxy data when resources are limited (Vleeshouwers et al., 1999). It is worth noting that the phenotypic data collected in DLA must be limited solely to above-ground response. Tuber resistance to late blight does not always correlate with foliar resistance (Kirk et al., 2001; Liu & Halterman, 2009; Mayton et al., 2010; Simko et al., 2006) despite evidence that foliar and tuber resistance are conferred by the same allele (Bradshaw et al., 2004). Tuber resistance data would provide a more complete picture of resistance for each clone and variety.

Moreover, the late blight pathogen, despite its voracity in the field, is difficult to maintain in vitro and experimental results are known to be inconsistent (e.g. Cai et al., 2018; Goodwin et al., 1995; Jansky, 2000) and can be difficult obtain even in the field (Tan et al., 2008). Additional assays with other late blight races would produce fuller, if more complex, late blight profiles of elite germplasm. The scale and scope of such an in-depth study would require a larger network or team of researchers focused on the problem. Projects like the Durable Resistance in potato against Phytophthora (DuRPH) program in the Netherlands model what late blight resistance studies could be with extensive cooperation and focus. Their output was prolific and useful for research and commercial application, and their success could be partially attributed to human and financial resources allocated to it. Additionally, the clones and varieties in this study were not examined for every known gene. At this point, however, not every R gene has a marker adequate for its identification; the R8 marker in this study is representative of that fact. As more markers become available, this germplasm panel could potentially be revisited.

The roles of R genes are not fully understood yet, although scientists are making headway both in terms of their molecular mechanism (Engelhardt et al., 2012) and the general function of the NLR protein class (Jubic et al., 2019; Lolle et al., 2020). In the late blight-potato system, genetic resistance sources may eventually need to be thought of in quantitative terms, where R genes are presented within context or relationship with other genes, especially when the effects are additive. Pyramiding is one form of this holistic thinking. The realization of the role that S- genes may have on resistance (Langner et al., 2018; Tian et al., 2015) underscores this position.

Furthermore, some genes have been correlated with late blight resistance but are not R genes.

Álvarez et al. (2017) discovered that StTL15A, producing a protein found in the thylakoid lumen, and StGP28, a stem glycoprotein, were associated with increased resistance, and allele

dosage affected the intensity of the effect, with homozygosity increasing resistance. None of their populations contained both, so they were unable to determine if there were additive effects via pyramiding. Trognitz et al. (2002) found that regulatory genes and general resistance genes had more significant effects on QTLs than R genes for disease resistance, including three WRKY genes and genes associated with the phenylpropanoid pathway. The combination of specific genes and allele dosage could be the best approach for conferring higher degrees of late blight resistance.

One of the overarching results of this study is that late blight gene pyramiding is not as straightforward as the model might suggest. Genetic context matters and a systematic study of clones’ genotypes require greater depth of genotypic exploration than presented here. A comprehensive study of R genes in selected clones is the logical next step, but those genes alone may not give a complete picture of the late blight resistance capacity of the clone.

2.5.1. Start of Late Blight Genetic Profile for Resistant Cultivars

Late blight continues to plague the potato industry as one of the worst diseases it faces.

While host resistance is an important component of integrated pest management, disease resistance often must be a secondary consideration relative to other desirable market traits like yield and dry matter content. This study was meant to begin the process of establishing a genetic profile for late blight resistance in potato varieties and clones known to have resistance to the disease. It may also be used to identify which varieties are susceptible to which late blight races.

In this study, one late blight resistant variety, Abnaki, was found to be susceptible to US-23.

Another, Pungo, is likely also susceptible to US-23. This finding does not mean that these varieties are no longer relevant; they still provide some field resistance (H. E. Stewart et al.,

2003b). While analysis of individual varieties have been pursued (e.g. Sarpo Mira by Rietman et al. (2012)) and differentials have been subjected to similar analyses (Zhu et al., 2015), this is the first study to scale and accrue findings for genetic sources of late blight resistance, although elite germplasm within breeding programs have been evaluated for sources of resistance to multiple diseases (Sharma et al., 2014). The results of these studies should be integrated with this study into a world-wide file for public access. This compilation may also be considered an “inventory” upon which breeders may build a pyramiding plan (Vossen et al., 2014). As technologies advance and new methods are introduced, complete profiles should become easier to build.

The value in such an inventory lies in potato breeders optimizing their crossing program for resistance against specific races, and to be able to reference other varieties and clones for their genetic combinations and their resistance or susceptibility against various races. Knowing that Yukon Gem is moderately resistant to US-8 and resistant to US-23, while Jacqueline Lee has high resistance to US-8 and partial susceptibility to US-23 might influence a breeder to make a cross between these two, rather than crossing Jacqueline Lee with another cultivar that is also partially resistant or susceptible to US-23, such as a Defender, which is resistant to US-8. Novel combinations will also need to be pursued to try to stay ahead of P. infestans; providing the late blight genetic backgrounds and more nuanced phenotypic data can inform potato breeders’ decisions.

2.6. Conclusion

Gene pyramiding has shown great promise for improving late blight resistance in potato.

Conventional potato breeders may want to utilize pyramiding in their breeding schemes, but such decisions cannot be made without knowing which genes are available in the parents, as well as knowing each cultivar’s resistance against new late blight races. The results of this genotyping

and phenotyping study indicate that the additive effects of pyramiding may play a role in some elite germplasm, but there is no apparent additive effect of R1 and R3b. The findings also reflect the complexity of genotype and phenotype relationships in the late blight-potato system and present provide genetic and phenotype profiles that will be useful for potato breeders. With a late blight genetic profile associated with each line, breeders may target their crosses for optimum pyramiding.

CHAPTER 3

ESTIMATION OF ALLELE DOSAGE OF TWO RESISTANCE GENES IN POTATO

CLONE NY121

3.1. Abstract

The availability of DNA-based markers provides an opportunity for Mendelian studies to aid in the breeding process. This can be especially useful in conventional potato breeding as it can provide breeders an estimate on the penetrance of these traits in the tetraploid setting. In this study, an elite tetraploid potato clone, NY121, is evaluated for the allege dosages of two agronomically important resistance traits based on linked DNA markers. NY121 is highly valued by potato breeders because of its resistance against late blight, Potato Virus Y (PVY), and potato cyst nematode (PCN). In NY121, PVY resistance is conferred by the Ryadg gene and PCN resistance is conferred by the H1 gene. Two crosses were performed with NY121 using susceptible clones, and the F1 progeny of each family were tested for markers linked to the Ryadg and H1 genes. Chi-square goodness of fit tests comparing the observed results with traditional

Mendelian segregation ratios reveal that NY121 carries one copy of each gene. These results indicate that a genotype-focused Mendelian study may be utilized for allele dosage resolution in tetraploid potato studies focused on trait studies for which suitable DNA markers are established

(e.g. PVY and PCN resistance). This approach may help potato breeders with specific mandates, such as breeding for PVY or late blight resistance, to maximize the trait in potential varieties. It may additionally provide utility in identifying varieties or elite breeding clones that could be selected for dihaploid development in diploid potato breeding schemes.

3.2. Introduction

Genetic sources of disease resistance in crops are an important component of integrated pest management for farmers. For late blight, resistance can be conferred by resistance genes (R genes) but resistance in the crop has been cited as incompletely dominant (Little, 1945), not durable (Ross, 1986), and plastic (Umaerus et al., 1983, per Mizubuti & Fry, 2006, and Wastie,

1991, per Mizubuti & Fry, 2006). Mendelian studies of tetraploid potato late blight resistance were therefore abandoned when no predictable or distinct phenotypic patterns were observed

(Meyer et al., 1998), leaving the matters of allele dosage and its effects on late blight resistance, if any, unknown. Allele dosage in tetraploids was not approached again until the genetic tools of

DNA markers and SNPs were available (Andrade et al., 2009; Barrell et al., 2013; Bradshaw et al., 2004; Meyer et al., 1998; Mosquera et al., 2016; Schmitz Carley et al., 2017; Simko, 2016).

Allele dosage is known to have an effect in other aspects of potato physiology (Jansky, 2009), including amylose content (Flipse et al., 1996), intensity of red color in skin (De Jong et al.,

2003), and maturity (Massa et al., 2015). RB-mediated resistance levels against late blight disease have been found to increase with increased transgene copies (Bradeen et al., 2009) and transcription levels (Kramer et al., 2009). For broad-spectrum R genes, which typically provide partial resistance, allele dosage may play a critical role in increasing late blight resistance.

Unlike late blight, resistance to other potato diseases may be conferred through alleles that exhibit complete dominance. Potato Virus Y (PVY) can be found worldwide and cause up to

80% crop loss (De Bokx & Huttinga, 1981, per Nolte et al., 2004). For PVY resistance, there are four known genes that confer extreme resistance: Rychc (Sato et al., 2006), Ryadg (Kasai et al.,

2000), Rysto (Y.-S. Song & Schwarzfischer, 2008), and Ry(o)phu (Torrance et al., 2020). Rychc and

Ry(o)phu have been mapped to two different loci on Chromosome 9 (Sato et al., 2006; Torrance et

al., 2020). Ryadg and Rysto have been located to the same hotspot on Chromosome 11 (Brigneti et al., 1997; Gebhardt & Valkonen, 2001; Hämäläinen et al., 1997), although Rysto has also been mapped to Chromosome 12 (Y.-S. Song & Schwarzfischer, 2008; Valkonen et al., 2008).

Herrera et al. (2018) suggested the Rysto gene mapped to Chromosome 11 is allelic to Ryadg.

Grech‐Baran et al. (2020) confirmed Rysto to be on Chromosome 12 in Alicja and identified its product as a nucleotide-binding leucine-rich repeat protein with a TIR N-terminal domain (TIR-

NRL).

For potato cyst nematode (PCN), the H1 gene confers near-immunity against Globodera rostochiensis (Finkers-Tomczak et al., 2011), specifically to the Ro1 and Ro4 pathotypes (Kort et al., 1977). PCN has been an issue in New York since World War II (USDA APHIS | Golden

Nematode, n.d.), and has recently become an issue in Idaho (USDA APHIS | Pale Cyst

Nematode, n.d.) and Quebec (De Koeyer et al., 2010). The H1 gene has been mapped to the distal end of Chromosome 5 (Gebhardt et al., 1993; Pineda et al., 1993), and while neither characterization of the gene nor its product has yet been established, several studies on its related markers have been deployed, with 90% - 98% accuracy (Biryukova et al., 2008;

Meiyalaghan et al., 2018; Park et al., 2018; Schultz et al., 2010; Schultz et al., 2012).

Diploid, breeding of potatoes has been proposed and pursued for decades as an alternative to the current breeding process of phenotypic recurrent selection of tetraploid plant material (Jansky & Spooner, 2018). Industrial diploid potato breeding has already begun in

Europe and scientists continue to make inroads for widespread application of diploid potato breeding (Jansky et al., 2016; Jansky & Spooner, 2018; Lindhout et al., 2011; Stokstad, 2019).

Therefore, screening tetraploid cultivars to determine their potential as diploids can help with diploid breeding efforts. The clone NY121 is a tetraploid parent that is valuable in public,

conventional potato breeding programs. Bred by Cornell, it is a chipping clone with good market traits and disease resistance against golden and pale cyst nematodes, late blight, and PVY

(Halseth & Cornell University, 2006). PVY resistance in NY121 is conferred by the Ryadg gene

(Kasai et al., 2000; Sagredo et al., 2009), and PCN resistance is conferred by the H1 gene

(Schultz et al., 2012; Whitworth et al., 2018). Late blight resistance appears to be conferred by a novel source (Mayton et al., 2010). While NY121 was not released as a cultivar, the offspring of

NY121 have shown great commercial viability (Plaisted et al., 2019).

This study assesses the tetraploid NY121 for allele dosages of two agronomically important genes, Ryadg and H1, through a Mendelian study focused on genotype. The intention of this approach is to benefit tetraploid and diploid breeding strategies and provide a framework for allele dosage studies in tetraploid potato late blight resistance research. Until recently there has not been an easy way to identify the number of alleles for late blight in a given potato clone or variety without resorting to working with diploids (Bonierbale et al., 1993; Gebhardt et al., 1991;

Jacobs et al., 1995; X. Li et al., 1998; Little, 1945; Meyer et al., 1998; van Eck et al., 1995) because no discernable phenotype patterns could be ascertained. DNA markers are able to deliver discrete results in tetraploid potato late blight resistance studies where phenotype cannot, and has already been shown to be an effective tool for determining allele dosage in Ryadg

(Andrade et al., 2009; Kneib et al., 2017), although in both cases these were investigations for the purpose of finding clones with multiple copies of the gene. DNA-based markers have enabled conventional breeding programs to make selection decisions that include knowledge of the genetic makeup of the progeny, something known as marker-assisted selection (MAS), but markers have wide application and in this case are used to estimate allele dosage of two disease

resistance genes using Mendelian genetics in an elite breeding clone. Here, rather than screening offspring for phenotype, segregation ratios are calculated based on DNA-based markers.

3.3. Methods

3.3.1. Plant Material

We selected crosses with NY121 as a parent from the 2017-2018 University of Maine potato breeding program. Two of these crosses, AF7505 (NY121 x Nicolet) and AF7506

(NY121 x AF4552-5) yielded 225 and 750 true potato seeds respectively. Nicolet and AF4552-5 are known to be phenotypically susceptible to PVY and PCN (G. Porter, personal comm.). Of the

750 seeds from the AF7506 family, 63 germinated and were selected for genotyping. Of the 225 seeds from AF7505, 68 germinated and 66 were selected for genotyping. Two individuals from family AF7505 were excluded due to low DNA concentrations in leaf extracts.

3.3.2. DNA Isolation

DNA was extracted using a modified Edwards protocol (Edwards et al., 1991). About 1 g of leaf tissue was ground in 800 µL of extraction buffer (200 mM Tris HCl pH 8.0, 250 mM

NaCl, 25 mM EDTA pH 8.0, 0.5% SDS, and 200 µg/mL RNAse A) for 2 minutes at its highest speed. It was incubated at 37°C for 30 min and centrifuged for 10 min. The supernatant (450 µL) was precipitated with 600 µL ice cold isopropanol and centrifuged for 15 min. The pellet was washed with 70% ethanol, centrifuged, aspirated, and dried. Elution was achieved with 120 µL

TE buffer (10 mM Tris HCl pH 8.0 and 0.5 mM EDTA pH 8.0) and an overnight incubation at

4°C. The samples were then vortexed and centrifuged, and 50 µL were aliquoted to 0.2 µL plates.

The concentrations of the samples were determined using the NanoDrop 1000 (Thermo

Scientific), and stored at -80°C until tested.

3.3.3. PCR Assay

The primers and PCR environments are listed in Table 3.1.

Table 3.1. Markers and primer pairs used for screening progeny of NY121.

Annealing Length Temp Gene Marker Sequence (5' - 3') (bp) (℃) H1 TG689 f: taa aac tct tgg tta tag cct at 141 55 Shultz et al. (2012) r: caa tag aat gtg ttg ttt cac caa

Ryadg RYSC3 f: ata cac tca tct aaa ttt gat gg 321 60 Kasai et al. (2000) r: agg ata tac ggc atc att ttt ccg a

Ryadg M6 f: aca tga tat aag ttg ata tgg aga at 994 60 Herrera et al. (2018) r: gtg ctt tgt ctt ttc tgc atg ta SUS3 SUS3 f: ctg caa gct aag cct gat ctt att atc 600 55 Harchenko (2014) r: agg ata tac ggc atc att ttt ccg a

The control marker SUS3 was implemented to confirm DNA presence and integrity (Fu & Park,

1995; Harchenko, 2014). The marker for H1 utilizes TG689F and TG689R primers described by

Schultz et al. (2010). To genotype for Ryadg, two markers were used: RYSC3 from Kasai et al.

(2000), and M6 marker from Herrera et al. (2018). PCR reactions were conducted in a total reaction volume of 20 μl, containing 10 μl Promega GoTaq® Green Master Mix (bacterially derived Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers), 4 μl of desired primer mix

(forward, reverse and Nuclease-Free Water), and 5 μl of nuclease-free water and 1 μl of corresponding DNA. The PCR conditions were followed as described in the literature (Table

3.1.) Gel electrophoresis was conducted with 0.8% agarose for RYSC3 and M6, and 2% agarose for TG689, with either ethidium bromide or GelGreen used for staining.

3.3.4. Statistical Analyses

Presence of PCR markers were scored by the appearance of a band on electrophoresis gels in the expected region. No band indicated an absence of the marker. Faint bands were

considered contamination and thus counted as absence of the marker. Chi-square goodness of fit tests were performed on the F1 progeny in Excel. Observed present and absent bands in each family were tested against expected values according to typical Mendelian segregation ratios in tetraploids for a simplex, duplex, and triplex or quadruplex progenitor. Nonsignificant results indicate observed results match expected results of the associated allele dosage. Significance level was set at a = 0.05.

3.4. Results

In family AF7505, about 53% individuals (35 of 66) (Table 3.2.) tested positive for the

TG689 marker (Figure 3.1.A.).

Table 3.2. Observed frequencies of markers in each family of NY121 progeny. The numbers of individuals per family that tested positive (+) or negative (-) for each marker are listed.

Marker Family (Gene) + - Total AF7505 TG689 (H1) 35 31 66 RYSC3 (Ryadg) 47 19 66 M6 (Ryadg) 37 29 66

AF7506 TG689 (H1) 27 36 63 RYSC3 (Ryadg) 34 29 63 M6 (Ryadg) 36 27 63

AF7505-1 AF7505-5 AF7505-2 AF7505-3 AF7505-4 AF7505-6 AF7505-7 AF7505-8 A. 1500 bp 1000 bp 700 bp 500 bp 400 bp 200 bp TG689

B. 1500 bp

1000 bp

700 bp 500 bp 400 bp RYSC3 300 bp

C. 5000 bp 3000 bp 2000 bp 1500 bp 1000 bp M6 700 bp 500 bp 400 bp

D. 1500 bp

1000 bp 700 bp SUS3 500 bp 400 bp 300 bp

Figure 3.1. Detection of the TG689, RYSC3 and M6 markers and the control SUS3 marker from 8 individuals of family AF7505. DNA ladders are shown in the left-most lane. (A) For marker TG689, individuals AF7505-1, AF7505-2, AF7505-5, and AF7505-6 were marked as present, while the remaining four individuals were marked as absent. (B) For marker RYSC3, individuals AF7505-2, AF7505-4, AF7505-5, and AF7505-7 were marked as present, and the remaining four individuals were marked as absent. (C) For marker M6, individuals AF7505-1, AF7505-4, AF7505-5, and AF7505-7 were marked as present, and the remaining four individuals were marked as absent. (D) In the control, all individuals were marked as present.

In the chi-square test, there is no significant difference between these results and the expected outcome for a single copy (p = 0.62246) (Table 3.2.), but there are significant differences when compared to the likelihood of two (p < 0.00001) or more (p < 0.00001) copies are in NY121.

About 43% of individuals (27 of 63) (Table 3.2.) tested positive in family AF7506. Similar to family AF7505, there appears to be no significant difference between the observed and expected results for a single copy (p = 0.25684), but the expected results for two or more copies are significant (p < 0.00001 for both). The results are consistent with NY121 having one copy of the

H1 gene based on the TG689 marker.

The copy number of Ryadg in NY121 was assessed using two PCR markers, RYSC3 and

M6. In family AF7505, 71.2% (47 of 66) (Table 3.2.) of the samples tested positive for the

RYSC3 marker and we observed a significant difference between the observed results and the expected results if there is a single copy of the allele (p = 0.00057) (Table 3.3.), two copies of the allele (p = 0.00823), as well as three or four copies (p < 0.00001).

Table 3.3. The p-values of the chi-square results comparing observed frequencies with expected frequencies if NY121 has one, two, or three or more copies of the respective alleles.

Marker Family (Gene) Goodness of Fit tests triplex or simplex duplex quadruplex AF7505 TG689 (H1) P = 0.62246 P < 0.00001 P < 0.00001 RYSC3 P = 0.00057 P = 0.00823 P < 0.00001 (Ryadg) M6 (Ryadg) P = 0.32476 P < 0.00001 P < 0.00001

AF7506 TG689 (H1) P = 0.25684 P < 0.00001 P < 0.00001 RYSC3 P = 0.52873 P < 0.00001 P < 0.00001 (Ryadg) M6 (Ryadg) P = 0.25684 P < 0.00001 P < 0.00001

Based on just the RYSC3 data from AF7505, we are not able to draw conclusions about the copy number of the Ryadg alleles in NY121 due to the lack of nonsignificant chi-square values.

However, when we tested the same family with the M6 marker (Figure 3.1.C.), the M6 marker was present in 56.0% (37 of 66) (Table 3.2.) of samples. Based on this data, the M6 marker in

Family AF7505 revealed no significant difference when compared to the expected ratio when only one copy of the allele is present (p = 0.32476) (Table 3.3.), but there are significant differences relative to expected results if multiple alleles are present; p < 0.00001 for two alleles, and p < 0.00001 if three or more alleles are present. Because the analysis of RYSC3 showed statistical differences between copy number states in the AF7505 (and therefore not informative based on the expected Mendelian ratios) we draw our analysis based on the M6 marker and infer that Ryadg is most likely present as a single copy in NY121.

We further tested our expectations using family AF7506, in which the RYSC3 marker revealed positive bands in 54.0% (34 of 63) (Table 3.2.) of samples. There is no significant difference between these results and the expected results of a single copy of the allele (p =

0.52873) (Table 3.3.). There is a significant difference between these results and the expected results for two or more copies of the allele (p < 0.0001 for both duplex and triplex/quadruplex).

Furthermore, the M6 marker was present in 57.1% (36 of 63) (Table 3.2.) of samples. There is no significant difference when these results are compared to what would be expected if one copy of the allele is present (p = 0.25684) (Table 3.3.). There are significant differences from expected results if there were two alleles present (p < 0.00001) and if there were three or four copies (p <

0.00001). Taken together, the combined results from the two families segregating for Ryadg from

NY121 indicate that there is most likely only one copy of the Ryadg allele in NY121.

3.5. Discussion

This study shows that genetic markers can be a useful tool in potato genetics and breeding when DNA markers linked to agronomically important genes are deployed inexpensively and efficiently for traits of interest (Barone, 2004). A progenitor that is multiplex for the resistance allele is of greater value, as it will produce more resistant progeny than a progenitor with one resistant allele. Determining the allele dosage of important genes through phenotyping in potato has been a challenge because polyploid species have more complex segregation ratios than diploid species, and that necessitates large populations to be reliably revealed by phenotype, which may be complicated if allelic effects are present (Jansky, 2009;

Little, 1945; Meyer et al., 1998). An additional complicating factor for potato includes segregation differences based on chromosomal location (Jansky & Spooner, 2018). DNA-based markers have been suggested as a way to determine allele dosage in polyploids (Andrade et al.,

2009; Barrell et al., 2013; Ortiz, 2020), especially when chromosomal location of the marker and gene is more proximal to the centromere.

As researchers and industry look to transition into diploid potato breeding, tetraploids with multiple copies of important genes can be targeted for dihaploid production to provide future varieties with predictable phenotypes (Caligari, 2007). A potato clone like NY121 is an attractive parent for breeding programs because of its good market traits and multiple resistances to disease. It may be of exceptional use as a dihaploid parent if it possesses multiple copies of the dominant resistance genes and can be introduced as homozygous for these alleles. The crosses made in this study revealed evidence of a single copy of the H1 gene in the NY121 clone, assuming random and independent assortment with an upper limit of 50%.

As H1 was mapped to the distal end of Chromosome 5 (Gebhardt & Valkonen, 2001), consideration of the validity of these results because of its location is warranted. Little (1945) observed that the upper limit of cross-over in tetraploids amounts rather to 43%, owing to chiasma frequency, distance between the gene and centromere, and quadrivalent formation. He was therefore a proponent of Mather's (1936) hypothesis and ratios and showed the accuracy of his theory in resolving a number of studies across polyploids. In a cross where one allele is present, Mather’s expected ratio is 11:13 present:absent rather than the Mendelian expected 1:1, and where two alleles are present, Mather’s expected ratio is 7:2 rather than Mendel’s expected

5:1. For the TG689 marker used for H1, chi-square tests using Mather’s ratios in family AF7505 yield p-values of 0.24062 and <0.00001, respectively. For family AF7506, the p-values against

Mather’s ratios are 0.63543 and <0.00001, respectively. In neither case is the conclusion altered.

Using Mather’s ratios may be beneficial when a Mendelian study is inconclusive or when the population is smaller than the present study, however Jansky (2009) warns that an exact ratio cannot be predicted because crossover events are unpredictable.

Both family sets yielded individuals showing no bands of the RYSC3 marker associated with Ryadg, eliminating the possibility that NY121 is triplex or homozygous for the resistant allele of Ryadg. The RYSC3 marker appears to behave differently in AF7505 and AF7506 families when using Mendel’s ratios, and requires further consideration. Applying Mather’s ratios to the RYSC3 observations in family AF7505, the segregation of RYSC3 appears to now be significant for simplex (p = 0.0004), but nonsignificant for duplex (p = 0.19987), indicating that NY121 can potentially be duplex for Ryadg. However, using Mather’s ratios did not alter the results of RYSC3 for family AF7506 (p = 0.19501 for simplex, p < 0.00001 for duplex).

Nonetheless, when Mather’s ratio was used on the M6 marker in both families, the chi-squared

analyses support our conclusion that NY121 as simplex for Ryadg (p = 0.09541for family AF7505 and p = 0.07161 for family AF7506).

The inconsistency between the RYSC3 and M6 marker data results in family AF7505 may be attributed to two things. First, false positives from human error cannot be ruled out.

Second, the population was too small to accurately reflect that NY121 is simplex for Ryadg, and this family simply had a higher-than-average number of progeny with the Ryadg gene. This conclusion is not necessarily in opposition with the M6 results; these markers test for different loci linked to the Ryadg gene, and crossing-over events between the markers may have occurred during meiosis. Phenotypic testing of the family could verify if this is the case. M6 is likely to be the better marker because it is located closer to the Ryadg gene at < 0.05 cM, whereas RYSC3 is about 0.2 cM away (Jara Vidalon, 2010, per Herrera et al., 2018). Additionally, a recombination point was found between M6 and RYSC3, which are 0.18 cM apart (Guzmán Escudero, 2010).

DNA-marker results used as a replacement for phenotypic observations in a testcross is merely one avenue of assessing allele dosage. Dosage may also be evaluated through quantitative or semi-quantitative PCR methods. Recent studies in high-resolution DNA melting (HRM) have shown great potential for potato, having been used in identifying allele dosage of genes in the carotenoid pathway (McCord et al., 2012) and resistance genes (De Koeyer et al., 2010;

Meiyalaghan et al., 2018; Nie et al., 2016), provided the samples have only one source of resistance and the probe is specific enough to target only the allele of interest (De Koeyer et al.,

2010). Similar to MAS programs that utilize electrophoresis, HRM requires that sequences have already been delineated (Barrell et al., 2013). Studies like this can be validated and eventually supplanted by HRM (Herrera et al., 2018), especially as marker sequences are refined

(Meiyalaghan et al., 2018); moreover, HRM is also capable of attaining the same MAS data as

an electrophoresis approach, and can be a tool used in mapping and genetic fingerprinting (De

Koeyer et al., 2010; McCord et al., 2012; Meiyalaghan et al., 2018; Nie et al., 2018; Simko,

2016; Villano et al., 2016; Yuan et al., 2009). However, these capabilities may be beyond the needs of conventional breeding programs, which are focused on quickly developing and releasing new varieties. For diploid breeding programs in need of developing new parents, HRM is likely the better option. If and when it becomes clear that important traits are governed by the presence of multiple alleles, such as case of the Zep1 gene (McCord et al., 2012), then HRM may be the best tool developed so far for most programs.

This research has some additional application for prospective diploid breeding programs.

As the future of potato breeding continues to make progress on the production of dihaploid clones, autotetraploid varieties and clones will need to be assessed for potential candidacy. In a diploid breeding system, clones that are homozygous for desirable traits will have the greatest value as breeding parents. This study shows data suggesting that the potato clone NY121 contains one allele each of Ryadg and H1. To maximize its potential as a dihaploid parent in a future diploid breeding program, it must be transformed for homozygosity for both alleles. As more markers become available, the clone NY121 will need to be tested for the presence and dosage of those alleles.

3.6. Conclusion

The elite breeding tetraploid clone NY121 was found to have a single copy each of Ryadg and H1. These results were discovered through a study based on Mendelian segregation but focused on genotype rather than phenotype. This type of study may have application in determining the allele dosage of late blight R genes, which confer a spectrum of resistance. Such

studies are the first step in establishing the role, if any, of allele dosage on late blight resistance, and furthering our understanding of late blight R genes.

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APPENDIX A

DETACHED LEAF ASSAY RESULTS AGAINST US-23

Figure A.1. Results of detached leaf assays against US-23 for each cultivar and clone in each trial. Leaflets were taken from the same plants for all three trials. The first experiment presented was measured on May 27, 2020, the second experiment was measured on June 1, 2020, and the third experiment was measured on June 12, 2020.

Abnaki Chaleur Dakota Trailblazer

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Defender Dorita Elba

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Figure A.1. Continued

Jacqueline Lee J117 Katahdin

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Figure A.1. Continued

Pungo Russet Burbank Sbu 8.5

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Figure A.1. Continued

Yukon Gem Superior

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APPENDIX B

LATE BLIGHT FIELD RATINGS

TABLE B.1. Late blight field ratings of potato varieties and clones in three states from 2011- 2019. The relative area under the disease progress curve (RAUDPC) and area under the disease progress curve (AUDPC) were averaged based on data provided by the respective locations (University of Maine, Aroostook Research Farm; The Pennsylvania State University, State College; Michigan State University, Clarksville Research Center), except for data from Pennsylvania in years 2016 and 2017 which were already averaged. The rating assessments were provided by the researchers overseeing the trials, except for those from Michigan. Those ratings were estimated in this study based on average RAUDPC, where calculations less than 10.00 were determined to be resistant, from 10.01 to 19.99 to be moderately resistant, and greater than 20.00 to be susceptible. These ratings are highlighted by a gray box. All other highlighted boxes are for ease of view. Some changes to ratings have been made for uniformity. a VS = very susceptible; S = susceptible; MS = moderately susceptible; MS/MR = moderately susceptible to moderately resistant; MR/R = moderately resistant to resistant; R = resistant; VR = very resistant.

Avg Avg Prevalent Year Location Line RAUDPC AUDPC Ratinga LB Race 2011 Maine Abnaki 2652.50 S 2011 Pennsylvania Abnaki 382.00 S US-8

2012 Maine Abnaki 1687.50 S 2012 Pennsylvania Abnaki 996.33 S US-23 2013 Maine Abnaki 1085.00 VS 2013 Pennsylvania Abnaki 513.75 S US-23 2014 Maine Abnaki 1067.50 S US-23

2014 Pennsylvania Abnaki 581.50 VS US-23 2015 Pennsylvania Abnaki 646.25 VS US-23 2016 Maine Abnaki 1058.75 S US-23 2016 Pennsylvania Abnaki 1013.50 VS US-23 2016 Pennsylvania Abnaki 0.25 S US-23

2017 Pennsylvania Abnaki 0.31 S US-23 2012 Maine AF2376-5 536.25 MR/R 2012 Pennsylvania AF2376-5 45.50 MR/R US-23 2013 Maine AF2376-5 455.00 MS 2013 Pennsylvania AF2376-5 12.00 MR-R US-23

2014 Maine AF2376-5 507.50 MR US-23 2014 Pennsylvania AF2376-5 118.25 MR US-23

Table B.1. Continued

2015 Pennsylvania AF2376-5 91.25 MR US-23 2016 Maine AF2376-5 586.25 MS/MR US-23

2016 Pennsylvania AF2376-5 95.25 MR US-23 2017 Pennsylvania AF2376-5 99.25 MR US-23 2018 Pennsylvania AF2376-5 175.00 MR US-23 2019 Pennsylvania AF2376-5 82.50 MR US-23 2010 Pennsylvania AF4303-1 57.00 R US-8

2011 Maine AF4303-1 1566.25 MR

2011 Pennsylvania AF4303-1 52.50 MR/R US-8 2010 Pennsylvania AF4329-4 92.83 MR US-8 2011 Maine AF4329-4 1355.00 MR/R 2011 Pennsylvania AF4329-4 69.33 MR/R US-8

2012 Maine AF4329-7 988.75 MS

2013 Maine AF4386-16 787.50 S 2013 Pennsylvania AF4386-16 477.50 S US-23 2014 Maine AF4386-16 1225.00 VS US-23 2014 Pennsylvania AF4386-16 648.75 VS US-23

2011 Maine AF4561-1 782.50 R

2011 Pennsylvania AF4561-1 57.67 MR/R US-8 2012 Maine AF4561-1 991.25 MS 2012 Pennsylvania AF4561-1 16.00 MR/R US-23 2011 Maine AF4624-1 1411.25 MR/R

2011 Pennsylvania AF4624-1 117.67 MR US-8

2011 Maine AF4626-3 1830.00 MR 2011 Pennsylvania AF4626-3 144.33 MS/MR US-8 2011 Maine AF4631-3 1443.75 MR/R 2011 Pennsylvania AF4631-3 29.00 MR/R US-8

2011 Maine AF4696-1 911.25 R

2011 Pennsylvania AF4696-1 24.00 R US-8 2012 Maine AF4696-1 151.25 R 2012 Pennsylvania AF4696-1 29.17 MR/R US-23 2013 Maine AF4696-1 52.50 R 2013 Pennsylvania AF4696-1 7.25 R US-23

2014 Maine AF4696-1 385.00 MR US-23 2014 Pennsylvania AF4696-1 6.00 VR US-23 2015 Pennsylvania AF4696-1 11.75 R US-23 2016 Maine AF4696-1 805.00 MS US-23

Table B.1. Continued

2016 Pennsylvania AF4696-1 6.75 VR US-23 2013 Maine AF5039-17 551.25 MS

2013 Pennsylvania AF5039-17 10.75 MR/R US-23 2014 Maine AF5039-17 647.50 MS US-23 2014 Pennsylvania AF5039-17 120.75 MR US-23 2015 Pennsylvania AF5039-17 64.25 R US-23 2016 Maine AF5039-17 980.00 S US-23

2016 Pennsylvania AF5039-17 25.00 R US-23

2014 Maine AF5382-12 971.25 S US-23 2014 Pennsylvania AF5382-12 311.50 MS US-23 2016 Maine AF5382-12 857.50 MS US-23 2016 Pennsylvania AF5382-12 176.50 MS/MR US-23

2014 Maine AF5400-2 542.50 MR US-23

2014 Pennsylvania AF5400-2 85.00 R US-23 2015 Pennsylvania AF5400-2 10.50 R US-23 2016 Maine AF5400-2 980.00 S US-23 2016 Pennsylvania AF5400-2 68.25 MR US-23

2014 Maine AF5403-3 691.25 MS US-23

2014 Pennsylvania AF5403-3 83.25 R US-23 2015 Pennsylvania AF5403-3 70.00 MR US-23 2016 Maine AF5403-3 883.75 MS US-23 2016 Pennsylvania AF5403-3 29.25 R US-23

2014 Maine AF5426-1 735.00 MS US-23

2014 Pennsylvania AF5426-1 176.50 MR US-23 2015 Pennsylvania AF5426-1 112.50 MR US-23 2016 Maine AF5426-1 980.00 S US-23 2016 Pennsylvania AF5426-1 146.75 MR US-23

2010 Michigan Atlantic 29.09 S US-22

2016 Pennsylvania Atlantic 0.38 S US-23 2017 Pennsylvania Atlantic 0.31 S US-23 Dakota 2010 Michigan Trailblazer 15.40 MR US-22 Dakota 2012 Michigan Trailblazer 2.17 R US-22 2011 Maine Defender 491.25 R 2011 Pennsylvania Defender 23.00 R US-8

2012 Maine Defender 430.00 MR/R

2012 Pennsylvania Defender 23.33 MR/R US-23

Table B.1. Continued

2013 Maine Defender 341.25 MR 2013 Pennsylvania Defender 20.75 MR/R US-23

2014 Maine Defender 210.00 R US-23 2014 Pennsylvania Defender 22.00 R US-23 2015 Pennsylvania Defender 11.75 R US-23 2016 Maine Defender 455.00 MR US-23 2016 Pennsylvania Defender 20.50 R US-23

2016 Pennsylvania Defender 0.01 R US-23

2017 Pennsylvania Defender 4.50 R US-23 2017 Pennsylvania Defender 0.00 R US-23 2018 Pennsylvania Defender 57.00 R US-23 2019 Pennsylvania Defender 20.00 R US-23

2016 Pennsylvania Dorita 0.04 MR US-23

2017 Pennsylvania Dorita 0.14 MR US-23 2016 Pennsylvania Elba 0.03 MR US-23 2017 Pennsylvania Elba 0.15 MR US-23 2011 Maine J117 400.00 R

2011 Maine J117 498.75 R

2011 Pennsylvania J117 13.17 R US-8 2012 Maine J117 46.25 VR 2012 Pennsylvania J117 3.50 R US-23 2013 Maine J117 0.00 VR

2013 Pennsylvania J117 0.00 R US-23

2014 Maine J117 61.25 VR US-23 2014 Pennsylvania J117 0.00 VR US-23 2016 Maine J117 52.50 VR US-23 2016 Pennsylvania J117 2.50 VR US-23

2017 Pennsylvania J117 0.00 R US-23

2018 Pennsylvania J117 0.00 VR US-23 2019 Pennsylvania J117 0.00 VR US-23 Jacqueline 2010 Michigan Lee 35.68 S US-22 Jacqueline 2011 Michigan Lee 9.48 R US-22 Jacqueline 2012 Michigan Lee 7.10 R US-22 Jacqueline 2014 Michigan Lee 0.04 R US-23

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Table B.1. Continued

Jacqueline 2016 Pennsylvania Lee 0.00 R US-23 Jacqueline 2017 Pennsylvania Lee 0.12 R US-23 2010 Michigan LBR9 0.60 R US-22 2012 Michigan LBR9 13.73 MR US-22

2010 Michigan M171-A 39.94 S US-22 2011 Michigan M171-A 11.56 MR US-22 2012 Michigan M171-A 10.43 MR US-22 2010 Michigan M182-1 13.25 MR US-22 2011 Michigan M182-1 3.21 R US-22

2013 Michigan M182-1 0.57 R US-23 2010 Michigan Missaukee 39.38 S US-22 2011 Michigan Missaukee 12.01 MR US-22 2012 Maine Missaukee 530.00 MR/R 2012 Michigan Missaukee 3.73 R US-22

2012 Pennsylvania Missaukee 7.83 R US-23 2013 Maine Missaukee 122.50 MR/R 2013 Pennsylvania Missaukee 5.00 R US-23 2014 Maine Missaukee 288.75 R US-23 2014 Michigan Missaukee 0.00 R US-23

2014 Pennsylvania Missaukee 3.00 VR US-23 2015 Pennsylvania Missaukee 10.50 R US-23 2016 Maine Missaukee 586.25 MS/MR US-23 2016 Pennsylvania Missaukee 21.50 R US-23 2017 Pennsylvania Missaukee 0.75 R US-23

2018 Pennsylvania Missaukee 10.00 VR US-23

2019 Pennsylvania Missaukee 8.00 R US-23 2010 Michigan NY121 4.90 R US-22 2011 Michigan NY121 5.68 R US-22 2013 Michigan NY121 0.13 R US-23

2014 Michigan NY121 7.92 R US-23

2015 Michigan NY121 1.70 R US-23 2010 Michigan Q086-3 45.65 S US-22 2011 Michigan Q086-3 34.04 S US-22 2012 Michigan Q086-3 18.90 MR US-22

2013 Michigan Q086-3 4.93 R US-23

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Table B.1. Continued

2014 Michigan Q086-3 33.10 S US-23 2014 Michigan Q086-3 33.15 S US-23 Russet 2013 Maine Burbank 621.25 S Russet 2013 Pennsylvania Burbank 338.75 MS US-23

Russet

2014 Maine Burbank 1032.50 S US-23

Russet

2014 Pennsylvania Burbank 360.00 MS US-23

Russet

2015 Pennsylvania Burbank 463.75 S US-23

Russet 2016 Maine Burbank 883.75 MS US-23 Russet 2016 Pennsylvania Burbank 320.25 MS US-23 Russet 2019 Pennsylvania Burbank 71.00 MR US-23 2016 Pennsylvania Sbu 8.5 2.50 VR US-23 2017 Pennsylvania Sbu 8.5 0.00 R US-23 2019 Pennsylvania Sbu 8.5 42.50 MR US-23 Saginaw 2010 Michigan Chipper 9.63 R US-22 Saginaw 2011 Michigan Chipper 1.87 R US-22 Saginaw 2012 Michigan Chipper 5.70 R US-22 Saginaw 2014 Michigan Chipper 11.61 MR US-23 Saginaw 2015 Michigan Chipper 3.63 R US-23

2010 Michigan Stirling 2.86 R US-22 2010 Michigan Stirling 1.49 R US-22 2011 Michigan Stirling 0.57 R US-22 2012 Michigan Stirling 0.80 R US-22 2014 Michigan Stirling 6.31 R US-23

2017 Pennsylvania Stirling 0.00 R US-23 2018 Pennsylvania Stirling 44.00 R US-23 2019 Pennsylvania Stirling 1.50 VR US-23 2011 Maine Superior 2628.75 S 2011 Pennsylvania Superior 385.33 S US-8

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Table B.1. Continued

2012 Maine Superior 2026.25 VS 2012 Pennsylvania Superior 734.67 S US-23

2013 Maine Superior 1032.50 VS 2013 Pennsylvania Superior 522.50 S US-23 2014 Maine Superior 1312.50 VS US-23 2014 Pennsylvania Superior 581.50 VS US-23 2015 Pennsylvania Superior 593.75 VS US-23

2016 Maine Superior 1050.00 S US-23

2016 Pennsylvania Superior 594.50 S US-23 2016 Pennsylvania Superior 0.58 S US-23 2017 Michigan Superior 33.77 S US-23 2017 Pennsylvania Superior 0.43 S US-23

2018 Pennsylvania Superior 736.00 S US-23

2019 Pennsylvania Superior 163.50 S US-23 2010 Michigan Torridon 0.37 R US-22 2012 Michigan Torridon 3.33 R US-22 2016 Pennsylvania Torridon 0.00 R US-23

2017 Pennsylvania Torridon 0.00 R US-23

2014 Maine Yukon Gem 236.25 R US-23 2014 Pennsylvania Yukon Gem 0.00 VR US-23 2015 Pennsylvania Yukon Gem 14.50 R US-23 2016 Maine Yukon Gem 428.75 MR US-23

2016 Pennsylvania Yukon Gem 7.00 VR US-23

2017 Pennsylvania Yukon Gem 0.00 R US-23 2018 Pennsylvania Yukon Gem 6.00 VR US-23 2019 Pennsylvania Yukon Gem 0.00 VR US-23

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APPENDIX C

DISCARDED ASSAYS

Whole plant assays

In addition to phenotyping late blight resistance the detached leaf assays (DLA), attempts were also made using whole-plant methods. Inoculated field studies of late blight are prohibited in Maine, so greenhouse whole-plant assays were planned to observe effects closer to a field response. This assay was conducted on 39 varieties with late blight race US-23.

Materials and Methods

This experiment was designed as a completely randomized block design with five replicates in a standard greenhouse environment. Additional lighting was implemented to achieve 16h of light for the duration of the experiment. The potatoes were planted in potting soil mixed with topsoil in three-gallon pots, and randomized weekly within their blocks until inoculation, about six weeks after planting. The blocks were then covered using tents made of opaque white plastic and inoculated by spraying. The zoospores were suspended in water at a rate of 20,000/mL. Whole-plant assessment and lesion measurements were taken at 1.5, 4, 7, and

11 dpi. The three largest lesions on each plant were measured and calculated as an elliptical lesion per Vleeshouwers et al. (1999).

The whole-plant assessment was measured according to Malcolmson (1976), and as described by Colton et al. (2006) and Karki et al. (2020). Assessment of whole plants is essentially the same as for DLA except for scale, and the phenotyping methods are further detailed in Chapter 2 of this study.

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Results and Conclusion

One whole-plant assay was successfully conducted. Additional assays were initiated the following two summers but were abandoned. The first year, the pathogen would not sporulate.

The second year, 40% of plants were lost to cockroaches. Detached leaf assays (DLA) were planned to supplement the whole-plant assays and the decision was made to focus on DLA.

Due to lack of replication, the results of the first assay are not included in Chapter 2; they are presented here in Figure C.1. Superior was included as the susceptible check, and had a mean resistance score of 8.211, indicating that it was resistant to US-23 in this trial. The high resistance rating of Superior was unexpected, and further replication is necessary to validate the whole-plant results of the other varieties and clones.

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Mean Resistance Estimate to US-23 9

4 Asessment Scale

1 Elba J117 LBR-9 NY121 Sbu8.5 Stirling Chaleur Barbara Tollocan Superior Torridon Defender AF2376-5 AF5401-1 AF5435-7 AF5403-3 AF5426-1 AF5040-4 AF5392-8 AF5400-2 AF5141-6 B3054A-7 B3054A-3 B3054A-9 MSQ086-3 Missaukee AF5395-10 AF5382-12 AF5039-17 B3054A-11 BD1258-H1 BD1258-H9 BD1258-H2 YukonGem BD1258-H18 BD1258-H14 Jacqueline Lee

Dakota Trailblazer Dakota

Figure C.1. The mean resistance of each variety and clone in a whole plant assay against late blight race US-23. Ratings between 7 and 9 are considered resistant, between 5 and 6.99 are considered partially resistant, and below 5 are considered susceptible.

Detached Leaf Assays

Assays were planned against several P. infestans races to develop a more robust profile of late blight (LB) resistance for the varieties and clones tested. P. infestans races US-8 were supplied by Cornell and Oregon State University, and US-11 and -24 were provided by Cornell. Isolates of all three races were prepared as inoculant in DLA but ultimately zoospore counts in assays utilizing races US-8, -11, and -24 were either low or non-existent, yielding DLA data that were inconsistent and unreliable (data not shown), despite multiple attempts over several years. Only assays with US-23 as inoculant yielded reliable data.

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BIOGRAPHY OF THE AUTHOR

Kristen Michelle Brown-Donovan was born in Biloxi, MS, and grew up in Pennsylvania. She graduated from Shikellamy Area High School in Sunbury, PA, and received a BA in history

(2004) and BS in ecology (2011) from Susquehanna University in Selinsgrove, PA. She received her MS in plant, soil, and environmental sciences (2013) from The University of Maine, and works as a research associate for The University of Maine. She is a candidate for the PhD degree in plant science from The University of Maine in December 2020.

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