Thesis Rahim Ahmadvand Keszthely, Hungary 2013

UNIVERSITY OF PANNONIA GEORGIKON FACULTY

DOCTOR OF PHILOSOPHY (PhD) THESIS

RAHIM AHMADVAND

KESZTHELY, HUNGARY

2013 2

UNIVERSITY OF PANNONIA

GEORGIKON FACULTY

DOCTORAL SCHOOL OF CROP PRODUCTION AND HORTICULTURAL SCIENCES PLANT BREEDING, GENETICS AND AGROBIOTECHNOLOGY PROGRAM

HEAD OF THE DOCTORAL SCHOOL

PROF. DR. LÁSZLÓ KOCSIS, DSC

ANALYSIS OF RESISTANCE GENES IN POTATO WITH SPECIAL ATTENTION TO EXPRESSIONAL APPROACHES

DOCTOR OF PHILOSOPHY (PhD) THESIS

WRITTEN BY RAHIM AHMADVAND

SUPERVISORS

DR. ZSOLT POLGÁR, PhD

AND

DR. JÁNOS TALLER, PhD

KESZTHELY, HUNGARY

2013

ANALYSIS OF RESISTANCE GENES IN POTATO WITH SPECIAL ATTENTION TO EXPRESSIONAL APPROACHES

Written By RAHIM AHMADVAND

Written at the University of Pannonia, Doctoral School of Crop Production and Horticultural Sciences, Plant Breeding, Genetics and Agrobiotechnology Program

Supervisors: Dr. Zsolt Polgár

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Dr. János Taller

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Table of Contents

ABSTRACT ...... 9

KIVONAT ...... 10

ABSTRAKT ...... 11

ABBREVIATIONS ...... 12

1. INTRODUCTION ...... 14

Research objectives ...... 17

2. LITERATURE REVIEW ...... 18

2.1. Origin and evolution of potato crop ...... 18 2.2. Potato production in Hungary ...... 19 2.3. History of potato research at Keszthely, Hungary ...... 19 2.4. Potato viruses ...... 20 2.4.1. Potato virus X ...... 21

2.4.2. Potato virus Y ...... 22

2.5. Genetics of resistance ...... 24 2.5.1. Structure and function of R genes ...... 25

2.5.2. Mechanism of virus resistance ...... 28

2.5.3. Signaling mediated resistance ...... 29

2.5.4. Gene silencing ...... 31

2.5.5. Types of resistance to potato viruses ...... 32

2.5.5.1. Hypersensitive reaction (HR) ...... 32 2.5.5.2. Extreme resistance (ER) ...... 33 2.5.5.3. Resistance to infection ...... 35 2.5.5.4. Resistance to virus accumulation ...... 36 2.5.5.5. Resistance to virus movement in plants ...... 36 2.5.5.6. Mature plant resistance ...... 37 2.5.5.7. Tolerance...... 37 2.5.5.8. Resistance to virus vectors ...... 38

2.6. The potato resistance genes Rx1 and Rx2 ...... 39 2.7. Resistance breeding and molecular markers ...... 40 2.8. Marker assisted selection ...... 41 2.9. Intron-targeting...... 42 2.10. Gene expression profiling in plant-virus interactions ...... 43 2.10.1. Subtraction Suppressive Hybridization (SSH) ...... 44

2.10.2. cDNA-AFLP ...... 46

2.10.3. Serial Analysis of Gene Expression ...... 46

2.10.4. Microarray ...... 47

2.10.5. Transcriptome analysis ...... 48

2.10.5.1 De novo transcriptome analysis ...... 51 2.10.5.2. Transcriptome analysis of the sequenced genomes ...... 51 3. MATERIALS AND METHODS ...... 54

3.1. Plant materials ...... 54 3.1.1. Plant materials used in PVX inoculation test ...... 54

3.1.2. Evaluation of the validity of developed specific primers for Rx1 and Rx2 genes ...... 54

3.1.3. Development of Intron targeting markers ...... 56

3.2. DAS-ELISA test ...... 57 3.3. PVX resistance tests ...... 57 3.3.1. Mechanical inoculation ...... 57

3.3.2. Graft inoculation ...... 57

3.4. Genomic DNA isolation ...... 58 3.4.1. Lysis of plant cells and protein denaturation ...... 58

3.4.2. Purification ...... 58

3.5. Identification of resistance gene to PVX...... 59 3.5.1. Marker analysis...... 59

3.5.2. Development of specific primers for Rx genes ...... 59

3.6. Development of a multiplex PCR for the Rx genes ...... 60 3.7. Transcriptome analysis ...... 60 3.7.1. Inoculation with pathogens ...... 61

3.7.1. 1. Inoculation with PVX and PVY ...... 61 3.7.1.2. Inoculation with Ph. infestans...... 61 3.7.2. mRNA isolation ...... 61

3.7.3. mRNA analysis ...... 62

3.7.4. mRNA preparation ...... 63

3.7.5. Suppression subtractive hybridization (SSH) ...... 63

3.7.5. 1. Construction of SSH library...... 63 3.7.5.2. Cloning and PCR screening of the SSH library ...... 64 3.7.5.3. Sequencing of cDNA clones and sequence analysis...... 64 3.7.6. NGS transcriptome sequencing ...... 65

3.8. Development of NGS derived intron-targeting markers ...... 65 3.8.1. PCR amplification of intron targeting markers ...... 66

3.8. 2. Data analysis ...... 66

4. RESULTS ...... 67 4.1. Development of functional markers and a multiple PCR for Rx1 and Rx2 resistance genes in potato ...... 67 4.1. 1. PVX resistance tests ...... 67 4.1.2. Identification of resistance gene to PVX...... 67 4.1.2.1. Results of the published PVX resistance markers ...... 67

4.1.2.2. Resistance gene analysis and development of gene-specific primers ...... 69

4.1.3. Testing of the Rx2 specific primer pair in the F1 populations ...... 73 4.1.4. Development of a multiplex PCR for the Rx genes ...... 74 4.2. Isolation of important genes related to resistance against PVX, PVY and Ph. infestans ..... 75 4.2.1. Construction of subtractive cDNA libraries in White Lady ...... 75

4.2.1.1. Analysis of PVY induced subtracted cDNA library ...... 75 4.2.1.2. Analysis of PVX induced subtracted cDNA library ...... 76 4.2.2. NGS based transcriptome analysis ...... 77

4.3. Development of Intron-targeting markers in potato ...... 80 4.3.1. IT polymorphism in the potato genotypes ...... 80

4.3.2. IT polymorphism in the wild Solanum species ...... 82

4.3.3. Localization of the IT markers in the potato genome ...... 84

5. DISCUSSION ...... 85 5.1. Development of functional markers and a multiple PCR for Rx1 and Rx2 resistance genes in potato ...... 85 5.2. Isolation of important genes related to resistance against PVX, PVY and Ph. infestans ..... 87 5.2.1. Analysis of PVY induced subtracted cDNA library ...... 87 5.2.2. Analysis of PVX induced subtracted cDNA library ...... 90

5.2.3. NGS based transcriptome analysis ...... 92

5.3. Development of intron-targeting markers in potato ...... 94 LIST OF NEW FINDINGS ...... 98

ÚJ TUDOMÁNYOS EREDMÉNYEK ...... 99

ACKNOWLEDGEMENTS ...... 100

PUBLICATION LIST ...... 102

Referred articles related to thesis ...... 102

Conference abstracts related to the thesis ...... 102

Other publications ...... 103

REFERENCES ...... 105

Appendix 1 ...... 124 Appendix 1 ...... 125 Appendix 2 ...... 126 Appendix 3 ...... 128 Appendix 4 ...... 130 Appendix 5 ...... 131 Appendix 6 ...... 152 Appendix 7 ...... 156 Appendix 8 ...... 169 Appendix 9 ...... 172 Appendix 10 ...... 175 Appendix 11 ...... 176 Appendix 12 ...... 178

ABSTRACT Potato virus X (PVX) is one of the main viruses infecting potatoes worldwide. Two extreme resistant genes, Rx1 and Rx2, against PVX have a 98% sequence similarity at the nucleotide level that makes it complicated to identify these genes and to distinguish them from other highly similar genes, like the Gpa2 or from paralogous sequences by a single PCR. Here, we report the development of functional markers for the simple and rapid identification of the Rx1 as well as the Rx2 gene. Furthermore, a multiplex PCR reaction was developed for the simultaneous detection of both genes in a single reaction. In further studies, we aimed to elucidate a better understanding of potato defense mechanisms against pathogens. To achieve this goal, two approaches, namely PCR-selected cDNA subtraction and RNA-Seq transcriptome were used to profile PVX, Potato virus Y (PVY) and Phytophthora infestans response genes in early stages of infection in the resistant potato cultivar, White Lady. Firstly, two subtracted cDNA libraries of White Lady in response to PVX and PVYNTN were constructed. We could identify 28 and 35 resistance response EST-s to PVX and PVYNTN in White Lady, respectively. Out of these 17 and 12 could be annotated in the PVX as well as in the PVY pool, while five EST-s were found to be common between the two libraries. The remaining genes are genes with unknown function or novel sequences. Secondly, we generated a global transcriptome profile of early response of White Lady to PVX, PVY and Ph. infestans infection using next generation sequencing (NGS). Alignment of fragments with 39 031 protein-coding genes of the genome of S. tuberosum group Phureja DM1-3 5116R44 identified 38 675 genes in the pathogen treated sample. Seven hundred and forty eight transcripts were recognized in the treated sample but not in control indicating stress response specific genes. The data set contains 141 NBS-LRR encoding genes. From these, an NBS-encoding and an LRR-type gene were identified that were expressed only in the treated samples and would be interesting for future studies. The generated dataset has provided comprehensive information of transcriptome dynamics that can serve as a blueprint to elucidate the resistance mechanisms for biologists and potato breeders. In addition, NGS derived transcript sequences were applied for the development of intron-targeting (IT) markers. The efficiency of the developed IT primers was experimentally analyzed. The results revealed the efficiency of NGS derived IT marker development and indicate their utility in diverse molecular analyses including their applicability for cross-species studies.

KIVONAT A burgonya X vírus (PVX – Potato virus X) világszerte elterjedt kórokozója a burgonyának. Két PVX extrém rezisztenciagén, a Rx1 és Rx2 ismert, melyek DNS szekvenciája 98%-os hasonlóságot mutat, ami megnehezíti e két gén azonosítását, illetve más, nagyon hasonló génektől, mint például a Gpa2 fonálféreg rezisztenciagéntől, vagy paralóg szekvenciáktól egyetlen PCR reakcióval történő elkülönítését. A jelen kutatási programban a Rx1 és a Rx2 gén egyszerű és gyors azonosítását lehetővé tevő funkcionális markereket fejlesztettünk ki. Továbbá, kifejlesztettünk egy multiplex PCR eljárást e két gén egyetlen reakcióban történő párhuzamos kimutatására. Kutatásaink fő területe a burgonya kórokozókkal szembeni védekezési mechanizmusainak megismerése volt. E célból két megközelítést, nevezetesen a cDNS szubtraktálást, valamint a RNS- Szek transzkriptom analízist alkalmaztuk a PVX, PVY vírus és a Ph. infestans fertőzés korai stádiumaiban kifejeződő gének vizsgálatára. Modellnövényként az e három kórokozóval szemben rezisztens White Lady fajtát választottuk. Első lépésben két, PVX, illetve PVYNTN fertőzésre szubtraktált cDNS klóntárat készítettünk. A PVX fertőzéssel szemben 28, míg a PVY-nal szemben 35 EST-t azonosítottunk. Ezek közül 17-et, illetve 12-t tudtunk annotálni a PVX, illetve a PVY klóntárban, míg 5 EST azonosnak mutatkozott a két klóntár között. A többi EST vagy ismeretlen funkciójú gén vagy új, az adatbázisokban nem található szekvencia volt. A következő lépésben elkészítettük a White Lady PVX, PVY és Ph. infestans fertőzésre adott teljes transzkriptom profilját ún. következő generációs szekvenálással (NGS – next generation sequencing). A S. tuberosum Phureja csoport DM1-3 511R44 genom 39 031 ismert fehérjekódoló génje alapján 38 675 gént tudtunk azonosítani a kezelt mintában, melyek közül 748 nem fordult elő a kontrollban, ami arra enged következtetni, hogy e gének specifikusan a stressz-válaszban fejeződnek ki. A transzkriptom adatbázis 141 NBS-LRR gént tartalmaz, melyek közül egy NBS-kódoló és egy LRR- típusú gén csak a kezelt mintában fejeződött ki. E két gén funkcionális vizsgálata a közeli jövőben tervezett. A generált adatbázis átfogó információkat nyújtott a génkifejeződési változásokról, ezért úgy gondoljuk jó kutatási eszköz lehet a rezisztencia mechanizmusok feltárásában. A fentiek mellett az NGS eljárással generált transzkriptomokat intron-targeting (IT) markerek fejlesztésére használtuk. A kifejlesztett IT primerek hatékonyságát kísérletesen elemeztük. Eredményeink azt mutatják, hogy az NGS alapú IT marker fejlesztés a különböző molekuláris vizsgálatokban, - a fajok közötti tanulmányokat is beleértve, - hatékonyan alkalmazható eljárás.

ABSTRAKT

Potato virus X ( PVX ) ist weltweit einer der wichtigsten Kartoffeln Viren. Die zwei bekannte PVX extreme Resistenzgenen Rx1 und Rx2 haben 98% Sequenzähnlichkeit zu der Nukleotid- Sequenz. Das kompliziert die Identifizierung diese Genen und auch die Unterscheidung von anderen sehr ähnliche Gene, wie die Gpa2 oder paraloge Sequenzen durch einen einzigen PCR. Hier berichten wir über die Entwicklung von funktionellen Markern für den einfachen und schnellen Identifizierung des Rx1 sowie die Rx2 Gen. Darüber hinaus wurde eine Multiplex-PCR- Reaktion zum gleichzeitigen Detektion beider Gene in einer einzigen Reaktion entwickelt. In weiteren Untersuchungen sollen wir zu einem besseren Verständnis der Kartoffel Abwehrmechanismen gegen Krankheitserreger aufzuklären. Um dieses Ziel zu erreichen, wurden zwei Ansätze, nämlich PCR ausgewählte cDNA Subtraktion und RNA-Seq Transkriptom zum Expression-profil PVX, Potato virus Y ( PVY ) und Phytophthora infestans response-Genen in frühen Stadien der Infektion in der resistenten Kartoffelsorte, White Lady verwendet. Erstens wurden zwei subtrahierte cDNA-Sammlungen von White Lady in Reaktion auf PVX und PVYNTN konstruiert. Wir konnten 28 und 35 EST-s zu PVX und PVYNTN in White Lady identifizieren, beziehungsweise. Von diesen 17 und 12 könnten in den PVX sowie in der PVY Pool annotiert, während fünf EST-s wurde gemeinsam in beiden gemeinsamen cDNA-Sammlungen. Die übrigen sind Gene mit unbekannter Funktion oder neue Sequenzen. Zweitens wir erzeugten eine globale Transkriptom Profil der frühen Reaktion der White Lady zu PVX , PVY und Ph. infestans Infektion mit Next Generation Sequencing (NGS ). Angleichung der Fragmente mit 39 031 Protein-kodierenden Genen des Genoms von S. tuberosum Gruppe phureja DM1-3 5116R44 identifizierte 38 675 Gene des Erregers behandelten Probe. Siebenhundertachtundvierzig Transkripte wurden in der behandelten Probe erkannt, aber nicht in der Kontrolle, die als Stressantwort spezifischen Genen erkannt wurden. Der Datensatz enthält 141 NBS-LRR kodierenden Gene. Von diesen wurden ein NBS- und ein LRR-Codierende Gen identifiziert, die wurden nur in den behandelten Proben exprimiert und sollen für zukünftige Studien interessant sein. Der erzeugte Datensatz hat ausführliche Information über die Transkriptom Dynamik, und kann als Blaupause verwendet, in die Resistenzmechanism Untersuchungen für Biologen und Kartoffelzüchtern. Darüber hinaus wurden NGS abgeleitet Transkript-Sequenzen für die Entwicklung von Intron- Targeting (IT) Marker aufgetragen. Die Effizienz der entwickelten IT Primere wurde experimentell untergesucht. Die Ergebnisse zeigten, die Effizienz der NGS abgeleitete IT Marker Entwicklung und zeigten ihren Nutzen in diverse molekulare Analysen einschließlich ihrer Anwendbarkeit für cross-species Studien.

ABBREVIATIONS

ACMV- African cassava mosaic virus HSP - Heat shock proteins AFLP - Amplified Fragment Length HR - Hypersensitive reaction Polymorphism IPM - Integrated pest management AOX- alternative oxidase IT - Intron targeting APAF-1 - Apoptotic protease-activating factor 1 JA - Jasmonic acid ARC - Apaf-1, R protein, CED-4 LRR - Leucine-rich repeat Avr - Avirulence gene MAPK - Mitogen-activated protein kinase BCMV- Bean common mosaic virus MAS - Marker assisted selection BLAST - basic alignment search tool mRNA - Messenger ribonucleic acid BLAT- The blast-like alignment tool NBS - Nucleotide-binding site BPB - Brome phenol blue NCBI - National Center for Biotechnology CAPS - Cleaved amplified polymorphic Information sequence NGS - Next generation sequencing CC - Coiled coil domain NO - Nitric oxide CDPK- Ca2+-dependent protein kinase ORFs- open reading frames cDNA - Complementary deoxyribonucleic acid ORMV - Oilseed rape mosaic virus CIA - Chloroform isoamyl alcohol PAMPs - Pathogen-associated molecular CP - Coat protein patterns CTR1-constitutive triple response PCL - Plant cell lysis DAS-ELISA – Double-antibody sandwich PIs- Proteinase inhibitors enzyme-linked immunosorbent assay PCR - Polymerase chain reaction DNA - Deoxyribonucleic acid PLRV - Potato leaf roll virus dsRNA - double-stranded RNA Potato-DM - Solanum tuberosum group EDTA - Ethylene diamine tetraacetic acid Phureja DM1-3 5116R44 ER - Extreme Resistance PMTV- Potato mop-top virus ERF- Ethylene-responsive transcription factor PR - Pathogenesis-related genes EST- Expressed sequence tag PRR - Pattern recognition receptors ETI - Effector triggered immunity PTNRD - Potato tuber necrotic ring spot disease ET - Ethylene PTI - PAMP triggered immunity

FST- Wright’s fixation index PVA - Potato virus A GMO - Genetically modified organism PVM - Potato virus M

HC- heterozygosity PVP - Polyvinylpyrrolidone

HC′- Shannon Wiener diversity index PVS - Potato virus S

HT- Nei’s total genetic diversity PVY - Potato virus Y

QTL - Qualitative trait loci SSH - Suppressive subtraction hybridization q-PCR - quantitative- PCR SSR - Simple sequence repeat R-Avr- Resistance- avirulence ssRNA - Single-stranded RNA RFLP - Restriction fragment length STS - Sequence tagged site polymorphism TBE - Tris-HCL, Boric acid, EDTA buffer RNA - Ribonucleic acid TC- transcriptomes ROS- Reactive oxygen species TE - Tris-HCL, EDTA buffer SA - Salicylic acid TGB - Triple gene block SAGE - Serial analysis of gene expression TIR - Toll interleukin-1 receptor domain SAR - systemic acquired resistance TIGR -The institute for genomic research SCAR - Sequence characterized amplified TMV- Tobacco mosaic virus region TNLs - TIR-NB–LRRs SCF- SKp1, Cullin, F-box protein TuMV-Turnip mosaic virus SGN- SOL genomics network TVCV- Turnip vein clearing virus siRNA- small interfering RNA TYLCV- Tomato yellow leaf curl virus SKP1- S phase kinase-associated protein 1 UN - United Nations SNP - Single nucleotide polymorphism VPg - viral protein linked to the genome SOLiD - Sequencing by oligonucleotide VSRs - Viral suppressors of RNA silencing ligation and detection

1. INTRODUCTION

Potato (Solanum tuberosum L.) is the third most important food crop after wheat and rice. According to FAOSTAT, potato production in 2009 reached 329.58 million tons produced from 18.65 million hectares at a mean fresh-weight yield of 17.6 t/ha. Potato is an excellent staple food crop and is grown as a vegetable for table use, or processed into French fries and crisps (chips), as well as it is used for dried products and starch production playing a main role in global food security (Kang and Priyadarshan, 2007). The nutritional value of potato per unit of land is 2-3 times more than that of cereals. Potatoes are a good source of carbohydrate and also provide a significant amount of protein, vitamins and minerals, such as calcium, potassium, phosphorus, magnesium, iron or zinc among others (Razdan and Mattoo, 2005; Storey, 2009). In addition, diverse non- food uses of potato are expanding, for example, potato as a source of starch for the production of biodegradable plastics (Doane 1994). In recognition of its important roles, the United Nations (UN) named 2008 as the International Year of the Potato. In recent years, there has been a shift in the end use of potatoes, with production for direct consumption being replaced by processing potatoes for the production of convenience foods such as French fries and potato chips (Kole, 2007). Potatoes are grown in 149 countries (Hijmans and Spooner, 2001). Top five potato producing countries are China, India, the Russian Federation, Ukraine and the United States (FAO statistics, 2008). This crop is susceptible to a wide range of fungal, bacterial, and viral diseases as well as various nematodes and pests. From these, beside Phytophthora infestans viruses are the most dangerous parasites of the potato (Ross, 1986). Viruses are important pathogens that can substantially decrease yield and quality of this crop. Up to now over 40 viruses have been reported which infect cultivated potatoes in the field influencing, the distribution, agricultural practices and/or the varieties that can be grown in a given region. Most important viruses are Potato leaf roll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus A (PVA), Potato virus S (PVS) and Potato virus M (PVM) considering their distribution and effect on yield. Other viruses occur in potato only occasionally or locally (Salazar, 2003). These

viruses can cause serious yield and quality loss depending on the genotype or variety. Chemical control of plant viruses is not commercially feasible; therefore, genetic resistance that decreases or prevents replication of the virus and the appearance of symptoms on the plant is the only solution to protect crops against viral infections. Resistant varieties are considered to be the most cost-effective and reliable approach to control viruses and prevent yield and quality losses. Introgression of resistance genes to main potato viruses from wild species to cultivated potato has been successfully achieved in classical breeding programs. One of the most successful resistance breeding programs in terms of combination of cultural quality traits with complex virus resistance is operated by the breeders of Potato Research Centre, University of Pannonia, Keszthely. During the last 50 years using considerable time, efforts and cost more than a dozen varieties with resistance to PVY, PVX, PVA and PLRV were developed in the program. However over due to the loss of some pedigree data the origin of the resistance genes is uncertain in several cases. In terms of genetic status, cultivated potato (Solanum tuberosum ssp. tuberosum) is an auto tetraploid (2n = 4x = 48) plant that displays tetrasomic inheritance. It is highly heterozygous due to inbreeding depression after repeated selfing. One to four different alleles are present per locus, resulting in one homozygous and four heterozygous genotypes (Gebhardt and Valkonen, 2001). Genetic knowledge of the potato has increased dramatically since the first molecular-marker map appeared in 1988 (Bonierbale et al., 1988). Molecular markers are considered as valuable tools for crop improvement, due to their usefulness in characterizing and manipulating genetic loci responsible for monogenic and polygenic traits. In addition, sequencing of potato genome has provided an important opportunity to accelerate isolation of important genes and identify their functions (PGSC, 2011). One of the most important viral diseases of potato is PVX, against which two extreme resistance genes have been widely incorporated into different cultivars and breeding line. These genes, the Rx1 and Rx2, have already been cloned (Bendahmane et al., 1999; Bendahmane et al., 2000). They originate from different species and reside on different chromosomes. Nevertheless, these two genes have a 98% sequence similarity at the nucleotide level, respectively. The high level of sequence similarity makes it

complicated to identify these genes and to distinguish them from other highly similar genes, like the Gpa2 or from paralogous sequences by a single PCR. In terms of the mechanism of virus resistance, the plants respond to the infection with general and virus-specific defense reaction, which also involves varieties of physiological changes. Physiological changes in the resistance response to viruses suggested that certain pathway(s) that confer a resistance response against the virus may be specifically activated in the resistant cultivar. In general, plant–virus interactions are the least studied among plant-pathogen interactions. Moreover, the very early response, which plays a main role in resistance to viruses, on the whole transcriptome level in these interactions is even less well understood (Baebler et al., 2009). Study on whole transcriptome response of resistant potato cultivars to viruses makes it essential to identify genes that play main roles in resistance. Several experimental approaches for investigation the changes in the transcriptional profiles induced by viral infection have been developed. Among them, suppression subtractive hybridization (SSH) and more recently next generation sequencing (NGS) technologies have considerable advantages over the other methods. The former, is a powerful approach to identify and isolate cDNA-s of differentially expressed genes since SSH allows the isolation of differentially expressed cDNA-s without prior knowledge of their sequence. The latter has provided a capability to simultaneously sequence hundreds of thousands of DNA fragments, dramatically changing the landscape of genetics studies. RNA-Sequencing (RNA-Seq) is one of the new applications of NGS technologies for transcriptome studies which determine accurately the expression levels of specific genes, differential splicing, and allele-specific expression of transcripts. All these attributes are not readily achievable from previously widespread hybridization-based (microarray platforms) or tag sequence-based approaches (Costa et al., 2010). An efficient method to generate gene-specific co-dominant markers for mapping in plants is the Intron Targeting (IT) method (Seres et al., 2007). On the other hand, high- throughput transcriptome sequencing has the advantage to generate large transcript sequence data sets for gene discovery and molecular marker development.

Research objectives The research objectives of the present study are including: 1) Evaluation of the reaction of Hungarian potato cultivars to PVX and identification of the type of resistance gene using molecular approaches.

2) Development of specific molecular markers for the cloned PVX extreme resistance genes, Rx1and Rx2.

3) Development of a multiplex PCR to detect Rx1 and Rx2 in a single reaction for practical utilization in marker assisted selection.

4) Partial gene expression study of a resistant potato cultivar in response to PVX and PVYNTN in early stages of inoculation using PCR-selected subtractive approach.

5) Whole transcriptome analysis of a resistance cultivar in response to PVX, PVYNTN and Ph. infestans using next generation sequencing.

6) Development of NGS based intron-targeting markers in potato.

2. LITERATURE REVIEW

2.1. Origin and evolution of potato crop Potato is a New World crop that was unknown to the rest of the world until the 1500's. The most obvious domestication originated in the Andes Mountains of South America. The potato of commerce today was first domesticated in present-day Peru and Bolivia, and played an important role in that society, as is witnessed by many representations of potato in ceramic artwork from the area (Bamberg and Del Rio, 2005). The 219 wild tuber-bearing Solanum species recognized by Hawkes (1990) are distributed from the southwestern United States to central Argentina and adjacent Chile and cover a great ecogeographical range (Hawkes, 1990). They form a polyploidy series from diploid (2n = 2x = 24) to hexaploid (2n = 6x = 72). The result of domestication was the diploid species S. stenotomum from which six other cultivated species were derived, including S. tuberosum, which became the most widely grown species in South America (Bradshaw, 2008). S. tuberosum is a tetraploid (2n = 4x = 48) species that displays tetrasomic inheritance. The short-day adapted landrace populations of the Andes and the long-day adapted ones of coastal Chile are genetically distinct groups that have been classified as separate subspecies (S. tuberosum subsp. andigena and subsp. tuberosum) and are also referred to as Andigena and Chilean Tuberosum potatoes (Raker and Spooner, 2002). Potatoes (tetraploid S. tuberosum) were introduced into Europe in the 1570s and they were exported from Europe and cultivated in many other parts of the world (Hawkes and Francisco-Ortega, 1993). The today potatoes are grown in 149 countries (Hijmans and Spooner, 2001) and that potatoes are the fourth most important food crop after wheat and rice (Lang, 2001). Their distribution reflects the adaptation of S. tuberosum first to the short summer days of the highland tropics and subtropics, then to the long summer days of lowland temperate regions and finally to the short winter days of the low land subtropics and tropics.

2.2. Potato production in Hungary The potato production area decreased during the last 15 years from 50.000 to 22.000 ha in Hungary. After Hungary joined the EU, the seed potato production area also significantly decreased from 1500 ha to 350 ha. The total production reached 600.000 Mts while 5000 Mts was only seed potato (FAO, 2010). The total production was only 1% of EU’s total potato production and could just cover the requests of local market. The national average yield is about 25-27 Mts/ha. Out of that less than 10% is consumed as processed food. The average consumption of potato is approximately 65 kg/year/capita in Hungary. According to FAO’s report, Hungary is in the 50th position in terms of production quantity. Twenty percent of the total production area is covered by Hungarian varieties; those were mainly bred at Keszthely. The leading varieties are named as: Red Scarlet (NL), Laura (D), Kondor (NL), Desiree (NL), Cleopatra (NL), Agria (D), as well as Balatoni Rózsa (HU), Hópehely (HU), Katica (HU), Démon (HU), Góliát (HU) and Rioja (HU).

2.3. History of potato research at Keszthely, Hungary Based on 200 years long tradition in agricultural research University of Pannonia (UP), modern potato research and breeding activities exists since 1950 at the UP, Potato Research Centre, Keszthely. The Centre operates under a university system and is the only institution dedicated to potato research and breeding in Hungary. It is an appreciated center of basic and applied research, breeding, extension and education of experts for potato. One of its major duties is the breeding of profitable potato varieties those suitable for Central European agro-ecological conditions due to their resistance against major potato pests, pathogens and extreme weather conditions. The research fields of the Centre starting from basic to applied are all dedicated toward this goal and try to cover all important issues of the potato sector. From the sixties till the middle of eighties of the previous century the Centre operated a consistent, large scale resistance-breeding program utilizing several wild species germplasm. There were years when 1.5 – 2 million of seedlings were produced and screened by artificial infection with major potato pathogens and pests (viruses, nematodes and late blight) to incorporate resistance genes into cultivated genetic background. In the

crossing program different accessions of S. stoloniferum, S. acaule, S. tub. ssp. andigenum, S. vernei and S. hougasii were most intensively used directly or through species hybrids. Due to the consistent resistance-breeding program utilizing wild species germplasm the Centre currently has 13 varieties on EU list (Arany Chipke, Démon, Balatoni rózsa, Katica, Lorett, Góliát, Rioja, Hópehely, White Lady, Vénusz Gold, Luca XL, Kánkán and Somogyi Kifli). These varieties except Somogyi Kifli due to their complex resistance, high yielding potential and outstanding consumption quality are unique of their type and some of them are especially advised for organic production. All the varieties show extreme resistance to the economically most important potato virus PVY and PVA as well as high field resistance to PLRV. Out of thirteen, 9 is resistant to common scab, potato wart and golden cyst nematodes, while two of them to potato late blight which feature makes those especially advised for organic production. Recently advanced parental line screening methods, somatic hybridization, genetic modification and markers assisted selection techniques are involved into the breeding methodology of the Centre. Current main research topics are: the development of marker assisted selection techniques for late blight and virus resistance genes; increasing the environmental and food safety of potato production by development of specialized production technologies integrated pest management (IPM), optimization of nitrogen use, etc.), screening and breeding for nitrogen utilization efficiency, breeding to combine multi-resistance with processing quality, and testing the virus resistance of genetically modified (GM) potato lines.

2.4. Potato viruses Most potato-infecting viruses have a positive-sense, single-stranded RNA (ssRNA) genome that replicates in cytoplasm by the viral RNA-dependent RNA polymerase. Virions are assembled from hundreds to thousands of viral coat protein (CP) molecules that encapsidate the viral RNA.

2.4.1. Potato virus X

Potato virus X (PVX) is one of the main potato viruses infecting potatoes worldwide. PVX can cause yield loss in the range of 5-20% depending upon the virus strain, the potato genotype and the simultaneous infection with other viruses like PVY and PVA. This virus belongs to the genus Potexvirus, a member of family Flexiviridae (Adams et al., 2004). Members of this family are characterized by flexuous, filamentous virions between 470 and 580 nm in length, built of subunits of a single coat protein (CP). Potexviruses have monopartite, positive-strand RNA genomes encoding five open reading frames (ORFs). The 5′ end has a methylguanosine cap and the 3′ end has a poly (A) tail (Huisman et al., 1988; Huang et al., 2004). The genomic RNA encodes replicase for viral RNA synthesis, triple gene block (TGB) proteins for virus cell-to-cell movement and CP that functions in assembly, cell-to-cell movement and as an elicitor for Rx- mediated PVX resistance. The protein encoded by the first triple gene block gene (TGBp1) modifies the plasmodesmata to allow larger particles pass through and was also shown to be involved in inhibiting a post transcriptional gene-silencing pathway which is one of the plant’s defense mechanisms against virus replication (Howard et al., 2004; Bayne et al., 2005). Replicase translated from the genome synthesizes minus- and plus- strand copies of the viral RNA and subgenomic RNA-s that are templates for translation of the TGB proteins and CP. PVX can be transmitted in a mechanical way only. The natural host range of PVX has so far included plant species of the families Amaranthaceae, Brassicaceae and Solanaceae. PVX isolates generally produce mild symptoms in the potato crop, but sometimes they cause severe mosaic in infected plants. PVX strains have been classified into four groups according to their reactions with the genes for localized hypersensitivity (Nb and Nx), and extreme resistance (Rx). Group 1 strains are sensitive to both the Nx and Nb genes. Group 2 strains are sensitive to Nx gene, whereas group 3 strains are sensitive to Nb genes. Group 4 strains overcome both Nx and Nb mediated resistance. Strain PVXHB, found in 7% of Bolivian clones of Solanum tuberosum subsp. andigena, resemble the normal group 4 strains in overcoming genes Nb and Nx but differs from these in its ability to overcome Rx genes

conferring extreme resistance to PVX (Cockerham, 1954; Moreira et al., 1980a; Querci et al., 1995). The most commonly occurring strains are in the group 3 strain.

2.4.2. Potato virus Y

Potato virus Y (PVY) nowadays is the most common and destructive virus of cultivated potato since the relative significance of Potato leaf roll virus (PLRV) has decreased all over the world. The yield loss caused by the virus depends on the interaction of specific virus strain and potato genotype varies between 30 and 80%. This virus is the type member of the genus Potyvirus in the family Potyviridae, the largest group of plant viruses (Hall et al., 1998). The viral genome consists of a single-stranded, positive-sense RNA molecule about 10 kb in length, with a VPg protein covalently attached to its 5′ end and a poly-A tail at its 3′ end. The viral RNA encodes a single large polypeptide, which is cleaved by three virus-encoded proteases into nine products (Dougherty and Carrington, 1988). PVY can be transmitted mechanically and by aphids in a non-persistent manner. Myzus persicae is probably the most efficient vector; and more than 40 other aphid species are known as vector belong to the Acyrtosyphon, Aphis, Aulacorthum, Brachycaudus, Capitophorus, Cavariella, Cerosipha, Dysaulacorthum, Hyadaphis, Idiopterus, Macrosiphoniella, Macrosiphum, Metopolophium, Myzus, Phorodon, Ropalosiphoninus, Ropalosiphum, Scisaphis genus and the number of known vectors has been increasing continuously. PVY generates a variety of symptoms with different intensity depending on the interaction of potato genotype and PVY strain. The most typical symptom is the ‘leaf-drop streak’ or necrosis along the veins of the underside of leaflets and leaf mosaic. In combination with Potato virus X it causes the disease called ‘rugose mosaic’. PVY has several genetic variants called strains. Strain groups are based on the host response and resistance gene interactions. Several distinct strain groups of PVY have been defined. These are: the common (ordinary) group (PVYO), the stipple streak group (PVYC), the tobacco veinal necrosis group (PVYN) and the tuber necrosis strain (PVYNTN) that induces potato tuber necrotic ring spot disease (PTNRD) (Beczner et al., 1984; Jones, 1990a; Blanco-Urgoiti et al., 1998a). Some strains can be distinguished by symptomatology in different indicator plants (Horvath, 1967), by

serological methods (monoclonal antibody), and via molecular genetic analysis. Two broad pathotypes of PVY are defined based on the symptoms induced in tobacco (DeBokx and Huttinga, 1981). Strains including PVYO and PVYC induce mosaic, vein clearing, and mild leaf mottling, while others including PVYN, PVYN-Wi, and PVYNTN induce mosaic and systemic vein necrosis. Some (mainly PVYNTN isolates) cause a tuber necrotic reaction in susceptible potato cultivars (Singh et al., 2008). PVYO strain group: PVYO in general causes severe symptoms such as crinkling, rugosity or leaf-drop streaks and stunting although the type and severity of symptoms may depend on potato genotypes. This strain does not cause veinal necrosis in tobacco but induces symptoms ranging from typical mild mosaic to systemic mottle. PVYC strain group: PVYC, the stipple streak group, was the first strain that was identified in the 1930s (Salaman, 1930). This strain causes mosaic patterns or stipple streak in susceptible potato. The PVYC isolates elicit a hypersensitive response (HR) in potato cultivars bearing the Nc resistance gene (Cockerham, 1970). Symptoms in tobacco are reported to be indistinguishable from those induced by PVYO isolates. Unlike the other strains of PVY, some PVYC strains are non-aphid transmissible (Horvath, 1966; Blanco-Urgoiti et al., 1998b). PVYN strain group: In the 1950s, a variant of PVY was detected in potatoes in many countries in Europe (Bode and Volk, 1957). It caused veinal necrosis in tobacco leaves and mild mottle symptoms in most potatoes and was referred to as ‘veinal necrosis virus’. Keller and Munster designated PVYN to describe the tobacco veinal necrosis strain (Keller and Münster, 1961). In Hungary PVYN was detected from tobacco by Szirmai, J. in 1958. The PVYN isolates induce necrosis in tobacco but do not induce necrosis in the presence of the genes Nc or Ny in potato cultivars. The virus exists in the Andes in native cultivars; and thus it is believed to originate in the Andes and remained undetected in Europe for many years. The strain PVYNTN was first reported in Hungary (Beczner et al., 1984) and was characterized at molecular level (Thole et al., 1993). Since then outbreaks have been reported in many regions of the world. In Hungary more than 90% of PVY isolates collected from potato fields belong to the PVYNTN strain (Wolf and Horvath, 2000). The strain that is not an apparently uniform entity causes severe mosaic leaf symptoms and ring like tuber necrosis (PTNRD) on tubers of several susceptible cultivars

but not on all. PVYNTN isolates characterized at the molecular level were found to be recombinants of PVYO and PVYN in the CP-encoding region. The virus probably spread to Europe and North America in the last two decades due to inappropriate application of new technologies during seed production, such as the use of monoclonal antibodies for its detection in seed programs (Salazar, 2003). In addition other groups have been identified including the PVYZ (reacted serologically to PVYO- specific antibodies and did not cause veinal necrosis symptoms in tobacco) (Kerlan et al., 1999) and PVYN-Wi (reacted serologically to PVYO-specific antibodies and cause veinal necrosis symptoms in tobacco, (Chrzanowska, 1991) and in North America called PVYN:O (Nie and Singh, 2002). Some PVYN-Wi isolates may also cause tuber necrosis (Piche et al., 2004). Researchers consider PVYN-Wi and PVYNTN as sub-strains of PVYN, and the identification of non-recombinant PVYNTN inducing PTNRD revealed that the recombinant structure of the genome is not a necessary prerequisite for PTNRD phenotype (Nie and Singh, 2003; Browning et al., 2004; Glais et al., 2005). Recently, Lorenzen et al. (2006) reported that strain variants of PVYN and PVYNTN from Europe were present in North America (Lorenzen et al., 2006). However, it is proposed that any newly found isolates should be described within the context of the original strain groups based on the original methods of distinguishing strains (Singh et al., 2008).

2.5. Genetics of resistance Plants live in complex environments in which they intimately interact with a broad range of microbial pathogens with different lifestyles and infection strategies. In general, plants defend themselves against pathogens by a combination of active and passive defense. In passive defense, structural characteristics act as physical barriers and inhibit the pathogen from gaining entrance and spreading through the plant and in active defense, biochemical reactions that take place in the cells and tissues of the plant and produce substances that are either toxic to the pathogen or create conditions that inhibit growth of the pathogen in the plant (Agrios, 2005). The evolutionary arms race between plants and their attackers provided plants with a highly sophisticated defense system that, like the animal innate immune system (active defense), recognizes pathogen molecules and responds by activating specific defenses

that are directed against the invader (Pieterse et al., 2009). Plants respond to infection using a two-branched innate immune system. The first branch recognizes and responds to molecules common to many classes of microbes, including non-pathogens. In this branch, the resistance is induced by the recognition of pathogen-associated molecular patterns (PAMPs) by plant cell surface pattern recognition receptors (PRR), which initiates PAMP triggered immunity, that usually prevent the infection of pathogens before invasion. Defense responses activated by PAMPs are collectively termed PAMP triggered immunity (PTI) or basal resistance (Jones and Dangl, 2006). In the majority of cases, PTI prevent pathogen growth at an early infection stage due to the induction of pathogen-responsive genes, production of reactive oxygen species, mitogen-activated protein kinase signaling and deposition of callose to reinforce the cell wall at sites of infection (Schwessinger and Zipfel, 2008). If a pathogen evades this line of defense, it must also overcome a second line of defense to become pathogenic. The second branch acts primarily inside the cell using disease resistance (R) proteins which recognize pathogen-delivered effectors or their effects on host proteins. R protein- mediated defenses are termed effector triggered immunity (ETI) or gene-for-gene resistance, in which the protein products of plant resistance (R) genes specifically recognize cognate pathogen avirulence (Avr) gene products and trigger a stronger resistance response. Direct or indirect recognition of effectors by R proteins initiates ETI, which is an amplified and accelerated PTI response resulting in disease resistance (Jones and Dangl, 2006). ETI usually induces a hypersensitive response (HR) with localized cell death and defense gene expression that suppresses the growth and spread of pathogens.

2.5.1. Structure and function of R genes

According to Sacco and Moffett (2009) over 70 R genes have already been cloned (Sacco and Moffett, 2009). However, the focus has been mainly on monogenic dominant resistance to fungal and bacterial pathogens. But, there is clear evidence that common mechanisms can be involved in virus resistance (Hammond-Kosack and Parker, 2003). The majority of R genes belong to the nucleotide-binding leucine-rich repeat (NB-LRR) family. NB-LRRs contain a C-terminal LRR domain and a central NB domain (Sacco and Moffett, 2009). The NB is part of a larger domain that is called the NB-ARC (Apaf-1, R

protein, CED-4) as it is shared between R proteins and the human apoptotic protease- activating factor 1 (APAF-1) and its Caenorhabditis elegans homolog CED- 4 (Takken et al., 2006). NB is proposed to act as a nucleotide dependent molecular switch regulating the conformation and signaling activity of these proteins. The LRR domain, positioned C- terminally to the NB-ARC, forms an arc-shaped conformation, forming a protein-protein interaction surface which provides recognition specificity (Fig 1.). Based on the identity of the N-terminal domain two main classes of NB-LRR, R proteins can be distinguished. Some contains toll interleukin-1 receptor (TIR) domain and these R proteins are called TIR-NB-LRRs or TNLs (Burch-Smith and Dinesh-Kumar, 2007). Non-TIR-NB-LRR proteins contain predicted coiled coil (CC) motifs, this family is referred as CC-NB- LRRs (Lupas, 1997). Direct interaction between the LRR and pathogen effectors has rarely been reported. The guard-theory describes an alternative recognition mechanism by which the NB-LRR proteins known as the guardee are the target of the Avr protein. When the recognition mechanism detects interference with the guardee protein, it activates resistance (Van Der Biezen and Jones, 1998; de Wit, 2002). The guard hypothesis suggests that the host- pathogen interaction is more likely an interaction between the Avr protein and a host recognition complex. This complex must be able to recognize the pathogen and signal a defense response. Complex levels and activation of signaling must be tightly regulated and the recognition complex must be poised to perceive and respond to pathogens. NBS-LRR encoded by the potato Rx gene is one of the best studied antiviral R proteins. Rx-mediated resistance to PVX and specificity of recognition depends on the presence of specific amino acid residues within the viral coat protein and properties of the Rx LRR domain (Farnham and Baulcombe, 2006). The NB domain of the Rx protein is found to be sufficient to induce defense responses (Rairdan et al., 2008). Whether this behavior extends to other CC-NB-LRR proteins remains to be determined. Mutations in the CP of resistance breaking strains of PVX were responsible for their evasion of Rx1-mediated recognition. Initiation of defense signaling by interaction between Rx and the PVX coat protein appears to involve a conformational change in the Rx polypeptide, allowing its conversion from an inhibited to an active state. It is not clear

if this reorganization of the Rx protein requires direct interaction with the elicitor (Rairdan and Moffett, 2006). More than 50 functional NB-LRR genes have been cloned from potato and related members of the Solanaceae (Hein et al., 2009). Recently, based on an amino acid motif based search of the annotated potato genome 438 NB-LRR type genes were identified among the ~39,000 potato gene models. Of the predicted genes, 77 contain an N-terminal toll/interleukin 1 receptor (TIR)-like domain, and 107 contain an N-terminal coiled-coil (CC) domain (Jupe et al., 2012). All of the R proteins encoded resistance genes against plant viruses belong to the nucleotide binding site-leucine-rich repeat (NBS-LRR) class and are located intracellular. However, there are no structural or other features of ‘‘antiviral’’ R proteins that sets them apart from the NBS-LRR proteins that confer resistance to bacteria, fungi, Oomycetes, or invertebrates (Jones and Dangl, 2006) and the signaling processes that they trigger are identical and lead to the activation of defenses against a broad spectrum of pathogens or pests, not only viruses (Murphy et al., 1999; Maule et al., 2007; Palukaitis and Carr, 2008; Moffett, 2009). Twelve dominant R genes conferring resistance to viruses expressed either as HR or extreme resistance (ER) have been cloned and sequenced (Maule et al., 2007; Palukaitis and Carr, 2008). In potato, thirteen functional NB-LRR genes have been identified in which Rx1 and Rx2 confer resistant to PVX (Bakker et al., 2011). Dominant genes Rx1 and Rx2, against PVX in potato, have been identified by map-based cloning as well by Agrobacterium transient expression system, respectively (Bendahmane et al., 1999; Bendahmane et al., 2000). Both genes belong to CC-NBS-LRR class. The potato gene Y- 1, an N gene homologue which confers HR to PVY, was also cloned and characterized (Vidal et al., 2002). After a virus is recognized by the LRR, the function of an R protein complex must switch from recognition to signal transduction. Intramolecular interactions, activation of the NBS domain, and changes in signaling components that may associate with the CC or TIR domain and LRR domain have all been implicated during early signaling (Fig 1.) (Caplan and Dinesh-Kumar, 2006; Sessa, 2013).

Fig 1. A Model for NB-LRR protein activation. In the resting state, an NB-LRR protein is kept in a closed and auto-inhibited state in which the LRR and N-terminal domain (CC/TIR) fold back on the NB-ARC core. Effector recognition, often aided by an accessory protein, likely occurs by an interface formed by the C-terminal half of the LRR and the CC/TIR domain. Effector recognition results in a conformational change that is transduced via the N-terminal part of the LRR to the ARC2. This change allows exchange of ADP for ATP, triggering a second conformational change in the NB-ARC resulting in a more open structure in which interfaces on either the NB or the N-terminal domain (CC/TIR) become exposed and activate defense signaling (Sessa, 2013).

2.5.2. Mechanism of virus resistance

To complete their life cycles, viruses undergo a multistep process that includes entry into plant cells, uncoating of nucleic acid, translation of viral proteins, replication of viral nucleic acid, assembly of progeny virions, cell-to-cell movement, systemic movement, and plant-to-plant movement (Carrington et al., 1996). Plant viruses typically initiate infection by penetrating through the plant cell wall into a living cell through wounds caused by mechanical abrasion or by vectors such as insects and nematodes. When virus particles enter a susceptible plant cell, the genome is released from the capsid, typically in the plant cytoplasm. Once the genome becomes available, it can be translated from mRNA-s to give early viral products such as viral replicase and other virus-specific proteins. Hereafter the virus faces various constraints imposed by the host and also

requires the involvement of many host proteins, typically diverted for function in the viral infection cycle (Kang et al., 2005). Successful infection of a plant by a virus therefore requires a series of compatible interactions between the host and a limited number of viral gene products. Absence of necessary host factors for virus replication or movement or mutation to incompatibility has been postulated to account for recessively inherited disease resistance (passive resistance). Also, it could occur from the presence of physical barriers to inoculation including cell walls, cuticules or leaf hairs (Fraser, 1990, 1992). In contrast, dominant resistance has been shown to result from an active recognition event that occurs between host and viral factors, resulting in the induction of host defense responses. Basically, passive or active resistance can function at any stage of the virus life cycle, although most known viral resistance mechanisms appear to target virus replication or movement (Kang et al., 2005). In active resistance, plant R gene interacts with pathogen-encoded avirulence (Avr) gene (gene-for-gene hypothesis). The plant specific R protein can recognize the pathogen Avr protein and initiate signaling that leads to defense response. (Caplan and Dinesh-Kumar, 2006).

2.5.3. Signaling mediated resistance

The response of plant to virus could also be divided into two major categories such as cellular stress and developmental defects. Comparison of Arabidopsis and N. benthamiana gene expression leads to the conclusion that virus infection causes characteristic changes in gene expression that is similar to stress and defense responses (Whitham et al., 2006). The stress responses are characterized by the induction of heat shock proteins (HSP) and defense responses by the induction of pathogenesis related (PR) genes and other genes associated with plant disease defense. The induction of HSP and PR genes represent cellular stress responses because of their non-specific nature and the lack of specific elicitors that induce them. Most viruses trigger these generic responses, which occur in the absence of typical gene-for-gene or resistance gene- avirulence gene interactions (Whitham et al., 2006). The expression of PR genes, mediated by salicylic acid (SA), is increased in many incompatible responses. In general, increasing of SA is required for the high accumulation of PR mRNA transcripts and

proteins that occur during resistance response to viruses but not in susceptible interactions (Malamy et al., 1990; Gaffney et al., 1993; Ryals et al., 1996). The defense-related genes include numerous pathogenesis-related (PR) genes such as PR-1, PR-2 (β-1,3 glucanase), PR-3 (chitinase), PR-4, PR-5 (thaumatin like protein), genes associated with redox status such as superoxide dismutase and GST (glutathione S- transferases), resistance gene homologs. (Robatzek and Somssich, 2001, 2002). Mitogen-activated protein kinase (MAPK) cascades play different roles in plant processes that include cytokinesis, phytohormone signaling, wound responses, osmotic stress, and pathogen resistance (Zhang and Klessig, 2001). Different transcription factors families such as TGA, MYB and WRKY have been implicated in disease resistance (Caplan and Dinesh-Kumar, 2006). In brief, some important signal transduction pathways involved in resistance to viruses are including: Salicylic acid (SA): SA plays a main role in the signal transduction pathway that results in the induction of systemic acquired resistance (SAR) and it is required for localization of viral and other pathogens during the HR. SA is required for the expression of a group of proteins that collectively are referred to as pathogenesis-related (PR) proteins. SA can induce inhibition of virus replication, cell-to-cell movement, and systemic movement but the precise effects of SA-induced resistance on the life cycle of a virus can differ between hosts and between viruses (Murphy and Carr, 2002; Love et al., 2007; Wang et al., 2007). SA biosynthesis is induced most strongly during HR lesion development. SA may be necessary to regulate the timing and extent of the HR. During the HR, SA forms a gradient, with SA accumulating to high levels at the center of the HR lesions, moderate levels at the lesion borders, and low levels in healthy tissue (Enyedi et al., 1992). Signaling mediated by Jasmonic acid and Ethylene: In spite of SA-dependent signaling pathways, the requirement of ethylene (ET) and jasmonic acid (JA) during R gene mediated resistance to viruses is more complex and variable. The crosstalk between ET-, JA- and SA-dependent signaling pathways can have synergistic or antagonistic effects on each other. ET and JA are secondary signaling molecules that function in microbial defense, wounding, and insect attack.

Signaling by reactive oxygen species, calcium, and nitric oxide: Reactive oxygen species (ROS) have been recognized as signals in defense, most notably during the oxidative burst or bursts that occur very early in the HR during a gene-for-gene response (Heath, 2000). ROS have several roles in the HR, but from the signaling point of view, perhaps two are the most important. Firstly, the oxidative burst activates Ca2+‏ ion influx across the plasma membrane via cyclic nucleotide-gated channels, in addition to mobilization of Ca2+ ions from intracellular stores (Torres and Dangl, 2005; Ma and Berkowitz, 2007). A second effect of changes in Ca2+‏- ion flux in the cytoplasm is triggering of the activity of calcium-dependent protein kinases, as well as highly complex MAPK cascades. ROS generated in the mitochondrion may also play roles in defensive signaling, particularly with respect to the induction by SA in resistance response to viruses. It has been proposed that alternative oxidase (AOX) is one of the factors that may influence this form, induction by SA, of defensive signaling (Ma and Berkowitz, 2007). Nitric oxide (NO) is an important signal in plant defense. For example, the relative levels of NO and H2O2 regulate programmed cell death during an HR (Delledonne et al., 2001), and regulate defense gene expression both at the point of infection and in distal tissues, in part by inducing the biosynthesis of SA (Song and Goodman, 2001). NO also may stimulate changes in nuclear gene expression and defensive signaling indirectly through inhibition of cytochrome oxidase (Huang et al., 2002).

2.5.4. Gene silencing

Plants have also evolved mechanisms to actively target viruses. Viral RNA-s can be targeted for degradation by the RNA silencing machinery of the plant. This involves the processing of viral double-stranded RNA (dsRNA) by Dicer like enzymes, into small interfering RNA-s (siRNA-s), which are subsequently incorporated into protein complexes that target viral RNA-s for degradation, through an endonucleolytic process. However, RNA silencing is generally insufficient to rapidly limit infections by viral pathogens, because most viruses encode viral suppressors of RNA silencing (VSRs) (Ding and Voinnet, 2007; Omarov et al., 2007). A rapid resistance response to viruses is afforded by gene-for-gene resistance. In addition to HR, many viral resistance genes

confer extreme resistance (ER), wherein cell death is not induced upon virus inoculation that occurs immediately after entry of the virus into plant cells and inhibits viral accumulation in the initially invaded cells (Ponz and Bruening, 1986; Kang et al., 2005).

2.5.5. Types of resistance to potato viruses

Resistant potato genotypes can react to the infection of viruses in the following ways.

2.5.5.1. Hypersensitive reaction (HR) Hypersensitivity is expressed as a development of local and sometimes systemic necrosis following virus infection. It is generally virus strain specific and becomes non- effective, when another, non-necrosis inducing strain of the virus is introduced or selected in the plant (Jones, 1990b). The HR prevents spread of the virus throughout the plant. Plants with HR show either local necrotic lesions, which prevent the infection from spreading further, or systemic necrosis. Virus can be detected in affected leaves in most cases. The HR reaction can be affected by environmental conditions or by physiology of the host plant (Loebenstein and Carr, 2006). Monogenic hypersensitive resistance to PVX, PVY and PVA has been reported in many potato cultivars and can confer useful field resistance to these viruses (Jones, 1990b). The hypersensitive resistance to the infection of PVX in potato is determined by two dominant genes, Nx and Nb (Cockerham,1955, 1970). Isolates of the PVX have been placed in strain groups according to their ability to elicit the hypersensitive response in potato cultivars carrying the dominant resistance genes Nb or Nx. The gene Nb confers resistance against PVX strains of the strain groups 1 and 2, whereas the gene Nx confers resistance against PVX strains of the strain groups 1 and 3 (Cockerham, 1954). The Nb gene has been localized to chromosome V to the same region that contains Rx2 (DeJong O et al., 1997). HR to PVY is expressed in genotypes containing the gene Nyadg but does N not confer resistance to PVY (Jones, 1990b; Valkonen et al., 1994). The gene Naadg confers HR to PVA in S. tuberosum subsp. andigena (Hamalainen et al., 1997; Hamalainen et al., 1998; Valkonen et al., 2000).

The gene Ns derived from S. tuberosum ssp. andigena confers resistance to PVS in potato (Marczewski et al., 1998). Rm gene descends from S. megistacrolobum induces a hypersensitive response in potato plants to Potato virus M. The locus Rm was placed on the short arm of chromosome XI (Marczewski et al., 2006). Hypersensitivity was not expressed in isolated protoplasts, suggesting that it is a tissue-related phenomenon and that cell to cell contact is required for the expression (Adams et al., 1986). The underlying mechanisms of HR resistance in potato are not yet clear, but some indications can be gathered from work done in tobacco. The N gene of tobacco mediates resistance to Tobacco mosaic virus (TMV) (Loebenstein and Carr, 2006). The N gene product is a cytoplasmically localized protein with a protein sequence motif known as ‘leucine-rich repeats’ (LRR) (Whitham et al., 1994). The N protein appears to recognize the presence of virus by binding, probably indirectly, to the TMV replicase protein. Subsequently, a series of steps is triggered that results in a hypersensitive response (HR) in the host plant. During the HR cells containing the virus die, resulting in a small necrotic lesion in the leaf.

2.5.5.2. Extreme resistance (ER) Extreme resistance is expressed as a low incidence of infection in intact plants with an extremely low virus titer in infected plants; no symptoms develop, except for limited systemic necrosis following graft inoculation in some cultivars. It is active against a broad spectrum of virus strains and breakdown of resistance in the field has not been documented, except for a limited area of the Andean region in South America where a resistance-breaking isolate of PVX occurred (Moreira et al., 1980b; Jones, 1985; Kavanagh et al., 1992). This type of resistance that functions at single cell level (Adams et al., 1986) is presumed as the best type of resistance to breed into potato. Rx gene upon recognition of PVX coat protein is highly effective in blocking of virus replication and also inhibit the translation of viral RNA-s by the host’s ribosomes (Bhattacharjee et al., 2009). Plants with ER to a virus show no symptoms, or limited necrosis (pinpoint lesions, flecks, or localized stem necrosis) when inoculated with virus. Only extremely low amounts of virus, if any, can be detected by sensitive techniques. ER is often regarded as

an immune response, i.e. plants cannot become infected no matter how intense the inoculum pressure and can be regarded as a ‘non-host’ of the virus. However, as indicated above, this may not always be strictly true. Monogenic extreme resistance to PVX, PVY and PVA has been detected in many tuber-bearing Solanum spp. and utilized in potato breeding programs. Extreme resistance to PVX is controlled by the dominant genes Rx1 and Rx2. The gene Rx1 is located on chromosome XII and the gene Rx2 on chromosome V. On the base of pedigree analysis, originating from diploid materials, it was assumed that Rx1 derived from Solanum tuberosum subsp. andigena and the Rx2 gene from Solanum acaule (Ritter et al., 1991). Rx confers extreme resistance to all strains of PVX , except a resistance-breaking strain HB, from South America (Moreira et al., 1980b). ER and HR to PVX and Potyviruses can be simply determined by sap-inoculation or graft- inoculation, observing the response and testing for infection. A connection between ER and HR has been suggested, because necrosis can sometimes occur in plants with ER genes (Ross, 1958; Cockerham, 1970; Delhfy, 1974).

The gene Ry confers ER type of resistance against PVY. Ryadg derived from Solanum tuberosum ssp. andigena and Rychc from S. chacoense were mapped on chromosome XI (Hamalainen et al., 1997) and IX (Hosaka et al., 2001), respectively. Another gene for extreme resistance to PVY, Rysto from S. stoloniferum has also been mapped to the same position as Ryadg on chromosome XI (Brigneti et al., 1997). Later, it was found that the pedigree of plant material might not be reliable (Gebhardt and Valkonen, 2001). Finally, using CAPS, STS, SSR, AFLP and intron-targeting markers, the gene was mapped to chromosome XII. (Flis et al., 2005; Song et al., 2005; Cernak et al., 2008).

The gene Ry-fsto has been obtained from S. stoloniferum mapped to chromosome XII

(Flis et al., 2005). It was suggested that the loci Ry-fsto and Rysto could represent two different genes for extreme resistance to PVY, both situated on chromosome XII (Song et al., 2005). The dominant gene Gm was derived from S. gourlayi and confers ER to PVM. This gene is mapped to a central region on potato chromosome IX (Marczewski et al., 2006). Hinrichs et al. (1998) reported that PVY replicated in initially infected leaf cells of inoculated plants of cultivars with the ER gene Rysto, and was transported into

neighboring cells, prior to a limited necrotic reaction after that the infection ceased

(Hinrichs et al., 1998). The ER gene Rxadg in cv. Cara has now been found to control separate virus resistance and cell death responses (Bendahmane et al., 1999), cell death does not normally occur when plants carrying Rxadg are inoculated with PVX because the ER is epistatic over the HR. When studying S. stoloniferum genes conferring resistance to PVY and PVA, Cockerham (1970) found genes for ER to be dominant or epistatic over genes for HR. Valkonen (1994) also found the ER gene Ryadg to be epistatic to the HR gene Nyadg in an Andigena-derived genotype. Extreme resistance to PVA is controlled by the dominant gene Ra (Cockerham, 1970; Ross, 1986; Barker, 1996). This gene is mapped on chromosome XI (Hamalainen et al., 1998).

2.5.5.3. Resistance to infection Varieties that are genetically susceptible to a virus, but under natural infection are less susceptible than highly susceptible genotypes called as field resistant genotypes. An alternative and less confusing term of describing this kind of resistance is the ‘resistance to infection’. This term describes the situation where the likelihood of infection, by natural means, is reduced in resistant plants, e.g. quantitative resistance to PLRV whereby fewer plants of resistant clones or cultivars become infected by aphids in field conditions (Davidson, 1980; Barker, 1987). Resistance to infection with PLRV can be the result of several different mechanisms that affect the vector and virus separately, and such resistance appears to be inherited polygenically (Ross, 1958; Baerecke, 1961; Davidson, 1980) probably as a result of the combination of several different mechanisms that contribute to the trait. Resistance to PLRV infection in some German cultivars appears to have been derived from introgression of genes from Solanum demissum (Ross, 1966; Davidson, 1980). Quantitative resistance to PVY infection, from S. phureja, has been reported (Davidson, 1980). It had been thought that this quantitative resistance to PVY might be more durable than major gene resistance, but extreme resistance (ER) and hypersensitive resistance (HR) conferred by single dominant genes have proved quite durable and offer a higher degree of protection.

2.5.5.4. Resistance to virus accumulation Plants with resistance to virus accumulation can be infected but the virus reaches only a relatively low concentration in the plant. This type of resistance has been shown for PLRV in a range of S. tuberosum breeding lines and other potato material (Barker and Harrison, 1985; Gase et al., 1988; Swiezyński et al., 1988; Van Den et al., 1993; Wilson and Jones, 1993). The most resistant tetraploid genotypes had 1-5% of the PLRV concentration found in susceptible clones. The advantage conferred by this type of resistance is that the virus is less likely to be acquired and spread to other plants by aphids (Barker and Woodford, 1992). To assess resistance to PLRV accumulation, plants can be graft-inoculated and the virus concentration can be determined by quantitative ELISA (Barker and Harrison, 1985). It was found that the most reliable and consistent results were obtained by testing tuber-progeny plants (secondary infection). However, at present, the result of quantitative- PCR (q-PCR) is more reliable. High level of resistance to accumulation of PVX and PVY, as well to PLRV in S. brevidens was found by (Gibson et al., 1990). In attempts to transfer this resistance into a S. tuberosum background by somatic hybridization, it was apparent that the gene controlling resistance to PLRV is different from those controlling resistance to PVY and PVX, and that genes conferring resistance to PVY and PVX are linked (Valkonen, 1994). The resistance to PVY and PVX in S. brevidens is thought to be associated with slow cell-to-cell spread of the viruses (Valkonen et al., 1991).

2.5.5.5. Resistance to virus movement in plants Resistance to virus movement occurs where the movement of virus through the plant is impeded, for example where a lower percentage of the tuber progeny of an infected plant become infected. This type of resistance has been recognized in certain potato clones with respect to PLRV (Hutton and Brock, 1953; Barker, 1987). In some cases it is associated with phloem necrosis as in cultivars Bismark (Hutton and Brock, 1953) and Apta (Golinowski et al., 1987). Resistance to phloem transport has been demonstrated in Bismark variety by Wilson and Jones (1992), who found it to be separate from resistance to accumulation and resistance to infection (Wilson and Jones, 1992). Phloem transport was not impeded in a number of clones studied (Derrick and Barker, 1992), but virus

accumulation was largely restricted to the internal phloem bundles (whereas in susceptible clones virus accumulated in internal and external phloem tissue). Swiezynski et al. (1989) found a high level of PLRV resistance associated with limited virus spread in four diploid potato clones (Swiezyński et al., 1988). HR could be regarded as a kind of resistance to virus movement, because movement is arrested or impeded by cell death. Phenotypic assessment of components of PLRV resistance showed that Solanum tuberosum subsp. andigena cultivars with high levels of resistance to infection were also resistant to the accumulation of PLRV and are able to restrict translocation of the virus to their tubers (Mihovilovich et al., 2007).

2.5.5.6. Mature plant resistance The more advanced the crop growth at the time of inoculation, the less likely it is that the daughter tubers will become infected. This is because virus replication at the site of inoculation and translocation to the tubers may be slower in plants showing mature plant resistance than in plants not showing such resistance. Long before crop maturity, virus ceases to move from the inoculated leaf (Beemster, 1987). Mature plant resistance has been demonstrated for PLRV, PVM, PVS, PVX and PVYO, but for some viruses (e.g. PVYN) such resistance develops later (Beemster, 1987) and may explain why PVYN is more difficult to control than PVYO. Mature plant resistance starts to develop at around the time of tuber initiation and can be complete 4 weeks later. It occurs in most cultivars, but its particular characteristics are cultivar specific and it differs according to virus, the virus strain and environmental conditions (Beemster, 1987). However, it is important to recognize that this type of resistance exists; it is unlikely that it could be easily exploited and manipulated in a breeding program.

2.5.5.7. Tolerance Although tolerance is not a form of virus resistance, this trait has been either deliberately or inadvertently selected in many breeding programs. Tolerant cultivars can be defined as those that show no symptoms or few obvious symptoms when plants are infected. Although tolerance may be seen as a valuable trait, it has a major disadvantage

since tolerant cultivars are in essence susceptible genotypes that exhibit less damage when infected than other susceptible genotypes, and importantly, they can act as virus reservoirs. The underlying causes of ‘tolerance’ are difficult to identify and may be related to many plant factors that influence the replication and movement of virus particles and the expression of disease symptoms. However, it is increasingly recognized that there are dangers in breeding for tolerance because of the risk of virus spread from infected symptomless stocks grown in proximity to healthy material, or of introducing a soil-borne virus to sites that were previously uncontaminated (Barker and Dale, 2006).

2.5.5.8. Resistance to virus vectors Resistance to aphid vectors has been sought in Solanum species and should be a promising trait for breeding because it should help diminish virus spread, and also because also aphids themselves may cause significant crop damage. A promising type of resistance is found in wild Bolivian potato species Solanum berthaultii. Plants of some accessions of this species possess two types of glandular hairs: A-type hairs, which when ruptured physically entrap aphids with their contents, and B-type hairs, which entangle aphids, making them struggle more and so rupture more A-type hairs (Tingey and Laubengayer, 1981). In addition, B-type hairs are sources of (E)-farnescene, which is the main component of the alarm pheromone for most aphids and the activity of this chemical can act to repel M. persicae and to induce rapid dispersal from the leaf (Gibson and Pickett, 1983). Rizvi and Raman (1983) investigated two accessions of S. berthaultii, one with type A and B hairs and one with type A only. Both accessions were susceptible to PVY and PLRV. In a field trial, both accessions were exposed to aphid-borne infection by the viruses and both were equally infected with PVY, indicating that type A and B hairs had no effect on PVY spread. However, the spread of PLRV was reduced significantly by B type hairs (22% spread in the accession with A- and B-type hairs and 84% in the accession with A-type hairs only (Rizvi and Raman, 1983). Unfortunately it proved to be difficult to incorporate the genes for the B-type hairs without also introducing undesirable characteristics. Kalazich and Plaisted (1991) found that in plants from backcrosses between S. bertaultii and S. tuberosum, there was a strong association between the

presence of the B-type trichomes and undesirable characteristics such as lower yields, fewer tubers and later maturity (Kalazich and Plaisted, 1991). There are no sources of resistance or tolerance to Potato mop-top virus (PMTV) that could be deliberately used in breeding programs, but resistance to Spongospora subterranea (the vector of PMTV) has been found in Solanum species and in certain S. tuberosum cvs, but there is no report whether this resistance could be developed to a level that would confer resistance to PMTV infection.

2.6. The potato resistance genes Rx1 and Rx2 Two dominant genes, Rx1 and Rx2, in potato confer extreme resistance to PVX without necrotic cell death. Rx-mediated resistance results in a very rapid arrest of PVX accumulation in the initially infected cell (Kohm et al., 1993). In contrast to HR- associated resistance, Rx-mediated resistance is active at protoplast level via mechanisms that either suppress virus replication or promote degradation of the viral RNA (Adams et al., 1986; Kohm et al., 1993). The Rx1 and Rx2 are functionally identical and almost identical in the C terminal region consistent with a role of leucine-rich repeat in recognition of PVX coat protein. Two amino acid chnages, located at positions 121 and

127, in the strain PVXHB coat protein, that was found in Bolivian clones of Solanum tuberosum subsp. andigena, are involved in breaking the resistance specified by Rx1 and Rx2 (Querci et al., 1995). Rx1 is located on chromosome XII and Rx2 on chromosome V (Ritter et al., 1991; Bendahmane et al., 1997). The Rx1 gene is tightly linked to the Gpa2 locus for resistance to the cyst nematode Globodera pallida (Van Der Vossen et al., 2000). Based on pedigree analysis of diploid materials, it is assumed that Rx1 derived from S. tuberosum subsp. andigena, while Rx2 from S. acaule (Ritter et al., 1991). The Rx1 gene was physically isolated through a map-based cloning strategy (Bendahmane et al., 1999). Based on the sequence of Rx1, a transient expression assay was then developed to identify and clone the Rx2 gene. In this assay, Rx homologues were cloned into an Agrobacterium vector and infiltrated into transgenic tobacco that expressed the PVX coat protein. The Rx homologue that elicited the HR upon Agroinfiltration into the transgenic tobacco was confirmed to be Rx2 (Bendahmane et al., 2000).

A sequence comparison between Rx1 and Rx2 revealed a close evolutionary relationship that the two R genes may have evolved through repeated sequence exchanges between these unlinked loci. The open reading frame (ORF) of the Rx1 and Rx2 genes show 95% sequence identity at the nucleotide level. Except a single extra triplet in the Rx2 gene, variations are dispersed in one to two nucleotides along the coding region. The similarity at the protein level is 96% of the 937, as well 938 amino acid long Rx1 and Rx2 genes, respectively. Although, they originate from different species this high level of sequence identity makes it somewhat difficult to separate the two genes from each other. One strategy could be the PCR-RFLP, where the PCR product is digested with point- mutation specific restriction enzymes. However, this method is rather laborious and costly. Consequently the identification of the two genes by a single PCR would be more beneficial for marker assisted selection. This would even allow the development of multiplex PCR reactions, where in the same reaction the presence of several different sequences can be analyzed. Multiplex PCR-s would really make the marker assisted selection economically competitive and effective.

2.7. Resistance breeding and molecular markers Numerous resistance genes have been discovered in Solanum species and introgressed into the cultivated potato. Conventional methods of plant breeding are based on the phenotypic selection of plants with favorite traits and finally desirable combinations of genes assemble in the new varieties. However, the interaction of environment and genotype can reduce the effectiveness of phenotypic selection and complicate the identification of superior genotypes. Therefore, the introgression of a resistance gene into a breeding line by traditional breeding is time-consuming and complicated by the need of performing artificial inoculation tests to assess the resistant phenotype (Jain and Brar, 2010). Molecular markers are important genetic tools for plant breeders to detect the genetic variation available in the germplasm collection. Since the 1980s, the introduction of molecular marker techniques has facilitated gene mapping and shifted orientation from phenotype-based resistance genetics to genotype-based approaches. Molecular markers

are now a well-established technology that can be used in large breeding programs to complement the traditional breeding process (Tuvesson et al., 2007). A number of loci conferring quantitative resistance and about forty single dominant resistance genes conferring qualitative resistance have been positioned on the potato molecular map. Until 2006, eight of the mapped R-genes have been isolated and molecularly characterized. The analysis of mapped and cloned resistance genes shows that they often occur in clusters and that some of them can respond to more than one elicitor. Indeed, similarly to other plant species, dominant resistance genes in potato tend to occur in clustered loci. Approximately three quarters of all resistance genes mapped in potato have been found in only five ‘resistance gene hotspots’ located on chromosomes V, XI and XII. Study on resistance gene evolution will help to understand the dynamic interaction between potato plants and pathogens and opens a new approach in development of resistant cultivars (Simko et al., 2007).

2.8. Marker assisted selection Since the advent of the first DNA markers, marker assisted selection (MAS) has been considered as a promising approach to simplify resistance breeding. Molecular markers linked to resistance genes can prevent the need for testing to identify resistant individuals from early generations, leading to an effective improvement of the breeding procedure. The most successful applications of MAS in plant breeding have been those for major disease resistance genes assisting backcrossing into elite cultivars and selecting alleles with major effects on high value traits with relatively simple inheritance (Jain and Brar, 2010). Molecular markers in desired genomic regions enable the selection of individuals with resistance to several pathogens simultaneously, unlike a phenotype-based selection that requires different independent experiments. When the percentage of genetic variation explained by the marker(s) is high, the efficiency of molecular marker(s) for MAS is also high. Therefore, MAS is the simplest and most effective method for R-gene-based resistance trait selection with markers tightly linked with, or residing within, the resistance gene itself. However, as the recombination frequency between the marker and the resistance locus increases, the value of the marker for MAS decreases. However, an important requirement for molecular

markers used in MAS is their universality in a wide genetic background, not just in a specific cross. In potato, molecular markers have been developed and successfully tested for a gene conferring extreme resistance to PVY. A sequence characterized amplified region

(SCAR) marker was developed for PVY resistance gene Ryadg. The marker was generated only in genotypes carrying Ryadg, when tested on 103 breeding lines and cultivars with diverse genetic backgrounds (Kasai et al., 2000). Other molecular markers linked to the Ns gene conferring resistance to PVS are used for selection in diploid breeding programs (Marczewski et al., 2002). With allele-specific primers, the presence of the R-gene can be followed even if other R-genes exist in the same plant material. Gebhardt et al. (2006) indicated that MAS could be efficiently used in resistance breeding programs. The authors applied screening with PCR-based molecular markers to develop breeding material that carries combination of four resistance genes: Ryadg for extreme resistance to PVY, Gro1 for resistance to G. rostochiensis, Rx1 for extreme resistance to PVX, or Sen1 for resistance to potato wart (Gebhardt et al., 2006). Furthermore, the anchor marker

Cat-in2, specific for Rystol, is effectively used in practical selection developed at the Potato Research Centre (University of Pannonia, Keszthely, Hungary) (Cernak et al., 2008).

2.9. Intron-targeting An efficient method to generate gene-specific co-dominant markers for mapping in plants is the Intron Targeting (IT) method (Seres et al., 2007). Introns are widespread and abundant in eukaryotic genomes. There can be various polymorphisms in introns. IT primer pairs are complementary to the sequences of the exons flanking the targeted intron. Since the targeted intron sequence is generally less conserved than the exons, the amplified product may display polymorphism due to length/nucleotide variation among introns in the alleles of the gene. On the other hand, the higher level of sequence conservation in the exons ensures that all alleles can be effectively amplified. The prerequisite of the method is that the genomic region is sequenced and mRNA, assembled EST consensus or at least EST sequences also exist. The genomic DNA and the EST

sequence of a gene in a model organism and the EST sequence from the studied plant can also be sufficient (Seres et al., 2007).

Intron marker Cat-in2 was used as an anchor marker to localize Rysto gene in the potato genome (Cernak et al., 2008). Twenty nine polymorphic intron targeting makers were also characterized in potato using Expressed sequence tag (EST) and NCBI database records (Poczai et al., 2010). Publication of the potato genome sequence in 2011 has provided new insights into potato genetics and breeding research study (PGSC, 2011). On the other hand, the advances in sequencing technology have also enabled deep sequencing of complete transcriptomes (RNA-Seq) from large collections of tissues, conditions and time points to study the temporal and spatial distribution of gene activity (Clarke et al., 2009).

2.10. Gene expression profiling in plant-virus interactions Virus infections induce various physiological changes in susceptible plants, like complex networks of signal transduction pathways and biosynthetic reaction chains which result in disease development. On the contrary, resistant plants respond to virus infection with general and virus-specific defense, which also involve a large number of physiological changes. A major challenge has been to identify host genes with altered mRNA transcription profiles and to elucidate how and why the changes are initiated. The ultimate goal is to use this information to investigate the functions of genes with altered expression profiles in plant-virus interactions. Research carried out in the past few years has been productive in identifying transcription factors that are important for regulating plant responses to these stresses. These studies have also revealed some of the complexity and overlap in the responses to different stresses, and are likely to lead to new ways to enhance crop tolerance to disease and environmental stress (Singh et al., 2002). The studies on the changes of gene expression in the host plant following infection are attempting to reveal the basis of the host susceptibility and resistance. Several techniques have been utilized to identify gene expression changes that occur after virus infection. These techniques are including in situ hybridization differential display, Suppression Subtractive Hybridization (SSH), cDNA Amplified Fragment Length

Polymorphism (cDNA-AFLP), Macro- and Microarrays and Serial Analysis of Gene Expression (SAGE), as well as whole transcriptome analysis (Whitham et al., 2006).

2.10.1. Subtraction Suppressive Hybridization (SSH)

To understand the molecular basis of biological processes such as cellular growth, organogenesis and also effect of biotic and abiotic stresses on the physiology of higher eukaryotes, the relevant subsets of differentially expressed genes of interest should be identified, cloned and studied in details. Subtractive cDNA hybridization has been a powerful approach to identify and isolate cDNA-s of differentially expressed genes. Numerous cDNA subtraction methods have been reported (Munir et al., 2004; Park et al., 2006; Liu et al., 2012; Rawat et al., 2012; Senthilkumar et al., 2012; Singh et al., 2013). In general, they involve hybridization of cDNA from one population (tester) to excess of mRNA (cDNA) from another population (driver) and then separation of the unhybridized fraction (target) from hybridized common sequences. Although, the traditional subtractive hybridization methods have been successful in some cases, they require several rounds of hybridization and are not well suited for the identification of rare messages (Hedrick et al., 1984; Duguid and Dinauer, 1990). SSH is a comprehensive, large-scale gene expression based approach that allows unbiased discovery of the genes induced or suppressed by a particular treatment. Further, since SSH allows the isolation of differentially expressed cDNA-s without prior knowledge of their sequence, it is highly desirable for studying differential gene expression in systems where information on the genomic sequence is scarce (Munir et al., 2004). A PCR-based cDNA subtraction method, termed suppression subtractive hybridization (SSH), could overcome the technical limitations of traditional subtraction methods and demonstrate its effectiveness. SSH is used to selectively amplify target cDNA fragments (differentially expressed) and simultaneously suppress non-target DNA amplification (Diatchenko et al., 1996; Gurskaya et al., 1996). However, microarray technology and its associated equipment are very expensive and beyond the reach of many laboratories. Compared with microarray, this method can be performed in the absence of sequence information with the possibility of finding novel genes (Zhang et al., 2007).

Combining normalization and suppression PCR steps in a single cycle, SSH makes it possible to equalize abundance of target cDNA-s in the subtracted population, and as a result, rare differentially expressed transcripts have been reported to be enriched by 1,000-fold to 5,000-fold (Diatchenko et al., 1996). Because of its high level enrichment, low background and normalized abundance of cDNA-s, SSH has been extensively used by researchers to identify differentially expressed genes that contribute to the molecular regulation of biological or pathological processes. The principle of the technique is shown in Fig. 2.

Fig.2. Suppression subtractive hybridization (SSH) (Clontech catalog No: 637401). Adaptor ligation: Two tester populations are created with different adaptors, Adaptors 1 and 2R. First hybridization: Each tester population hybridizes separately with driver cDNA generating the type a, b, c, and d molecules in each sample. Only type a molecules are significantly enriched for differentially expressed sequences. Second hybridization: Two primary hybridization samples are mixed and form a, b, c, d and e. Primary and secondary PCR: Only type e molecules have Adaptors 1 and 2R with different annealing sites for first and second (nested) primers on their 5' and 3' ends. Therefore, only e type molecules will be amplified exponentially.

2.10.2. cDNA-AFLP

The cDNA-AFLP is sensitive and able to identify low-abundance targets (Bachem et al., 1996). This technique was used to study gene expression from stolon formation to sprouting in a range of different tissues during the potato tuber life cycle. In this experiment, about 18000 transcript-derived fragments were detected. The sequence similarities of these transcriptomes to known genes give insights into the kinds of processes occurring during tuberization, dormancy and sprouting. (Bachem et al., 1996; Bachem et al., 2000). This method has been used to perform a large scale survey of genes differentially expressed during the tuber life cycle and the isolation of some of their promoter regions (Trindade et al., 2004). Using this technique, 48 differentially expressed transcript fragments were identified in the susceptible Bintje and in the resistant Sarpo Mira potato cultivars to Ph. infestans (Orlowska et al., 2011). In another study, twelve inducible expressed gene fragments were detected using cDNA-AFLP in resistance tobacco in response to Tobacco mosaic virus (TMV) (Chen et al., 2012). The gene expression of resistant and susceptible genotype of beans (Phaseolus vulgaris) upon inoculation with Bean common mosaic virus (BCMV, genus Potyvirus) was compared by cDNA-AFLP and 17 unique transcripts were found (Cadle-Davidson and Jahn, 2006). One of the disadvantages of cDNA-AFLP is that it does not provide gene sequence information and requires isolation of gene fragments from polyacrylamide gels for sequencing (Bryan and Hein, 2008). It is also expensive, technically difficult and suffers from false positive results.

2.10.3. Serial Analysis of Gene Expression

The Serial Analysis of Gene Expression (SAGE) allows the quantitative analysis of a large number of gene transcripts (Velculescu et al., 1995; Matsumura et al., 1999). This approach is based on release and analysis of short fragments (tags) adjacent to the Nla III restriction site closest to the 3’ end of a cDNA. The relative numbers of copies of the different tags detected by this approach reflect the relative differences in the transcript expression levels. As the tag sequences can be used to identify the corresponding genes, especially in species of which the whole genome has been sequenced, the method can

provide information about the relative expression levels of genes with known or predicted functions. This technique has been used for analysis of gene expression in potato tubers. Of 58322 generated sequence tags, with the length of 19 nucleotides, 22233 fragments were found to be unique (Nielsen et al., 2005). SAGE was used in cassava to identify genes induced in resistant genotypes to African cassava mosaic virus (ACMV, genus Begomovirus). The genes identified belonged to two main functional groups including systemic acquired resistance (SAR) and genes involved in cell cycle activities and intra- and inter-cellular virus trafficking (Fregene et al., 2004). This approach has the advantage over microarray technology due to its potential to discover new transcripts. Nevertheless, the technique is limited by the requirement of a reliable genome annotation.

2.10.4. Microarray

Microarray technology is the most common approach used for gene expression profiling. Microarrays make use of the information created by genome sequencing and from expressed sequence tags (EST-s) which generated from genes expressed in specific cells, tissues organs at a particular time and under particular conditions. The Arabidopsis Gene Chip microarrays (Affymetrix), containing 8734 probe sets, was used to monitor a large scale changes in host genes after infection with five different viruses including Turnip vein clearing virus (TVCV, genus Tobamovirus), Oilseed rape mosaic virus (ORMV, tentative Tobamovirus member), PVX, CMV, and Turnip mosaic virus (TuMV, genus Potyvirus) (Whitham et al., 2003). The changes in gene expression were general for different viruses or virus-specific and putatively associated with defense or stress responses. In the first genome-wide study on host gene expression following a virus-specific defense response, a genotype of Arabidopsis, carrying the gene RCY1 for HR to CMV, was inoculated with strain Y of CMV. The analysis of results revealed 80 defense-responsive genes that might participate in R gene mediated defense against both viral and bacterial pathogens (Marathe et al., 2004b). In most potato microarray experiments, the information produced by The Institute for Genomic Research (TIGR), contains about 10,000 cDNA clones has been used. In addition, the same organization offered a transcription profiling service to allow the

evaluation of the arrays by a wide range of users working on different Solanaceous plant species asking different biological questions. This allowed generation of massive microarray data that is publicly available (http://www.jcvi.org/potato/#AProcedure) (Bryan and Hein, 2008; Davies et al., 2008). Due to high degree of allelic heterozygosity in potato, the use of short oligonucleotide arrays may result in misinterpretations. Therefore, long oligonucleotide arrays have also been used in some experiments in potato. For this purpose, the potato oligo chip initiative (POCI) has selected the Agilent “44K feature platform” system. This system is very flexible and allows for redesign of the array as more gene sequence information becomes available (Bryan and Hein, 2008). Platforms such as the existing potato cDNA and oligonucleotide based arrays are limited by lack of the full gene complement being interrogated on the platform. Recent advances in high-throughput sequencing technologies have overcome these limitations. This hybridization-based technology is largely restricted to known genes and has a limited range of quantification. In the other word, they are limited in that only information can be obtained for probes that are on the chip. Only information for organisms for which chips are available can be obtained, and they suffer from the problems of hybridizing large numbers of molecules (differing in hybridization temperatures). Furthermore, hybridization and cross-hybridization artifacts, dye-based detection issues and design constraints that preclude or seriously limit the detection of RNA splice patterns and previously unmapped genes (Okoniewski and Miller, 2006; Casneuf et al., 2007). These issues have made it difficult for standard array designs to cover all possible genes or reliable detection of all RNA-s of all prevalence classes, including the least abundant ones that are physiologically relevant (Mortazavi et al., 2008).

2.10.5. Transcriptome analysis

Large scale transcriptome data allow examining the expression of tens of thousands of genes over time or over a set of conditions under study such as biotic and abiotic stresses. In recent years, numerous technologies have been developed to analyze and quantify the transcriptome. Initially, a traditional sequencing method was used, but this

approach was costly and time-consuming. Because it involved cDNA library construction, cloning and labor intensive Sanger sequencing. The advent of the remarkable technology, Next Generation Sequencing (NGS), allowed direct and cost- effective sequencing of DNA in an impressive speed. The massive parallel sequencing platforms, Next Generation Sequencing (NGS), were introduced in 2004. The pioneer, the Roche (454) Genome Sequencer (GS) (http://www.454.com/), was able to simultaneously sequence several hundred thousand DNA fragments, with a read length greater than 100 base pairs (bp). The current GS FLX Titanium produces greater than 1 million reads in excess of 400 bp. It was followed in 2006 by the Illumina Genome Analyzer (GA) capable to generate tens of millions of 32- bp reads. Today, the Illumina GAIIx produces 200 million 75–100 bp reads. The last to arrive in the marketplace was the Applied Biosystems platform based on Sequencing by Oligo Ligation and Detection (SOLiD) capable of producing 400 million 50-bp reads, and the Helicos BioScience HeliScope, the first single-molecule sequencer that produces 400 million 25–35 bp reads (Table 1 ). The Applied Biosystems SOLiD system, which is used in this study, is based on emulsion PCR in combination with sequencing by ligation with dye labeled oligonucleotides (Shendure et al., 2005). It produces up to one billion short reads (up to 50 bases) per run, leading to a total sequence output of up to 30 gigabases per single read run. As templates it uses fragmented double-stranded DNA. Fragment sizes for the construction of paired end sequencing libraries can be up to 10 kb. The sequences produced are 99.94% accurate http://www3.appliedbiosystems. com/AB_Home/; Metzker 2010).

Table 1. Comparison of available NGS technologies.

Platform Template Sequencing Read Total Company homepage preparation chemistry length output per (bp) run (Gbp) Genome Sequencer Emulsion Pyrosequencing 400 0.4 http://www.454.com/index.asp FLX from 454 Life PCR Sciences

Illumina Genome Solid Phase Sequencing by 76 6 http://www.solexa.com PCR synthesis Analyzer

Applied Biosystems Emulsion Sequencing by 50 30 http://www3.appliedbiosystems.co PCR ligation m/ AB_Home/ SOLiD tSMS by Helicos Single Sequencing by 32 21 http://www.helicosbio.co molecule synthesis Bioscience

SMRT by Pacific Single Real time >900 ? http://www.pacificbiosciences.com/ Biosciences molecule Reference: (Bräutigam and Gowik, 2010).

One of the applications of these technologies is sequencing the complete transcriptome (RNA-Seq). RNA-Seq has been used as a powerful and cost-efficient tool for advanced research in many areas, including gene discovery, gene functional studies and molecular marker development, resequencing, microRNA expression profiling, DNA methylation and de novo transcriptome sequencing for non-model organisms. RNA-Seq is appropriate for comparative gene expression studies because it verifies direct transcript profiling without compromise and potential bias, thus allowing for more sensitive and accurate profiling of the transcriptome that more closely resembles the biology of the cell (Wang et al., 2009; Garber et al., 2011). The RNA-Seq method generates absolute information, rather than relative gene expression measurements; thus, it avoids many of the limitations of microarray analysis (Xu et al., 2012). RNA-Seq is also free from many of the limitations of microarray, such as the dependence on prior knowledge of the organism. This technology has been used in transcriptome profiling studies for various organisms, including maize, rice, and soybean (Eveland et al., 2010; Luo et al., 2011). Many NGS transcriptome projects aim to lay a foundation for future experiments and

create a sequence resource (Bräutigam and Gowik, 2010). The workflow for transcriptome analysis depends on the plant with or without sequenced genome.

2.10.5.1 De novo transcriptome analysis De novo transcriptome assembly is often the preferred method to study non-model organisms, since it is cheaper and easier than building a genome, and reference-based methods are not possible without an existing genome. The transcriptomes of these organisms can thus reveal novel proteins and their isoforms that are implicated in such unique biological phenomena. To create a transcriptome database in plant species without sequenced genome (de novo sequencing), many researchers have used 454 NGS since it produces the longest reads among current NGS technologies. Because during the assembly of contigs, single reads are assessed for their overlapping sequence. The assembly becomes increasingly more difficult when the read length gets shorter and shorter (Pop and Salzberg, 2008). Indeed, two different strategies are possible for de novo sequencing. If the non-model species is closely related to a species with a sequenced genome, the sequence reads can be mapped onto the reference. In this case, the type of mapping software can influence the results. In the second strategy, the normalized cDNA libraries from all conditions to be analyzed later and non-normalized libraries from these conditions should be assembled into a reference transcriptome. Using a long read sequencing technology for at least the normalized cDNA library will facilitate the assembly (Pop and Salzberg, 2008).

2.10.5.2. Transcriptome analysis of the sequenced genomes Prior to the development of transcriptome assembly computer programs, transcriptome data were analyzed primarily by mapping on to a reference genome, which is a robust way of characterizing transcript sequences. Transcriptome analysis of species with a fully sequenced genome identifies novel transcripts and identifies splicing isoforms. A much higher sequencing depth at comparable cost can be achieved using short read technology such as the Illumina Genome Analyser or the Applied Biosystems SOLiD system, producing over 100 million sequencing reads. These reads are directly

mapped to the genome sequence (Cloonan et al., 2008; Lister et al., 2008; Mortazavi et al., 2008). Splice isoforms can be identified by reads reaching over predicted exon boundaries (Mortazavi et al., 2008). Novel genes and incorrectly annotated 5′ or 3′ untranslated regions are discovered if reads map to genomic regions for which no elements were annotated. The abundance of a transcript can be measured simply by counting how many map onto a given gene reads. In contrast to microarray experiments, which report a ratio of fluorescence in arbitrary units, NGS measurements are absolute. To compare the abundance of transcripts within a sequence library these read counts are often normalized to the transcript length, e.g. reads per kilobase (RPK) of transcript. To compare the abundance of transcripts in two different libraries generated in two different conditions of an organism, the read counts are normalized to one million reads. According to this method, the abundance of a certain transcript in a certain cDNA population ⁄ sequence library, obtained by NGS, is generally given as reads per kilobase per million mapped sequence read (RPKM), meaning reads counted per 1000 bp of this transcript and per one million total reads from the sequence library (Mortazavi et al., 2008). This way, not only relative but absolute abundance values are determined for a given condition. Mapping the huge amounts of short read sequences produced by NGS to a given reference sequence is challenging. A traditional and well established sequence alignment tool like basic alignment search tool (BLAST) (Altschul et al., 1997) can be used for mapping these short reads, but BLAST is not optimized to cope with high numbers of reads; therefore such mappings are very time consuming. The blast-like alignment tool (BLAT) was developed to perform alignment tasks much faster (Kent, 2002). BLAT is suitable to map reads from the 454 platform, but short read sequencing technologies can produce over ten times more data within a single run, thus new bioinformatics tools capable of dealing with such huge amounts of data have been developed (Flicek and Birney, 2009). Currently, there is much progress in the development of such software, leading to the publication of several new programs within the last 2 years. Since experience with these programs and also comparative investigations is limited at the moment, it is difficult to predict if and which of these tools will become accepted as the standard. Perhaps it will turn out that, depending on the amount of reads, read length and

genome complexity of the organism investigated, a different program is favored (Palmieri & Schlotterer 2009). The high throughput, short read NGS systems have been successfully used in several studies for quantitative and qualitative transcriptome analysis in animal, plant and microbial model systems (Cloonan et al. 2008; Mortazavi et al. 2008; Nagalakshmi et al. 2008; Sultan et al. 2008; Wilhelm et al. 2008). An example of a particularly comprehensive study comes from Lister et al. (2008). By combining different techniques, they assessed the strand-specific transcriptome, small RNA-s and cytosine methylation in Arabidopsis on the genome scale, using short read sequencing with the Illumina Genome Analyser. The comparison of wild-type plants, DNA methyl transferase and DNA demethylase mutants allowed analysis of the interactions between DNA methylation, small RNA function and effects on transcriptional regulation within the experiment. Publication of the potato genome sequence has provided the possibility to apply genome sequence as a reference sequence for the transcriptome analysis of this crop. In this study, transcriptome profile of the resistant potato cultivar, White Lady in response to PVX, PVY and Ph. infestans was generated by NGS, 5500 XL, SOLiD Life Technology platform. The procedure of sequencing by this platform is summarized in the Fig. 3.

Fig. 3. The Procedure of preparation and sequencing RNA using NGS, 5500 XL, SOLiD Life Technologies.

3. MATERIALS AND METHODS

3.1. Plant materials 3.1.1. Plant materials used in PVX inoculation test

To examine the reaction of Hungarian potato genotypes to PVX, virus free minitubers of 16 genotypes (Table 2.) were used in this experiment. In addition, two F1 populations, developed at the Potato Research Centre (University of Pannonia, Keszthely, Hungary), resulted from crosses cv. White Lady × cv. Kuroda and cv. Luca XL × W1100 with 75 and 96 progenies, respectively, were grown under vector free greenhouse conditions. Cultivars White Lady and Luca XL are extreme resistant to PVX, while Kuroda and the breeding line W1100 are susceptible to it. Beside these, because of their known pedigree cv. Cara was used as a reference cultivar for the Rx1 gene and cv. Bzura for the Rx2 gene. Growing and testing conditions were all the same for all the plants.

Table 2. Cultivars and breeding lines

Cultivar Cultivar Breeding lines Cara Rioja 01.536 Bzura Démon 06.62 White Lady Góliát 06.256 Luca XL Balatoni Rózsa 06.325 Lorett Somogy Kifli 76.9104 Hópehely Katica W1100 Vénusz Gold

3.1.2. Evaluation of the validity of developed specific primers for Rx1 and Rx2 genes

Forty-eight commercial cultivars from different genetic backgrounds were used to examine the presence/absence of the Rx1 as well as Rx2 specific markers, developed in this study (Table 3.).

Table 3. Examined potato varieties with three developed primer pairs 1Rx1, 5Rx1 and 106Rx2 of this study

1Reaction 1Reaction Cultivar Country of origin 2 Pedigree Cultivar Country of origin 2 Pedigree to PVX to PVX Ditta Austria - Unknown Premier The Netherlands - CPC 1673-11 Laura Germany - Unknown Santé The Netherlands - CPC(adg) 1673-20

Romina Austria - CPC 1673-20 Saturna The Netherlands Sc CPC 1673-1

Shepody Canada Sa Unknown Spunta The Netherlands - Unknown

Raritan Canada - Unknown Lady Rosetta The Netherlands - CPC 1673-20

Franceline France - Unknown Amalia The Netherlands - CPC 1673-11

Agria Germany - Unknown Mondial The Netherlands Rd CPC 1673-20)

Bellarosa Germany Sa Unknown Courage The Netherlands - CPC 1673-20

Bettina Germany - Unknown Divina The Netherlands - Cara

Franzi Germany - Aquila Raja The Netherlands - CPC 1673-24)

Gala Germany - Unknown Multa The Netherlands Rc CPC 1673-1

Natascha Germany - Unknown Alcmaria The Netherlands Rc CPC 1673-20

Panda Germany - Aquila Amaryl The Netherlands Rc CPC 1673-20

Rosita Germany Sb Unknown Justa Poland - Unknown

Somogyi Sárga Hungary - Unknown Nativ Romania - Aquila

Somogyi Korai Hungary - Unknown Kuba Poland - Bzura

Sarolta Hungary - Aquila Eridia Slovakia - Unknown

Agata The Netherlands - Unknown Irati Spain - CPC 1673-20

Asterix The Netherlands - CPC 1673-20 Atlantic The United States Rd CPC 1673

Ausonia The Netherlands - CPC 1673-20 Kennebec The United States - Unknown

Carlita The Netherlands - CPC 1673-11 Rhinered The United States - Unknown

Desiree The Netherlands Sa Unknown Russet Burbank The United States - Unknown

Idol The Netherlands - CPC 1673-11 Snowden The United States - Unknown

Mozart The Netherlands - CPC 1673-1 Wauseon The United States - Unknown 1: Based on literatures; 2: Possible PVX resistance origin retrieved from http://www.plantbreeding.wur.nl/potatopedigree; R: indicates extreme Resistance; S: indicates Susceptible; a: tested in PRC-UP; b: data from (Bonierbale and F., 2007); c: data from (Rouppe van der Voort et al., 1999a); d: data from (Wilson and Jones, 1995).

3.1.3. Development of Intron targeting markers

To analyze the applicability of potato NGS derived IT markers, 24 individuals of a

tetraploid F1 potato population originated from a cross between cultivar White Lady as female and the breeding line S440 as male parent were used. Twenty four potato cultivars from different origins were also involved in the analysis (Table 4.).

Table 4. Potato cultivars and their country of origin.

Cultivar Country of origin Cultivar Country of origin Cultivar Country of origin

Ditta Austria Natasha Germany Santé The Netherlands Laura Germany White Lady Hungary Desiree The Netherlands Shepody Canada Katica Hungary Bzura Poland Victoria England Luca XL Hungary Justa Poland Franceline France Lorett Hungary Irati Spain Gala Germany Démon Hungary Eridia Slovakia Rosita Germany Vénusz Gold Hungary Kennebec The United States Agria Germany Agata The Netherlands Snowden The United States

To demonstrate the utility of developed markers in the related Solanum species, three populations from wild Solanum species were also examined. The species and their ploidy level of each population are given in Table 5.

Table 5. Accession of non-potato related Solanum species used in this study.

sect. Solanuma Ploidy Accession sect. Archaesolanuma Ploidy Accession sect. Ploidy Accession (population 1) (2n) No. (population 2) (2n)b No. Solanumc (2n) No. (population S. scabrum 6x = 72 824750011 S. aviculare var. latifolium x = 46 844 750 003 S.3) nigrum 6x = 72 UPG0013.1- UPG0013.10 S. chenopodioides 2x = 24 HUGEO09027 S. aviculare var. albiflorum x = 46 904750109

S. retroflexum 4x = 48 904750228 S. laciniatum x = 92 A24750 011

S. opacum 6x = 72 884750223 S. vescum x = 46 904750 174

S. americanum 2x = 24 904750023 S. multivenosum x = 92 Symon 13889 S. villosum 4x = 48 804750186

a: one individual from each species was used; b: members of Archaesolanum are anomalous aneuploid; c: eleven individuals were used.

3.2. DAS-ELISA test DAS-ELISA experiment was carried out based on the method of Clark and Adams (Clark and Adams, 1977) using commercially available polyclonal antibody supplied by Loewe Biochem, Germany. Assays were conducted according to the instructions of the producer. The enzymatic reaction was recorded at 405 nm using a Spectro Star Nano (BMG Labtech, Germany) reader. Samples were considered as positive when absorbance at 405 nm was at least two fold of the average of three negative controls (threshold value) after incubation for 60 to 120 min at room temperature in a dark place.

3.3. PVX resistance tests 3.3.1. Mechanical inoculation

Firstly, the Hungarian PVX, isolate Ny (kindly provided by Pál Salamon), was propagated on Nicotiana tabacum cv. xanthi. Before inoculation of potato, tobacco plants were tested by DAS-ELISA to ensure their infection with the virus. The experimental potato plants, at four to six leaf stages, were dusted with carborundum powder (300 mesh). PVX infected tobacco leaf tissues was grounded in phosphate buffer (pH 7.5; 0.1 M K2HPO4, 0.025 M KH2PO4) and was rub inoculated to leaves and rinsed with tap water after 5 minutes of infection. The cultivars and breeding lines were tested in five replications while in three replications for the F1 progenies. Negative controls were inoculated with sap from healthy leaves of N. tabacum cv. xanthi and cultivar Hermes was applied as susceptible control. Four weeks post-inoculation, virus infection was monitored by DAS-ELISA test.

3.3.2. Graft inoculation

Non-infected (ELISA negative, putative PVX resistant) genotypes in mechanical inoculation test were applied for graft inoculation to identify the type of resistance (extreme resistance or hypersensitivity resistance). Tomato, cv. Rutgers, was applied as the donor of PVX. Tomato leaves were inoculated mechanically by PVX. Three weeks after inoculation, infection of the tomato plants with the virus was confirmed by DAS- ELISA. Graft inoculation was carried out by reciprocal grafting: 1) infected tomato

scions were grafted onto healthy potato stems, and 2) healthy potato shoots were grafted onto infected tomato root stocks. For each putative resistant genotype the reciprocal grafting was done in three replications. Four weeks post-inoculation, the grafted plants were monitored by the same DAS-ELISA method as in the case of mechanically inoculated plants.

3.4. Genomic DNA isolation Genomic DNA was extracted from 80 mg of leaf and stem tissue of in vitro, greenhouse and field grown plants using the method of (Walbot and Warren, 1988) as follows:

3.4.1. Lysis of plant cells and protein denaturation

1. Grind 50-100 mg plant tissue in 1.5 ml microfuge tube with minimal volume of quartz sand using a pestle. 2. Add 1200 µl PCL solution (100 mM Tris-HCl (pH 8), 50 mM EDTA (pH 8), 1M NaCl and 1% PVP (polyvinyl-pyrrolidone) and 80 µl 10% SDS to grounded material. 3. Vortex and shake the tubes for a few times. Incubate at 65oC for 45 minutes. During incubation samples are gently mixed (2-3 times). 4. Centrifuge at 10000 rpm for 10 min at room temperature. 5. Transfer the supernatant (800 µl) to fresh tubes and add 500 µl isopropanol and 150 µl protein precipitation solutions (7.5 M ammonium-acetate). Samples are mixed by gentle inversion and incubated at -20oC for at least 30 minutes. The incubation should not exceed 12 hours. 6. Centrifuge at 15000 rpm for 10 minutes.

3.4.2. Purification

7. Discard supernatant and resolve the DNA pellet in 600 µl TE (10 mM Tris-HCl, pH 8; 1 mM EDTA, pH 8) buffer. 8. Add 500 µl CIA (chloroform and isoamyl alcohol (24:1)) and mix by vortexing (10 min with 90 rpm).

9. Centrifuge at 15000 rpm for 5 minutes. 10. Pipette 400 µl from the upper phase to a new 1.5 Eppendorf tube (carefully). 11. Add 1 ml 99.5% ethanol and 50 µl 3M sodium-acetate then gently inversion the tubes. 12. Centrifuge at 15000 rpm for 10 minutes 13. Discard the liquid phase and resolve the pellet in 400 µl 70% ethanol. 14. Centrifuge at 15000 rpm for 5 minutes. 15. Discard supernatant and dry pellet completely (upside down of the tubes on paper-towel). 16. Dissolve the pellet in 100-200 µl TE buffer

3.5. Identification of resistance gene to PVX 3.5.1. Marker analysis

Published markers linked to the Rx1 as well to the Rx2 gene were examined in the first step to check their applicability and to determine the source of PVX extreme resistance in the analyzed genotypes. For Rx1 the following CAPS markers were tested: 77L (AluI), 77R (HaeIII), 221R, 218R (AluI), IPM3 (DdeI), IPM4 (TaqI) (Kanyuka et al., 1999); CP60 (DdeI), and GP34 (TaqI) (Bendahmane et al., 1997). For Rx2 the CAPS marker GP21 (AluI) and the marker TG432 (DeJong et al., 1997) were tested. PCR and restriction digestion conditions were as described in the literatures.

3.5.2. Development of specific primers for Rx genes

Sequence specific primers were designed based on the alignment of the Rx1 (NCBI Acc. No. AJ011801) and Rx2 (NCBI Acc. No. AJ249448) sequences, as well on sequences obtained during the experiment. Besides the alignment based primer design, the Primer 3 (v. 0.4.0) program (Rozen and Skaletsky, 2000) and NCBI (National Center for Biotechnology Information, USA) primer blast was also applied. PCR was carried out in a final reaction volume of 12 μl. PCR reaction mixture contained 40 ng DNA template, 10 pmol of each of the primers, 200 μM dNTP (Fermentas, Lithuania), 1.5 μl 10x PCR Dream Taq Green Buffer provided by the

manufacturer (Fermentas, Lithuania) and 0.1 µl of 5 U µl-1 Dream Taq DNA polymerase. The reactions were performed in an Eppendorf Mastercycler ep384 (Eppendorf, Germany) with the following profile: 3 min at 94ºC, followed by 30 cycles at 94ºC for 20 s, annealing for 15 s (temperature for the selected primer pairs can be seen in Table 8.), and 72ºC for 1 min. A final extension step at 72°C for 5 min was applied. To confirm that really the expected sequences were amplified the PCR products were cloned using the pJET 1.2 PCR Cloning kit (Fermentas, Lithuania) following the manufacturer’s instructions, and E. coli cells, strain DH5α, were used for transformation. Sequencing was performed with a 3500 Genetic Analyzer (life Technologies, USA) sequencer machine using a standard protocol. Sequenced fragments were aligned using MEGA5 software (Tamura et al., 2011) and also by NCBI, BLASTN.

3.6. Development of a multiplex PCR for the Rx genes To develop a multiplex PCR method for the simultaneous detection of the Rx1 and Rx2 genes, those primer pairs were selected which gave clear and characteristically different size bands in the single reactions in both genes. Combinations of different concentrations of primer pairs ranging from 0.1 µM - 1.5 µM for each primer were tested. The amount of Dream Taq DNA polymerase also was increased to 0.2 µl of 5 U µl-1. The other component of PCR reaction and PCR situation was the same as those of single PCR. The varieties Cara, Bzura, the Hungarian resistant cultivars and two susceptible cultivars Démon and Katica were involved in the experiments. DNA mixture of Cara (Rx1) and Bzura (Rx2) was used to test the reliability of multiplex PCR to model the identification of potential hetero duplex resistant genotypes. The optimal profile was determined by gradient PCR.

3.7. Transcriptome analysis For transcriptome analysis, the potato cultivar White Lady, which is extreme resistant to PVX, PVY and resistant to Ph. infestans, was used. Inoculation was performed in three independent experiments with three replications in each time point of sampling.

3.7.1. Inoculation with pathogens

3.7.1. 1. Inoculation with PVX and PVY Virus free minitubers of White Lady were grown under vector free greenhouse conditions. Fully expanded leaves of 4-week-old plants were mechanically inoculated either with tobacco leaves sap infected with PVX isolate Ny, or PVYNTN, isolate D-10 in three replications for each time point of sampling. In each experiment, control plants (mock inoculation) were inoculated with a buffered suspension of healthy tobacco leaves sap. At 0, 5, 10 and 30 min; 1, 2, 4, 6, 8, 12, 24 and 48 hours; 1 and 2 weeks after inoculation, leaves of treated and control plants were simultaneously harvested and frozen immediately in liquid nitrogen for mRNA extraction. To confirm the pathogenicity of viruses, Gomphrena globosa and N. tabacum cv. xanthi were inoculated by PVX and PVY, respectively. Three days post inoculation, G. globosa was inspected for local lesions and the infection of tobacco to PVY was tested with DAS-ELISA four weeks after inoculation.

3.7.1.2. Inoculation with Ph. infestans Ph. infestans, isolate H12/10, was propagated on susceptible potato tuber slices of Hópehely cultivar and sporangia were rinsed with sterile distilled water and the concentration of suspension was adjusted to 1.5×104 spores/ml. To induce zoospore formation, suspension was maintained at 4oC and room temperature for 2 hours and 20 minutes, respectively. After rinsing the detached leaves with sterile distilled water, 50 µl of sporangia suspension was dropped to the lower surface of the leaves. Inoculated detached leaves were incubated in a humid plastic chamber. Control plants were inoculated with sterile distilled water. Leaves were incubated in culture room with 16/8 day/night period at 21oC. Samples were collected at 1, 4, 16, 24, 30, 48 and 72 hours; 6 days post inoculation and frozen in liquid nitrogen for mRNA extraction.

3.7.2. mRNA isolation

RNAzol (MRC, USA) was used to isolate mRNA. RNAzol®RT separates RNA from other molecules in a single-step based on the interaction of phenol and guanidine with

cellular components. No chloroform-induced phase separation is necessary to obtain pure RNA. A biological sample is homogenized or lysed in RNAzol®RT. DNA, proteins, polysaccharides and other molecules are precipitated from the homogenate/lysate by the addition of water and removed by centrifugation. The pure RNA is isolated from the resulting supernatant by alcohol precipitation, followed by washing and solubilization. Protocol for isolation of mRNA, with some modifications, is as follows: 1. Homogenization: Add 1200 µl RNAzol to up to 100 mg tissue in a 2 ml Eppendorf tube. 2. DNA/protein precipitation: Add 400 µl of water per 1 ml of RNAzol. Shake the resulting mixture vigorously for 15 second and incubate at room temperature for 15 minutes. Then centrifuge for 20 minutes at 12,000 g (14500 rpm). 3. mRNA precipitation: Prepare three new 0.5 ml Eppendorf tubes and transfer 360 µl of supernatant carefully to each tube. Then precipitate mRNA with 150 µl of 75% ethanol. Incubate the tubes at room temperature for 10 minutes. Centrifuge for 10 minutes at 14500 rpm. RNA precipitate forms a white pellet at the bottom of tube. Discard supernatant carefully. It is safe to collect up to 85% of the supernatant. 4. mRNA washes: Dissolve mRNA pellet with 360 µl 75% ethanol and centrifuge for 4 minutes at 10500 rpm. Discard supernatant carefully and remove the alcohol solution using the micropipette. Repeat this step twice. 5. RNA solubilisation: Dissolve the RNA pellet without drying (it greatly decreases RNA solubility) in 10 µl of nuclease free water to approach 1-2 µg/µl. For solubilisation in water, vortex the RNA pellet at room temperature for 2-5 minutes. Drying the RNA pellet is not recommended.

3.7.3. mRNA analysis

The concentration and quality of mRNA was determined by measuring the ratios of absorbance A260 nm/A280 nm and A260 nm/A230 nm which should be between 2 and 3 using the Nanodrop 2000 (Thermo Scientific, USA) spectrophotometer. The integrity of the mRNA was tested by load 5 µl of mRNA with 3 µl of BPB in a 1% agarose gel.

3.7.4. mRNA preparation

In each experiment, one pooled sample from each treated and control mRNA was prepared represented all genes which are expressed in all sampling stages after inoculation (pathogen or mock inoculation). In PVX and PVY experiments, mRNA-s was divided into two sections, one used for PCR-selected subtractive hybridization experiment and the second applied for NGS transcriptome sequencing. For NGS sequencing, two pooled samples were created, representing equal quantities of mRNA from the treated and control plants respectively of all three experiments and these samples were used for NGS transcriptome sequencing.

3.7.5. Suppression subtractive hybridization (SSH)

The RNA preparation and handling, first-strand cDNA synthesis, second-strand cDNA synthesis, RsaI digestion, adaptor ligation, first hybridization, second hybridization and PCR amplification were performed based on PCR-SelectTM cDNA subtraction protocol (PCR-select™ cDNA subtraction Kit user manual, Cat. No. 637401) with minor modifications.

3.7.5. 1. Construction of SSH library Extracted mRNA from PVX and/or PVY inoculated samples was used as the “tester” and mRNA from control (mock inoculated plants) served as the “driver”. Differentially expressed cDNA-s are present in the ‘tester’ cDNA but are either absent or present in very low levels in the ‘driver’ cDNA-s. The construction of the libraries was performed according to the SSH procedure using the PCR-select cDNA subtraction kit (Clontech). Equal amounts of mRNA from each of the tester and driver populations were converted to double-stranded cDNA by reverse transcription and followed by digestion with RsaI to produce shorter blunt-ended fragments. The digested tester cDNA was subdivided into two populations each of which was ligated with a different adaptor provided in the cDNA subtraction kit (Clontech). After ligation, a series of two hybridization steps was performed. For the first hybridization, an excess of driver was added to each tester,

denatured, and allowed to anneal. The target sequences in the tester became significantly enriched for differentially expressed genes. In the second hybridization step, the two reaction products from the first hybridization were mixed with each other and with fresh denatured driver cDNA. The populations of normalized and subtracted single-stranded target cDNA-s anneal with each other, forming double-stranded hybrids with different adaptor sequences at their 5′ ends. The adaptor ends were then filled with DNA polymerase and the subtracted molecules were specifically amplified by nested PCR using adaptor-specific primer pairs.

3.7.5.2. Cloning and PCR screening of the SSH library CloneJET™ PCR cloning kit was used for cloning. Transformation of cloned cDNA into bacterial cells was carried out based on procedure suggested by Bioline (www.bioline.com). The transformed fragments were screened for size of insert by colony PCR using pJET (Fermentas, Lithuania) primers. Amplification was performed with an initial denaturation at 95°C for 3 min, followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min/kb, and a final extension of 5 min at 72°C.

3.7.5.3. Sequencing of cDNA clones and sequence analysis After plasmid purification of recombinant clones using Gene Jet Plasmid Miniprep Kit (Fermentas, Lithuania), sequencing was carried out by a 3500 Genetic Analyzer (life Technologies, USA) sequencer machine using a standard protocol. Sequences were assembled into contigs using CodonCode Aligner (CodonCode Corperation). The sequenced EST-s were analyzed for similarity with BLASTx (non-redundent proteins) against reference sequences of S. tuberosum group Phureja DM1-3 5116R44 (hereafter referred as potato-DM) with the E-value of less than 10-5 up to July 2013 in the SOL Genomics Network. Furthermore, the NCBI database using BLASTx (non- redundent protein sequences) with the E-value < 10-5 up to July 2013.

3.7.6. NGS transcriptome sequencing

mRNA samples were sent to Baygen company for RNA-Seq sequencing in Szeged. mRNA fragmentation, ligation to adaptors, construction and amplification of cDNA library and SOLiD template beads preparation for emulsion PCR was carried out using Life Tech SOLiD RNA Sequencing Kit (Life Technologies, USA) according to the manufacturer’s instructions. NGS sequencing was performed using a 5500 XL SOLiD (Life Technologies) sequencer. All sequences were filtered to remove low-quality and broken sequences. The remaining sequence reads were assembled into contigs, normalized and the fold change and the number of reads per thousand bases per million mapped reads (RPKM) (Mortazavi et al., 2008) was analyzed by CLC Genomics Workbench 4.8 (64 bit) software. The functional annotation of transcript sequences was performed using reference sequences of Potato-DM with identified 39031 protein-coding genes is available in the SOL Genomics network (SGN). The ratio of RPKM-treated/ RPKM-control value was applied for fold change with the threshold of ≥ + 2 and ≤ -2, in treated and control samples was considered for up- and down-regulated genes, respectively. Log 2 fold change values were also used to prepare heat maps in Cluster 3.0 (Eisen et al., 1998) using uncentered correlation and the complete linkage method and the results visualized in Treeview software (Saldanha, 2004).

3.8. Development of NGS derived intron-targeting markers Genes from generated transcript data set were selected and aligned with potato genome sequence in NCBI database using BLASTN (nr and Whole-genome shotgun contigs) with the E-value 10-20 up to June 2012. The program Sim4 (Florea et al., 1998) was applied to the corresponding genes in potato in order to find the putative intron regions. In addition, the SGN (Mueller et al., 2005) was used to predict the intron regions. Intron-targeting primers were designed on the exon sequences flanking the putative introns with the PRIMER3 v. 0.4.0 software (Rozen and Skaletsky, 2000).

3.8.1. PCR amplification of intron targeting markers

Final volume of the PCR reaction mixture was 12 μl, and contained 40 ng DNA template, 0.5 μM of each primer, 2 mM dNTP (Fermentas, Lithuania), 1.5 μl 10 x PCR Dream Taq Green Buffer provided by the manufacturer (Fermentas, Lithuania) and 0.1 µl of 5 U/µl Dream Taq DNA polymerase. The PCR conditions were as follows: 3 min 94ºC for initial denaturation, 35 cycles of 20 s denaturation at 94ºC, a 20 s annealing at the temperature indicated for each primer pair (Appendix 1), 1 min extension at 72ºC, and a final extension at 72ºC for 5 min. PCR products were separated on 1.5% agarose gel, visualized after ethidium-bromide staining with a GenGenius Bio Imaging System (Syngene, UK).

3.8. 2. Data analysis

The banding patterns were scored based on the size and presence/absence of a band. The genetic statistics were calculated for all population by the program POPGENE version 1.31 (Yeh et al., 1997) designed for haploid and diploid data analysis. In addition, ATETRA v. 1.2 software (Van Puyvelde et al., 2010), designed to analyze tetraploid microsatellite data, was applied to estimate the same genetic parameters. The SGN was applied to determine the chromosomal location of each polymorphic intron marker (Mueller et al., 2005).

4. RESULTS

4.1. Development of functional markers and a multiple PCR for Rx1 and Rx2 resistance genes in potato 4.1. 1. PVX resistance tests Results of the DAS-ELISA test of mechanical and graft inoculation revealed that the cultivars Cara, Bzura, White Lady, Luca XL, Lorett and Hópehely are extreme resistant to PVX, while the other tested cultivars and breeding lines are susceptible (Table 6.).

F1 population of the White Lady × Kuroda and of the Luca XL × W1100 crosses both showed a 1:1 segregation for PVX resistance (39:36 and 54:42 resistant to susceptible genotypes, respectively), indicating that the PVX extreme resistance gene in White Lady and in Luca XL is in simplex form (x2=0.12, P=0.72 and x2= 1.5, P=0.22, respectively). Nevertheless, it was not clear whether the Rx1 or the Rx2 gene is present in the tested plants.

Table 6. PVX resistance of the cultivars and breeding lines

Resistant cultivars Susceptible cultivars Breeding lines - all are susceptible Cara Rioja 01.536 Bzura Démon 06.62 White Lady Góliát 06.256 Luca XL Balatoni Rózsa 06.325 Lorett Somogy Kifli 76.9104 Hópehely Katica W1100 Vénusz Gold

4.1.2. Identification of resistance gene to PVX 4.1.2.1. Results of the published PVX resistance markers

For Rx1 gene the CAPS markers 77L (AluI), 77R (HaeIII), 221R, 218R (AluI), IPM3 (DdeI), IPM4 (TaqI) (Kanyuka et al., 1999); CP60 (DdeI), and GP34 (TaqI) (Bendahmane et al., 1997) and for Rx2 the GP21 (AluI) and TG432 (DeJong et al., 1997) tightly linked markers were tested. These specific markers for the Rx1 gene could not

amplify the desired fragments in the Hungarian PVX resistant genotypes, except the CP60 and 221R markers. For the CP60 diagnostic marker (Gebhardt et al., 2006) beside Cara, a similar molecular weight band was also detected in Bzura, White Lady and in the susceptible cultivar Hermes (Fig. 4a.). Sequencing of these fragments revealed 99% similarity between the White Lady and Hermes sequences, while the fragment of Cara derived from a different region of the corresponding sequence (Acc. No. AEWC01029415.1) (Appendix 2). However, the expected band for the 221R, Rx1 specific marker was present in Cara; it could also be detected in Bzura and White Lady (Fig 4b.). Sequencing and alignment of this amplified fragment of the three cultivars showed 100% identity between White Lady and Bzura and 97% identity with Cara (see Appendix 3). The Rx2 specific GP21 marker beside the cultivar Bzura could also be detected in the susceptible cultivar Hermes (Fig. 5). Alignment of the sequenced fragments indicated 97% identity between them and both represented the same region of the corresponding potato-DM sequence (Acc. No. AEWC01009533.1). Multi-alignment of their PCR product with the relevant sequence in potato genome sequence is shown in Appendix 4.

Fig 4. Electrophoretic pattern of the DdeI digested CP60 CAPS marker (a) and marker 221R (b). Samples from left to right: M: 100 bp plus DNA ladder, 1: Cara (Rx1), 2: Bzura (Rx2), 3: White Lady, 4: Luca XL, 5: Hermes (susceptible)

Fig 5. Electrophoretic pattern of the AluI digested GP21 CAPS marker. Samples from left to right: M: 100 bp plus DNA ladder, 1: Luca XL (Extreme resistance to PVX), 2: W1100, 3: (Bzura (Rx2), 4: Hermes (susceptible)

4.1.2.2. Resistance gene analysis and development of gene-specific primers

Alignment of Rx1 and Rx2 genes shows 98% sequence identity at the nucleotide level. Except a single extra triplet in the Rx2 gene, variations are dispersed in one to two nucleotides along the coding region. Although, they originate from different species this high level of sequence identity makes it somewhat difficult to separate the two genes from each other. One strategy could be the PCR-RFLP, where the PCR product is digested with point-mutation specific restriction enzymes. However, this method is more laborious and more costly. It is considered that identification by a single PCR would be more beneficial for marker assisted selection. Based on sequence differences of the Rx1 and Rx2 genes and using primer blast in NCBI, eight primer pairs were designed for the Rx1 gene, and three for the Rx2 gene. For Rx1 two primer pairs, 1Rx1 and 5Rx1 amplified the expected 974 bp as well 186 bp fragment, respectively, in the cultivar Cara and in 16 further cultivars including all those for which according to their pedigree they are carrying the Rx1 gene. These bands were not amplified in Bzura and in the other cultivars which are carrying the Rx2 gene and also not in the PVX susceptible cultivars (Fig. 6.; Table 7.). Cloning, sequencing and alignment with NCBI, BLASTN of the amplified 1Rx1 and 5Rx1 fragments of all 17 cultivars indicated their identity with the relevant regions of the Rx1 gene (Gene Bank Accession No. AJ011801).

With these R-gene specific sequences, we expected only one out of four haplotypes from a tetraploid cultivar to yield a PCR amplicon. Hence in the text hereafter the sequence of a cultivar is equal to the sequence of a single haplotype. For cultivars such as Asterix, Sante, Alcmaria, Amaryl, Mondial, Irati and Ditta which amplified by 1Rx1; 100% sequence homology was observed relative to Cara. On the other hand, the cultivars Lady Rosetta, Wauseon, Amalia and Atlantic showed one SNP and a number of SNPs were observed in Agria, Rhinered, Eridia, Multa and Divina (Appendix 5). SNPs were also detected in the amplified fragment of the promoter region targeted by the 5Rx1 primer pair, in Lady Rosetta, Amaryl, Multa, Agria and Rhinered. In cultivars Amalia, Divina, Multa, Eridia and Wauseon an Indel was identified (Appendix 6). The observed differences in the sequenced amplified fragments could be the result of the CPC1673 seedlings used as the entry points. Across the seedlings from this accession (1, 11, 20, 24)(Table 7), there may be allelic variation in the Rx1 gene. Perhaps individual seedlings could be duplex for Rx1, and may display different haplotypes. Hereafter we refer to “Rx1 specificity” as being diagnostic for Rx1 even in the presence of SNPs, or Indels that annihilate functionality.

Fig. 6. Detection of the Rx1 gene. The electrophoretograms show in 17 cultivars the expected 974 bp, as well 186 bp size fragments obtained with the two different primer pairs 1Rx1 (a) and 5Rx1 (b), respectively. Samples from left to right: M: 100 bp plus DNA ladder, 1: Cara (Rx1), 2: Bzura (Rx2), 3: Sante, 4: Lady Rosetta, 5: Ditta, 6: Asterix, 7: Agria, 8: Amalia, 9: Alcmaria, 10: Amaryl, 11: Divina, 12: Mondial, 13: Multa, 14: Atlantic, 15: Wauseon, 16: Rhinered 17: Irati, 18: Eridia, 19: White Lady, 20: Luca XL, 21: Lorett, 22: Hópehely, 23: Démon (susceptible), 24: Rioja (susceptible)

Table 7. Potato cultivars in which Rx1 gene was detected by 1Rx1 and 5Rx1 primers

Cultivar Country of origin 1Possible PVX resistance origin based on Pedigree

Cara Irland adg- hybrid

Agria Germany Unknown

Ditta Austria Unknown

Asterix The Netherlands CPC 1673-20 (adg)

Sante The Netherlands CPC 1673-20 (adg)

Amalia The Netherlands CPC 1673-11 (adg)

Lady Rosetta The Netherlands CPC 1673-20 (adg)

Alcmaria The Netherlands CPC 1673-20 (adg)

Amaryl The Netherlands CPC 1673-20 (adg)

Divina The Netherlands Cara

Mondial The Netherlands CPC 1673-20 (adg)

Multa The Netherlands CPC 1673-1 (adg)

Atlantic The United States CPC 1673 (adg)

Wauseon The United States CPC 1673 (adg)

Rhinered The United States Unknown

Irati Spain CPC 1673-20 (adg)

Eridia Slovakia Unknown

1: Available at http://www.plantbreeding.wur.nl/potatopedigree

Furthermore, alignment of the sequenced fragments with the highly homologous Gpa2 gene and other published homologous sequences (Bakker et al., 2011; Jupe et al., 2012) indicated the Rx1 specificity of the developed 1Rx1 and 5Rx1 primers (Appendix 5 and 6). The 10Rx2 primer pair, designed to detect the Rx2 gene, successfully amplified the expected 1095 bp fragment in resistant cultivars, but gave no PCR product in PVX susceptible cultivars or in Rx1 containing cultivars such as Cara. By exception a PCR

product was amplified in cv. Démon. Cloning and sequencing confirmed that the PCR product of the resistant genotypes is 100% similar to the relevant region of the Rx2 gene. Considering the above results it was concluded that similarly to Bzura, the Hungarian PVX resistant cultivars as well as Nativ and Courage possess the Rx2 but not the Rx1 gene. The fragment obtained with the 10Rx2 primer pair in the PVX susceptible cultivar Démon showed high similarity both to the Rx1 and the Rx2 genes. It is suggested that this fragment of the susceptible cultivar Démon may derive from the recessive allele (rx) or it may be a resistance gene analogue (RGA). Hence, by aligning all sequences of this study we could observe suitable SNPs as they correlated with the absence and presence of a PVX resistant phenotype expected to be derived from a functional Rx2 gene. These SNPs could be exploited to design nine new reverse primers which could be used to avoid amplification of a product in Démon. Screening with these new reverse primer candidates showed that the 106Rx2 reverse primer used in pair with the 10Rx2 forward primer (from here referred as 106Rx2 primer pair) amplifies a 543 bp fragment only in the resistant cultivars except Rx1 carrying cultivars, but no product is obtained in any of the susceptible cultivars or breeding lines (Fig. 7). Sequencing and alignment of the PCR product of each cultivar with the Rx2 gene and other paralogous sequences from NCBI and published literature showed that the expected region of the Rx2 gene was amplified (Bakker et al., 2011; Jupe et al., 2012). Moreover, Alignment of amplified fragments with Rx2 gene identified SNPs in cultivars Nativ and Hópehely (Appendix 7).

Fig. 7. Gel image of a 543 bp PCR product from the 106Rx2 primer pair, indicative of the Rx2 gene. PVX resistant and susceptible genotypes from left to right: M: 100 bp plus DNA ladder, 1: Bzura (Rx2), 2: Cara (Rx1), 3: White Lady, 4: Luca XL, 5: Lorett, 6: Hópehely, 7: Nativ, 8: Courage, 9: Bellarosa, 10: Shepody, 11: Desiree, 12: Démon, 13: Rioja, 14: Katica, 15: Góliát, 16: Balatoni Rózsa, 17: Somogy Kifli, 18: Vénusz Gold, 19: 06.62, 20: 06.256, 21: 06.325, 22: 76.9104, 23: 01.536, 24: W1100

4.1.3. Testing of the Rx2 specific primer pair in the F1 populations

Segregation of the 96 F1 genotypes of the Luca XL × W1100 and of the 75 F1 genotypes of the White Lady × Kuroda crosses were analyzed with the developed Rx2 specific primer pair, 106Rx2. The results revealed complete matching of the phenotypic and genotypic tests (Fig. 8.). The expected band (543 bp) appeared in all genotypes which, according to the DAS-ELISA test were resistant, but no PCR product could be detected in the PVX susceptible genotypes.

Fig. 8. Co-segregation between PVX resistance and the “Rx2 specific” PCR product that was obtained with the 106Rx2 primer pair. M: 100 bp plus DNA ladder; P1: Luca XL (resistant parent); P2: W1100 (susceptible parent); lanes 1-11: resistant progeny, lanes 12-22: susceptible progeny

It is concluded that specific primers have been successfully developed for the identification of the Rx2 gene. The marker obtained with the 106Rx2 primer pair could be effectively applied in potato breeding for selection of PVX extreme resistant genotypes. Also, the Rx1 specific markers obtained with the 1Rx1 and 5Rx1 primer pairs may be used for similar reasons, i.e. to follow up the inheritance of the Rx1 gene. Primer pairs that can be effectively used for the detection of the Rx1 as well for the Rx2 gene are shown in Table 8.

Table 8. Specific primer pairs for the Rx1 as well for the Rx2 gene

′ ′ 1 Primer Sequence (5 -3 ) Annealing region Ta Size of the expected product name (oC) (bp)2 1Rx1 F3: GGAGAAATCCTGCAATATAAT Exon 1 60 974 R4: CGACCGAACTTACATTTTCCC

5 Rx1 F: TCAGGGCAAAACCCTAACAC Promoter 62 186 R: ATCGGCCTAGAGTGACATCG

10Rx2 F: GGAGAAATCCTGCAATGTAAC Exon 1 64 1095 R: GAAATCCGTTCATCCTCTGC

106Rx2 F: GGAGAAATCCTGCAATGTAAC Exon 1 66 543 R: CTTGTCAAAGAAAGAAGGCCT 1: annealing temperature; 2: base pairs; 3: forward primer; 4: reverse primer.

4.1.4. Development of a multiplex PCR for the Rx genes A series of setting combinations were tried to optimize the multiplex PCR reaction. From the different settings the 5Rx1 and 106Rx2 primer pairs gave the best results when 0.75 µM for 106Rx2 and 0.125 µM for 5Rx1 primer concentrations and 66oC annealing temperature were used (Fig. 9.). The expected pattern could be detected by the applied method and equipment described in Materials and Methods.

Fig. 9. Simultaneous and independent identification of the Rx1 and Rx2 genes with the 5Rx1 and 106Rx2 primer pairs in a multiplex PCR reaction. M: 100 bp plus DNA ladder, 1: Cara (Rx1), 2: Mixture of Cara (Rx1) and Bzura (Rx2), 3: Bzura (Rx2), 4: White Lady (Rx2), 5: Luca XL (Rx2), 6: Lorett (Rx2), 7: Hópehely (Rx2), 8: Démon (S), 9: Katica (S). S – Susceptible

4.2. Isolation of important genes related to resistance against PVX, PVY and Ph. infestans 4.2.1. Construction of subtractive cDNA libraries in White Lady

To isolate differentially or highly expressed genes in early stages of inoculation of PVX and PVYNTN from White Lady, the suppression subtractive hybridization (SSH) method was chosen as one approach. In two independent experiments for each virus, two mRNA pools from different time courses after inoculation were prepared for the so called testers and drivers. Following cDNA synthesis, restriction digestion with the RsaI endonuclease and adaptor ligation, the common cDNA population between the tester and driver fragments was subtracted by two rounds of hybridizations. The obtained fragments represented differentially expressed genes, which were subjected to two rounds of PCR amplifications. The PCR products were ligated into pJET 1.2/blunt vector and transformed into E. coli, DH5α, by heat shock transformation.

4.2.1.1. Analysis of PVY induced subtracted cDNA library To identify differentially expressed genes in the subtracted cDNA library, 452 colonies were randomly selected. The fragment size after PCR amplification with pJet primers (which flank in the plasmid pJET Vector) varied between 290-1300 bp. PCR products were separated on a 1.5% agarose gel. Sixty-nine differentially expressed clones were sequenced and vectors as well as adaptors were trimmed from the raw sequence data. The resulted overlapped similar sequences were assembled into contigs. With the resulting sequences in total, 35 EST-s were identified and applied for homology analysis in databases. The BLAST search of sequenced fragments with 39 031 protein-coding genes of the potato genome using SGN database revealed that 12 clones have high similarity with annotated genes in potato and 10 matched to genes with unknown function or conserved genes of unknown function in potato. The remaining 13 EST-s showed no significant matches with SGN database records (Appendix 8). Analysis of BLASTx results in NCBI also indicated similar functions, except for clone No. 389. The proposed identity for this EST in NCBI is available since March 2013, while the SGN database is updated in June 2012.

In total, BLAST results showed that these 13 EST-s have no significant matches in neither of the two public databases and were therefore defined as novel genes (Appendix 8). Considering the result of BLAST search in both databases, only 13 genes (approximately 37%) of the analysed 35 genes showed significant similarity with known functional proteins and 22 genes were defined as novel genes or genes with unknown function.

4.2.1.2. Analysis of PVX induced subtracted cDNA library From the PVX induced subtracted cDNA library 486 colonies were randomly isolated and the presence of cDNA inserts was confirmed by colony PCR. Size of the cDNA inserts ranged from 370 to 1300 bp (Fig 10.).

Fig 10. Colony PCR of the PVX induced subtracted cDNA library. M: 100 bp plus DNA ladder, 1-24 randomly selected colonies

The different size insert of 62 colonies were sequenced, and the vector as well as adaptor sequences were excluded from further analysis. The similar EST-s (subtracted sequences) were assembled into contigs and 28 EST-s were finally identified in the public databases. Out of these 28 EST-s, 14 were assigned to genes with known function, while seven showed high similarity with genes of unknown function or conserved genes with unknown function compared to annotated proteins of potato-DM using the SGN database. No function could be defined to seven isolated EST-s in potato-DM indicating absence of these genes in the double monoploid potato-DM (see Appendix 9). Homology search in NCBI with BLASTx identified 17 known functional genes and 10 hypothetical or uncharacterized proteins. The clone 323-2 was not match to any functional gene in these databases, hence it was considered as a novel EST.

4.2.2. NGS based transcriptome analysis

To obtain a global transcriptome profile of early response of White Lady to PVX, PVY and Ph. infestans, mRNA was extracted from both pathogen and mock inoculated plants, hereafter referred to as treated and control, respectively, in three independent experiments. Then, two pooled samples were created, representing equal quantities of the three mRNA samples of the treated and control plants. Finally, one pool represented the treated and the other one the control samples. These two samples were then sequenced upon our ordering by Baygen, Szeged on an Applied Biosystem 5500 XL SOLiD platform. After quality assessment and data filtering by CLC Genomics Workbench 4.8 (64 bit) software, broken pairs were ignored and an intact pair was counted as one fragment. With this 12 060 751 as well as 9 861651 sequence reads were obtained in the treated as well as in control samples, respectively. High-quality reads of each sample were assembled into contigs and mapped to the potato-DM sequence that was used as the reference sequence. After trimming non-specific fragments, the reads matching more than once to the reference sequence resulted 4 604 328 as well as 4 482 928 uniquely mapped fragments in the treated and in the control samples, respectively. The mapping and match specificity statistics are given in Table 9 and Fig. 11. About 58% and 51% of specific fragments could not be mapped to the reference sequence in treated and control, respectively; indicating the absence of these genes in the sequenced genome of the potato-DM.

Table 9. Summary of mapping statistics of the RNA-Seq data

Data Treated mRNA Control mRNA Total number of sequence reads 12 060 751 9 861 651 Non- specific fragments 1 119 619 6 66 745 Specific fragments 10 941 132 9 194 906 Counted unique fragments 4 604 328 4 482 928 Uncounted fragments 6 336 804 4 711 978

Fig 11. Match specificity of sequence reads in treated (a) and control (b) samples. The number of reads that are mapped zero (0.0) times includes the number of reads that cannot be mapped to reference (uncounted fragments) and the number of reads that are mapped one (1.0) include specific match reads that are only mapped to one position in the reference (counted unique fragments). The number of reads that are equally matched to other places in the reference (2.0, 3.0, 4.0 and 5.0 positions) are considered non-specific fragments.

The abundance of mapped reads for each transcript was normalized using the RPKM value (Mortazavi et al., 2008) and defined as expression value for transcripts. The functional annotation of transcript sequences was performed using reference sequences from the SOL Genomics Network (SGN). Alignment of fragments with 39 031 known protein-coding genes of the potato-DM identified 38 675 and 37 927 genes in the treated and control samples, respectively, representing over 99% of the 39 031 protein-coding genes in potato-DM. By comparison of fold change, the ratio of RPKM values in treated to control transcripts, 8466 and 4670 up as well as down-regulated genes were identified, respectively. From among the up-regulated genes, 748 transcripts were recognized only in treated samples but not in the control indicating stress response specific genes. Out of these, 57% encoded proteins of unknown genes or conserved genes with unknown function. The pathogen specific genes may play a role in the resistance response to any or all of the three examined pathogens. The transcriptome data set also contains 141 NBS- LRR encoding genes with 13 Toll Interleukin-like receptor (TIR) and 50 Coiled-coil (CC) types in the treated samples. The number of identified genes in treated and control samples has been summarized in Table 10.

Table 10. Identified genes in treated and control samples

Data Pathogen inoculated mRNA Mock inoculated mRNA Total annotated genes 38 675 37 927 Up-regulated genes 8 466 - Down-regulated genes 4 670 - Genes with unknown function 2 600 - (up-regulated) Genes with unknown function 1 535 - (down-regulated) NBS-LRR 141 139 Pathogen response specific genes 748 -

The ratio of Log 2 treated RPKM to control RPKM values were used to cluster the up-and the down-regulated genes in Cluster 3.0, and the results were visualized using Treeview (Fig. 12.).

a b

Fig.12. A snapshot of Heat map of some up-regulated (a) and down-regulated (b) genes in potato induced by inoculation with PVX, PVY and Ph. infestans. Red colour indicates up-regulated genes and green colour indicates down-regulated genes. The log2 fold change is used as a scale for intensity of the colours

These results suggest that PVX, PVY and Ph. infestans induce large sets of up- and down-regulated genes in the potato cultivar, White Lady that result in resistance in this cultivar. However, it would be important to analyze the effect of these three pathogens in independent experiments.

4.3. Development of Intron-targeting markers in potato From the 38 675 transcriptomes (TC) obtained after assembling the NGS reads 250 were randomly chosen and analyzed for the presence of introns. Comparing these transcriptomes with sequences in the NCBI as well in the SOL Genomics Network database 144 transcript sequences were identified as potentially harboring introns. Locus specific primer pairs were designed for each sequence, and 40 randomly chosen loci were experimentally analyzed for the assumed intron length polymorphisms. While for all 40 loci PCR products were obtained, in total 25 loci showed polymorphism in the analyzed populations.

4.3.1. IT polymorphism in the potato genotypes

The results of primer screening indicated that 17 out of 40 designated putative markers showed different level of polymorphism both in the F1 population and among the 24 potato cultivars, while the remaining 23 loci showed to be monomorphic. The number of alleles per locus ranged from 2 to 5 in both populations (Fig. 13). The highest number of polymorphic bands was obtained with the primer pair Antiv1, which is an anti-virus transcriptional factor, while the lowest numbers were obtained with the primer pairs for RPB36 and Cat which mark a late blight resistance gene, Rpi-blb2a as well as a catalase gene, respectively.

Fig. 13. A representative gel image of amplified intron regions in F1 (a) and potato cultivars (b) with primer pair PGRSH.

The analysis of the polymorphic markers by the ATETRA software showed a heterozygosity (Hc) ranging from 0.08 to 0.79 in the F1 population and 0.12 to 0.78 in the cultivars. The PGRSH marker was the most informative in both populations (Hc= 0.79 and 0.77 in F1 and cultivars, respectively) and the marker, Cat, was the least informative (Hc= 0.08). The values of Shannon Wiener diversity index (Hc′) ranged from 0.24 to 1.26 in the F1 population, while in the cultivars it ranged from 0.27 to 1.30. The same genetic diversity parameters were calculated under Hardy–Weinberg equilibrium by Popgene package using the dominant marker option, a method that may be challenged in the future. Results of statistical analysis obtained with the ATETRA as well with the POPGENE software on heterozygosity are shown in Appendix 10. Applying POPGENE package for F-statistic analysis indicated that Nei’s total genetic diversity was HT = 0.27, while the average diversity within populations was HS = 0.24. The overall Wright’s fixation index displayed a moderate genetic differentiation in populations (FST = 0.13) (Table 11.). With the exception of markers TREPSH, AVTPSH, Cin and TRP77 which showed a high value of FST (ranging from 0.19 to 0.47), the other markers indicated low value of

FST (ranging from 0.5 to 0.11). Because the selected potato cultivars originated from different genetics backgrounds, the diversity within the population was high (HS=0.24) (Table 11.).

4.3.2. IT polymorphism in the wild Solanum species All 40 IT primer pairs which were designed based on the potato transcriptome sequences resulted one or more PCR product in one, two or in all three wild Solanum groups. Out of the 40 analyzed loci the number of polymorphic loci was 21 in sect. Archaesolanum, 22 in sect. Solanum and 16 in accessions of S. nigrum (See Appendix 11). The number of polymorphic bands per locus ranged from 2 to 10. Interestingly, while polymorphism could not be detected in the potato populations for the Mresis, Cad, Winsh, Str, PT11, Pe54, Cop12 and Cunf34 loci, they showed variation among wild Solanum species. On the other hand, for the LBR57 and RP3a loci, which were polymorphic in the potato populations, only a monomorphic band was observed among wild Solanum-s. The locus RPB36 displayed polymorphism only in Archaesolanum species. In contrary, loci R1L333 and Winsh were only monomorphic in sect. Archaesolanum. Most of the IT markers amplified significant length variation among the three wild Solanum populations (Fig 14.; Appendix11). The overall Wright’s fixation index, FST of total wild Solanum population was 0.33 suggesting great population differentiations, which is not surprising as these groups represented distantly related lineages. Statistical summary of the potato- and wild Solanum populations obtained with the ATETRA and POPGENE software are shown in Table 11.

Fig.14. An example for IT polymorphism of wild Solanum populations using PKF11 primer pair. Samples from left to right: M: 100 bp plus DNA ladder, sect. Archaesolanum species including lane 1: S. aviculare var. latifolium, lane 2: S. aviculare var. albiflorum, lane 3: S. laciniatum, lane 4: S. vescum, lane 5: S. multivenosum, sect. Solanum species including lane 6: S. scabrum, lane 7: S. chenopodioides, lane 8: S. retroflexum., lane 9: S. opacum, lane 10: S. americanum, lane 11: S. villosum, lanes 12-22: S. nigrum individuals

Table 11. Statistical summary of all populations using the ATETRA and the POPGENE software

Marker No. Genotype ATETRA analysis POPGENE analysis Hc H′c h I H H Fst individuals T S

PKF11 48 F1 & cultivars 0.51 0.91 0.20 0.33 0.20 0.19 0.05 22 Wild Solanum 0.83 1.86 0.25 0.38 0.25 0.15 0.40

LBR57 48 F1 & cultivars 0.73 1.20 0.40 0.59 0.40 0.38 0.05 Wild Solanum -a ------

AVTPSH 48 F1 & cultivars 0.70 1.21 0.24 0.37 0.24 0.17 0.29 22 Wild Solanum 0.80 1.63 0.18 0.30 0.18 0.17 0.06

PGRSH 48 F1 & cultivars 0.78 1.19 0.45 0.64 0.45 0.40 0.11 22 Wild Solanum 0.55 1.46 0.25 0.39 0.28 0.17 0.39

RP3a35 48 F1 & cultivars 0.75 1.34 0.35 0.53 0.35 0.32 0.09 Wild Solanum ------

NB89 48 F1 & cultivars 0.51 0.84 0.22 0.37 0.38 0.35 0.08 22 Wild Solanum 0.68 1.55 0.20 0.34 0.21 0.19 0.10

PTA-83-1 48 F1 & cultivars 0.65 1.13 0.33 0.48 0.34 0.33 0.03 22 Wild Solanum 0.75 1.50 0.33 0.49 0.37 0.16 0.57

RL333 48 F1 & cultivars 0.52 0.94 0.18 0.27 0.18 0.17 0.06 22 Wild Solanum 0.15 0.71 0.05 0.11 0.06 0.04 0.33

ATP-218 48 F1 & cultivars 0.57 1.06 0.27 0.42 0.27 0.25 0.07 22 Wild Solanum 0.42 0.90 0.20 0.33 0.24 0.13 0.46

Cin 48 F1 & cultivars 0.66 1.18 0.26 0.40 0.26 0.21 0.19 22 Wild Solanum 0.80 1.53 0.35 0.52 0.38 0.11 0.71

TSWVP 48 F1 & cultivars 0.59 1.04 0.26 0.39 0.26 0.24 0.08 22 Wild Solanum 0.50 0.69 0.30 0.48 0.37 0.13 0.65

TREPSH 48 F1 & cultivars 0.66 1.05 0.15 0.21 0.15 0.08 0.47 22 Wild Solanum 0.44 0.91 0.13 0.23 0.12 0.11 0.08

TRP77 48 F1 & cultivars 0.51 0.89 0.13 0.21 0.13 0.08 0.38 Wild Solanum 0.80 1.57 0.24 0.38 0.23 0.17 0.26

Nitsh 48 F1 & cultivars 0.42 0.73 0.19 0.31 0.20 0.19 0.05 21 Wild Solanum 0.79 2.22 0.16 0.28 0.17 0.12 0.29

RPB36 48 F1 & cultivars 0.41 0.60 0.32 0.49 0.32 0.31 0.03 22 Wild Solanum 0.19 0.92 0.14 0.27 0.20 0.15 0.25

Cat 48 F1 & cultivars 0.10 0.21 0.05 0.11 0.05 0.05 0.00 20 Wild Solanum 0.60 1.81 0.13 0.24 0.14 0.12 0.14

Table 11. (Continued)

Marker No. Genotype ATETRA analysis POPGENE analysis Hc H′c h I H H Fst individuals T S

Mresis 48 F1 & cultivars ------22 Wild Solanum 0.47 0.65 0.23 0.32 0.25 0.12 0.52

Cad 48 F1 & cultivars ------13 Wild Solanum 0.58 0.98 0.31 0.48 0.39 0.21 0.46

Winsh 48 F1 & cultivars ------22 Wild Solanum 0.65 1.06 0.25 0.34 0.25 0.13 0.48

Str 48 F1 & cultivars ------20 Wild Solanum 0.46 0.92 0.18 0.30 0.15 0.13 0.13

PT11 48 F1 & cultivars ------22 Wild Solanum 0.61 1.01 0.24 0.36 0.27 0.21 0.22

Pe54 48 F1 & cultivars ------20 Wild Solanum 0.78 1.67 0.19 0.32 0.20 0.18 0.10

Antiv1 48 F1 & cultivars 0.74 1.15 0.34 0.52 0.34 0.33 0.03 21 Wild Solanum 0.83 1.34 0.31 0.47 0.31 0.29 0.06

Cop12 48 F1 & cultivars ------21 Wild Solanum 0.77 1.67 0.19 0.32 0.20 0.14 0.30

Cunf34 48 F1 & cultivars ------21 Wild Solanum 0.88 1.70 0.23 0.39 0.24 0.18 0.25

Mean F1 & cultivars 0.58 0.98 0.26 0.39 0.27 0.24 0.13 Wild Solanum 0.62 1.32 0.22 0.35 0.24 0.15 0.33

′ HC: Heterozygosity corrected for sample size; Hc : Shannon index corrected for sample size; h: Expected heterozygosity or Nei’s gene diversity; I: Shannon’s information index of phenotypic diversity; HT: panmictic heterozygosity or total genetic diversity; HS: intra-population genetic diversity; FST: Wright’s fixation index; a: Monomorphic.

4.3.3. Localization of the IT markers in the potato genome Using the Solanum Genomic Network database (SGN), the genomic location of 21 out of the 25 polymorphic loci was determined. These markers resided on potato chromosomes I to XII (Appendix 1) the only exception being chromosome X. SGN based map location of the remaining 15 loci that were monomorphic in each populations are given in Appendix 12.

5. DISCUSSION

5.1. Development of functional markers and a multiple PCR for Rx1 and Rx2 resistance genes in potato In this study specific primers have been developed to distinguish the Rx1 and Rx2 genes, which confer extreme resistance to the PVX in potato. In spite of that the two genes originate from different species and reside on separate chromosomes (Querci et al., 1995) they show a high level of similarity, 98% at the nucleotide and 96% at the amino acid sequence, indicating the highly conserved nature of these almost 3 kbp large genes (Bendahmane et al., 2000). These loci are also suitable for analysis of R gene evolution because they are in clusters of R genes for which more recognition specificities have been identified (Bendahmane et al., 1999). On chromosome XII where the Rx1 locus resides, also the potato cyst nematode gene Gpa2, the PVY extreme resistance gene Rysto, and further Rx1 homologues can be found (Rouppe van der Voort et al., 1997; Song et al., 2005). On chromosome V where the Rx2 locus resides several R genes including the late blight resistance gene R1 (Leonards-Schippers et al., 1992), a hypersensitive PVX resistance gene Nb (DeJong et al., 1997), a single dominant gene H1 conferring resistance to G. rostochiensis pathotype Ro1 (Gebhardt et al., 1993; Pineda et al., 1993), a major quantitative trait locus (QTL) for resistance to Ph. infestans (Leonards-Schippers et al., 1994; Visker et al., 2003) and quantitative resistance loci (QRL) for root cyst nematodes G. rostochiensis and G. pallida were localized (Kreike et al., 1994; Rouppe van der Voort et al., 1998; Rouppe van der Voort et al., 2000). This region on chromosome V is one of the most important hot spots for resistances in the potato genome. Moreover, this region contains several genes which control plant vigour, plant maturity, tuber yield, tuber starch and sugar content (Ballvora et al., 2007). Because of the known genomic location of the Rx1 and Rx2 genes, the markers developed in this study could also be utilized as anchor markers in mapping studies. From a practical point of view, the introgression of PVX extreme resistance genes into potato cultivars would be beneficial both for producer and consumer. Potato cultivars are bred by the intercrossing of hundreds of genotypes of various pedigrees (Gebhardt, 2005) resulting in different gene pools in the different breeding programs, where markers

may be inherited linked to a locus in one pool but this would not be implicit for another. In the present study several published markers have been tested for the detection of the Rx genes. While these markers could clearly distinguish some of the genotypes, since their non-functional nature regarding the PVX resistance their applicability is genetic background dependent (Andersen and Lübberstedt, 2003). It is evidenced that the Rx1 gene has been introgressed from a single wild species clone, S. tuberosum subsp. andigena CPC 1673 (Rouppe van der Voort et al., 1999a). Based on the pedigree data of 13 out of 17 cultivars (Table 7.) in which Rx1 was detected by 1Rx1 and 5Rx1 markers, it was known that the Rx1 gene was introgressed into these cultivars via the CPC 1673 clone. But the source of resistance of the cultivars Agria, Ditta, Rhinered and Eridia was unknown (Table 7.). Sequencing and alignment of amplified fragments by the 1Rx1 and 5Rx1 markers in these cultivars indicated that the source of their PVX resistance is also the Rx1 gene. Beside the Rx1 gene, the cultivars Cara, Alcmaria, Multa, Amaryl, Atlantic and Wauseon are carrying the Gpa2 gene (Wilson and Jones, 1995; Rouppe van der Voort et al., 1999b; Asano et al., 2012). In spite of the presence of this highly similar gene, the developed primers amplified only the expected region of the Rx1 gene indicating the specificity of the primers. The known sequence of the Rx1 and Rx2 genes offers an excellent opportunity to develop functional markers and utilize them by marker-assisted selection (MAS). Nevertheless, for the development of markers for MAS both practical and economic aspects should be considered. In the present study because of the high similarity of the Rx1 and Rx2 genes the development of a PCR-RFLP would have been easier than that of a simple PCR based method. From a practical point of view, this opportunity was not preferred here, because in the selection processes the application of PCR-RFLP is more time consuming and is significantly more expensive than a single PCR based detection method. Furthermore, PCR based markers, in a case dependent manner, can be used in multiplex PCR, where more than one marker can be detected in the same reaction. This would make the MAS process more effective and economic. Here, we reported the development of primers by which the Rx1 and the Rx2 genes can be easily distinguished

in a single multiplex PCR reaction and they are ready to be applied in potato breeding in mapping experiments as well as in research studies of resistance.

5.2. Isolation of important genes related to resistance against PVX, PVY and Ph. infestans To date, few studies have been published on the transcriptome changes of the early incompatible plant-virus interaction (Ishihara et al., 2004; Marathe et al., 2004a; Baebler et al., 2009; Vuorinen et al., 2010). Transcriptome of resistant potato cultivar would elucidate a better understanding of potato defense mechanisms against pathogens. To achieve this goal, two technical approaches including PCR-selected cDNA subtraction and RNA-Seq transcriptome were used to profile PVX, PVY and Ph. infestans response genes.

5.2.1. Analysis of PVY induced subtracted cDNA library It is well documented that the majority of resistance genes substantially change in their expression levels 48 h after inoculation of the resistant plant with viruses (Ishihara et al., 2004; Oh et al., 2006; Milavec et al., 2008; Baebler et al., 2009; Peng et al., 2012). Therefore, to isolate the higher number of expressed genes in our experiment, sampling was performed covering with 12 sampling times points this 0-48 h period. The expression of 35 genes after inoculation, including Cytochrome P450, Senescence-associated protein, Zinc-knuckle family protein, F-box protein, Pectin esterase, CTR1 kinase, Relative to APETALA, SKP1 1, Lipoxygenase, Kunitz-type proteinase inhibitor, Calcium-dependent protein kinase, suggest their role in the resistant potato - PVYNTN interaction. The function of some identified gene in our experiment is briefly discussed as follow: In the present study, cytochrome P450 was over-expressed following inoculation of PVYNTN. In White Lady we obtained similar results as Baebler et al. (2009), who observed an increase in expression of photosynthesis-related genes in resistant cultivar Sante upon inoculation with PVY (Baebler et al., 2009). The reason for up-regulation may be the elevated energy demand of the plant needed for the first response to the stress.

Narusaka et al., (2011) found that the expression of cytochrome P450 genes was induced in response to Alternaria and various abiotic stresses in Arabidopsis (Narusaka et al., 2004). A senescence-associated protein was also identified in the present experiment. The combined activities of senescence-associated proteins and phytoalexins are necessary for programmed cell death triggered by resistance (R) proteins in response to avirulent pathogen isolates and in restricting the growth of virulent pathogens (Feys et al., 2005). Three genes including F-box protein, F-box domain-containing protein and SKP1 (S phase kinase-associated protein 1) were induced following in our experiment inoculation of PVYNTN. The genes are the components of the SCF (SKp1, Cullin, F-box protein) complex, which mediates ubiquitination of proteins targeted for degradation by the proteasome, playing an essential role in many cellular processes and also can be used in the defense response by the host (Correa et al., 2013). A pectin-esterase inhibitor was identified in our PVY induced cDNA library. Although a direct connection of this gene in plant response to virus infection has not yet been shown, the binding of plant viruses to pectin-methyl-esterase enables transportation of viruses through the cell wall (Giovane et al., 2004). It could be speculated that expression of pectin-esterase inhibitor gene contributes to the restriction of virus movement in the plant. Up-regulation of pectin methyl esterase gene was detected also in the resistant potato cultivar Sante after inoculation with PVYNTN (Kogovšek et al., 2010) that is in agreement with our results. On the other hand, its expression level dramatically decreased in response to Turnip mosaic virus (TuMV) infection in a compatible reaction (Yang et al., 2007). CTR1(constitutive triple response) was also isolated from the cDNA library. CTR1 is a member of mitogen-activated protein kinases (MAPK) which play a pivotal role in signaling plant defense against pathogen attack (Frye et al., 2001; Meng and Zhang, 2013). An EST with a high level of homology with APETALA2 gene was identified in the cDNA library. It is a gene coding for a member of a large family of transcription factors, the AP2/EREBP family. In addition to their important role in the plants throughout their

life cycle, they contribute to the adaptive mechanisms underlying the response to various biotic and abiotic stresses (Gu et al., 2002; Liang et al., 2010). Another identified gene was lipoxygenase in our experiment. Rapid increases in lipoxygenase enzyme activity and/or mRNA and protein levels are specifically associated with resistance-avirulence (R-Avr) gene mediated incompatibility (Hammond-Kosack and Jones, 1996). The study of transgenic lines made clear that lipoxygenase is not only important for the synthesis of jasmonic acid, but also of a number of other products that have specific roles in development and in response to stresses. Some product of lipoxygenase metabolism is required to induce the HR in incompatible interaction that limits pathogen growth (Porta and Rocha-Sosa, 2002). Kunitz-type protease inhibitor is a member of Proteinase inhibitors (PIs), one of the most important classes of defense proteins. It can be induced in response to pathogens (De Leo et al., 2002). In agreement with our results, over-expression of oryza cystatin, a rice cysteine proteinase inhibitor gene, in tobacco could enhance the resistance to PVY. It was concluded that cysteine proteinase inhibitor acts against cysteine proteinase which is essential in processing of polyprotein for replication of Potyviruses (Gutierrez-Campos et al., 1999). Ca2+-dependent protein kinase (CDPK) and MAPK pathways are known to be involved in signaling of abiotic and biotic stress in animals, yeast and plant cells. These two type of kinases present two major pathways that have been identified as central components mediating plant immunity (Asai et al., 2002; Wurzinger et al., 2011). The expression of CDPK in this experiment indicates the possible role of this gene in resistance response to PVY. The expression of a zinc-finger protein gene in the PVY induced subtracted cDNA library was also increased. Many of the (putative) zinc-finger transcription factors have been implicated in important biological processes. Mutations in some of the genes coding for zinc-finger proteins have been found to cause profound developmental aberrations or defective responses to environmental cues.

5.2.2. Analysis of PVX induced subtracted cDNA library

Up to now we identified 28 genes from our PVX induced subtracted cDNA library that was constructed from the cultivar White Lady. Five out of these 28 putative genes, including cytochrome P450, senescence-associated protein, F-box protein, F-box domain- containing protein and zinc-finger family protein were also identified in the PVY induced subtracted cDNA library and were discussed above. The function of some identified genes in our experiment is explained as follow: Permease 1 was one of the expressed genes in response to PVX. This gene may be involved in trafficking of macromolecules and signaling. Up-regulation of this gene has been reported in tomato in response to Tomato yellow leaf curl virus (TYLCV). In this experiment, permease I-like protein was preferentially expressed in the resistant tomato compared to susceptible plants and was strongly up-regulated upon TYLCV inoculation. It is also shown that silencing of this gene can lead to the loss of TYLCV resistance in tomato plants (Czosnek et al., 2013). A putative peroxidase gene was also isolated from our cDNA library. Peroxidases play a key role in controlling reactive oxygen species (ROS) concentration, leading to oxidative signal transductions (Park et al., 2006). The expression of peroxidase has been investigated by Milavec et al. (2008) in cultivar Sante after inoculation with PVYNTN. The first changes were observed at three hours after inoculation and after 24 hours the increase was twice than that in the control plants (Milavec et al., 2008). WRKY was expressed following inoculation with PVX. The members of the WRKY transcription factor family, which is a type of zinc-finger family proteins, are involved in the regulation of various physiological processes that are unique to plants including pathogen defense and senescence (Eulgem, 2005). They are important regulators of salicylic acid-dependent defense responses. It is demonstrated that they bind to promoter elements of defense-related genes and regulate, activate or repress, their expression (Eulgem et al., 2000). WRKY3 and WRKY4 are induced at a high level by salicylic acid and also during the N-mediated response to Tobacco mosaic virus (TMV) (Chen and Chen, 2000). An ethylene-responsive transcription factor (ERF) was also induced in this experiment. ERFs are members of a novel family of transcription factors that are specific

to plants. Up-regulation of this gene has also been described in grapevine and potato in response to Phytoplasma (Albertazzi et al., 2009; Longoria-Espinoza et al., 2012). However, induction of this gene is not yet reported in virus-plant interactions. Zinc-finger proteins have diverse functions, which include DNA/RNA recognition and binding to proteins or lipids, and have been shown to be critically involved in stress tolerance of higher plants (Ströher et al., 2009). Zinc-finger proteins were also reported to be up-regulated during phytoplasma infection on grapevine and potato (Albertazzi et al., 2009; Longoria-Espinoza et al., 2012).

Several sequences in both the PVX and PVY cDNA libraries did not have significant homology to coding sequences in the SGN and NCBI database and therefore could not be annotated by similarity. These sequences may contain some novel coding sequences that have not previously been isolated. However, it is unrealistic to suggest that all of the undiscovered EST-s encode for novel proteins with no significant homology to those in public sequence databases. Because some of the EST sequences such as Cont5 and 252-2 in the PVY induced subtracted cDNA library contain short stretches, 110 bp and 121 bp in length, respectively, which are insufficient to accurately assign an annotation to them based on homology in public databases. Furthermore, among both subtracted cDNA libraries, several EST-s, 37% in PVY and 26% in PVX library did not show significant similarity to potato-DM sequence (see Appendix 8 and 9) which is consistent with our results in RNA-Seq analysis in which only 42% and 49% of sequenced fragments were mapped to the reference sequence in the treated and control mRNA samples, respectively (see section 4.2.2.). In conclusion, although the construction and sequencing of subtractive cDNA libraries is time consuming and labor intensive that limits the number of samples that can be surveyed by this technology in each study, the result of this approach showed that it could be used as a cost-effective method that requires only basic research infrastructure of a molecular genetics laboratory.

5.2.3. NGS based transcriptome analysis

The application of RNA-Seq transcriptome was demonstrated in tetraploid potato in response to viruses, PVX and PVY, and Ph. infestans. Up to our knowledge, only one NGS-based transcriptome study has focused on tetraploid potato (Gao et al., 2013). In that study the interaction between potato and Ph. infestans was investigated. In our study, we presented RNA-Seq results of tetraploid potato that beside Phytophthora was also challenged with two different viruses. In our experiment, the SOLiD platform was used that generates relatively shorter (max. 50 nucleotides) but higher number of reads compared to the 454 ⁄ Roche technology with which longer sequence reads can be obtained (Bräutigam and Gowik, 2010). It was suggested that the potato-DM genome sequence what we used for reference and the genome of the tetraploid White Lady differ significantly, especially in their resistance gene content that most interested us. It was known that the choice of mapping software can influence the resulting contigs and that among the commercially available programs the CLC software is the least affected by differences in reads compared to the reference (Palmieri and Schlötterer, 2009). Considering these, the CLC genomics workbench software was applied for data analysis. One of the main goals of RNA sequencing is to compare gene expression levels between samples (ex. treated and control). To achieve a consistent result of expression level regardless of the length of transcripts, RPKM of transcripts were measured in our study that allows transcript levels to be compared among samples (Mortazavi et al., 2008). Only 42% and 49% of sequenced fragments were mapped to the reference sequence in the treated and control mRNA samples, respectively; suggesting that the resistance genes (genes which are relevant to resistance) of our interest of the cultivar White Lady may not be present in the potato-DM genome. While potato-DM is a homozygote doubled monoploid S. tuberosum group Phureja genotype, commercial cultivars such as White Lady are highly heterozygous tetraploid carrying different resistance genes which mainly originate from different wild Solanum species. Publication of the tomato genome sequence is proposed by the end of year 2013 (Prof. Richard Visser, personal communication). De novo assembling of unmapped White Lady sequences in relation to the tomato genome sequence may discover more genes in the

dataset. We plan to do this new analysis since tomato would be the closest relative of potato for which the genome sequence is available. Further, in the assembling of the tomato genome more than one-hundred genotypes are involved, which implies higher probability for the appearance of rare genes/alleles. The idea of this new analysis is justified also because for one-third of the up-regulated genes of mapped sequences annotation was not possible, since these sequences matched proteins of unknown functions (Table 10.). Structure based prediction of the function of these sequences is difficult and risky, since homologous proteins often have distinct and sometimes multiple functions. It would be very important to determine the role of these genes in the pathogen-potato interaction, since White Lady is well known as a cultivar with complex resistance, which is provided by a scale of different resistance genes originating mainly from different wild potato species. The identified resistance genes could be utilized in marker assisted potato breeding. Pathogen infection caused gene expressional changes of mapped genes suggest their role in resistance to at least to one of the examined pathogens, i.e.: to PVX, PVY or Ph. infestans. About 22% and 13% of annotated genes were up- or down-regulated indicating their relevance to stress response. Nevertheless, the expression level (fold change) of over 65% of these mapped genes had little variation after inoculation with pathogens, which indicates that these genes may have basically housekeeping function or are involved in normal physiological processes. In the present study, 141 NBS-LRR genes, which are related to resistance, were identified. Out of them an NBS-encoding resistance gene (PGSC0003DMG401029961) and an LRR type gene (PGSC0003DMG400002279) was expressed in the treated plant but not in the control. Since most R genes are constitutively expressed at low levels resulting in low concentrations of R proteins in the cell (Lukasik and Takken, 2009), expression of these genes only in the treated plant is somewhat noteworthy. Functional study of these two genes by over-expression in the susceptible potato cultivar, Somogy- Kifli and their silencing in White Lady is planned to get an answer about their function. Summarizing the above, the present study revealed that many genes related to disease resistance were up- or down-regulated in response to the examined pathogen infections. The generated dataset can be utilized to determine targets for future investigations to

explore the role of genes involved in resistance response to pathogens. Hence, our future functional analyses will focus on the search for candidate genes involved in biotic stress response of potato.

5.3. Development of intron-targeting markers in potato The advent of NGS platforms has provided the possibility to sequence whole genome and transcriptome. This sequence information can be used to obtain genetic variation among individuals within a population through the comparison of sequences at a given locus or loci. Due to the presence of highly repetitive transposable elements and paralogous sequences, especially in polyploid species, plant genome sequences are complicated. Therefore, detection of genetic variation via NGS has mainly focused on transcript sequences (Deschamps and Campbell, 2010). Although single nucleotide polymorphism (SNP) and single sequence repeats (SSR) are commonly identified by NGS, IT markers can serve as a suitable alternative because of simplicity in detection (agarose-based), locus specificity, codominant nature and efficiency in genome coverage. IT markers are associated with functional genes and have the potential to generate specific functional markers directly related to a given phenotype (Poczai et al., 2013) and could be used in gene tagging, comparative mapping and genetic map construction. The intron marker Cat-in2 was developed earlier to localize the Rysto gene in the potato genome (Cernak et al., 2008). In our experiments, polymorphism was detected in 11 out of 12 chromosomes by investigating 40 IT-loci. Eighty-eight percent of polymorphic IT markers detected in potato genotypes showed transferability to non-potato species. The successful transferability of primers to the other Solanum species indicates relative conservation of exon-intron junctions across solanaceous plants. This amount of conservation in Solanaceae genomes may result from few genome rearrangements and duplications and it can be concluded that the gene content and order of solanaceous species is very similar (Mueller et al., 2005). This phenomenon provides a potential to apply IT markers in other important solanaceous crops and wild Solanum species. Moreover, discovering an IT marker linked to important traits in the plant can be traced in other related species which is useful for breeders to monitor valuable traits in other Solanum species. In another study, the developed intron targeting makers from potato

expressed sequence tag (EST) and NCBI database records could successfully be applied for the detection of genetic variability in another solanaceaus species S. nigrum which is in consistence with our results (Poczai et al., 2010). One to four alleles per IT-locus were observed in a tetraploid potato genotype indicating polyploidy that prevents the calculation of a range of standard genetic statistics, including deviations from the Hardy–Weinberg principle and from linkage equilibriums (Thurlby et al., 2011). The level of calculated genetic diversity by the POPGENE program was lower than that of ATETRA analyzed data under Monte-Carlo simulations. The reason for this discrepancy might be that POPGENE estimated the values under diploid and dominant marker condition, while the analyzed potato samples are auto tetraploid and tetrasomic in which the allelic combinations are produced in equal frequencies (Ronfort et al., 1998). The other non-potato species consisted of different ploidy level species from diploid to octaploid. The product size of primers varied from 100 to 1200 bp and 100 to 2000 bp in the potato genotypes (F1 population and the cultivars) as well as in the wild Solanum-s, respectively. In general, the allele size ranges in F1 population and cultivars were different from that of the other Solanum species. While the F1 population is a specific cross for breeding purposes with a restricted distribution and the cultivars are derivatives of potato breeding, the other examined Solanum species are not closely related to potato and have variable ploidy levels even accompanied with aneuploid losses, which facts may influence the size patterns. In a large-scale analysis of 3008 species, it was shown that genome downsizing is a biological response to polyploidy (Leitch and Bennett, 2004). However, various factors such as insertion of transposable elements (Bartolomé et al., 2002) and deletion events can result into changes in size of introns (Petrov et al., 2000). Generating transcriptome with a reference sequence has the advantage to localize the marker in the chromosome. The developed IT markers were identified on chromosomes I, II, III, IV, V, VI, VII, VIII, IX, XI and XII in potato genotypes, using SGN, and could be used as anchor markers in mapping studies. The higher number of polymorphic IT markers in non-potato species compared to the potato genotypes (23 compared to 17 IT

markers) might be due to genetics bottlenecks phenomenon occurred in domestication process of modern cultivated potato (Tanksley and McCouch, 1997). In general, the intronic regions are more polymorphic than exonic ones, thus IT markers are increasingly used as fingerprinting tools, as it has been reported for example for species such as Rhododendron (Wei et al., 2005; De Keyser et al., 2009), Lolium, Festuca (Tamura et al., 2009) and species of the Rosaceae family (Sargent et al., 2009). On the other hand, sequencing large number of genes in different crops suggested higher frequency of single nucleotide polymorphism (SNP) in intronic regions of the genes (Ching et al., 2002; Rajesh and Muehlbauer, 2008). Hence, sequencing and multi- alignment of the identified intron regions could also be used for SNP detection. Introns can be exploited for the construction of genetic maps, because they directly reflect variation occurring within genes (Han et al., 2006). Our results demonstrated the potential of developed IT markers for genetic studies in potato and other non-potato related Solanum species. The cross-species applicability of potato IT markers has also been demonstrated by Poczai et al. (2010). IT markers from transcribed regions provide functional markers which directly reflect the variation within the genes and would be more valuable in gene tagging, genetic map construction and comparative mapping studies (Gupta et al., 2012). Additionally, they will increase marker resources and marker density at important genomic regions (Choudhary et al., 2012). There is an evidence that IT may have an effect on phenotypic diversity in eukaryotes (Sureshkumar et al., 2009; Kumar et al., 2010). By characterization of the function and the effect of polymorphic genes on the phenotype, they could be used as markers in populations without mapping, in mapped populations without risk of information loss owing to recombination and to better represent the genetic variation in natural or breeding populations. IT markers could also be applied in plant breeding, for the selection of parental materials to produce segregating populations, as well as the subsequent selection of inbred lines (Andersen and Lübberstedt, 2003). Observed levels of polymorphisms and genetic diversity suggest that the developed markers are fully adequate for characterizing of genetic variation and provide efficient tools in potato genetic studies namely DNA fingerprinting, marker-assisted selection and genetic mapping and diversity analysis. They could also be applied as anchor markers of different

chromosomes in the potato genome. The number of IT markers could be rapidly increased by examining more transcript sequences.

LIST OF NEW FINDINGS

1) It was revealed that the Rx2 gene is responsible for the extreme resistance to PVX in the Hungarian potato cultivars. One specific primer pair for the Rx2 and two specific primer pairs for the other known PVX extreme resistance gene, the Rx1 gene were developed. Further, a multiplex PCR method for the simple distinguishing of these two highly similar genes in a single reaction was developed. 2) A subtracted cDNA library of the White Lady in response to PVX was constructed and 28 resistance response EST-s were isolated from it. 3) A subtracted cDNA library of the White Lady in response to PVYNTN was constructed and 35 resistance response EST-s were isolated from it. 4) An NGS based transcriptome dataset of White Lady in response to PVX, PVYNTN and Ph. infestans was generated and 748 transcripts were recognized only in treated samples but not in the control indicating stress response specificity of these genes. Out of these, 57% encoded proteins of unknown genes or conserved genes with unknown function. It was found that the transcriptome data set contains 141 NBS-LRR encoding genes with 13 Toll Interleukin-like receptor (TIR) and 50 Coiled-coil (CC) types in the treated samples. 5) The utility of NGS-based transcriptome sequences for the development of Intron- targeting (IT) markers, which are potential anchor markers was demonstrated and the effective transferability of these IT markers to the other wild Solanum species was experimentally proven.

ÚJ TUDOMÁNYOS EREDMÉNYEK

1) Megállapítottuk, hogy a magyar PVX rezisztens burgonyafajták az Rx2 gént tartalmazzák. Egy specifikus primer párt fejlesztettünk ki az Rx2 azonosítására, és két specifikus primer párt a másik ismert PVX extrém rezisztenciagén, az Rx1 azonosítására. Továbbá, kifejlesztettünk egy multiplex PCR eljárást e két, nagyon hasonló szekvenciájú gén egyetlen reakcióban történő megkülönböztetésére. 2) Létrehoztunk egy, a White Lady fajtában PVX fertőzésre kifejeződő géneket tartalmazó cDNS klóntárat, és ebből 28 rezisztenciaválasz EST-t izoláltunk. 3) Létrehoztunk egy, a White Lady fajtában PVYNTN fertőzésre kifejeződő géneket tartalmazó cDNS klóntárat, és ebből 35 rezisztenciaválasz EST-t izoláltunk. 4) Létrehoztunk egy NGS eljárással generált, PVX, PVYNTN and Ph. infestans fertőzésen alapuló transzkriptóm adatbázist a White Lady fajtában. Az adatbázisból 748 transzkriptomot azonosítottunk, melyek csak a kezelt mintában fejeződtek ki, ami e gének rezisztencia válasz specifikusságára enged következtetni. E gének 57%-a ismeretlen géneket vagy ismeretlen funkciójú konzervált géneket kódolt. A kezelt mintában 141 NBS-LRR típusú gént azonosítottunk, melyek közül 13 a Toll Interleukin-szerű receptor (TIR), és 50 pedig az ún. Coiled-coil (CC) típusba tartozott. 5) Kísérletesen igazoltuk az NGS eljárással generált transzkriptóm szekvenciákon alapuló és anker markerként alkalmazható, ún. intron-targeting markerek fejlesztésének hatékonyságát, valamint demonstráltuk e markerek más Solanum fajokban való alkalmazhatóságát.

ACKNOWLEDGEMENTS

First of all, I pray to Allah to show me the straightway, the way of those on whom He has bestowed His Grace, those whose (portion) is not wrath, and who go not astray. I am grateful to my kind supervisors, Dr. Zsolt Polgár and Dr. János Taller, for their guidance, support and being as a friend throughout the past three years. Without their enlightening guidance, consistent support, and unreserved trust, I would not have been able to overcome the obstacles I encountered, and would not have been able to comprehend and appreciate the beauty of science. Their numerous comments, criticisms and suggestions during the preparation of this thesis are gratefully acknowledged. I would like to express my sincere appreciation to my Iranian friends and colleagues Dr. Ahmad Mousapour Gorji and Mr. Ramin Hajianfar, who gave me valuable discussions and hands-on help in this research program. Sincere appreciation is extended to my Egyptian friend, Dr. Antar Elbana, who taught me the necessary experimental knowledge and skills for genetic engineering. With his help, I started to establish my confidence in this field. I would miss the weekends and late nights that we spent in the lab to prepare constructs for plant transformation. I express gratitude to all my Hungarian friends, Mrs. Mátyás Kinga, Dr. Kincső Decsi, Dr. István Cernák and Dr. Péter Poczai, in the Biotechnology laboratory for providing a friendship and enjoyable atmosphere in the lab and their effort to help me in daily life to solve the problems with Hungarian language. I sincerely thank to Dr. Kolics Balázs for his excellent assistance to provide materials and facilities that led to the successful completion of my study. Many thanks go to Mr. Wolf István, Mr. Vaszily Zsolt and Mrs. Ildiko for organizing and assistance in greenhouse experiments. I also would like to express my gratitude to staff members in the Potato Research Center and Department of Biotechnology for their generous help and support. Also many thanks to Dr. Tallerné Barna Piroska and Miss Bődör Beáta for their excellent help in various administrative letters during my study. I acknowledge the Agricultural Research, Education and Extension Organization (AREEO) for approving the educational mission to continue my Ph.D. study and I am deeply indebted to Dr. Mostafa Aghaee and Dr. Niaz Ali Sepahvand, the former and the

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present Director general of Seed and Plant Improvement Research Institute (SPII) and their deputy Dr. Rajab Chokan for their attempts to obtain the approval of AREEO for this educational mission. My special appreciation goes my in-laws for their love, support, and wishes. It was definitely impossible to fulfill my PhD without their support during past three years. The last but the best part my life are my family. I am lucky that I have a happy family, where I can always find support, understanding, encouragement and hope. You spent hard times during my study, especially when you had to live thousands of kilometers far from me in Iran. My deepest gratitude goes to you for your patience. I would like to say to my little son, Armin, now I have time to spend with you. I apologize all the times that I was extremely busy and no time to stay at home at the weekends and holidays or working in front of laptop at home in the nights. Please do not hate my laptop anymore. In life, every ending is just a new beginning. Enriched with experiences and wisdom during these years of my study, I look forward to newer and better experiences in the future.

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PUBLICATION LIST

Referred articles related to thesis

1. Ahmadvand, R., Takács, A., Taller, J., Wolf, I., and Polgár, Z. (2012). Potato viruses and types of resistance to these viruses in potato. Acta Agronomica Hungarica, 60(3), 283-298.

2. Ahmadvand, R., Wolf, I., Mousapour Gorji, A., Polgár, Z, and Taller, J. Development of molecular tools for distinguishing between the highly similar Rx1 and Rx2 PVX extreme resistance genes in tetraploid potato. Potato Research. (accepted)

3. Ahmadvand, R., Poczai, P., Hajianfar, R., Mousapour Gorji, A., Polgár, Z, and Taller, J. Next generation sequencing based development of intron-targeting markers in tetraploid potato and their transferability to other Solanum species. GENE. (accepted with revision)

Conference abstracts related to the thesis

1. Ahmadvand, R., Hajianfar, R., Mousapour Gorji, A., El-Banna, A., Polgár, Z, and Taller, J. (2013). Development of intron-targeting markers as a tool for molecular breeding in response to pathogens. Proceeding of 8th plant breeding international conference, 6-7 May, Egypt. 2. Ahmadvand, R., Taller, J.,Wolf, I., and Polgár, Z. (2012). Identification of the resistance gene to PVX in Hungarian potato cultivars. 54 th Georgikon Scientific conference (Georgikon napok), October, 11-12. 3. Ahmadvand, R., Hajianfar, R., Polgár, Z, and Taller, J. (2013). Transcriptome and functional marker study in potato. "Jövőnk" konferencia. TÁMOP-4.2.3- 12/1/KONV-2012-0001. Keszthely, 2013. Május 15. Összefoglalók p:31. 4. Ahmadvand, R., Hajianfar, R., Mousapour Gorji, A., Cernák, I, Polgár, Z, and Taller, J. (2013). Transcriptome analysis of White Lady in response to PVX, PVY and Phytophthora infestans using next generation sequencing. EAPR - EUCARPIA

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Congress "The challenges of improving both quality and resistance to biotic and abiotic stresses in potato", June 30 - July 04. 2013, Hévíz, Hungary. Pp: 26. 5. Elbana, A., Ahmadvand, R., Hajianfar, R., Mousapour Gorji, A., Cernák, I, Polgár, Z, and Taller, J. (2013). Isolation and functional analysis of resistance response genes in potato and the development of molecular markers. EAPR - EUCARPIA Congress "The challenges of improving both quality and resistance to biotic and abiotic stresses in potato", June 30 - July 04. 2013, Hévíz, Hungary. Pp: 27. 6. Hajianfar, R., Ahmadvand, R., Mousapour Gorji, A., Cernák, I, Polgár, Z, and Taller, J. (2013). Next generation sequencing based analysis of genes for resistance to Phytophthora infestans in cultivar White Lady. EAPR - EUCARPIA Congress "The challenges of improving both quality and resistance to biotic and abiotic stresses in potato", June 30 - July 04. 2013, Hévíz, Hungary. Pp: 26. 7. Hajianfar, R., Ahmadvand, R., Polgár, Z., Wolf,I, and Taller, J. (2013) Allelic variation of the R1 late blight (Phytophthora infestans) resistance gene in White Lady variety. "Jövőnk" konferencia. TÁMOP-4.2.3-12/1/KONV-2012-0001. Keszthely, 2013. Május 15. Összefoglalók p:31.

Other publications

1. Reza Hessan Sajedi, Hossein Nader-manesh, Khosro Khajeh, Rahim Ahmadvand, Bijan Ranjbar, Ahmad Asoodeh., and Fatemeh Moradian (2005) Ca-independent α- amylase that is active and stable at low pH from the Bacillus sp. KR-8104 Enzyme and Microbial Technology 36: 666-671.

2. Fatima Moradian, Khosro Khajeh, Hossein Nader-manesh, Rahim Ahmadvand, Reza Hassan Sajedi.,and Majid Sadeghizadeh (2006) Thiol-Dependent Serine Alkaline Proteases From Bacillus sp. HR-08 and KR-8102 Applied Biochemistry and Biotechnology 134:77-87.

3. Ahmadvand, R., and Rahimian, H. (2005).Study on phenotypic and electrophoretic characteristic of pectobacteriums infecting of corn in Mazandaran. Iranian Journal of Phytopathology 2:41, 271-290.

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4. Ahmadvand, R., and Hasan- Abady, H. (2010). Evaluation of Resistance of Potato Promising Clones to PVX, PVY and PVA in Greenhouse. Seed and Plant Improvement Journal 25-1:517-531.

5. Ahmadvand, R., and Zarbakhsh, A. (2008). Identification and Determination Physiological Races of Tomato Wilt Agent in Major Cultivation Areas. Agricultural Research Journal 18-3:156-173.

6. Translated book entitled “Plant virus evolution”. By Roossinck, M. J. (2008). Springer. Translated by:Nasaj-Hosseini, Ahmadvand, R.

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Appendix 1

Characteristics of the polymorphic loci and their intron-targeting primer sequences

1 1 Locus Putative function Primer sequence (5′-3′) Ta Transcript ID Ch

PKF11 LATD/NIP F: C AAAGCTCCTTGAACCATCC 5 PGSC0003DMG40001 V

R: TGCAAGCTCTATTCAAACACCT 8 3511 LBR57 Late blight resistance protein F: TGAACTTCAAATCTTTACTCTTTGC 5 PGSC0003DMG40003 IV R: TTTGTTTGAAAAACATATGAGCTAAGT 4 6257 AVTPSH Anti-virus transcriptional factor F: TCTCCAGTAGTGGATGCAATAAGA 5 PGSC0003DMG40001 XII R: TTTCAAGTGTACACCAAATCTTCAA 4 1831 PGRSH PGR5 1A, chloroplastic F: TGTCCCTGCTGCTGTTGTT 5 PGSC0003DMG40001 VIII R: TCTCGAATCCAGTCAAATCG 5 2494 RP3a35 Disease resistance protein R3a F: TGAAAATGCTTCACTCCACA 5 PGSC0003DMG40203 XI R: TTGTTCTTTCCGTTTTTCAGTG 8 0235 NB89 NBS-LRR type resistance F: GCTCTTCAAGATGTGGCAACT 5 PGSC0003DMG40101 - R: GCCATCTTCCATGTCTGGTT protein 5 7089 PTA-83 Late blight resistance protein Rpi- F: GACCATTTGGGAAAGAGTCG 5 PGSC0003DMG40103 VIII R: GGGACAAAAGAAAATGATGAGA pta1 2 0883 R1L333 Late blight resistance protein F: CCAGAACACAAGGAACAAATAGAA 5 PGSC0003DMG40003 V homolog R1C-3 R: GCTAGCCTCAATTAAAGCATGA 8 7333 ATPb-218 ATP binding / kinase/ protein F: CATATGAAAAATTGACTGGGATGA 5 PGSC0003DMG40201 III R: TGGAAAATAAATGGAACAAACAA kinase 4 4218 Cin Cinnamyl alcohol dehydrogenase F: TCTTTCCATTACTTATGGGTGAG 5 PGSC0003DMG40001 XI R: GATCCCACTTCCAGCTACGA 4 6115 TREPSH Tospovirus resistance protein E F: CCCGAGTATATGGCTTTGGA 5 PGSC0003DMG40000 IX R: CAAACCTACCTGGCTGCTGT 6 3794

TSWVP Tomato spotted wilt virus F: TTGATGCTTGCTTTCGCTTA 5 PGSC0003DMG40004 V protein R: GGACAGGAGAATGAGCATGG resistance 4 6201 TRP77 Transcriptional regulator family F: AAGAGGCTAGATATCCATTTTCCA 5 PGSC0003DMG40002 II protein R: CCAATCCATTTTGGCTCAAT 7 8477 Nitsh Nitrate transporter F: TCGCCATATGAAACAGAGGTC 6 PGSC0003DMG40101 V R: GCCTTGAGTGATTCAACTCGT 0 5590 RPB36 Late blight resistance protein F: CTTCAAGGTTGGGCAGTCTC 5 PGSC0003DMG40002 VI blb2 R: ATCGGCCATGGGATTTTTAC Rpi- 4 0736 Cat Catalase F: ATCGTGTCAGGGAACGACTT 5 PGSC0003DMG40002 XII R: CCGAGTCAATCAGGGACATT 6 9408 Antiv1 Putative anti-virus transcriptional F: GTTGGAGTTGCAATCGGAAT 5 PGSC0003DMG40002 III factor R: CTTTCTGACGGCATGTTCAG 7 4467

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Appendix 1

(Continued)

1 1 Locus Putative function Primer sequence (5′-3′) Ta Transcript ID Ch

Mresis Multidrug resistance pump F: GCGACCAATCTGATCTTCGT 55 PGSC0003DMG400030304 - R: AGTTTGACGCTCAAGTTCACC

Cad Calcium-dependent protein F: CAATCCGTACCAGCCTTCAT 56 PGSC0003DMG400033335 - kinase 8 R: GCTGGAATGAAGGAAGCATT

Winsh Wound-inducible F: CCATGAACGAGTTAGAAGGACA 54 PGSC0003DMG402027687 VI carboxypeptidase R: AGCCACTCATTTCTCCTCAAG

Str Structural constituent of cell F: CTTTCCCTCCACTTCAACGA 52 PGSC0003DMG400021585 VI wall R: CAACCTTAGCAGTGATCTTGAAA

PT11 Potassium transporter 11 F: GAGAGAAGTATTGAAAACACCCCTAA 55 PGSC0003DMG400010431 II R: TTTCATTCCCGAACTATTGACA

Pe54 Peroxidase 7 F: AAAGTTCATACCAGCCTCTTCA 53 PGSC0003DMG400013654 II R: TCGATCAGTGCGAGTTTGTT

Cop12 Cop9 signalosome complex F: AATCTATGGCCAAACATGAGC 55 PGSC0003DMG400022712 I subunit R: CAGGAAGAACAAGATGTTGATATAGA

Cunf34 Conserved gene of unknown F: GAGTCCGAACACGATGACCT 54 PGSC0003DMG400005234 - function R: AAGCAGAGTTGAGAATTTTGTATGA

º Ta: annealing temperature in C; Ch: Location on potato chromosome; 1: Available in SOL Genomics Network (http://sgn.cornell.edu/).

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Appendix 2 Multi-alignment of the CAPS marker CP60 with the corresponding S. tuberosum group Phureja DM 1-3 516 R44 sequence scf00283-28, - whole genome shotgun sequence (Acc. No. AEWC01029415.1).

49990 50000 50010 50020 50030 50040 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM ACATGAAAAATCAGTCCCAAGAAAAACTTGGGCGTTCCAGTGACATATTGTTATAGAGAG Cara (Rx1) ------TTGGGTGTTCCAGTGAAATATTGTTATAGAGAG White Lady (Rx2) ------Hermes (Susceptible) ------

50050 50060 50070 50080 50090 50100 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM GTTTGACTGTTATCAGAGCAATCAATCGTGTATCCAACTATAAGTTCAATGGTTTGGTAG Cara (Rx1) GTCTGACTGTAATTAGAGCAATCAATCATGTATCCAACTATGGGTTCAATAGTTTGGTAG White Lady (Rx2) ------Hermes (Susceptible) ------

50110 50120 50130 50140 50150 50160 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM AACCTAGTGTTTCGTTGTTGAATTCTATATATACGTGCAAAAATCAAGTGTAGACCATGT Cara (Rx1) AAGCTAGTGTTTCGTTATTGAATTCTATGTATACGTGCAAAAAACAAGTGTAGACCATGT White Lady (Rx2) ------Hermes (Susceptible) ------

50170 50180 50190 50200 50210 50220 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM CTAAATTCTCGAAATTTACTAGTCGTGAAGTCAATTGGATTAACCGTAGTTATTGAAATC Cara (Rx1) CTAAATTCTCGAAATTTACTAGTCGTTAAGTCAATGGATTAAGAATACGATATTACTACT White Lady (Rx2) ------Hermes (Susceptible) ------

50230 50240 50250 50260 50270 50280 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM ACTTGTCAGGAAACACTAACACATTGGCTGGAAATGTACCTCCAAAGGCCAACCTCGGGG Cara (Rx1) CCAGTGGGGGACTTATCGTTGTTACTGAAATCACATATCAGGAAACACAAACACATTGGC White Lady (Rx2) ------Hermes (Susceptible) ------

50290 50300 50310 50320 50330 50340 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM ACTTGAGGGGTTTGGCAAGAGGGCTCGATGGACTTAACAGCTCAGCCATGATTGATGGTG Cara (Rx1) TGGAAATGTACCTCCAAAGGCCAACCTCGGGGACTTGAGGGGTTTGGCAAGAGGGCTCGA White Lady (Rx2) ------GCCATGATTGATGGTG Hermes (Susceptible) ------GATTCAGCCATGATTGATGGTG

50350 50360 50370 50380 50390 50400 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM GTGACTTTGATGGTGCCAAAGCATACAAAGACAGCAAGGTTTGCAATATGCTCACTATGC Cara (Rx1) TGGACTTAACAGCTC------White Lady (Rx2) GTGACTTTGATGGTGCCAAAGCATACAAAGACAGCAAGGTTTGCAATATGCTCACTATGC Hermes (Susceptible) GTGACTTTGATGGTGCCAAAGCATACAAAGACAGCAAGGTTTGCAATATGCTCACTATGC

50410 50420 50430 50440 50450 50460 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM AGGAATTCCATAGACGATACCACGAGGAGACTGGCATCACATTTGCTTCTCTATACCCTG Cara (Rx1) ------White Lady (Rx2) AGGAATTCCATAGACGATACCATGAGGAGACTGGCATCACATTTGCCTCTCTATACCCTG Hermes (Susceptible) AGGAATTCCATAGACGATACCATGAGGAGACTGGCATCACATTTGCCTCTCTATACCCTG

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50470 50480 50490 50500 50510 50520 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM GCTGCATCGCGACAACAGGGCTATTCAGGGAGCATATCCCCTTGTTTAGGCTCCTTTTCC Cara (Rx1) ------White Lady (Rx2) GCTGCATCGCGACAACAGGGCTATTCAGGGAGCATATCCCTTTGTTTAGGCTCCTTTTCC Hermes (Susceptible) GTTGCATCGCGACAACAGGGCTATTCAGGGAGCATATCCCTTTGTTTAGGCTCCTTTTCC

50530 50540 50550 50560 50570 50580 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM CTCCATTCCAGAAGTTTATCACCAAGGGATTCGTCTCCGAGGCAGAATCTGGAAAGAGAC Cara (Rx1) ------White Lady (Rx2) CTCCATTCCAGAAGTTTATCACCAAAGGATTCGTCTCCGAGGCAGAATCTGGAAAGAGAC Hermes (Susceptible) CTCCATTCCAGAAGTTTATCACCAAAGGATTCGTCTCCGAGGCAGTATCTGGAAAGAGAC

50590 50600 50610 50620 50630 50640 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM TTGCACAGGTAATAATAATCAGTGTCAGTT-CACGTATAATCACTGAACTTTATCCACTA Cara (Rx1) ------White Lady (Rx2) TTGCACAGGTAATAATAATCAGTGTCAGTTTCACGTATAATCACTGAACTTTATCCACTA Hermes (Susceptible) TTGCACAGGTAATAATAATCAGTGTCAGTTTCACGTATAATCACTGAACTTTATCCGCTA

50650 50660 50670 50680 50690 50700 ....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM TAACAATAA--CCATAAAGATATGACACATGGGCCTAACTCAACCCCAAAAGCTAGCTCA Cara (Rx1) ------White Lady (Rx2) TAACAGTGAAGTCTGTAAAATATGATA------Hermes (Susceptible) TAACAGTGAAGTCTGTAAAATATGATACCTGA------

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Appendix 3 Multi-alignment of the 221R marker with the corresponding sequence of S. tuberosum resistance gene cluster, - complete sequence (Acc. No. AF265664.1) (Green color regions show annealing region of primers).

103960 103970 103980 103990 104000 104010 104020 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance ACAATTAAAGTGAACTCTATATTAATTAGTGAAGAAAGCTTTGGACAATGAAGCTTACATTTGCTCGAAG Cara (Rx1) ------GCTTACATTTGCTCGAAG Bzura (Rx2) ------GCTTACATTTGCTCGAAG White Lady (Rx2) ------CTTACATTTGCTCGAAG

104030 104040 104050 104060 104070 104080 104090 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance AAGCCACTTGTCGAGCAAACTGTGACATTCTTGTGGCAATTTGTTTGGAGCGAGAAATTACTGTAACATC Cara (Rx1) AAGCCACTTGTCGAGCAAACTGTGACATTCTTGTGGCAATTTGTTTGGAGCGAGAAATTACTGTAACATC Bzura (Rx2) AAGCCACTTGTCAAGCAAACTGTGACATTCTTGTGGCAATTTATTTGGAGCGAGAAATTACTGTAACATC White Lady (Rx2) AAGCCACTTGTCAAGCAAACTGTGACATTCTTGTGGCAATTTATTTGGAGCGAGAAATTACTGTAACATC

104100 104110 104120 104130 104140 104150 104160 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance CGACAATTTGAAATAACTATGAAGAGGCTTAGAAATGGAAATAGTCATTTTTTTTGAAAGAATTTAAGTC Cara (Rx1) CGACAATTTGAAATAACTATGAAGAGGCTTAGAAATGGAAATAGTCATTTTTTTTGAAAGAATTTAAGTC Bzura (Rx2) CGACAATTTGAAATAACTATGAAGAGGCTTAGAAATGGAAATAGTCATTTTTTTTGAAAGAATTTAAGTC White Lady (Rx2) CGACAATTTGAAATAACTATGAAGAGGCTTAGAAATGGAAATAGTCATTTTTTTTGAAAGAATTTAAGTC

104170 104180 104190 104200 104210 104220 104230 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance TGGAAAAAAATTTGGTTAAGTATGGGAAAAAGTGAGTTTTTGGCCAACTTTGAACAGTTATAACTCCTAG Cara (Rx1) TGGAAAAAAATTTGGTTAAGTATGGGAAAAAGTGAGTTTTTGGCCAACTTTGAACAGTTATAACTCCTAG Bzura (Rx2) TGGAAAAAAATTTGGTCAAGTATGGGAAAAAGTGAGTTTTTGGCCAACTTTGAACAGTTATAACTCCTAG White Lady (Rx2) TGGAAAAAAATTTGGTCAAGTATGGGAAAAAGTGAGTTTTTGGCCAACTTTGAACAGTTATAACTCCTAG

104240 104250 104260 104270 104280 104290 104300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance CTCAGGATGACTTGGGAGTAGTTCCAGTTATGTTTGTGAAGCCCGTGGAATGATCTTTCCAACGCCGCTG Cara (Rx1) CTCAGGATGACTTGGGAGTAGTTCCAGTTATGTTTGTGAAGCCCGTGGAATGATCTTTCCAACGCCGCTG Bzura (Rx2) CTCAGGATGATTTGGGAGTAGTTCCAGTTATGTTTGTGAAGCCCGTGGAATGATCTTTCCAACGCCGCTG White Lady (Rx2) CTCAGGATGATTTGGGAGTAGTTCCAGTTATGTTTGTGAAGCCCGTGGAATGATCTTTCCAACGCCGCTG

104310 104320 104330 104340 104350 104360 104370 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance ATTTTTCTCGATTCCGAGTTCGTATGAGTGAGTTATGCCCTTTCGAAGTTGGGCTTTTGGTAAGGGAAGT Cara (Rx1) ATTTTTCTCGATTCCGAGTTCGTATGAGTGGGTTATGCCCTTTCGAAGTTGGGCTTTTGGTAAGGGAAGT Bzura (Rx2) AGTTTTCTCAATTCCGAGTTCGTATGAGTGAGTTATGCCCTTTCGAAGTTGGGCTTTTGGTAAGGGAAGT White Lady (Rx2) AGTTTTCTCAATTCCGAGTTCGTATGAGTGAGTTATGCCCTTTCGAAGTTGGGCTTTTGGTAAGGGAAGT

104380 104390 104400 104410 104420 104430 104440 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance CAGTCCGGAAATTGTAAAGGTATTTTGGTCTTTTCCCTAGCCTTGTCTTTTGGATATATATTAAGGTGTT Cara (Rx1) CAGTCCGGAAATTGTAAAGGTATTTTGGTCTTTTCCCTAGCCTTGTCTTTTGGATATATATTAAGGTGTT Bzura (Rx2) CAGTCCGGAAATTGTAAGGGTATTTTGGTCTTTTCCCTAGCCTTGTCTTTTGGATATATATTAAGGTGTT White Lady (Rx2) CAGTCCGGAAATTGTAAGGGTATTTTGGTCTTTTCCCTAGCCTTGTCTTTTGGATATATATTAAGGTGTT

104450 104460 104470 104480 104490 104500 104510 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance AGACTGTTTTTAAATTAGTTTTCCCTTTTATAAGAGTG-GAGTAAGGGTTTGTGAGTGAAGAGGAGAAGA Cara (Rx1) AGACTGTTTTTAAATTAGTTTTCCCTTTTATAAGAGTG-GAGTAAGGGTTTGTGAGTGAAGAGGAGAAGA Bzura (Rx2) AGACTGTTTTTAAATTAGTTTTCCCTTTTATAAGAGTGAGAGTAAGGGTTTGTGAGTGAAGAGGA----- White Lady (Rx2) AGACTGTTTTTAAATTAGTTTTCCCTTTTATAAGAGTGAGAGTAAGGGTTTGTGAGTGAAGAGGA-----

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104520 104530 104540 104550 104560 104570 104580 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance AGAGGAGAAAGAGAAGATCAAGCTAAGTTTCGTCAAAGATCGTCGTGGATATCGTCGGGGGTGATCCCTA Cara (Rx1) AGAGGAGAAAGAGAAGATCAAGCTAAGTTTCGTCAAAGATCGTCGTGGATATCGTCGGGGGTGATCCCTA Bzura (Rx2) ------GAAAGAGAAGATCAAGCTAAGTTTCGTCAAAGATCGTCGTGGATATCGTCGGGGGTGATCCCTA White Lady (Rx2) ------GAAAGAGAAGATCAAGCTAAGTTTCGTCAAAGATCGTCGTGGATATCGTCGGGGGTGATCCCTA

104590 104600 104610 104620 104630 104640 104650 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance CTAGGTATGTGAGTTCATAGTGTTGGGTTAGTCCTTTCCCCCACACGCTAAACTCATTTTAAATTTCGAG Cara (Rx1) CTAGGTATGTGAGTTCATAGTGTTGGGTTAGTCCTTTCCCCCACACGCTAAACTCATTTTAAATTTCGAG Bzura (Rx2) CTAGGTATGTGAGTTCATAGTGTTGGGTTAGTCCTTTCCCCCACACGCTAAACTCATTTTAAATTTCGAG White Lady (Rx2) CTAGGTATGTGAGTTCATAGTGTTGGGTTAGTCCTTTCCCCCACACGCTAAACTCATTTTAAATTTCGAG

104660 104670 104680 104690 104700 104710 104720 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance AAAAGATTGGTTATGTTTGTTGAAGTTTGTGGTTGTGTTTGTTGAAGTTTGGTTGGTGAGGTTGTGGTTG Cara (Rx1) AAGAGATTGGTTATGTTTGTTGAAGTTTGTGGTTGTGTTTGTTGAAGTTTGGTTGGTGAGGTTGTGGTTG Bzura (Rx2) AAAAGATTGGTTATGTTTGTTGAAGTTTGTGGTTGTGTTTGTTGAAGTTTGGTTGGTGAGGTTGTGGTTG White Lady (Rx2) AAAAGATTGGTTATGTTTGTTGAAGTTTGTGGTTGTGTTTGTTGAAGTTTGGTTGGTGAGGTTGTGGTTG

104730 104740 104750 104760 104770 104780 104790 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance TGTTGTTGAAGTTCTTGTGGATTTGAGACATGTTTTAG-TTGTGTTTTCGAGTTGAATCTATTGATTATT Cara (Rx1) TGTTGTTGAAGTTCTTGTGGATTTGAGACATGTTTTAG-TTGTGTTTTCGAGTTGAATCTATTGATTATT Bzura (Rx2) TGTTGTTGAAGTTCTTGTGGATTTGAGACATGTTTTAGGTTGTGTTTTCGAGTTGAATCTATTGATTATT White Lady (Rx2) TGTTGTTGAAGTTCTTGTGGATTTGAGACATGTTTTAGGTTGTGTTTTCGAGTTGAATCTATTGATTATT

104800 104810 104820 104830 104840 104850 104860 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum resistance AAGGGTTTTAATGATCCCAAGTGTTTGGGGAAAATAACCTTTGAAGTTAAGGGAGTTTAGAGTTGGTAAC Cara (Rx1) AAGG------Bzura (Rx2) AAGG------White Lady (Rx2) AAGG------

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Appendix 4 Multi-alignment of the CAPS marker GP21 with the corresponding S. tuberosum group Phureja DM 1-3 516 R44 sequence scf00051-63, - whole genome shotgun sequence Acc. No. AEWC01009533.1)

31090 31100 31110 31120 31130 31140 31150 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM CCATGTTATGAAGTAAATTAGGCTGACTTGAGCTGATATAAGCTATGCTATCTGTTAAATCTTTCATATA Bzura (Rx2) ------CTATGCTATCTGTTAAATCTTTCATATA Hermes(susceptible) ------CTATGCTAACTGTTAAATCTTTCATATA

31160 31170 31180 31190 31200 31210 31220 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM CTAAGACACTTACTGTGTTAAATTTCTTATTAGTTTTTTGTATT---AATCTCTGATTGAAAGTAACATG Bzura (Rx2) CTAAGACACTTACTGTGTTAAATTTCTTATTAGTCTTTTGTATTCATAATCTCTGACTGAAAGTAACATG Hermes(susceptible) CTAAGACACTTGCTGTGTTAAATTTCTTATTAGTCTTTTGTATTCAGAATCTCTGACTGAAAGTAACATG

31230 31240 31250 31260 31270 31280 31290 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM TATTATCAGCAATGGAGTAACAAATTCATCAGTAGTTTAATACCCTATATTTCTTTTGCAGGTTGAATCT Bzura (Rx2) TATTATCAGCAATGGAGTAACAAATTCATCAGTAGTTTAATACCCTATATTTCTTTTGCAGGTTGAATCT Hermes(susceptible) TATTATCAGCAATGGAGTAACAAATTCATCAGTAGTTTAATACCCTATATTTCTTTTGCAGGTTGAATCT

31300 31310 31320 31330 31340 31350 31360 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM ATTTTTTGTCTCTCTCATTTGTTTTATACTAGTAAACGTGAACTTTCATACTTAATTTAAGTATAAGTGA Bzura (Rx2) ATTTTTTGTCTCTCTCATTTGTTTTATACTAGCAAATGTAAACTTTCATACTTAATTTAAGTGTAAGTGA Hermes(susceptible) ATTTTTTGTCTCTCTCATTTGATTTATACTAGTAAACGTAAACTTTCATACTTAATTTAAGTATAAGTGA

31370 31380 31390 31400 31410 31420 31430 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM CTTCAGAGATGCTCGTCAAAAAACAAACTTCAGTGTGTCAAATTTGCTGATTTCTGATACTATTAGAGCA Bzura (Rx2) CTTCAGAGATACTCGTCAAAAAACAAACTTCAGTGTGTCAAATTTGCTGATTTCTGATACTATTAGAGCA Hermes(susceptible) CTTCAGTGATACTCGTCAAAAAACAAACTTCAGTGTGTCAAATTTGCTGATTTCTGATACTATTAGAGCA

31440 31450 31460 31470 31480 31490 31500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM GTGCATTTTAAATCAGTTCTTTCTATCAAATACATGGCCATTTAGAAAGTCAATGAGCAAGCAAGTAATG Bzura (Rx2) GTGCATTTTAAATCAGTTCTTTCTATCAAATACGTGGCCATTTAGAAAGTCAATGAGCAAGCAAGTAATG Hermes(susceptible) GTGCATTTTAATTCAGTTCTTTCTGTCAAATACGTGGCCATTTAGAAAGTCAATGTGCAAGCAAGTAATG

31510 31520 31530 31540 31550 31560 31570 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| Solanum tuberosum cultivar DM CTATGCTGCCTCACTAATGTGGTGAACTATGACCACTTCCTCATAGACATGAAAGTACGAGTTTTAAAAG Bzura (Rx2) CTATGCTGGCTCACTATCTTC------Hermes(susceptible) CTATGCTGGCTCACT------

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Appendix 5 Multi-alignment of PCR products of 17 potato cultivars amplified by 1Rx1 primer with the sequence of Rx1 , Rx2, Gpa2 and 34 other paralogous genes in potato-DM (Green colour regions show annealing region of primers). All the sequences are aligned with Rx1 (Acc. No. AJ011801).

11950 11960 11970 11980 11990 12000 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Asterix-amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Atlantic-amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Cara- amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Ditta-amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Irati- amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Lady Rosetta-amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Rhinered- amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Sante-amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Alcmaria -amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Amaryl -amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Mondial -amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Eridia -amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Amalia -amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA wauseon -amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Divina -amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA Multa -amplified by 1Rx1 ------GGAGAAATCCTGCAATATAATGGGCGATCATGA AJ011801_Rx_gene AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA Solanum_tuberosum_rx_gene. AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AJ011801_Rx_gene_join_(11849-1 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA Solanum acaule Rx2.ac15 AAAGCTCGAATCTTTAAGAGCTATTCTGGAGAAATCCTGCAATGTAACGGGCGATCATGA Solanum tuberosum GPA2 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AJ249448_S.acaule_Rx2.ac15 AAAGCTCGAATCTTTAAGAGCTATTCTGGAGAAATCCTGCAATGTAACGGGCGATCATGA AF195939_Gpa2 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AJ249449_GPA2 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AF266747_RGC1 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 AAAGATCGAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA EU352874_SH-RGH6_gene AAAGATCGAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC237866_RH137K16_2833_ch.12 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC237866_RH137K16_36092_ch.12 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC237866_RH137K16_46342-ch.12 AAAGCTTGAATCTTTGAGAGCTATTATGGAGAAATCCTGCAATATAACGGGAGATCATGA AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC238225_RH125I04_29334 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC238225_RH125I04_65982_ch.5 AAAGCTCGAATCTTTAAGAACTATTCTGGAGAATTCCTGCAATATAACGGGCGATCATGA AC238225_RH125I04_89012 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC238291_RH153N17_34642_ch.12 AAAGATCGAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC238291_RH153N17_96301_ch.12 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC238387_RH192P22_23420_ch.12 AAAGATCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA AC238387_RH192P22_66050_ch.12 AAAGATCGAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA PGSC0003DMB000000063:400004-40 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAACCTTGGAAAGCAACAGACGATCTCGA PGSC0003DMB000000116:773867.77 AAAGCTCGAATCTTTAAGAACTATTCTGGAGAATTCCTGCAATATAACGGGCGATCATGA PGSC0003DMB000000116:833883.85 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA PGSC0003DMT400007570-Gpa2 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAACCTTGGAAAGCAACAGACGATCTCGA PGSC0003DMT400010966-Gpa2 ------PGSC0003DMT400010970-Gpa2 AAAGCTTGAATCGTTGATAGCTATTATGGAGAAACCTTGCAGCATAATAGGCGATCTCGA PGSC0003DMT400010987-NBS-RGA ACAAAATTGCCCTTTAA----TCTTGTGGGCTTAAAC------ATGCCA---CGTGG PGSC0003DMT400020342-Gpa2 AAAGCTCGAATCTTTAAGAACTATTCTGGAGAATTCCTGCAATATAACGGGCGATCATGA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 AAAGCTCGAATCTTTGAGAGCTATTCTAGAGAAATCCTGCAATATAACGGGCGATCATGA PGSC0003DMT400020360-Gpa2 AAAGCTTGAATCTTTGAGAGCTATTATGGAGAAATCCTGCAATATAACGGGAGATCATGA PGSC0003DMT400036058-Gpa2 AAAGTTTGAATCTTTGAGAGCTATTCTGGAGAAACAC------ATGGGAGATCTTGA PGSC0003DMT400036104-NBS-LRR ATCGATGGAAGTTACTTGATATAATTTTCAATCGTTCTATAAGAAGCTTGAATCCCTGAG PGSC0003DMT400062366-Gpa2 AAAGCTTGAATCCTTGAGAGCTATTATGGAAAAATCTTGCACGATAACGGGTGATCTTGA PGSC0003DMT400071638-PSH-RGH7 AAAGCTTGAATCTTTGAGAGCTAATCTGGAGAAACC------GATAGGCGATCTTGA PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 AAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCCTGCAATATAATGGGCGATCATGA

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12010 12020 12030 12040 12050 12060 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Asterix-amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Atlantic-amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Cara- amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Ditta-amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Irati- amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Lady Rosetta-amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Rhinered- amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Sante-amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Alcmaria -amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Amaryl -amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Mondial -amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Eridia -amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Amalia -amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT wauseon -amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Divina -amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Multa -amplified by 1Rx1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT AJ011801_Rx_gene GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Solanum_tuberosum_rx_gene. GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT AJ011801_Rx_gene_join_(11849-1 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT Solanum acaule Rx2.ac15 GGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTAGCATACACAACAGAAGATATGGT Solanum tuberosum GPA2 GGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTAGCATACACAACAGAAGATATGGT AJ249448_S.acaule_Rx2.ac15 GGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTAGCATACACAACAGAAGATATGGT AF195939_Gpa2 GGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTAGCATACACAACAGAAGATATGGT AJ249449_GPA2 GGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTAGCATACACAACAGAAGATATGGT AF266747_RGC1 GGGGTTAACAATCTTAAAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 GGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTAGCATACACAACAGAAGGTATGGT EU352874_SH-RGH6_gene GGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTAGCATACACAACAGAAGATATGGT AC237866_RH137K16_2833_ch.12 GGAGTTAACAATCTTGGAAGTTGAAATTGTAGAGGTAGCATACACAGCAGAAGATATGGT AC237866_RH137K16_36092_ch.12 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT AC237866_RH137K16_46342-ch.12 GGGGTTAACAACCTTGGAAGTTGAAATCGCGGAGGTAGCATACAAAGCAGAAGACATGGT AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT AC238225_RH125I04_29334 GGAGTTAACAATCTTGGAAGTTGAAATTGTAGAGGTAGCATACACAGCAGAAGATATGGT AC238225_RH125I04_65982_ch.5 GGAGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT AC238225_RH125I04_89012 GGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTAGCATACACAACAGAAGATATGGT AC238291_RH153N17_34642_ch.12 GGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTAGCATACACAACAGAAGATATGGT AC238291_RH153N17_96301_ch.12 GGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTAGCATACACAACAGAAGATATGGT AC238387_RH192P22_23420_ch.12 GGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT AC238387_RH192P22_66050_ch.12 GGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTAGCATACACAACAGAAGATATGGT PGSC0003DMB000000063:400004-40 GGCATTAACAAACTTGGAAGCTGAAATCTCAGAGGTAGCATACAGTGCAGGGGATATGGT PGSC0003DMB000000116:773867.77 GGAGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT PGSC0003DMB000000116:833883.85 GGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTAGCATACACAACAGAAGATATGGT PGSC0003DMT400007570-Gpa2 GGCATTAACAAACTTGGAAGCTGAAATCTCAGAGGTAGCATACAGTGCAGGGGATATGGT PGSC0003DMT400010966-Gpa2 ------PGSC0003DMT400010970-Gpa2 GGCATTGGCAAGCTTGGAAGTAAAAATCGCGGGGGTTGCATACAGAGCAGAAGATGAGAT PGSC0003DMT400010987-NBS-RGA AAAGTTAAAA-----GTAAAATGTTACCAAAAAAGGAA-AGGGGCCATTCTTTTTGAAAC PGSC0003DMT400020342-Gpa2 GGAGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTAGCATACACAACAGAAGATATGGT PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 GGGGTTAACAACCTTGGAAGTTGAAATCACGGAGGTAGCATTCAAAGCAGAAGACATGGT PGSC0003DMT400020360-Gpa2 GGGGTTAACAACCTTGGAAGTTGAAATCGCGGAGGTAGCATACAAAGCAGAAGACATGGT PGSC0003DMT400036058-Gpa2 TGCATTGAAAAGCTTGGAAGCTGAAATCATAGAACTTGTATGCACTACAGAAGATATTTT PGSC0003DMT400036104-NBS-LRR AGCTATTATGAGAAACCTTGCAATATAACAGGTGATCTTGAAGTATTGAAAAGATTGGAA PGSC0003DMT400062366-Gpa2 GGCATTAACAAGCTTGGAAGCTGAAATTGCAGCGGTAGCCTACAGCACAGAAGATATGAT PGSC0003DMT400071638-PSH-RGH7 GGCATTGATAAGCTTGGAAGCTGAAATCATAGAGGTTGTATGCACCACAGAACATTTTTT PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 GGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTAGCATACACAACAGAAGATATGGT

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12070 12080 12090 12100 12110 12120 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Asterix-amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Atlantic-amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Cara- amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Ditta-amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Irati- amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Lady Rosetta-amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Rhinered- amplified by 1Rx1 TGACTCGGAATCAAGAAATGCTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Sante-amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Alcmaria -amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Amaryl -amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Mondial -amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Eridia -amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTCTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Amalia -amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA wauseon -amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Divina -amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAA-GGCTA Multa -amplified by 1Rx1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-AGCTA AJ011801_Rx_gene TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Solanum_tuberosum_rx_gene. TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AJ011801_Rx_gene_join_(11849-1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA Solanum acaule Rx2.ac15 TGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAATTTGGAGGAAAGAAACAG-GGCTA Solanum tuberosum GPA2 TGACTCGGAATCAAGAAATGTTTTTTTAGCACGGAATGTGGGGAAAAGAAGCAG-GGCTA AJ249448_S.acaule_Rx2.ac15 TGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAATTTGGAGGAAAGAAACAG-GGCTA AF195939_Gpa2 TGACTCGGAATCAAGAAATGTTTTTTTAGCACGGAATGTGGGGAAAAGAAGCAG-GGCTA AJ249449_GPA2 TGACTCGGAATCAAGAAATGTTTTTTTAGCACGGAATGTGGGGAAAAGAAGCAG-GGCTA AF266747_RGC1 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAAAATTTGGAGGAAAGAAGCAG-GGCTA AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA EU352874_SH-RGH6_gene TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC237866_RH137K16_2833_ch.12 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC237866_RH137K16_36092_ch.12 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC237866_RH137K16_46342-ch.12 TGATTCGAAATCAAGAAAAGTTTCTTCCGCAGAAACTGTAATTACACGAAGCAA-AGCTT AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC238225_RH125I04_29334 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC238225_RH125I04_65982_ch.5 TGACTTGGAATCAAGAAGTGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC238225_RH125I04_89012 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC238291_RH153N17_34642_ch.12 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC238291_RH153N17_96301_ch.12 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA AC238387_RH192P22_23420_ch.12 TGACTCGGAATCAAGAAATGTTGTTTTGGCACAAAATTTGGAGGAAAGAAGCAG-GGCTA AC238387_RH192P22_66050_ch.12 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA PGSC0003DMB000000063:400004-40 TGACTTGAAATCAAGAAATGTTCTTTTTGCACAAAAGGCAAAGA-ATAAAGCAAGAGCTT PGSC0003DMB000000116:773867.77 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA PGSC0003DMB000000116:833883.85 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA PGSC0003DMT400007570-Gpa2 TGACTTGAAATCAAGAAATGTTCTTTTTGCACAAAAGGCAAAGA-ATAAAGCAAGAGCTT PGSC0003DMT400010966-Gpa2 ------PGSC0003DMT400010970-Gpa2 TGACTCTAAATCAATAGAAGTTATACGCGCAAAAACAGATACTTTGCGAGGGAA-AGCTT PGSC0003DMT400010987-NBS-RGA AGACTAAAAAGGAAAGGAGGTC------ATTCTTTTTGAAACG-GAGGGGTAGCTT PGSC0003DMT400020342-Gpa2 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 TGATTCGAAATCAAGAAAAGTTTCTTCTGCAGAAACTGTAATTACACGAAGCAA-AGCTT PGSC0003DMT400020360-Gpa2 TGATTCGAAATCAAGAAAAGTTTCTTCCGCAGAAACTGTAATTACACGAAGCAA-AGCTT PGSC0003DMT400036058-Gpa2 GGACTTGGAATCAAGAAATGTT------AAAAATCCAATTTCAAGAATAATAGCTT PGSC0003DMT400036104-NBS-LRR GCTGAAGTCGTAGAGCTTGTATGCAGCACAGAAGATATTGTGGATTTGGAATCA-AGAAA PGSC0003DMT400062366-Gpa2 TGAATCGGAATCAAGAAAAGTTTCCTTAGTAAAAACCTCGGTTACACGAAGAAT-AGATT PGSC0003DMT400071638-PSH-RGH7 GGACTCGGAATCAAGAAATGTTA------AAAATCCAAT--TTCACAAATAAT-AGCTT PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 TGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAATTTGGAGGAAAGAAGCAG-GGCTA

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12130 12140 12150 12160 12170 12180 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Asterix-amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Atlantic-amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Cara- amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Ditta-amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Irati- amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Lady Rosetta-amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Rhinered- amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Sante-amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Alcmaria -amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Amaryl -amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Mondial -amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Eridia -amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Amalia -amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC wauseon -amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Divina -amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCGGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Multa -amplified by 1Rx1 TGTGGGAGATTTTTTTCGTCCGGGAACAAGCACTAGAACGCATTGATTCCACCGTGAAAC AJ011801_Rx_gene TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Solanum_tuberosum_rx_gene. TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AJ011801_Rx_gene_join_(11849-1 TGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Solanum acaule Rx2.ac15 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC Solanum tuberosum GPA2 TGTGGGGGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AJ249448_S.acaule_Rx2.ac15 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AF195939_Gpa2 TGTGGGGGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AJ249449_GPA2 TGTGGGGGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AF266747_RGC1 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC EU352874_SH-RGH6_gene TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AC237866_RH137K16_2833_ch.12 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGCTTCCACCGTGAAAC AC237866_RH137K16_36092_ch.12 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATACATTGATTCCACCGTGAAAC AC237866_RH137K16_46342-ch.12 TCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGAATGCATTGATTCCTTCATGATGC AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATACATTGATTCCACCGTGAAAC AC238225_RH125I04_29334 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGCTTCCACCGTGAAAC AC238225_RH125I04_65982_ch.5 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AC238225_RH125I04_89012 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATACATTGATTCCACCGTGAAAC AC238291_RH153N17_34642_ch.12 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AC238291_RH153N17_96301_ch.12 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATACATTGATTCTACCGTGAAAC AC238387_RH192P22_23420_ch.12 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC AC238387_RH192P22_66050_ch.12 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC PGSC0003DMB000000063:400004-40 TCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGAACGCATTGATTCCCTCATGATGC PGSC0003DMB000000116:773867.77 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC PGSC0003DMB000000116:833883.85 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGCTTCCACCGTGAAAC PGSC0003DMT400007570-Gpa2 TCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGAACGCATTGATTCCCTCATGATGC PGSC0003DMT400010966-Gpa2 ------ATGATCA PGSC0003DMT400010970-Gpa2 TTTGGAAACTGTGTTGTTTCTTGGAACAAGTGGTAGAACACATTGATTCCATCATGAAGG PGSC0003DMT400010987-NBS-RGA TTTGGAAACTTCATTCTCTCTTGAAACAAGCAGTAGGACGCGTTGATTCCATGCTCAATG PGSC0003DMT400020342-Gpa2 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGATTCCACCGTGAAAC PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 TCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGAATGTATTGATTCCTTCATGATGC PGSC0003DMT400020360-Gpa2 TCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGAATGCATTGATTCCTTCATGATGC PGSC0003DMT400036058-Gpa2 TTTGGAAACTTCATTCTCTCTTGAAACAAGCAGTAGGACGCATTGATTCCACAATGAACA PGSC0003DMT400036104-NBS-LRR TGTTGAAAATCCAATTTCAAGAATTAATAACTTTTTGGAAACTTCATTTTCTCTTGAAAC PGSC0003DMT400062366-Gpa2 TTTGGGAACTTTGTTTCTCCCTGAAATAAGCAGTAGAACACATTGGTTGCACAATGAACA PGSC0003DMT400071638-PSH-RGH7 CTTGGAAATTTCATCATCTCTTGGAACATGCCGTTGGAGACATTGATTCCAGAGTCAACA PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 TGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGAATGCATTGCTTCCACCGTGAAAC

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12190 12200 12210 12220 12230 12240 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 AGTGGATGGCAAC------AT Asterix-amplified by 1Rx1 AGTGGATGGCAAC------AT Atlantic-amplified by 1Rx1 AGTGGATGGCAAC------AT Cara- amplified by 1Rx1 AGTGGATGGCAAC------AT Ditta-amplified by 1Rx1 AGTGGATGGCAAC------AT Irati- amplified by 1Rx1 AGTGGATGGCAAC------AT Lady Rosetta-amplified by 1Rx1 AGTGGATGGCAAC------AT Rhinered- amplified by 1Rx1 AGTGGATGGCAAC------AT Sante-amplified by 1Rx1 AGTGGATGGCAAC------AT Alcmaria -amplified by 1Rx1 AGTGGATGGCAAC------AT Amaryl -amplified by 1Rx1 AGTGGATGGCAAC------AT Mondial -amplified by 1Rx1 AGTGGATGGCAAC------AT Eridia -amplified by 1Rx1 AGTGGATGGCAAC------AT Amalia -amplified by 1Rx1 AGTGGATGGCAAC------AT wauseon -amplified by 1Rx1 AGTGGATGGCAAC------AT Divina -amplified by 1Rx1 AGTGGATGGCAAC------AT Multa -amplified by 1Rx1 AGTGGATGGCAAC------AT AJ011801_Rx_gene AGTGGATGGCAAC------AT Solanum_tuberosum_rx_gene. AGTGGATGGCAAC------AT AJ011801_Rx_gene_join_(11849-1 AGTGGATGGCAAC------AT Solanum acaule Rx2.ac15 AGTGGATGGCAAC------AT Solanum tuberosum GPA2 AGTGGATGGCAAC------AT AJ249448_S.acaule_Rx2.ac15 AGTGGATGGCAAC------AT AF195939_Gpa2 AGTGGATGGCAAC------AT AJ249449_GPA2 AGTGGATGGCAAC------AT AF266747_RGC1 AGTGGATGGCAGC------AT AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 AGTGGATGGCAGC------AT EU352874_SH-RGH6_gene AGTGGATGGCAAC------AT AC237866_RH137K16_2833_ch.12 AGTGGATGGCAGC------AT AC237866_RH137K16_36092_ch.12 AGTGGATGGCAGC------AT AC237866_RH137K16_46342-ch.12 AGTGGTTGGCAAT------AT AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 AGTGGATGGCAGC------AT AC238225_RH125I04_29334 AGTGGATGGCAGC------AT AC238225_RH125I04_65982_ch.5 AGTGGATGGCAGC------AT AC238225_RH125I04_89012 AGTGGATGGCAAC------AT AC238291_RH153N17_34642_ch.12 AGTGGATGGCAAC------AT AC238291_RH153N17_96301_ch.12 AGTGGATGGCAGC------AT AC238387_RH192P22_23420_ch.12 AGTGGATGGCAAC------AT AC238387_RH192P22_66050_ch.12 AGTGGATGGCAGC------AT PGSC0003DMB000000063:400004-40 AGTGGTTGGCAAT------AT PGSC0003DMB000000116:773867.77 AGTGGATGGCAGC------AT PGSC0003DMB000000116:833883.85 AGTGGATGGCAGC------AT PGSC0003DMT400007570-Gpa2 AGTGGTTGGCAAT------AT PGSC0003DMT400010966-Gpa2 TTGCTGAGAAGAA------CA PGSC0003DMT400010970-Gpa2 AGTGGATGGCAATCCGGGACGGGTGCAGCAACATCAAAGAGTATATGATTGTCTCAGAAT PGSC0003DMT400010987-NBS-RGA AGTGGATGGAAAT------AC PGSC0003DMT400020342-Gpa2 AGTGGATGGCAGC------AT PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 AGTGGTTGGCAAT------AT PGSC0003DMT400020360-Gpa2 AGTGGTTGGCAAT------AT PGSC0003DMT400036058-Gpa2 AGTGGATGGAAAT------GC PGSC0003DMT400036104-NBS-LRR AAGTAGTAGGGCG------CG PGSC0003DMT400062366-Gpa2 AGTGGAAGGAAAT------GC PGSC0003DMT400071638-PSH-RGH7 AGTGGATGAAAAT------PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 AGTGGATGGCAGC------AT

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12250 12260 12270 12280 12290 12300 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 CGGACAG------Asterix-amplified by 1Rx1 CGGACAG------Atlantic-amplified by 1Rx1 CGGACAG------Cara- amplified by 1Rx1 CGGACAG------Ditta-amplified by 1Rx1 CGGACAG------Irati- amplified by 1Rx1 CGGACAG------Lady Rosetta-amplified by 1Rx1 CGGACAG------Rhinered- amplified by 1Rx1 CGGACAG------Sante-amplified by 1Rx1 CGGACAG------Alcmaria -amplified by 1Rx1 CGGACAG------Amaryl -amplified by 1Rx1 CGGACAG------Mondial -amplified by 1Rx1 CGGACAG------Eridia -amplified by 1Rx1 CGGACAG------Amalia -amplified by 1Rx1 CGGACAG------wauseon -amplified by 1Rx1 CGGACAG------Divina -amplified by 1Rx1 CGGACAG------Multa -amplified by 1Rx1 CGGACAG------AJ011801_Rx_gene CGGACAG------Solanum_tuberosum_rx_gene. CGGACAG------AJ011801_Rx_gene_join_(11849-1 CGGACAG------Solanum acaule Rx2.ac15 CGGACAG------Solanum tuberosum GPA2 CGGACAG------AJ249448_S.acaule_Rx2.ac15 CGGACAG------AF195939_Gpa2 CGGACAG------AJ249449_GPA2 CGGACAG------AF266747_RGC1 CGGACAG------AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 CGGACAG------EU352874_SH-RGH6_gene CGGACAG------AC237866_RH137K16_2833_ch.12 CGGACAG------AC237866_RH137K16_36092_ch.12 CGGACAG------AC237866_RH137K16_46342-ch.12 GGGACTG------AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 CGGACAG------AC238225_RH125I04_29334 CGGACAG------AC238225_RH125I04_65982_ch.5 CGGACAG------AC238225_RH125I04_89012 CGGACAG------AC238291_RH153N17_34642_ch.12 CGGACAG------AC238291_RH153N17_96301_ch.12 CGGACAG------AC238387_RH192P22_23420_ch.12 CGGACAG------AC238387_RH192P22_66050_ch.12 CGGACAG------PGSC0003DMB000000063:400004-40 GGAACTGGTACA------PGSC0003DMB000000116:773867.77 CGGACAG------PGSC0003DMB000000116:833883.85 CGGACAG------PGSC0003DMT400007570-Gpa2 GGAACTGGTACA------PGSC0003DMT400010966-Gpa2 TGTACATGACAA------PGSC0003DMT400010970-Gpa2 CGAGCAAAAGTTTTTTGGCAAAATCTAAAATGAGACAAGGACTAGCGTTTTGGAAACGAG PGSC0003DMT400010987-NBS-RGA ACAACATGTACA------PGSC0003DMT400020342-Gpa2 CGGACAG------PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 GGGACTGGTACA------PGSC0003DMT400020360-Gpa2 GGGACTGGTACA------PGSC0003DMT400036058-Gpa2 AGAACATGTACA------PGSC0003DMT400036104-NBS-LRR TTGATTCCATGG------PGSC0003DMT400062366-Gpa2 AGAACAAGTACA------PGSC0003DMT400071638-PSH-RGH7 ---GCAAAAATT------PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 CGGACAG------

136

12310 12320 12330 12340 12350 12360 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 ------Asterix-amplified by 1Rx1 ------Atlantic-amplified by 1Rx1 ------Cara- amplified by 1Rx1 ------Ditta-amplified by 1Rx1 ------Irati- amplified by 1Rx1 ------Lady Rosetta-amplified by 1Rx1 ------Rhinered- amplified by 1Rx1 ------Sante-amplified by 1Rx1 ------Alcmaria -amplified by 1Rx1 ------Amaryl -amplified by 1Rx1 ------Mondial -amplified by 1Rx1 ------Eridia -amplified by 1Rx1 ------Amalia -amplified by 1Rx1 ------wauseon -amplified by 1Rx1 ------Divina -amplified by 1Rx1 ------Multa -amplified by 1Rx1 ------AJ011801_Rx_gene ------Solanum_tuberosum_rx_gene. ------AJ011801_Rx_gene_join_(11849-1 ------Solanum acaule Rx2.ac15 ------Solanum tuberosum GPA2 ------AJ249448_S.acaule_Rx2.ac15 ------AF195939_Gpa2 ------AJ249449_GPA2 ------AF266747_RGC1 ------AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 ------EU352874_SH-RGH6_gene ------AC237866_RH137K16_2833_ch.12 ------AC237866_RH137K16_36092_ch.12 ------AC237866_RH137K16_46342-ch.12 ------AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 ------AC238225_RH125I04_29334 ------AC238225_RH125I04_65982_ch.5 ------AC238225_RH125I04_89012 ------AC238291_RH153N17_34642_ch.12 ------AC238291_RH153N17_96301_ch.12 ------AC238387_RH192P22_23420_ch.12 ------AC238387_RH192P22_66050_ch.12 ------PGSC0003DMB000000063:400004-40 ------G------TA PGSC0003DMB000000116:773867.77 ------PGSC0003DMB000000116:833883.85 ------PGSC0003DMT400007570-Gpa2 ------G------TA PGSC0003DMT400010966-Gpa2 ------A------AA PGSC0003DMT400010970-Gpa2 CAGTAGGACACATTGATTCTATGATAAACAACAGGATGATGAAGCAGAGCATGTACACCA PGSC0003DMT400010987-NBS-RGA ------CCA PGSC0003DMT400020342-Gpa2 ------PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 ------A------CA PGSC0003DMT400020360-Gpa2 ------A------CA PGSC0003DMT400036058-Gpa2 ------CCA PGSC0003DMT400036104-NBS-LRR ------T------GA PGSC0003DMT400062366-Gpa2 ------A------CA PGSC0003DMT400071638-PSH-RGH7 ------GTACACCA PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 ------

137

12370 12380 12390 12400 12410 12420 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Asterix-amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Atlantic-amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Cara- amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Ditta-amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Irati- amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Lady Rosetta-amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Rhinered- amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Sante-amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Alcmaria -amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Amaryl -amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Mondial -amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Eridia -amplified by 1Rx1 -CACGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Amalia -amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG wauseon -amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Divina -amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Multa -amplified by 1Rx1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG AJ011801_Rx_gene -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Solanum_tuberosum_rx_gene. -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG AJ011801_Rx_gene_join_(11849-1 -CATGAAAGATCTAAAACCACAAACTAGCTCGCTTGTC---AGTTTACCTGAA---CATG Solanum acaule Rx2.ac15 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAC---CATG Solanum tuberosum GPA2 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AJ249448_S.acaule_Rx2.ac15 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAC---CATG AF195939_Gpa2 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AJ249449_GPA2 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AF266747_RGC1 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 -CATGAAAGATCTAAAACAACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG EU352874_SH-RGH6_gene -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC237866_RH137K16_2833_ch.12 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC237866_RH137K16_36092_ch.12 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC237866_RH137K16_46342-ch.12 -CTTTAAAGATTTCAAAGCACAAAATTTTTGTCTATCC---AAGATACCTGAA---CGTG AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC238225_RH125I04_29334 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC238225_RH125I04_65982_ch.5 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC238225_RH125I04_89012 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC238291_RH153N17_34642_ch.12 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC238291_RH153N17_96301_ch.12 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC238387_RH192P22_23420_ch.12 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG AC238387_RH192P22_66050_ch.12 -CATGAAAGATCTAAAACAACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG PGSC0003DMB000000063:400004-40 AAATCAAAGATTTGAAAGCACGAAAGTTTTCTCTAGCC------GAA---CATG PGSC0003DMB000000116:773867.77 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG PGSC0003DMB000000116:833883.85 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG PGSC0003DMT400007570-Gpa2 AAATCAAAGATTTGAAAGCACGAAAGTTTTCTCTAGCCCCTAGTATACCTGAA---CATG PGSC0003DMT400010966-Gpa2 ACAAAGATAATATTATTGCCAATACATCGTCATCTCAA------CATGCCTTA---TTAG PGSC0003DMT400010970-Gpa2 AAAGCAAAGATGTAGAAGGACAGAATTTGACTCTTGCC---AGTACATCTCGA---CATT PGSC0003DMT400010987-NBS-RGA AAAGCAAAGATGAAGAAGCACAT------CTTGCTAGTACTACATCGATATCTCAAC PGSC0003DMT400020342-Gpa2 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 ACATTAAAGATTTCAAAGCACAAAATTTCTGTCTATCC---AAGATACCTGAA---CTTG PGSC0003DMT400020360-Gpa2 ACTTTAAAGATTTCAAAGCACAAAATTTTTGTCTATCC---AAGATACCTGAA---CGTG PGSC0003DMT400036058-Gpa2 AAAGGAAAGATGAAGAAGCACATAACTTGGATCTTGCTAGTACTACATCGATGTCTCAAC PGSC0003DMT400036104-NBS-LRR ACAAGTGGAAAGATGTAGAAGCACATAACTTGGCTTTC------ACCAGTACT---ACAT PGSC0003DMT400062366-Gpa2 ATATCAAAGACCTCAAAGTACAAAGCATGTCTCTTGGC------GATGTATCT---CGAC PGSC0003DMT400071638-PSH-RGH7 GAACCAAAGATGTACAAGGGAATAACTCGGCTCTCGCC---AGTACATCTCAA---CATG PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 -CATGAAAGATCTAAAACCACAAACTAGCTCACTTGTC---AGTTTACCTGAA---CATG

138

12430 12440 12450 12460 12470 12480 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Asterix-amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Atlantic-amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTAAAAATGAATTTGAGATGAT-- Cara- amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Ditta-amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Irati- amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Lady Rosetta-amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Rhinered- amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Sante-amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Alcmaria -amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Amaryl -amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Mondial -amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Eridia -amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Amalia -amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- wauseon -amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Divina -amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Multa -amplified by 1Rx1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AJ011801_Rx_gene ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Solanum_tuberosum_rx_gene. ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AJ011801_Rx_gene_join_(11849-1 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Solanum acaule Rx2.ac15 CTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- Solanum tuberosum GPA2 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AJ249448_S.acaule_Rx2.ac15 CTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AF195939_Gpa2 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AJ249449_GPA2 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AF266747_RGC1 ATGTTGAGCAGCCCGATAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- EU352874_SH-RGH6_gene CTTTTGAGCAGCCCGAGAAT---ATAATGGTTGGCTATGAAAATGAATTTGAGATGAT-- AC237866_RH137K16_2833_ch.12 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AC237866_RH137K16_36092_ch.12 CTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AC237866_RH137K16_46342-ch.12 CTGTTGAGCGGTCCGAGGAT---ATAATGGTTGGCTATGAAAATGAATTCAAGATGAT-- AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 CTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AC238225_RH125I04_29334 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AC238225_RH125I04_65982_ch.5 CTGTTGAACAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AC238225_RH125I04_89012 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AC238291_RH153N17_34642_ch.12 CTTTTGAGCAGCCCGAGAAT---ATAATGGTTGGCTATGAAAATGAATTTGAGATGAT-- AC238291_RH153N17_96301_ch.12 CTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AC238387_RH192P22_23420_ch.12 CTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- AC238387_RH192P22_66050_ch.12 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- PGSC0003DMB000000063:400004-40 CTGTAGGGAAGCCTGAGAAT---ATAATGGTTGGCCATGAAAATGAATTCGAGATGAT-- PGSC0003DMB000000116:773867.77 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- PGSC0003DMB000000116:833883.85 CTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- PGSC0003DMT400007570-Gpa2 CTGTAGGGAAGCCTGAGAAT---ATAATGGTTGGCCATGAAAATGAATTCGAGATGAT-- PGSC0003DMT400010966-Gpa2 AGCCTGATGAGAACATGAAT------ATGGTTGGTGATGAAAATGAATTCGAGATGAT-- PGSC0003DMT400010970-Gpa2 CCTTGGAGCATGAGAATA------TGATGGTTGGCCATGAAAATGAATTCGAGATGAT-- PGSC0003DMT400010987-NBS-RGA ATGTTGTGGAGCCTCAAGAT---ATGATGGTTGGACATGAAAATGAACTCGAGATGATCA PGSC0003DMT400020342-Gpa2 ATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT-- PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 CTGTTGAGCGGTCCGAGGTT---ATAATGGTTGGCTATGAAAATGAATTCAAGATGAT-- PGSC0003DMT400020360-Gpa2 CTGTTGAGCGGTCCGAGGAT---ATAATGGTTGGCTATGAAAATGAATTCAAGATGAT-- PGSC0003DMT400036058-Gpa2 ATGTTGTGGAGCCTCAGGAT---ATGATGGTTGGACATGAAAATGAATTCGAGATAATCA PGSC0003DMT400036104-NBS-LRR CTCAACATGTTGTGGAGCCG------ATTGTTGGCCATAAAAATGAATTCGAGATGAT-- PGSC0003DMT400062366-Gpa2 TTGCTGTAGAGACTGAGAAT---GTGATGGTTGGCCATGAAAATGAATTTGAGATGAT-- PGSC0003DMT400071638-PSH-RGH7 TTGTGGAGCCTGAGGATAATTATATGATGGTTGGTCATGAAAATGAACTCGAGATGAT-- PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 CTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCGTGAAAATGAATTTGAGATGAT--

139

12490 12500 12510 12520 12530 12540 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Asterix-amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Atlantic-amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Cara- amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Ditta-amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Irati- amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Lady Rosetta-amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Rhinered- amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Sante-amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Alcmaria -amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Amaryl -amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Mondial -amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Eridia -amplified by 1Rx1 -GCTGGATCTACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Amalia -amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG wauseon -amplified by 1Rx1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Divina -amplified by 1Rx1 -GCTGGACCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCGATCGTAGGGATGG Multa -amplified by 1Rx1 -GCTGGACCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AJ011801_Rx_gene -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Solanum_tuberosum_rx_gene. -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AJ011801_Rx_gene_join_(11849-1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Solanum acaule Rx2.ac15 -GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG Solanum tuberosum GPA2 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AJ249448_S.acaule_Rx2.ac15 -GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AF195939_Gpa2 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AJ249449_GPA2 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AF266747_RGC1 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 -GCTGGATCAACTTGCAAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG EU352874_SH-RGH6_gene -GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC237866_RH137K16_2833_ch.12 -GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC237866_RH137K16_36092_ch.12 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC237866_RH137K16_46342-ch.12 -GCTGGATCAACTTGCTAGAGGAGAAAGAGAACTAGCAGTTGTATCAATTGTAGGTATGG AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC238225_RH125I04_29334 -GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC238225_RH125I04_65982_ch.5 -GCTGGATCAACTTGTTAGAGGAGGAAGGGAGCTAGAAGTTGTCTCAATCGTAGGGATGG AC238225_RH125I04_89012 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC238291_RH153N17_34642_ch.12 -GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC238291_RH153N17_96301_ch.12 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC238387_RH192P22_23420_ch.12 -GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG AC238387_RH192P22_66050_ch.12 -GCTGGATCAACTTGCAAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG PGSC0003DMB000000063:400004-40 -GCTCAATCAAGTTGCTGGAGGAGAAAGGGAACTAGAAGTTGTCTCAATTGTAGGTATGG PGSC0003DMB000000116:773867.77 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG PGSC0003DMB000000116:833883.85 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG PGSC0003DMT400007570-Gpa2 -GCTCAATCAAGTTGCTGGAGGAGAAAGGGAACTAGAAGTTGTCTCAATTGTAGGTATGG PGSC0003DMT400010966-Gpa2 -GCAGGGGAGACTTACAAGAGGAGGAAGGGAACTAAAAGTTGTCTCGATTGTCGGTATGG PGSC0003DMT400010970-Gpa2 -GCAGGACCAACTTACTAGAGGTGCCAGTGATCTAGAAATTGTCTCAATCGTTGGGATGG PGSC0003DMT400010987-NBS-RGA TGCAGGATCAGCTTGCTAGAGGAGCAAGTGAACTTGAAGTTGTCTCCATTGTAGGTATTG PGSC0003DMT400020342-Gpa2 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 -GCTGGATCAACTTGCTAGAGGAGAAAGAGAACTAGAAGTTGTATCAATTGTAGGTATGG PGSC0003DMT400020360-Gpa2 -GCTGGATCAACTTGCTAGAGGAGAAAGAGAACTAGCAGTTGTATCAATTGTAGGTATGG PGSC0003DMT400036058-Gpa2 TGGAGGATCAGCTTGCTAGAGGAGCAAGTGAACTTGAAGTTGTCTCCATTGTAGGTATGG PGSC0003DMT400036104-NBS-LRR -GCAGGATCAACTTGTCAGAGGAGCAAGTGAACTAGAAGTTGTCTCAATTGTAGGTATGG PGSC0003DMT400062366-Gpa2 -GCAGGATCAAATTGCTAGAGGATCAAATGAACTAGAAGTTGTCTCAATTGTCGGGATGG PGSC0003DMT400071638-PSH-RGH7 -GCAGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATTGT------PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 -GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGAAGTTGTCTCAATCGTAGGGATGG

140

12550 12560 12570 12580 12590 12600 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Asterix-amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Atlantic-amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Cara- amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Ditta-amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Irati- amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Lady Rosetta-amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Rhinered- amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTGTAGTGATCCGTGCATTATGTCTC Sante-amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Alcmaria -amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Amaryl -amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Mondial -amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Eridia -amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Amalia -amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC wauseon -amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Divina -amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Multa -amplified by 1Rx1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC AJ011801_Rx_gene GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Solanum_tuberosum_rx_gene. GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC AJ011801_Rx_gene_join_(11849-1 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCGTGCATTATGTCTC Solanum acaule Rx2.ac15 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC Solanum tuberosum GPA2 GAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTATAGTGATCCTTACATTATGTCTC AJ249448_S.acaule_Rx2.ac15 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC AF195939_Gpa2 GAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTATAGTGATCCTTACATTATGTCTC AJ249449_GPA2 GAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTATAGTGATCCTTACATTATGTCTC AF266747_RGC1 GAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTATAGTGATCCTTACATTATGTCTC AF266746_RGC3_pseudogene ------AAAACAACTTTGGCTACAAAAC EU352875_SH-RGH7 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTATATAGTGATCCTTACATTATGTCTC EU352874_SH-RGH6_gene GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTGCATTATGCCTC AC237866_RH137K16_2833_ch.12 GAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTATAGTGATCCTTACATTATGTCTC AC237866_RH137K16_36092_ch.12 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC AC237866_RH137K16_46342-ch.12 GAGGCATTGGCAAGACAACTTTGGCTACGAAACTCTACAACGATCCATGCATGATGTATC AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC AC238225_RH125I04_29334 GAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTATAGTGATCCTTACATTATGTCTC AC238225_RH125I04_65982_ch.5 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC AC238225_RH125I04_89012 GAGGCATTGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC AC238291_RH153N17_34642_ch.12 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTGCATTATGCCTC AC238291_RH153N17_96301_ch.12 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGAACCTTACATTATGTCTC AC238387_RH192P22_23420_ch.12 GAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTGCATTATGTCTC AC238387_RH192P22_66050_ch.12 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTATATAGTGATCCTTACATTATGTCTC PGSC0003DMB000000063:400004-40 GAGGCATTGGCAAAACAACTTTGGCTACAAAACTCTATAGAGATCCACGCATTATGTCTC PGSC0003DMB000000116:773867.77 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC PGSC0003DMB000000116:833883.85 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC PGSC0003DMT400007570-Gpa2 GAGGCATTGGCAAAACAACTTTGGCTACAAAACTCTATAGAGATCCACGCATTATGTCTC PGSC0003DMT400010966-Gpa2 GTGGCATTGGCAAGACAACTTTGGCTAACAAAATCTATAGTGATCCATTCATTATGTCTC PGSC0003DMT400010970-Gpa2 GGGGCTTAGGCAAGACAACTTTGGCTAACAAAATTTTCTGTGACCCATTCGTTATGTCTT PGSC0003DMT400010987-NBS-RGA GGGGCATCGGTAAGACAACTTTGGCTGACAAAATTTATAATGATCCATTCATAATGTCAC PGSC0003DMT400020342-Gpa2 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 GAGGCATTGGCAAGACAACTTTGGCAACAAAACTCTACAACGATCCATGCATGATGTATC PGSC0003DMT400020360-Gpa2 GAGGCATTGGCAAGACAACTTTGGCTACGAAACTCTACAACGATCCATGCATGATGTATC PGSC0003DMT400036058-Gpa2 GGGGCATCGGTAAGACAACTTTGGCTGACAAAATTTATAATGATCCATTCATAATGTCAC PGSC0003DMT400036104-NBS-LRR GTGGCATCGGTAAAACAACTTTGGCTAACAAAATTTACAATGATTCATTCATTATGTCTC PGSC0003DMT400062366-Gpa2 GAGGCATTGGCAAGACAACTTTGGCTAACAAAGTTTACAGTGATCCATTCATTATGTCTC PGSC0003DMT400071638-PSH-RGH7 -AGGCATCGGTAAGACAACTTTGGCGAACAAAATCTACAATGATCCATTCATAATGTCAC PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 GAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTATAGTGATCCTTACATTATGTCTC

141

12610 12620 12630 12640 12650 12660 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Asterix-amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Atlantic-amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Cara- amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Ditta-amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Irati- amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Lady Rosetta-amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Rhinered- amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Sante-amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Alcmaria -amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Amaryl -amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Mondial -amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Eridia -amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Amalia -amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC wauseon -amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Divina -amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTC- Multa -amplified by 1Rx1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTC- AJ011801_Rx_gene GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Solanum_tuberosum_rx_gene. GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC AJ011801_Rx_gene_join_(11849-1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Solanum acaule Rx2.ac15 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC Solanum tuberosum GPA2 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC AJ249448_S.acaule_Rx2.ac15 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC AF195939_Gpa2 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC AJ249449_GPA2 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC AF266747_RGC1 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTATTCC AF266746_RGC3_pseudogene TCTATACTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCAC EU352875_SH-RGH7 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC EU352874_SH-RGH6_gene GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTAC AC237866_RH137K16_2833_ch.12 GATTTGATATTCGTGCAAAAGTAACTGTTTCGCAGGAGTATTGTGTGAGAAATGTAATCC AC237866_RH137K16_36092_ch.12 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTAATCC AC237866_RH137K16_46342-ch.12 GTTTTGACATTCGTGCTAAAGCTACTGTTTCACAAGAGTATTGTGTGAGAAATGTTTTCC AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTAATCC AC238225_RH125I04_29334 GATTTGATATTCGTGCAAAAGTAACTGTTTCGCAGGAGTATTGTGTGAGAAATGTAATCC AC238225_RH125I04_65982_ch.5 GATTTGATATTCGTACAAAAGTAACTGTTTCACAGGAGTATTGTGTGAGAAATGTAATCC AC238225_RH125I04_89012 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTAATCC AC238291_RH153N17_34642_ch.12 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTAC AC238291_RH153N17_96301_ch.12 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTTC AC238387_RH192P22_23420_ch.12 GATTTGATATTCATGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC AC238387_RH192P22_66050_ch.12 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCC PGSC0003DMB000000063:400004-40 ACTTTGACATTCTTGCAAAAGCTACTGTTTCGCAAGAGTACTGTGTGAGAAATGTACTCC PGSC0003DMB000000116:773867.77 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTAATCC PGSC0003DMB000000116:833883.85 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTAATCC PGSC0003DMT400007570-Gpa2 ACTTTGACATTCTTGCAAAAGCTACTGTTTCGCAAGAGTACTGTGTGAGAAATGTACTCC PGSC0003DMT400010966-Gpa2 ACTTTGACATTCGTGGAAACGTAACTGTTTCACAAGAGTATTGTAGGGAATATGTACTCC PGSC0003DMT400010970-Gpa2 GTTTTGATATACGTGCAAAAGTCACCATCTCACAAGAGTATTGTGTGAGAAATGTACTCT PGSC0003DMT400010987-NBS-RGA ACTTTGACATTCGTGCAAAAGCTACTGTTTCACAAGAGTATTGTGCGAAAAAAGTACTCC PGSC0003DMT400020342-Gpa2 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTAATCC PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 GCTTTGACATACGTGCAAAAGCTACTGTTTCACAAGAGTATTGTGTGAGAAATGTTTTCC PGSC0003DMT400020360-Gpa2 GTTTTGACATTCGTGCTAAAGCTACTGTTTCACAAGAGTATTGTGTGAGAAATGTTTTCC PGSC0003DMT400036058-Gpa2 ACTTTGACATTCGTGCAAAAGCTACTGTTTTACAAGAGTATTGTGCGAAAAAAGTACTCC PGSC0003DMT400036104-NBS-LRR ACTTTGACGTTCGTGCAAAAGCTACTGTTTCACAAGAGCATTGTGTGAGAAATGTACTCT PGSC0003DMT400062366-Gpa2 GCTTTGACATCCGTGCAAAAATTACTGTCTCACAAGAGTATTGTGCAAGAAATGTACTTC PGSC0003DMT400071638-PSH-RGH7 ATTTTGACATTCGTGCAAAAGCTACTGTTTCACAAGAGTATTGCGAGAAAAATGTA---- PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 GATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTAATCC

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12670 12680 12690 12700 12710 12720 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Asterix-amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Atlantic-amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Cara- amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Ditta-amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Irati- amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Lady Rosetta-amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGGTGAACCTGATGAT------CAGCTA---- Rhinered- amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Sante-amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Alcmaria -amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Amaryl -amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Mondial -amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Eridia -amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Amalia -amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- wauseon -amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Divina -amplified by 1Rx1 TAGGCCTTCTTTCCTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Multa -amplified by 1Rx1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- AJ011801_Rx_gene TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Solanum_tuberosum_rx_gene. TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- AJ011801_Rx_gene_join_(11849-1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- Solanum acaule Rx2.ac15 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAACTA---- Solanum tuberosum GPA2 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTAT------CAGCTA---- AJ249448_S.acaule_Rx2.ac15 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAACTA---- AF195939_Gpa2 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTAT------CAGCTA---- AJ249449_GPA2 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTAT------CAGCTA---- AF266747_RGC1 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGAT------CAGCTA---- AF266746_RGC3_pseudogene AAGAGTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTG EU352875_SH-RGH7 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTAT------CAGCTA---- EU352874_SH-RGH6_gene AAGGCCTTCTTTCTTCGATAAGTGATGAACCTGATGAT------CAGCTA---- AC237866_RH137K16_2833_ch.12 TAGGCCTTCTTTCTTCGATAAGTGATGAACCTGAGAAT------CAACTA---- AC237866_RH137K16_36092_ch.12 TAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGAT------CAGCTA---- AC237866_RH137K16_46342-ch.12 TAGACCTTCTTTCTTGTATAAGTGATAAACCTTATGAT------CAGCTA---- AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 TAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGAT------CAGCTA---- AC238225_RH125I04_29334 TAGGCCTTCTTTCTTCGATAAGTGATGAACCTGAGAAT------CAACTA---- AC238225_RH125I04_65982_ch.5 TAGGCCTTCTTTCTTCGATAAGTAATGAACCTGATGAT------CAGCTA---- AC238225_RH125I04_89012 TAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGAT------CAGCTA---- AC238291_RH153N17_34642_ch.12 AAGGCCTTCTTTCTTCGATAAGTGATGAACCTGATGAT------CAGCTA---- AC238291_RH153N17_96301_ch.12 AAGGCCTTCTTTCTTCGATAAGTGATGAACCTGATGAT------CAGCTA---- AC238387_RH192P22_23420_ch.12 AAGGCCTTCTTTCTTCGATAAGTGATGAACCTGATGAT------CAGCTA---- AC238387_RH192P22_66050_ch.12 TAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTAT------CAGCTA---- PGSC0003DMB000000063:400004-40 TTGCCCTTCTTGCTTCGACAAGTGAGGAACCTGATGAC------CAACTA---- PGSC0003DMB000000116:773867.77 TAGGCCTTCTTTCTTCGATAAGTAATGAACCTGATGAT------CAGCTA---- PGSC0003DMB000000116:833883.85 TAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGAT------CAGCTA---- PGSC0003DMT400007570-Gpa2 TTGCCCTTCTTGCTTCGACAAGTGAGGAACCTGATGAC------CAACTA---- PGSC0003DMT400010966-Gpa2 TAGGTCTTCTTTCTTCTGTAAGTGGAATGAGTAGTCATGAATTTTATGAGCAACAAGATG PGSC0003DMT400010970-Gpa2 TATACCTTCTTTATTCCGTACGTGGAAAGACTGATGCTCAAACTTATGTGGAGCAAAATG PGSC0003DMT400010987-NBS-RGA TAAGTCTTCTTTCTTCGACTAGTGGAAAGATCGATGAG------CATCAAGATG PGSC0003DMT400020342-Gpa2 TAGGCCTTCTTTCTTCGATAAGTAATGAACCTGATGAT------CAGCTA---- PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 TAGACCTTCTTTCTTGTATAAGTGATAAACCTTATGAT------CAGCTACATG PGSC0003DMT400020360-Gpa2 TAGACCTTCTTTCTTGTATAAGTGATAAACCTTATGAT------CAGCTACATG PGSC0003DMT400036058-Gpa2 TAAGTCTTCTTTCTTCGACTAATGGAAAGATCGATGAG------CACCAAGATG PGSC0003DMT400036104-NBS-LRR TAACCCTTCTTTCTTGTATTAGTGTAAAGACTGATGAATCTGATGATAAGTGTCAAGAGG PGSC0003DMT400062366-Gpa2 TAGGCCTTCTTTCTTCTGTAAGTGGAAAGGCTGATGA---ACTTTATGAGCAGCAAGATG PGSC0003DMT400071638-PSH-RGH7 ------TGCGCAA-----TGAGCATCAAGATG PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 TAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGAT------CAGCTA----

143

12730 12740 12750 12760 12770 12780 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGCAG Asterix-amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Atlantic-amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Cara- amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Ditta-amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Irati- amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Lady Rosetta-amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Rhinered- amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Sante-amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Alcmaria -amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Amaryl -amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Mondial -amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Eridia -amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Amalia -amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG wauseon -amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Divina -amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Multa -amplified by 1Rx1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AJ011801_Rx_gene ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Solanum_tuberosum_rx_gene. ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AJ011801_Rx_gene_join_(11849-1 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Solanum acaule Rx2.ac15 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG Solanum tuberosum GPA2 ------GCGGACCAACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AJ249448_S.acaule_Rx2.ac15 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AF195939_Gpa2 ------GCGGACCAACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AJ249449_GPA2 ------GCGGACCAACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AF266747_RGC1 ------GCGGACAGACTGCAAAAGCATTTGAAAGGCAGG---AGATACTTGGTAG AF266746_RGC3_pseudogene ATTATCAGCTAGCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG EU352875_SH-RGH7 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG EU352874_SH-RGH6_gene ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AC237866_RH137K16_2833_ch.12 ------GCGGACCGACTGCAAAAG------AC237866_RH137K16_36092_ch.12 ------GCGGACCGATTGCAGAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AC237866_RH137K16_46342-ch.12 ------GCAGATCGACTACAAAAGCTTCTAAAAGGTGAG---AGATATTTGGTAG AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 ------GCGGACCGATTGCAGAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AC238225_RH125I04_29334 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AC238225_RH125I04_65982_ch.5 ------GCGGACCGACTGCAAAAGAATCTGAAAGGCAGG---AGATACTTGGTAG AC238225_RH125I04_89012 ------GCGGACCGATTGCAGAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AC238291_RH153N17_34642_ch.12 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AC238291_RH153N17_96301_ch.12 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG AC238387_RH192P22_23420_ch.12 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTAGTAG AC238387_RH192P22_66050_ch.12 ------GCGGACCGACTGCAAAAGCATCTGAAAGGCAGG---AGATACTTGGTAG PGSC0003DMB000000063:400004-40 ------GCAGACCAACTGCAAAAGCGTCTAAAAGGCAAG---AGATACTTGGTAG PGSC0003DMB000000116:773867.77 ------GCGGACCGACTGCAAAAGAATCTGAAAGGCAGG---AGATACTTGGTAG PGSC0003DMB000000116:833883.85 ------GCGGACCGATTGCAGAAGCATCTGAAAGGCAGG---AGATACTTGGTAG PGSC0003DMT400007570-Gpa2 ------GCAGACCAACTGCAAAAGCGTCTAAAAGGCAAG---AGATACTTGGTAG PGSC0003DMT400010966-Gpa2 ATGGGGAACTAGCAGAACAATTGCAAAAGCTTCTAAAAGGCAGA---AGATACTTGGTAG PGSC0003DMT400010970-Gpa2 ATGGGGAACTAGCAGACCAATTGCAAAAGCTTCTAAAACGCGGG---AGATACTTGGTAG PGSC0003DMT400010987-NBS-RGA ATGGGCAACTAGCTGACCGATTACAAAAAAGTCTAAAAGGGAGG---AGGTATTTGATAG PGSC0003DMT400020342-Gpa2 ------GCGGACCGACTGCAAAAGAATCTGAAAGGCAGG---AGATACTTGGTAG PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 ACGGGCAACTAGCAGATCGACTGCAAAAGCTTCTAAAAGGCAAG---AGATATTTGGTAG PGSC0003DMT400020360-Gpa2 ACGGGCAACTAGCAGATCGACTACAAAAGCTTCTAAAAGGTGAG---AGATATTTGGTAG PGSC0003DMT400036058-Gpa2 ATGGGCAACTAGCTGACCGATTACAAAAAAGTCTAAAAGGGAGG---AGGTATTTGGTAG PGSC0003DMT400036104-NBS-LRR ATGGGCAACTAGCATATCGATTGCAAAAGCTTCTAAAAGGCAGGGGGAGATTCTTGGTAG PGSC0003DMT400062366-Gpa2 ATGGACAACTAGCGGACCGACTGCAAAAGCTTCTGAAAGGTAGG---AGGTACTTGATAG PGSC0003DMT400071638-PSH-RGH7 ATGGGCAACTAGCTGATCGACTGCAAAAAAGTCTAAAAGGGAGG---CGGTATTTGGTAG PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 ------GCGGACCGATTGCAGAAGCATCTGAAAGGCAGG---AGATACTTGGTAG

144

12790 12800 12810 12820 12830 12840 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Asterix-amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Atlantic-amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Cara- amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Ditta-amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Irati- amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Lady Rosetta-amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Rhinered- amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Sante-amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Alcmaria -amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Amaryl -amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Mondial -amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Eridia -amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Amalia -amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT wauseon -amplified by 1Rx1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Divina -amplified by 1Rx1 TCATTGATGACATATGGACTACGGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Multa -amplified by 1Rx1 TCATTGATGACATATGGACTACGGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AJ011801_Rx_gene TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Solanum_tuberosum_rx_gene. TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AJ011801_Rx_gene_join_(11849-1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT Solanum acaule Rx2.ac15 TCATTGATGACATATGGACTACAAAAGCTTGGGATGGTATAAAACTATGTTTCCCAGACT Solanum tuberosum GPA2 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AJ249448_S.acaule_Rx2.ac15 TCATTGATGACATATGGACTACAAAAGCTTGGGATGGTATAAAACTATGTTTCCCAGACT AF195939_Gpa2 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AJ249449_GPA2 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AF266747_RGC1 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AF266746_RGC3_pseudogene TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACCATGTTTCCCAGACT EU352875_SH-RGH7 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT EU352874_SH-RGH6_gene TCATTGATGACATATGGACTACAGAAACTTGGGATGATATAAAACTATGTTTCCCAGACT AC237866_RH137K16_2833_ch.12 ------AC237866_RH137K16_36092_ch.12 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AC237866_RH137K16_46342-ch.12 TCATTGATGACATATGGACTACAAAAGCTTGGGATGATATACAAATATGTTTCCCAGACT AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AC238225_RH125I04_29334 TCATTGATGACATATGGACTGTAT------AC238225_RH125I04_65982_ch.5 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACAATGTTTCCCAGACT AC238225_RH125I04_89012 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AC238291_RH153N17_34642_ch.12 TCATTGATGACATATGGACTACAGAAACTTGGGATGATATAAAACTATGTTTCCCAGACT AC238291_RH153N17_96301_ch.12 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AC238387_RH192P22_23420_ch.12 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT AC238387_RH192P22_66050_ch.12 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT PGSC0003DMB000000063:400004-40 TAATTGATGACATATGGACTAAAGGAGCTTGGGATGATATAAGACAATGTTTCCCAGACT PGSC0003DMB000000116:773867.77 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACAATGTTTCCCAGACT PGSC0003DMB000000116:833883.85 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT PGSC0003DMT400007570-Gpa2 TAATTGATGACATATGGACTAAAGGAGCTTGGGATGATATAAGACAATGTTTCCCAGACT PGSC0003DMT400010966-Gpa2 TCATTGATGACATATGGACTAGGGAAACTTGGGATGATATACAATTATGTTTCCCAGATT PGSC0003DMT400010970-Gpa2 TCATTGATGACATATGGACTAAAGGAGCTTGGGATGATATAAAACTATGTTTCCCAGATT PGSC0003DMT400010987-NBS-RGA TCATTGATGACATATGGACCGAACGAGCTTGGGATGATATCAACCTATGTTTCCCAGATT PGSC0003DMT400020342-Gpa2 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACAATGTTTCCCAGACT PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 TCATTGATGACATATGGACTACAAAAGCTTGGGATGATATACAAATATGTTTCCCAGACT PGSC0003DMT400020360-Gpa2 TCATTGATGACATATGGACTACAAAAGCTTGGGATGATATACAAATATGTTTCCCAGACT PGSC0003DMT400036058-Gpa2 TCATTGATGACATATGGACCGAACAAGCTTGGGATGATATTAAGCTATGTTTCCCAGATT PGSC0003DMT400036104-NBS-LRR TCATTGATGACATATGGACTAGAAAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT PGSC0003DMT400062366-Gpa2 TCATTGATGACATATGGACTACAGATTCTTGGGATGATGTAAAACTATGTTTTCCAGACT PGSC0003DMT400071638-PSH-RGH7 TCATTGATGACATATGGACCGAACGAGCTTGGGATGATATGAAACTATGATTCCCAGATT PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 TCATTGATGACATATGGACTACAGAAGCTTGGGATGATATAAAACTATGTTTCCCAGACT

145

12850 12860 12870 12880 12890 12900 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Asterix-amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Atlantic-amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Cara- amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Ditta-amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Irati- amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Lady Rosetta-amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Rhinered- amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Sante-amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Alcmaria -amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Amaryl -amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Mondial -amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Eridia -amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Amalia -amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA wauseon -amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Divina -amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Multa -amplified by 1Rx1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AJ011801_Rx_gene GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Solanum_tuberosum_rx_gene. GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AJ011801_Rx_gene_join_(11849-1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Solanum acaule Rx2.ac15 GTTATAAGGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA Solanum tuberosum GPA2 GCGATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AJ249448_S.acaule_Rx2.ac15 GTTATAAGGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AF195939_Gpa2 GCGATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AJ249449_GPA2 GCGATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AF266747_RGC1 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAAAATGCTA AF266746_RGC3_pseudogene GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA EU352875_SH-RGH7 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA EU352874_SH-RGH6_gene GTAATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AC237866_RH137K16_2833_ch.12 ------AC237866_RH137K16_36092_ch.12 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATGTGCTA AC237866_RH137K16_46342-ch.12 TTCATAATGGAAGCCGAATACTCTTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATGTGCTA AC238225_RH125I04_29334 ----TAATGGAAGCAAAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AC238225_RH125I04_65982_ch.5 GTAATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AC238225_RH125I04_89012 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATGTGCTA AC238291_RH153N17_34642_ch.12 GTAATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AC238291_RH153N17_96301_ch.12 ATTATAATGGAAGCAGAATACTCCTGACTGCTCGGAATGTGGAAGTGGCTGAATATGCTA AC238387_RH192P22_23420_ch.12 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA AC238387_RH192P22_66050_ch.12 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA PGSC0003DMB000000063:400004-40 GTAATAATAGAAGCCGAATACTCTTGACTACTCGGAATGTGGAAGTGGCTGGATATGCTC PGSC0003DMB000000116:773867.77 GTAATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA PGSC0003DMB000000116:833883.85 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATGTGCTA PGSC0003DMT400007570-Gpa2 GTAATAATAGAAGCCGAATACTCTTGACTACTCGGAATGTGGAAGTGGCTGGATATGCTC PGSC0003DMT400010966-Gpa2 GTAACAATGGAAGCCGAATACTCATGACAACTCGAAATATGGATGTGGCTAAATATGCTA PGSC0003DMT400010970-Gpa2 GCAACTGTAGAAGCCGAATACTCATGACTACTCGGAATATGGAGGTGGCTGAATATGCTA PGSC0003DMT400010987-NBS-RGA GTAACTGTGGAAGCAGAATACTGCTGACCACTCGGAATATGGAAGTTGCTGAATATGCTA PGSC0003DMT400020342-Gpa2 GTAATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 TTCATAATGGAAGCCGAATACTCTTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA PGSC0003DMT400020360-Gpa2 TTCATAATGGAAGCCGAATACTCTTGACTACTCGGAATGTGGAAGTGGCTGAATATGCTA PGSC0003DMT400036058-Gpa2 GTAACTGTGGAAGCAGAATACTGCTGACCACTCGGAATATGGAAGTTGCTGAATATGCTA PGSC0003DMT400036104-NBS-LRR GTAATAATGGAAGCCGAATACTCATGATTACTCGGAATGTGGAAGTAGCTGAATATGCTA PGSC0003DMT400062366-Gpa2 GTGACCGTGGAAGCCGAATACTCATGACTACTCGGAATATGGAGGTGGCTGAATATGCTA PGSC0003DMT400071638-PSH-RGH7 GTAACTGTGGAAGCAGAATACTGCTGACAACTCGGAATATGGAAGTAGCTAAGTATGCTA PGSC0003DMT400020350-Gpa2 ------PGSC0003DMT400020353-Gpa2 GTTATAATGGAAGCAGAATACTCCTGACTACTCGGAATGTGGAAGTGGCTGAATGTGCTA

146

12910 12920 12930 12940 12950 12960 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Asterix-amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Atlantic-amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Cara- amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Ditta-amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Irati- amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Lady Rosetta-amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Rhinered- amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Sante-amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Alcmaria -amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Amaryl -amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Mondial -amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Eridia -amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Amalia -amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT wauseon -amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Divina -amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Multa -amplified by 1Rx1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AJ011801_Rx_gene GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Solanum_tuberosum_rx_gene. GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AJ011801_Rx_gene_join_(11849-1 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Solanum acaule Rx2.ac15 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT Solanum tuberosum GPA2 GCTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AJ249448_S.acaule_Rx2.ac15 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AF195939_Gpa2 GCTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AJ249449_GPA2 GCTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AF266747_RGC1 GCTCAGGTAAGCCT---CCTCATCACATGCGCATCATGAATTTTGACGAAAGTTGGAATT AF266746_RGC3_pseudogene GTTCAGGTAAGCCT---CCTCATAACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT EU352875_SH-RGH7 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTCTATCGAAAGTTGGAATT EU352874_SH-RGH6_gene GTTCAGGTAAGCCT---CCTCATCATATGCGCCTCATGAATTTTGAAGAAAGTTGGAATT AC237866_RH137K16_2833_ch.12 ------AC237866_RH137K16_36092_ch.12 GGTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AC237866_RH137K16_46342-ch.12 GCTCAGGTAAGCCT---CCTTATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAGTT AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 GGTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AC238225_RH125I04_29334 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AC238225_RH125I04_65982_ch.5 GTTCAGGTAAGCCT---CCTCATCATATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AC238225_RH125I04_89012 GGTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AC238291_RH153N17_34642_ch.12 GTTCAGGTAAGCCT---CCTCATCATATGCGCCTCATGAATTTTGAAGAAAGTTGGAATT AC238291_RH153N17_96301_ch.12 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT AC238387_RH192P22_23420_ch.12 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAAGTTTGACGAAAGTTGGAATT AC238387_RH192P22_66050_ch.12 GTTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTCTATCGAAAGTTGGAATT PGSC0003DMB000000063:400004-40 GCTCAGGTAAACCT---CCTTATCACATGCGCGTCATGGATTTTGATGAAAGTTGGAGTT PGSC0003DMB000000116:773867.77 GTTCAGGTAAGCCT---CCTCATCATATGCGCCTCATGAATTTTGACGAAAGTTGGAATT PGSC0003DMB000000116:833883.85 GGTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT PGSC0003DMT400007570-Gpa2 GCTCAGGTAAACCT---CCTTATCACATGCGCGTCATGGATTTTGATGAAAGTTGGAGTT PGSC0003DMT400010966-Gpa2 GCTCAGGTAACACTACTCCTTATCAAATGAGCCTTTTGGATTTTAAAAGAAGTTGGAATT PGSC0003DMT400010970-Gpa2 GCTCAGGTAGGCCT---CCTTATCAAATGCGCCTTATGAATTTTGATGAAAGTTGGAGTT PGSC0003DMT400010987-NBS-RGA GCGCAGGTAAGCCT---CCTAATCAAATGCGACTCTTAAATTTTGATGAAAGTTGG---- PGSC0003DMT400020342-Gpa2 GTTCAGGTAAGCCT---CCTCATCATATGCGCCTCATGAATTTTGACGAAAGTTGGAATT PGSC0003DMT400020346-NBS-LRR ------GCCTCATGAATTTTGACGAAAGTTGGAATT PGSC0003DMT400020349-Gpa2 GCTCAGGTAAGCTT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT PGSC0003DMT400020360-Gpa2 GCTCAGGTAAGCCT---CCTTATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAGTT PGSC0003DMT400036058-Gpa2 GCTCAGGTAAGCCT---CCTAATCAAATGCGACTCTTAAATGTTGATGAAAGTTGGAAGT PGSC0003DMT400036104-NBS-LRR GCTCAGGTATTCCT---CCTTTTCAAATGTGCCTCATGAATTTTGATGAAAGTTGGAGTT PGSC0003DMT400062366-Gpa2 GTTCAGGTAAGCCT---CCTTGTCAAATGTCCCTCATGAATTTTGATGAAAGTTGGAGCT PGSC0003DMT400071638-PSH-RGH7 GCTCAGGTAAGCCT---CCTAATCAAATGCGACTATTGAATATTGATGAAAGTTGGAAGT PGSC0003DMT400020350-Gpa2 ------ATGCGCCTCATGAATTTTGACGAAAGTTGGAATT PGSC0003DMT400020353-Gpa2 GGTCAGGTAAGCCT---CCTCATCACATGCGCCTCATGAATTTTGACGAAAGTTGGAATT

147

12970 12980 12990 13000 13010 13020 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Asterix-amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Atlantic-amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Cara- amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Ditta-amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Irati- amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Lady Rosetta-amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Rhinered- amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Sante-amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Alcmaria -amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Amaryl -amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Mondial -amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Eridia -amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Amalia -amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG wauseon -amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Divina -amplified by 1Rx1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Multa -amplified by 1Rx1 TACCACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AJ011801_Rx_gene TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Solanum_tuberosum_rx_gene. TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AJ011801_Rx_gene_join_(11849-1 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Solanum acaule Rx2.ac15 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG Solanum tuberosum GPA2 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AJ249448_S.acaule_Rx2.ac15 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AF195939_Gpa2 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AJ249449_GPA2 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AF266747_RGC1 TACTACACAAAAAGATCTTTGAAACAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AF266746_RGC3_pseudogene TACTACACAAAATGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG EU352875_SH-RGH7 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG EU352874_SH-RGH6_gene TACTATACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC237866_RH137K16_2833_ch.12 ------AC237866_RH137K16_36092_ch.12 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC237866_RH137K16_46342-ch.12 TATTGTATGAAAAAGTCTTTACGAAAGA---CTCTTTCTCCCCTGAATATGAACAACTTG AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC238225_RH125I04_29334 TACTACACAAAAAGATCTTTGAAACAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC238225_RH125I04_65982_ch.5 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC238225_RH125I04_89012 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC238291_RH153N17_34642_ch.12 TACTATACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC238291_RH153N17_96301_ch.12 TACTACACAAAATGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC238387_RH192P22_23420_ch.12 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG AC238387_RH192P22_66050_ch.12 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG PGSC0003DMB000000063:400004-40 TATTGTACGAAAAGGTCTTTGCGAAAGA---CTCTTTTCCCTCTGAATTTGAGCAACTTG PGSC0003DMB000000116:773867.77 TACTACACAAAA-GATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG PGSC0003DMB000000116:833883.85 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG PGSC0003DMT400007570-Gpa2 TATTGTACGAAAAGGTCTTTGCGAAAGA---CTCTTTTCCCTCTGAATTTGAGCAACTTG PGSC0003DMT400010966-Gpa2 TACTGCACACAAAGGTCTTTGATAAAGAATCATGTGTTTCTCATGAATTTAAAGAAATTG PGSC0003DMT400010970-Gpa2 TATTGTATGAAAAGGTCTTTGCGAAGGA---TTGTTTTCCCCCTGAATTTGATGAAGTTG PGSC0003DMT400010987-NBS-RGA ------PGSC0003DMT400020342-Gpa2 TACTACACAAAA-GATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG PGSC0003DMT400020346-NBS-LRR TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG PGSC0003DMT400020349-Gpa2 TATTATATGAAAAAGTCTTTACGAAAGA---CTCTTTCTCCCCTGAATATGAACAACTTG PGSC0003DMT400020360-Gpa2 TATTGTATGAAAAAGTCTTTACGAAAGA---CTCTTTCTCCCCTGAATATGAACAACTTG PGSC0003DMT400036058-Gpa2 TACTGCAGAGAGAAGTCTTTGTAAAAAA---CTGTTTCTCCCCTGAATTTGAACAACTTG PGSC0003DMT400036104-NBS-LRR TACTGTATGAAAAGGTCTTTGTTGTGAGAGATTCGTTTCCCCCAGAATTTGAACAACTTG PGSC0003DMT400062366-Gpa2 TATTGTTCGAAAAGGTCTTTGTGAAAGATAATTT---TTCCCCTGAATTTGAACAACTTG PGSC0003DMT400071638-PSH-RGH7 TACTACAGAGTAGAGTCTTTGTAAAACA---CTGTTTCTCCCCTGAATTTGAACAACTTG PGSC0003DMT400020350-Gpa2 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG PGSC0003DMT400020353-Gpa2 TACTACACAAAAAGATCTTTGAAAAAGAAGGTTCTTATTCTCCTGAATTTGAAAATATTG

148

13030 13040 13050 13060 13070 13080 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 GGAAACAAATTG-CATTAAAACGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Asterix-amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Atlantic-amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Cara- amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Ditta-amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Irati- amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Lady Rosetta-amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Rhinered- amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Sante-amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Alcmaria -amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Amaryl -amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Mondial -amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Eridia -amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGGG Amalia -amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGGG wauseon -amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGGG Divina -amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGACTGCTGG- Multa -amplified by 1Rx1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGACTGCTGG- AJ011801_Rx_gene GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Solanum_tuberosum_rx_gene. GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- AJ011801_Rx_gene_join_(11849-1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Solanum acaule Rx2.ac15 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- Solanum tuberosum GPA2 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTTTGATTGCTGG- AJ249448_S.acaule_Rx2.ac15 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- AF195939_Gpa2 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTTTGATTGCTGG- AJ249449_GPA2 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTTTGATTGCTGG- AF266747_RGC1 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- AF266746_RGC3_pseudogene GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- EU352875_SH-RGH7 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- EU352874_SH-RGH6_gene GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- AC237866_RH137K16_2833_ch.12 ------AC237866_RH137K16_36092_ch.12 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- AC237866_RH137K16_46342-ch.12 GAAAGCAAATTG-CATTGAAATGCGGAGGATTGCCTCTAGCAATTGTTGTGATCGCGGG- AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- AC238225_RH125I04_29334 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGACTGCTGG- AC238225_RH125I04_65982_ch.5 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- AC238225_RH125I04_89012 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGAAATTACTTTGATTGCTGG- AC238291_RH153N17_34642_ch.12 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- AC238291_RH153N17_96301_ch.12 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- AC238387_RH192P22_23420_ch.12 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- AC238387_RH192P22_66050_ch.12 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- PGSC0003DMB000000063:400004-40 GTAAACAAATTG-CATTGAAATGTGGAGGATTGCCTCTAGCAATTATTGTGATCGCGGG- PGSC0003DMB000000116:773867.77 GGAAACAAATTGACATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- PGSC0003DMB000000116:833883.85 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- PGSC0003DMT400007570-Gpa2 GTAAACAAATTG-CATTGAAATGTGGAGGATTGCCTCTAGCAATTATTGTGATCGCGGG- PGSC0003DMT400010966-Gpa2 GGAAACAAATTG-CATTGAAATGCGGAGGATTACCTCTAGCAATTACTGTGATTGCTGG- PGSC0003DMT400010970-Gpa2 GGAAACAAATTG-CATTAAAATGCAAAGGATTACCTCTAACGATTGTTGTTATAGCTGG- PGSC0003DMT400010987-NBS-RGA ------PGSC0003DMT400020342-Gpa2 GGAAACAAATTGACATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG- PGSC0003DMT400020346-NBS-LRR GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGAAATTACTTTGATTGCTGG- PGSC0003DMT400020349-Gpa2 GAAAGCAAATTG-CATTGAAATGCGGAGGATTGCCTCTAGCAATTGTTGTGATCGCGGG- PGSC0003DMT400020360-Gpa2 GAAAGCAAATTG-CATTGAAATGCGGAGGATTGCCTCTAGCAATTGTTGTGATCGCGGG- PGSC0003DMT400036058-Gpa2 GGAAGCAAATTG-CTCTTAAATGCGGGGGATTACCTCTAGCAATTGTTGTTATTGCTGG- PGSC0003DMT400036104-NBS-LRR GGAAGCTAATTG-CATTAAAATGCGGAGGATTACCTCTAGCAATTGTTGCAACTGCTGG- PGSC0003DMT400062366-Gpa2 GTAAACAAATTG-CATTAGAATGCAAAGGATTGCCTCTAACTATTGTTGTCATTGCTGG- PGSC0003DMT400071638-PSH-RGH7 GGAAACAAATTG-CTCTTAAATGCGGGGGATTACCTTTAGCTATTATTGTTATTGCTGG- PGSC0003DMT400020350-Gpa2 GGAAACAAATTG-CATTAAAATGTGGAGGATTACCTCTAGCAATTACTGTGACTGCTGG- PGSC0003DMT400020353-Gpa2 GGAAACAAATTG-CATTAAAATGTGGAGGGTTACCTCTAGCAATTACTGTGATTGCTGG-

149

13090 13100 13110 13120 13130 13140 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Asterix-amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Atlantic-amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Cara- amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Ditta-amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Irati- amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Lady Rosetta-amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Rhinered- amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Sante-amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Alcmaria -amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Amaryl -amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Mondial -amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Eridia -amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Amalia -amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAGGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG wauseon -amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Divina -amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Multa -amplified by 1Rx1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG AJ011801_Rx_gene ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Solanum_tuberosum_rx_gene. ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG AJ011801_Rx_gene_join_(11849-1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTAAG Solanum acaule Rx2.ac15 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG Solanum tuberosum GPA2 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTACG AJ249448_S.acaule_Rx2.ac15 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG AF195939_Gpa2 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTACG AJ249449_GPA2 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTACG AF266747_RGC1 ACTTCTCTCCAAAATGGGTCAAAGATTAGATAAGTGGCAAAGAATTGCGGAGAATGTAAG AF266746_RGC3_pseudogene ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGAAAAATGTTGCGGAGAATGTAAG EU352875_SH-RGH7 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTAAG EU352874_SH-RGH6_gene ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG AC237866_RH137K16_2833_ch.12 ------AC237866_RH137K16_36092_ch.12 ACTTCTCTCCAAAATCAGTAAAACATTGGGTGTGTGGCAATATGTTGCGGAGAATGTAAG AC237866_RH137K16_46342-ch.12 ACTTCTCACTAAAATTGACAAAGCATTGAGTGAGTGGCAAAGTGTTGCTGAAAATGTAAG AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 ACTTCTCTCCAAAATCAGTAAAACATTGGGTGTGTGGCAATATGTTGCGGAGAATGTAAG AC238225_RH125I04_29334 ACTTCTCTCCAAAATCGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTAAG AC238225_RH125I04_65982_ch.5 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG AC238225_RH125I04_89012 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG AC238291_RH153N17_34642_ch.12 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG AC238291_RH153N17_96301_ch.12 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTAAG AC238387_RH192P22_23420_ch.12 ACTTCTCTCCAAAATCGGTCAAAGATTAGATAAGTGGCAAAGAATTGTGGAGAATGTAAG AC238387_RH192P22_66050_ch.12 ACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTAAG PGSC0003DMB000000063:400004-40 ACTTCTCTCCAAAACCGGCAAAACTTTGGATGTGTGGCAAAGTATTGCCGAGAATGTAAG PGSC0003DMB000000116:773867.77 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG PGSC0003DMB000000116:833883.85 ACTTCTCTCCAAAATCAGTAAAACATTGGGTGTGTGGCAATATGTTGCGGAGAATGTAAG PGSC0003DMT400007570-Gpa2 ACTTCTCTCCAAAACCGGCAAAACTTTGGATGTGTGGCAAAGTATTGCCGAGAATGTAAG PGSC0003DMT400010966-Gpa2 AGTTCTCTCCAAAATAGGTAAATCATTGGGTGAGTGGCAAAGTGTTGCCGAGAACGTAAG PGSC0003DMT400010970-Gpa2 ACTTCTTTCCAAAATTGGTAAAGCATTGGATGAATGGAAAAGTGTTGCCGCGAATGTAAG PGSC0003DMT400010987-NBS-RGA ------TGAAGCATTCGATGAATGGACAAGTGTTGCCGAGAATGTAAG PGSC0003DMT400020342-Gpa2 ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG PGSC0003DMT400020346-NBS-LRR ACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTAAG PGSC0003DMT400020349-Gpa2 ACTTCTCACTAAAATTGACAAAGCATTGAGTGAGTGGCAAAGTGTTGCTGAAAATGTAAG PGSC0003DMT400020360-Gpa2 ACTTCTCACTAAAATTGACAAAGCATTGAGTGAGTGGCAAAGTGTTGCTGAAAATGTAAG PGSC0003DMT400036058-Gpa2 ACTTCTTTCCAAAATTGTTGAAGCATTCGATCAATGGACTAGTGTTGCTGAGAATGTAAG PGSC0003DMT400036104-NBS-LRR AGTTCTCTCGAAAAGTGGTAAAACATTGAACGTTTGGAGAAGTGTTGCTGAGAATGTAAG PGSC0003DMT400062366-Gpa2 ACTTCTATCAAAAGTTGGTAAAACATTGAATGAGTGGACAAGTGTTGCCAAGAATGTAAG PGSC0003DMT400071638-PSH-RGH7 AGTTCTGTCTAATATTGGTGAATCATTCGATGAATGGACAAGTGTTGCGGAGAATGTAAG PGSC0003DMT400020350-Gpa2 ACTTCTCTCCAAAATCGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTAAG PGSC0003DMT400020353-Gpa2 ACTTCTCTCCAAAATCAGTAAAACATTGGGTGTGTGGCAATATGTTGCGGAGAATGTAAG

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13150 13160 13170 13180 13190 13200 ....|....|....|....|....|....|....|....|....|....|....|....| Agria-amplified by 1Rx1 TTCGGTCG------Asterix-amplified by 1Rx1 TTCGGTCG------Atlantic-amplified by 1Rx1 TTCGGTCG------Cara- amplified by 1Rx1 TTCGGTCG------Ditta-amplified by 1Rx1 TTCGGTCG------Irati- amplified by 1Rx1 TTCGGTCG------Lady Rosetta-amplified by 1Rx1 TTCGGTCG------Rhinered- amplified by 1Rx1 TTCGGTCG------Sante-amplified by 1Rx1 TTCGGTCG------Alcmaria -amplified by 1Rx1 TTCGGTCG------Amaryl -amplified by 1Rx1 TTCGGTCG------Mondial -amplified by 1Rx1 TTCGGTCG------Eridia -amplified by 1Rx1 TTCGGTCG------Amalia -amplified by 1Rx1 TTCGGTCG------wauseon -amplified by 1Rx1 TTCGGTCG------Divina -amplified by 1Rx1 TTCGGTCG------Multa -amplified by 1Rx1 TTCGGTCG------AJ011801_Rx_gene TTCGGTCGTTAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA Solanum_tuberosum_rx_gene. TTCGGTCGTTAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA AJ011801_Rx_gene_join_(11849-1 TTCGGTCGTTAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA Solanum acaule Rx2.ac15 TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA Solanum tuberosum GPA2 TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA AJ249448_S.acaule_Rx2.ac15 TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA AF195939_Gpa2 TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA AJ249449_GPA2 TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA AF266747_RGC1 TTCGGTGGTAAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA AF266746_RGC3_pseudogene TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA EU352875_SH-RGH7 TTCGGTGGTAAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA EU352874_SH-RGH6_gene TTCGGTGGTAAGCACAGATCTTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA AC237866_RH137K16_2833_ch.12 ------AC237866_RH137K16_36092_ch.12 TTCAGTGGTAAGCACAGATCTTGAAGCAAAGTGCATGAGAGTGTTGGCTTTGAGTTACCA AC237866_RH137K16_46342-ch.12 TTCAGCAGTAAGCACAGATGTTGGTGTCCAATGCATGAGGGTGTTGGCAATGAGTTACCA AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplifyS.acaul ------AC238225_RH125I04_13358_ch.5 TTCAGTGGTAAGCACAGATCTTGAAGCAAAGTGCATGAGAGTGTTGGCTTTGAGTTACCA AC238225_RH125I04_29334 TTCGGTGGTAAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA AC238225_RH125I04_65982_ch.5 TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA AC238225_RH125I04_89012 TTCGGTGGTAAGCAGAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA AC238291_RH153N17_34642_ch.12 TTCGGTGGTAAGCACAGATCTTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA AC238291_RH153N17_96301_ch.12 TTCGGTGGTAAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA AC238387_RH192P22_23420_ch.12 TTCGGTGGTAAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA AC238387_RH192P22_66050_ch.12 TTCGGTGGTAAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA PGSC0003DMB000000063:400004-40 TTCAGTGGTAAGCACAGATCTTGAAGCCCAATGCATGAGAGTTTTGGCTTTGAGTTACCA PGSC0003DMB000000116:773867.77 TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA PGSC0003DMB000000116:833883.85 TTCAGTGGTAAGCACAGATCTTGAAGCAAAGTGCATGAGAGTGTTGGCTTTGAGTTACCA PGSC0003DMT400007570-Gpa2 TTCAGTGGTAAGCACAGATCTTGAAGCCCAATGCATGAGAGTTTTGGCTTTGAGTTACCA PGSC0003DMT400010966-Gpa2 TTCAGTGGTAAGCACAGATGTTGATGTTCAATGCATGCAAGTGTTGGCATTGAGTTATCA PGSC0003DMT400010970-Gpa2 TTCAGTGGTGAGCACAGATCTTGATGTCCAATGCATGAGAGTATTGACTTTGAGTTACCA PGSC0003DMT400010987-NBS-RGA TTCAGTGGTAAGCACAG-TCATAATGTTCAATGCATGAGAGTTTTGGCATTGAGTTACCA PGSC0003DMT400020342-Gpa2 TTCGGTGGTAAGCACAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA PGSC0003DMT400020346-NBS-LRR TTCGGTGGTAAGCAGAGATCTTGAAGCAAAATGCATGAGAGTGTTGGCTTTGAGTTACCA PGSC0003DMT400020349-Gpa2 TTCAGCAGTAAGCACAGATGTTGGTGTCCAATGCATGAGGGTGTTGGCAATGAGTTACCA PGSC0003DMT400020360-Gpa2 TTCAGCAGTAAGCACAGATGTTGGTGTCCAATGCATGAGGGTGTTGGCAATGAGTTACCA PGSC0003DMT400036058-Gpa2 TTCAGCAGTAAGCACAGATCATAATGTTCAATGCATGAGAGTTTTGGCATTGAGTTACCA PGSC0003DMT400036104-NBS-LRR TTTAGCAGTAAGCACTGATCTTGAAGTCCAATGCATGACAGTGCTAGCTTTGAGTTACCA PGSC0003DMT400062366-Gpa2 TTCTAAGGCAAGCATAGATCTTGATATCCAATGTATGAGAGTGTTGTCTTTAAGTTACCA PGSC0003DMT400071638-PSH-RGH7 TTCAGTGGTAAGCACAGATCACAATGGTCAATGCATGAGAGTGTTGGCGTTGAGTTATCA PGSC0003DMT400020350-Gpa2 TTCGGTGGTAAGCACAGATCCTGAAGCACAATGCATGAGAGTGTTGGCTTTGAGTTACCA PGSC0003DMT400020353-Gpa2 TTCAGTGGTAAGCACAGATCTTGAAGCAAAGTGCATGAGAGTGTTGGCTTTGAGTTACCA

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Appendix 6 Multi-alignment of PCR products of 17 potato cultivars amplified by 5Rx1 primer with the sequence of Rx1 , Rx2, Gpa2 and 34 other paralogous genes in potato-DM (Green colour regions show annealing region of primers). All the sequences are aligned with Rx1 (Acc. No. AJ011801)

4450 4460 4470 4480 4490 4500 ....|....|....|....|....|....|....|....|....|....|....|....| Cara-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Amaryl-amplified by 5Rx1 ------TTAGGGCAAAACCCTAACACTC Divina-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Amalia-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Asterix-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Atlantic-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Ditta-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Lady Rosetta-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Rhinered-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Sante-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Agria-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Eridia-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Irati-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Wauseon-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Alcmaria-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Mondial-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Multa-amplified by 5Rx1 ------TCAGGGCAAAACCCTAACACTC Solanum_tuberosum_rx_gene. CTTTAAAGAAAAAATAAACTTCAAATAAGATAAGAAAATCAGGGCAAAACCCTAACACTC AJ011801_Rx_gene_complete_cds GGACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTA AJ011801_Rx_gene_join_(11849-1 GGACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGGGGAAAATGTA Solanum acaule Rx2.ac15 -NBS-L GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA Solanum tuberosum GPA2-NBS-LRR GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA AJ249448_S.acaule_Rx2.ac15_exo GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA AF195939_Gpa2_gene_complete_cd GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA AJ249449_GPA2_exons_1-3 GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA AF266747_RGC1 GGACTTCTCTCCAAAATGGGTCAAAGATTAGATAAGTGGCAAAGAATTGCGGAGAATGTA AF266746_RGC3_pseudogene GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGAAAAATGTTGCGGAGAATGTA EU352875_SH-RGH7 GGACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTA EU352874_SH-RGH6_gene GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA AC237866_RH137K16__2833_ch.12 ------AGAACCTTGCATTTTCTGTTTTCTTAAGAACTGAAAA AC237866_RH137K16_36092-ch.12 GGACTTCTCTCCAAAATCAGTAAAACATTGGGTGTGTGGCAATATGTTGCGGAGAATGTA AC237866_RH137K16_46342_ch.12 GGACTTCTCACTAAAATTGACAAAGCATTGAGTGAGTGGCAAAGTGTTGCTGAAAATGTA AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplify_AJ2494 ------AC238225_RH125I04_13358_ch.5 GGACTTCTCTCCAAAATCAGTAAAACATTGGGTGTGTGGCAATATGTTGCGGAGAATGTA AC238225_RH125I04_29334 GGACTTCTCTCCAAAATCGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTA AC238225_RH125I04_65982_ch.5 GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA AC238225_RH125I04_89012 GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA AC238291_RH153N17_34642_ch.12 GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA AC238291_RH153N17_96301_ch.12 GGACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTA AC238387_RH192P22_23420-ch.12 GGACTTCTCTCCAAAATCGGTCAAAGATTAGATAAGTGGCAAAGAATTGTGGAGAATGTA AC238387_RH192P22_66050-ch.12 GGACTTCTCTCCAAAATGGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTA PGSC0003DMB000000063:400004.40 GGACTTCTCTCCAAAACCGGCAAAACTTTGGATGTGTGGCAAAGTATTGCCGAGAATGTA PGSC0003DMB000000116:773867.77 GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA PGSC0003DMB000000116:833883.85 GGACTTCTCTCCAAAATCAGTAAAACATTGGGTGTGTGGCAATATGTTGCGGAGAATGTA PGSC0003DMT400007570-Gpa2 GGACTTCTCTCCAAAACCGGCAAAACTTTGGATGTGTGGCAAAGTATTGCCGAGAATGTA PGSC0003DMT400010966-Gpa2 GGAGTTCTCTCCAAAATAGGTAAATCATTGGGTGAGTGGCAAAGTGTTGCCGAGAACGTA PGSC0003DMT400010970- Gpa2 GGACTTCTTTCCAAAATTGGTAAAGCATTGGATGAATGGAAAAGTGTTGCCGCGAATGTA PGSC0003DMT400010987-NBS-RGA ------TGAAGCATTCGATGAATGGACAAGTGTTGCCGAGAATGTA PGSC0003DMT400020342-Gpa2 GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA PGSC0003DMT400020346-NBS-LRR GGACTTCTCTCCAAAATCAGTAAAACATTGGATGAGTGGCAAAATGTTGCGGAGAATGTA PGSC0003DMT400020349- Gpa2 GGACTTCTCACTAAAATTGACAAAGCATTGAGTGAGTGGCAAAGTGTTGCTGAAAATGTA PGSC0003DMT400020360- Gpa2 GGACTTCTCACTAAAATTGACAAAGCATTGAGTGAGTGGCAAAGTGTTGCTGAAAATGTA PGSC0003DMT400036058-Gpa2 GGACTTCTTTCCAAAATTGTTGAAGCATTCGATCAATGGACTAGTGTTGCTGAGAATGTA PGSC0003DMT400036104-NBS-LRR GGAGTTCTCTCGAAAAGTGGTAAAACATTGAACGTTTGGAGAAGTGTTGCTGAGAATGTA PGSC0003DMT400062366-Gpa2 GGACTTCTATCAAAAGTTGGTAAAACATTGAATGAGTGGACAAGTGTTGCCAAGAATGTA PGSC0003DMT400071638-PSH-RGH7 GGAGTTCTGTCTAATATTGGTGAATCATTCGATGAATGGACAAGTGTTGCGGAGAATGTA PGSC0003DMT400020350-Gpa2 GGACTTCTCTCCAAAATCGGTCAAAGATTAGATGAGTGGCAAAGAATTGCGGAGAATGTA PGSC0003DMT400020353- Gpa2 GGACTTCTCTCCAAAATCAGTAAAACATTGGGTGTGTGGCAATATGTTGCGGAGAATGTA

152

4510 4520 4530 4540 4550 4560 ....|....|....|....|....|....|....|....|....|....|....|....| Cara-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Amaryl-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Divina-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Amalia-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Asterix-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Atlantic-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Ditta-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Lady Rosetta-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Rhinered-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Sante-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Agria-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTCGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Eridia-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Irati-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Wauseon-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Alcmaria-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Mondial-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT Multa-amplified by 5Rx1 TCTTCGATCCCTTACTCTTATTTTGTGCCTTAATCATCCAAGTGAATTTGTTTTCTTTCT Solanum_tuberosum_rx_gene. TCTTCGATCCCTTACTCTTATTTTGTGCCTCAATCATCCAAGTGAATTTGTTTTCTTTCT AJ011801_Rx_gene_complete_cds AGTTCGGTCGTTAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA AJ011801_Rx_gene_join_(11849-1 AGTTCGGTCGTTAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA Solanum acaule Rx2.ac15 -NBS-L AGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA Solanum tuberosum GPA2-NBS-LRR CGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA AJ249448_S.acaule_Rx2.ac15_exo AGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA AF195939_Gpa2_gene_complete_cd CGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA AJ249449_GPA2_exons_1-3 CGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA AF266747_RGC1 AGTTCGGTGGTAAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA AF266746_RGC3_pseudogene AGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA EU352875_SH-RGH7 AGTTCGGTGGTAAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA EU352874_SH-RGH6_gene AGTTCGGTGGTAAGCACAGATCTTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA AC237866_RH137K16__2833_ch.12 ATTACATAACTTGTGGCAATTTGTCCATTTTCAT-AC-TGAGAGATATTTCTATTTTTTG AC237866_RH137K16_36092-ch.12 AGTTCAGTGGTAAGCACAGATCTTGAAGCAAAGT-GCATGAGAGTGTTGGCTTTGAGTTA AC237866_RH137K16_46342_ch.12 AGTTCAGCAGTAAGCACAGATGTTGGTGTCCAAT-GCATGAGGGTGTTGGCAATGAGTTA AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplify_AJ2494 ------GGCAATTTGTTCATTTTCAT-AC-TGAGAGA------CTTGGTTTG AC238225_RH125I04_13358_ch.5 AGTTCAGTGGTAAGCACAGATCTTGAAGCAAAGT-GCATGAGAGTGTTGGCTTTGAGTTA AC238225_RH125I04_29334 AGTTCGGTGGTAAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA AC238225_RH125I04_65982_ch.5 AGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA AC238225_RH125I04_89012 AGTTCGGTGGTAAGCAGAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA AC238291_RH153N17_34642_ch.12 AGTTCGGTGGTAAGCACAGATCTTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA AC238291_RH153N17_96301_ch.12 AGTTCGGTGGTAAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA AC238387_RH192P22_23420-ch.12 AGTTCGGTGGTAAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA AC238387_RH192P22_66050-ch.12 AGTTCGGTGGTAAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA PGSC0003DMB000000063:400004.40 AGTTCAGTGGTAAGCACAGATCTTGAAGCCCAAT-GCATGAGAGTTTTGGCTTTGAGTTA PGSC0003DMB000000116:773867.77 AGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA PGSC0003DMB000000116:833883.85 AGTTCAGTGGTAAGCACAGATCTTGAAGCAAAGT-GCATGAGAGTGTTGGCTTTGAGTTA PGSC0003DMT400007570-Gpa2 AGTTCAGTGGTAAGCACAGATCTTGAAGCCCAAT-GCATGAGAGTTTTGGCTTTGAGTTA PGSC0003DMT400010966-Gpa2 AGTTCAGTGGTAAGCACAGATGTTGATGTTCAAT-GCATGCAAGTGTTGGCATTGAGTTA PGSC0003DMT400010970- Gpa2 AGTTCAGTGGTGAGCACAGATCTTGATGTCCAAT-GCATGAGAGTATTGACTTTGAGTTA PGSC0003DMT400010987-NBS-RGA AGTTCAGTGGTAAGCACAG-TCATAATGTTCAAT-GCATGAGAGTTTTGGCATTGAGTTA PGSC0003DMT400020342-Gpa2 AGTTCGGTGGTAAGCACAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA PGSC0003DMT400020346-NBS-LRR AGTTCGGTGGTAAGCAGAGATCTTGAAGCAAAAT-GCATGAGAGTGTTGGCTTTGAGTTA PGSC0003DMT400020349- Gpa2 AGTTCAGCAGTAAGCACAGATGTTGGTGTCCAAT-GCATGAGGGTGTTGGCAATGAGTTA PGSC0003DMT400020360- Gpa2 AGTTCAGCAGTAAGCACAGATGTTGGTGTCCAAT-GCATGAGGGTGTTGGCAATGAGTTA PGSC0003DMT400036058-Gpa2 AGTTCAGCAGTAAGCACAGATCATAATGTTCAAT-GCATGAGAGTTTTGGCATTGAGTTA PGSC0003DMT400036104-NBS-LRR AGTTTAGCAGTAAGCACTGATCTTGAAGTCCAAT-GCATGACAGTGCTAGCTTTGAGTTA PGSC0003DMT400062366-Gpa2 AGTTCTAAGGCAAGCATAGATCTTGATATCCAAT-GTATGAGAGTGTTGTCTTTAAGTTA PGSC0003DMT400071638-PSH-RGH7 AGTTCAGTGGTAAGCACAGATCACAATGGTCAAT-GCATGAGAGTGTTGGCGTTGAGTTA PGSC0003DMT400020350-Gpa2 AGTTCGGTGGTAAGCACAGATCCTGAAGCACAAT-GCATGAGAGTGTTGGCTTTGAGTTA PGSC0003DMT400020353- Gpa2 AGTTCAGTGGTAAGCACAGATCTTGAAGCAAAGT-GCATGAGAGTGTTGGCTTTGAGTTA

153

4570 4580 4590 4600 4610 4620 ....|....|....|....|....|....|....|....|....|....|....|....| Cara-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Amaryl-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Divina-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Amalia-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Asterix-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Atlantic-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Ditta-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Lady Rosetta-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTCATGAGGG Rhinered-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Sante-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Agria-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTACGAGGG Eridia-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Irati-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Wauseon-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Alcmaria-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Mondial-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Multa-amplified by 5Rx1 AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG Solanum_tuberosum_rx_gene. AAAACGATATATAGATTTGCAAAAATCAAAGATCTGTAATTTCAAAATCATTTATGAGGG AJ011801_Rx_gene_complete_cds CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCA-CAGAGG AJ011801_Rx_gene_join_(11849-1 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCA-CAGAGG Solanum acaule Rx2.ac15 -NBS-L CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTATATTTTGCAATTTTCG-CAGAGG Solanum tuberosum GPA2-NBS-LRR CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG AJ249448_S.acaule_Rx2.ac15_exo CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTATATTTTGCAATTTTCG-CAGAGG AF195939_Gpa2_gene_complete_cd CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG AJ249449_GPA2_exons_1-3 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG AF266747_RGC1 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTATATTTTGCAATTTTCG-CAGAGG AF266746_RGC3_pseudogene CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG EU352875_SH-RGH7 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTATATTTTGCAATTTTCG-CAGAGG EU352874_SH-RGH6_gene CCATCACTTGCCTTCTCACCTAAAATCGTGTTTTCTATATTTTGCAATTTTCG-CAGAGG AC237866_RH137K16__2833_ch.12 --GATATATGGCTTATGCTGCTGTTACTTCCCTTATGAGAACCATACATCAATCAATGGA AC237866_RH137K16_36092-ch.12 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG AC237866_RH137K16_46342_ch.12 CCATTACTTGCCTCACCACCTAAAACCATGCTTTTTATATTTCGCAATCTTCC-CAGAGG AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplify_AJ2494 --AATACATGGCCAATT------AC238225_RH125I04_13358_ch.5 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG AC238225_RH125I04_29334 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG AC238225_RH125I04_65982_ch.5 CCATCACTTGCCTTCTCACCTAAAACCATGTTTTCTGTATTTTGCAATTTTCG-CAGAGG AC238225_RH125I04_89012 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG AC238291_RH153N17_34642_ch.12 CCATCACTTGCCTTCTCACCTAAAATCGTGTTTTCTATATTTTGCAATTTTCG-CAGAGG AC238291_RH153N17_96301_ch.12 CCATCACTTGCCTTCTCACCTAAAACCATGTTTTCTATATTTTGCAATTTTCG-CAGAGG AC238387_RH192P22_23420-ch.12 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTATATTTTGCAATTTTCG-CAGAGG AC238387_RH192P22_66050-ch.12 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTATATTTTGCAATTTTCG-CAGAGG PGSC0003DMB000000063:400004.40 CCATCACTTGCCTTCTCACCTAAAACCGTGCTTTCTATATTTTGCAATTTTCC-CAGAGG PGSC0003DMB000000116:773867.77 CCATCACTTGCCTTCTCACCTAAAACCATGTTTTCTGTATTTTGCAATTTTCG-CAGAGG PGSC0003DMB000000116:833883.85 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG PGSC0003DMT400007570-Gpa2 CCATCACTTGCCTTCTCACCTAAAACCGTGCTTTCTATATTTTGCAATTTTCC-CAGAGG PGSC0003DMT400010966-Gpa2 TCATCACTTGCCTCAACACCTAAAGCCGTGTTTCCTATATTTTGCAATCTTCC-CAGAAG PGSC0003DMT400010970- Gpa2 CCATCACTTGCCTCATCACCTAAGAGCGTGCTTTCTATATTTTGCACTCTTCC-CAGAGG PGSC0003DMT400010987-NBS-RGA CCATTACTTGCCAAATCACCTAAGACCGTGCTTTCTATATTTTGCAATCTGCCATGTTAG PGSC0003DMT400020342-Gpa2 CCATCACTTGCCTTCTCACCTAAAACCATGTTTTCTGTATTTTGCAATTTTCG-CAGAGG PGSC0003DMT400020346-NBS-LRR CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG PGSC0003DMT400020349- Gpa2 CCATTACTTGCCTCACCACCTAAAACCATGCTTTTTATATTTCGCAATCTTCC-CAGAGG PGSC0003DMT400020360- Gpa2 CCATTACTTGCCTCACCACCTAAAACCATGCTTTTTATATTTCGCAATCTTCC-CAGAGG PGSC0003DMT400036058-Gpa2 CCATCACTTACCAAATCACCTAAGAGTGTGCTTTCTATATTTTGCAATGTTCC-CGGAAG PGSC0003DMT400036104-NBS-LRR CCATCACTTGCCTCGTCACCTAAAACCGTGCTCTCTGTATTTTGCAATCTTCC-CAGAGG PGSC0003DMT400062366-Gpa2 CCATCACTTGCCACGTCACCTAAAATCGTGTTTTCTGTATTTTGCAATCTTTC-CAGAAG PGSC0003DMT400071638-PSH-RGH7 TCATCACTTACCACATCACTTGAGAGCGTGTTTTCTATATTTTGCAATATTCC-CGGAGG PGSC0003DMT400020350-Gpa2 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG PGSC0003DMT400020353- Gpa2 CCATCACTTGCCTTCTCACCTAAAACCGTGTTTTCTGTATTTTGCAATTTTCG-CAGAGG

154

4630 4640 4650 4660 4670 4680 ....|....|....|....|....|....|....|....|....|....|....|....| Cara-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Amaryl-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Divina-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Amalia-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Asterix-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Atlantic-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Ditta-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Lady Rosetta-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Rhinered-amplified by 5Rx1 ACCCAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Sante-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Agria-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Eridia-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Irati-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Wauseon-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Alcmaria-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Mondial-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Multa-amplified by 5Rx1 ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGAT------Solanum_tuberosum_rx_gene. ACCTAATTATTTTCTCCTCCAACACGATGTCACTCTAGGCCGATTGACAAAGAATAAGTT AJ011801_Rx_gene_complete_cds ATGAACAGATTTCTGTAAATGAACTTGTTGAGTTATGGCCTGTAGAGGG-ATTTTTGAAT AJ011801_Rx_gene_join_(11849-1 ATGAACAGATTTCTGTAAATGAACTTGTTGAGTTATGGCCTGTAGAGGG-ATTTTTGAAT Solanum acaule Rx2.ac15 -NBS-L ATGAACGGATTTCTGTAACTAAACTTGTTGAGTTATGGGCTGTAGAGGG-GTTTTTGAAT Solanum tuberosum GPA2-NBS-LRR ATGAACGGATTTATGTAAATAAACTTGTTGAGTTATGGGCCGTAGAGGG-GTTTTTGAAT AJ249448_S.acaule_Rx2.ac15_exo ATGAACGGATTTCTGTAACTAAACTTGTTGAGTTATGGGCTGTAGAGGG-GTTTTTGAAT AF195939_Gpa2_gene_complete_cd ATGAACGGATTTATGTAAATAAACTTGTTGAGTTATGGGCCGTAGAGGG-GTTTTTGAAT AJ249449_GPA2_exons_1-3 ATGAACGGATTTATGTAAATAAACTTGTTGAGTTATGGGCCGTAGAGGG-GTTTTTGAAT AF266747_RGC1 ATGAAGAGATTTATGTAAATAAACTTGTTGACTTATGGGCTGTAGAGGG-GTTTTTGAAT AF266746_RGC3_pseudogene ATGAACGGATTTATGTAAATAAACTTGTTGAGTTATGGGCCGTAGAGGG-GTTTTTGAAT EU352875_SH-RGH7 ATGAACGGATTTATGTACATGAACTTGTTGAGTTATGGCCTGTAGAGGG-GTTTTTGAAT EU352874_SH-RGH6_gene ATGAACAGATTTATGTAAATAACCTTGTTGAGTTATGGGGTGTAGAGGG-GTTTTTGAAT AC237866_RH137K16__2833_ch.12 ACTTACTGGATGTGATTTGCAACCGTTTTATGAAAAGCTCAAATCTTTGAGAGCTATTCT AC237866_RH137K16_36092-ch.12 ATGAACAGATTTATGTAAATAAACTTGTTGAATTATGGGCCGTAGAGGG-GTTTTTGAAT AC237866_RH137K16_46342_ch.12 ATAAACTGATTTTTGTCGATAAACTTATGATATTGTGGGAAACAGAGGG-ATTTTTGAAG AC237866_RH137K16_50554_ch.12 ------Primers_used_to_amplify_AJ2494 ------AC238225_RH125I04_13358_ch.5 ATGAACAGATTTATGTAAATAAACTTGTTGAATTATGGGCCGTAGAGGG-GTTTTTGAAT AC238225_RH125I04_29334 ATGAACGGATTTTTGTAAATAAACTTGTTGAGTTATGGGCCGTAGAGGG-GTTTTTGAAT AC238225_RH125I04_65982_ch.5 ATGAACAGATTTATGTAAGTGAACTTGTTGAGTTATGGGCTGTAGAGGG-GTTTTTGAAT AC238225_RH125I04_89012 ATGAACGGATTTATGTAAATGCACTTGTTGAGTTATGGGCCGTAGAGGG-GTTTTTGAAT AC238291_RH153N17_34642_ch.12 ATGAACAGATTTATGTAAATAACCTTGTTGAGTTATGGGGTGTAGAGGG-GTTTTTGAAT AC238291_RH153N17_96301_ch.12 ATGAAGAGATTTATGTAAATAAACTTGTTGAGTTATGGGCCGTAGAGGG-GTTTTTGAAT AC238387_RH192P22_23420-ch.12 ATGAATGGATTTTTGTAAATAAACTTGTTGAGTTATGGTCCGTAGAGGG-GTTTTTGAAT AC238387_RH192P22_66050-ch.12 ATGAACGGATTTATGTACATGAACTTGTTGAGTTATGGCCTGTAGAGGG-GTTTTTGAAT PGSC0003DMB000000063:400004.40 ATAGGCTGATTTTTGTCAATAAACTTTTGGACTTATGGGTTGCAGAGGG-ATTTTTGAAG PGSC0003DMB000000116:773867.77 ATGAACAGATTTATGTAAGTGAACTTGTTGAGTTATGGGCTGTAGAGGG-GTTTTTGAAT PGSC0003DMB000000116:833883.85 ATGAACAGATTTATGTAAATAAACTTGTTGAATTATGGGCCGTAGAGGG-GTTTTTGAAT PGSC0003DMT400007570-Gpa2 ATAGGCTGATTTTTGTCAATAAACTTTTGGACTTATGGGTTGCAGAGGG-ATTTTTGAAG PGSC0003DMT400010966-Gpa2 ATGAAGTGATTTTTGTCGATAAACTTATGGAGTTATGGGTTGTAGAAGG-ATTTTTGAAG PGSC0003DMT400010970- Gpa2 ATAAATTGATTTTTGTGAATAAACTTGTGAAATTATGGGCAGCAGAAGG-TTTTCTGAAG PGSC0003DMT400010987-NBS-RGA GTACCACCATAAGCTTGGAGGTAAGGTCTATGTCCTCCTCTATGTATTTTGTTTTTG--- PGSC0003DMT400020342-Gpa2 ATGAACAGATTTATGTAAGTGAACTTGTTGAGTTATGGGCTGTAGAGGG-GTTTTTGAAT PGSC0003DMT400020346-NBS-LRR ATGAACGGATTTATGTAAATGCACTTGTTGAGTTATGGGCCGTAGAGGG-GTTTTTGAAT PGSC0003DMT400020349- Gpa2 ATAAACTGATTTTTGTCAATAAACTTATGATATTGTGGGAAACAGAGGG-ATTTTTGAAG PGSC0003DMT400020360- Gpa2 ATAAACTGATTTTTGTCAATAAACTTATGATATTGTGGGAAACAGAGGG-ATTTTTGAAG PGSC0003DMT400036058-Gpa2 ATACAGTGATTTTTGTTAATAAACTTGTGAAGTTATGGGCAGCAGAGGG-GTTTTTGAAG PGSC0003DMT400036104-NBS-LRR ATGAAGTGATTTTTGTCGATAAACATGTGGAGTTATGGGTTGTAGAAGG-TTTCTTGAAG PGSC0003DMT400062366-Gpa2 ATAAATTATTTTTTGTCAATGAACTTATGGAATTATGGACTGTGGAGGG-ATTTTTGAAG PGSC0003DMT400071638-PSH-RGH7 ATACAGTGATTTTTGTGAATAAACTTGTGAAATTATGGACAGCAGAGGG-TTTTTTGAAG PGSC0003DMT400020350-Gpa2 ATGAACGGATTTTTGTAAATAAACTTGTTGAGTTATTGGCCGTAGAGGG-GTTTTTGAAT PGSC0003DMT400020353- Gpa2 ATGAACAGATTTATGTAAATAAACTTGTTGAATTATGGGCCGTAGAGGG-GTTTTTGAAT

155

Appendix 7 Multi-alignment of PCR products of 7 potato cultivars amplified by 106Rx2 primer with the sequence of Rx1 , Rx2, Gpa2 and 34 other paralogous genes in potato-DM (Yellow colour regions show annealing region of primers). All the sequences are aligned with Rx2 (Acc. No. AJ249448).

430 440 450 460 470 480 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 ------GGAGAAATCC Lorret -amplified by 106Rx2 ------GGAGAAATCC Courage- amplified by 106Rx2 ------GGAGAAATCC Luca -amplified by 106Rx2 ------GGAGAAATCC Nativ -amplified by 106Rx2 ------GGAGAAATCC White Lady -amplified by 106Rx ------GGAGAAATCC Bzura-amplified by 106Rx2 ------GGAGAAATCC AJ249448_S.acaule_Rx2 TGATTTGCAACCGTTTTA--TGAAAAGCTCGAATCTTTAAGAGCTATTCTGGAGAAATCC AJ011801_Rx_gene_complete_cds TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AJ011801_Rx_gene_join_(11849-1 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AF195939_Gpa2_gene_complete_cd TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AJ249449_GPA2_exons_1-3 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AF266747_RGC1 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 TGATTTGCAACCGTTTTA--TGAAAAGATCGAATCTTTGAGAGCTATTCTGGAGAAATCC EU352874_SH-RGH6_gene_complete TGATTTGCAACCGTTTTA--TGAAAAGATCGAATCTTTGAGAGCTATTCTGGAGAAATCC AC237866_RH137K16__2833_ch_12 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AC237866_RH137K16_36092-ch_12 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AC237866_RH137K16_46342-ch_12 TGATTTGCAACCATTCTA--TGAAAAGCTTGAATCTTTGAGAGCTATTATGGAGAAATCC AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AC238225_RH125I04_29334 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AC238225_RH125I04_65982_ch_5 TGATTTGCAACCGTTTTA--TGAAAAGCTCGAATCTTTAAGAACTATTCTGGAGAATTCC AC238225_RH125I04_89012 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AC238291_RH153N17_34642_ch_12 TGATTTGCAACCGTTTTA--TGAAAAGATCGAATCTTTGAGAGCTATTCTGGAGAAATCC AC238291_RH153N17_96301_ch_12 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC AC238387_RH192P22_23420_ch_12 TGATTTGCAACCGTTTTA--TGAAAAGATCAAATCTTTGAGAGCTATTCTGGAGAAATCC AC238387_RH192P22_66050_ch_12 TGATTTGCAACCGTTTTA--TGAAAAGATCGAATCTTTGAGAGCTATTCTGGAGAAATCC PGSC0003DMB000000063:400004..4 TGATTTGCAACCGTTCTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAACCT PGSC0003DMB000000116:773867..7 TGATTTGCAACCGTTTTA--TGAAAAGCTCGAATCTTTAAGAACTATTCTGGAGAATTCC PGSC0003DMB000000116:833883..8 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC PGSC0003DMT400007570-Gpa2 TGATTTGCAACCGTTCTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAACCT PGSC0003DMT400010966-Gpa2 ------PGSC0003DMT400010970-Gpa2 TAATTTACAATCGTTCTA--CGAAAAGCTTGAATCGTTGATAGCTATTATGGAGAAACCT PGSC0003DMT400010987-NBS-codin TAAATTAAAGTTATGTCAAATGTACAAAATTGCCCTTTAA----TCTTGTGGGCTTAAAC PGSC0003DMT400020342-Gpa2 TGATTTGCAACCGTTTTA--TGAAAAGCTCGAATCTTTAAGAACTATTCTGGAGAATTCC PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 TGATTTGCAACCGTTTTA--TGAAAAGCTCGAATCTTTGAGAGCTATTCTAGAGAAATCC PGSC0003DMT400020360- Gpa2 TGATTTGCAACCATTCTA--TGAAAAGCTTGAATCTTTGAGAGCTATTATGGAGAAATCC PGSC0003DMT400036058-Gpa2 TAATTTGCAATCATTCTA--TGAAAAGTTTGAATCTTTGAGAGCTATTCTGGAGAAACAC PGSC0003DMT400036104-NBS-LRR TAATTTTCAATCGTTCTA--TAAGAAGCTTGAATCCCTGAGAGCTATTATG-AGAAACCT PGSC0003DMT400062366-Gpa2 TAATTTGCAATCATCCTA--TGAAAAGCTTGAATCCTTGAGAGCTATTATGGAAAAATCT PGSC0003DMT400071638- PSH-RGH7 TGATTTGCAATCATTCAA--TGAAAAGCTTGAATCTTTGAGAGCTAATCTGGAGAAACC- PGSC0003DMT400020353-Gpa2 TGATTTGCAACCGTTTTA--TGAAAAGCTCAAATCTTTGAGAGCTATTCTGGAGAAATCC

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490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 TGCAATGTAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTA Lorret -amplified by 106Rx2 TGCAATGTAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTA Courage- amplified by 106Rx2 TGCAATGTAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTA Luca -amplified by 106Rx2 TGCAATGTAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTA Nativ -amplified by 106Rx2 TGCAATGTAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTA White Lady -amplified by 106Rx TGCAATGTAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTA Bzura-amplified by 106Rx2 TGCAATGTAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTA AJ249448_S.acaule_Rx2 TGCAATGTAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCTTAGAGGTA AJ011801_Rx_gene_complete_cds TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTA AJ011801_Rx_gene_join_(11849-1 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTA AF195939_Gpa2_gene_complete_cd TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTA AJ249449_GPA2_exons_1-3 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTA AF266747_RGC1 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTAAAAGTTGAAATCGTAGAGGTA AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTA EU352874_SH-RGH6_gene_complete TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTA AC237866_RH137K16__2833_ch_12 TGCAATATAATGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATTGTAGAGGTA AC237866_RH137K16_36092-ch_12 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTA AC237866_RH137K16_46342-ch_12 TGCAATATAACGGGAGATCATGAGGGGTTAACAACCTTGGAAGTTGAAATCGCGGAGGTA AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTA AC238225_RH125I04_29334 TGCAATATAATGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATTGTAGAGGTA AC238225_RH125I04_65982_ch_5 TGCAATATAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTA AC238225_RH125I04_89012 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTA AC238291_RH153N17_34642_ch_12 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTA AC238291_RH153N17_96301_ch_12 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTA AC238387_RH192P22_23420_ch_12 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTA AC238387_RH192P22_66050_ch_12 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCATAGAGGTA PGSC0003DMB000000063:400004..4 TGGAAAGCAACAGACGATCTCGAGGCATTAACAAACTTGGAAGCTGAAATCTCAGAGGTA PGSC0003DMB000000116:773867..7 TGCAATATAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTA PGSC0003DMB000000116:833883..8 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTA PGSC0003DMT400007570-Gpa2 TGGAAAGCAACAGACGATCTCGAGGCATTAACAAACTTGGAAGCTGAAATCTCAGAGGTA PGSC0003DMT400010966-Gpa2 ------PGSC0003DMT400010970-Gpa2 TGCAGCATAATAGGCGATCTCGAGGCATTGGCAAGCTTGGAAGTAAAAATCGCGGGGGTT PGSC0003DMT400010987-NBS-codin ------ATGCCA---CGTGGAAAGTTAAAA-----GTAAAATGTTACCAAAAAAGGA PGSC0003DMT400020342-Gpa2 TGCAATATAACGGGCGATCATGAGGAGTTAACAATCTTGGAAGTTGAAATCGTAGAGGTA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 TGCAATATAACGGGCGATCATGAGGGGTTAACAACCTTGGAAGTTGAAATCACGGAGGTA PGSC0003DMT400020360- Gpa2 TGCAATATAACGGGAGATCATGAGGGGTTAACAACCTTGGAAGTTGAAATCGCGGAGGTA PGSC0003DMT400036058-Gpa2 ------ATGGGAGATCTTGATGCATTGAAAAGCTTGGAAGCTGAAATCATAGAACTT PGSC0003DMT400036104-NBS-LRR TGCAATATAACAGGTGATCTTGAAGTATTGAAAAGATTGGAAGCTGAAGTCGTAGAGCTT PGSC0003DMT400062366-Gpa2 TGCACGATAACGGGTGATCTTGAGGCATTAACAAGCTTGGAAGCTGAAATTGCAGCGGTA PGSC0003DMT400071638- PSH-RGH7 ------GATAGGCGATCTTGAGGCATTGATAAGCTTGGAAGCTGAAATCATAGAGGTT PGSC0003DMT400020353-Gpa2 TGCAATATAATGGGCGATCATGAGGGGTTAACAATCTTGGAAGTTGAAATCGCAGAGGTA

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550 560 570 580 590 600 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAAT Lorret -amplified by 106Rx2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAAT Courage- amplified by 106Rx2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAAT Luca -amplified by 106Rx2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAAT Nativ -amplified by 106Rx2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAAT White Lady -amplified by 106Rx GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAAT Bzura-amplified by 106Rx2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAAT AJ249448_S.acaule_Rx2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAGTGTTTTTTTAGCACAGAAT AJ011801_Rx_gene_complete_cds GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AJ011801_Rx_gene_join_(11849-1 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AF195939_Gpa2_gene_complete_cd GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACGGAAT AJ249449_GPA2_exons_1-3 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACGGAAT AF266747_RGC1 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAAAAT AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 GCATACACAACAGAAGGTATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT EU352874_SH-RGH6_gene_complete GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AC237866_RH137K16__2833_ch_12 GCATACACAGCAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AC237866_RH137K16_36092-ch_12 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AC237866_RH137K16_46342-ch_12 GCATACAAAGCAGAAGACATGGTTGATTCGAAATCAAGAAAAGTTTCTTCCGCAGAAACT AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AC238225_RH125I04_29334 GCATACACAGCAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AC238225_RH125I04_65982_ch_5 GCATACACAACAGAAGATATGGTTGACTTGGAATCAAGAAGTGTTTTTTTAGCACAGAAT AC238225_RH125I04_89012 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AC238291_RH153N17_34642_ch_12 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AC238291_RH153N17_96301_ch_12 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT AC238387_RH192P22_23420_ch_12 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTGTTTTGGCACAAAAT AC238387_RH192P22_66050_ch_12 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT PGSC0003DMB000000063:400004..4 GCATACAGTGCAGGGGATATGGTTGACTTGAAATCAAGAAATGTTCTTTTTGCACAAAAG PGSC0003DMB000000116:773867..7 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT PGSC0003DMB000000116:833883..8 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT PGSC0003DMT400007570-Gpa2 GCATACAGTGCAGGGGATATGGTTGACTTGAAATCAAGAAATGTTCTTTTTGCACAAAAG PGSC0003DMT400010966-Gpa2 ------PGSC0003DMT400010970-Gpa2 GCATACAGAGCAGAAGATGAGATTGACTCTAAATCAATAGAAGTTATACGCGCAAAAACA PGSC0003DMT400010987-NBS-codin A-AGGGGCCATTCTTTTTGAAACAGACTAAAAAGGAAAGGAGGTC------ATTCT PGSC0003DMT400020342-Gpa2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 GCATTCAAAGCAGAAGACATGGTTGATTCGAAATCAAGAAAAGTTTCTTCTGCAGAAACT PGSC0003DMT400020360- Gpa2 GCATACAAAGCAGAAGACATGGTTGATTCGAAATCAAGAAAAGTTTCTTCCGCAGAAACT PGSC0003DMT400036058-Gpa2 GTATGCACTACAGAAGATATTTTGGACTTGGAATCAAGAAATGTT------AAAAA PGSC0003DMT400036104-NBS-LRR GTATGCAGCACAGAAGATATTGTGGATTTGGAATCAAGAAATGTT------GAAAATC PGSC0003DMT400062366-Gpa2 GCCTACAGCACAGAAGATATGATTGAATCGGAATCAAGAAAAGTTTCCTTAGTAAAAACC PGSC0003DMT400071638- PSH-RGH7 GTATGCACCACAGAACATTTTTTGGACTCGGAATCAAGAAATGTTA------AAAATCC PGSC0003DMT400020353-Gpa2 GCATACACAACAGAAGATATGGTTGACTCGGAATCAAGAAATGTTTTTTTAGCACAGAAT

158

610 620 630 640 650 660 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 TTGGAGGAAAGAAACAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA Lorret -amplified by 106Rx2 TTGGAGGAAAGAAACAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA Courage- amplified by 106Rx2 TTGGAGGAAAGAAACAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA Luca -amplified by 106Rx2 TTGGAGGAAAGAAACAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA Nativ -amplified by 106Rx2 TCGGAGGAAAGAAACAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA White Lady -amplified by 106Rx TTGGAGGAAAGAAACAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA Bzura-amplified by 106Rx2 TTGGAGGAAAGAAACAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AJ249448_S.acaule_Rx2 TTGGAGGAAAGAAACAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AJ011801_Rx_gene_complete_cds TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGA AJ011801_Rx_gene_join_(11849-1 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCCTGGAACAAGCACTAGA AF195939_Gpa2_gene_complete_cd GTGGGGAAAAGAAGCAG-GGCTATGTGGGGGATTTTTTTCGTCTTGGAACAAGCACTAGA AJ249449_GPA2_exons_1-3 GTGGGGAAAAGAAGCAG-GGCTATGTGGGGGATTTTTTTCGTCTTGGAACAAGCACTAGA AF266747_RGC1 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA EU352874_SH-RGH6_gene_complete TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC237866_RH137K16__2833_ch_12 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC237866_RH137K16_36092-ch_12 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC237866_RH137K16_46342-ch_12 GTAATTACACGAAGCAA-AGCTTTCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGA AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC238225_RH125I04_29334 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC238225_RH125I04_65982_ch_5 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC238225_RH125I04_89012 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC238291_RH153N17_34642_ch_12 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC238291_RH153N17_96301_ch_12 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC238387_RH192P22_23420_ch_12 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA AC238387_RH192P22_66050_ch_12 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA PGSC0003DMB000000063:400004..4 GCAAAGA-ATAAAGCAAGAGCTTTCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGA PGSC0003DMB000000116:773867..7 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA PGSC0003DMB000000116:833883..8 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA PGSC0003DMT400007570-Gpa2 GCAAAGA-ATAAAGCAAGAGCTTTCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGA PGSC0003DMT400010966-Gpa2 ------ATGATCATTGCTGA PGSC0003DMT400010970-Gpa2 GATACTTTGCGAGGGAA-AGCTTTTTGGAAACTGTGTTGTTTCTTGGAACAAGTGGTAGA PGSC0003DMT400010987-NBS-codin TTTTGAAACG-GAGGGGTAGCTTTTTGGAAACTTCATTCTCTCTTGAAACAAGCAGTAGG PGSC0003DMT400020342-Gpa2 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 GTAATTACACGAAGCAA-AGCTTTCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGA PGSC0003DMT400020360- Gpa2 GTAATTACACGAAGCAA-AGCTTTCTGGGAACTCTGTTGTTCCTTGGAACAAGAGGTAGA PGSC0003DMT400036058-Gpa2 TCCAATTTCAAGAATAATAGCTTTTTGGAAACTTCATTCTCTCTTGAAACAAGCAGTAGG PGSC0003DMT400036104-NBS-LRR CAATTTCAAGAATTAAT-AACTTTTTGGAAACTTCATTTTCTCTTGAAACAAGTAGTAGG PGSC0003DMT400062366-Gpa2 TCGGTTACACGAAGAAT-AGATTTTTGGGAACTTTGTTTCTCCCTGAAATAAGCAGTAGA PGSC0003DMT400071638- PSH-RGH7 AAT--TTCACAAATAAT-AGCTTCTTGGAAATTTCATCATCTCTTGGAACATGCCGTTGG PGSC0003DMT400020353-Gpa2 TTGGAGGAAAGAAGCAG-GGCTATGTGGGAGATTTTTTTCGTCTTGGAACAAGCACTAGA

159

670 680 690 700 710 720 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------Lorret -amplified by 106Rx2 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------Courage- amplified by 106Rx2 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------Luca -amplified by 106Rx2 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------Nativ -amplified by 106Rx2 GTGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------White Lady -amplified by 106Rx ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------Bzura-amplified by 106Rx2 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AJ249448_S.acaule_Rx2 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AJ011801_Rx_gene_complete_cds ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AJ011801_Rx_gene_join_(11849-1 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AF195939_Gpa2_gene_complete_cd ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AJ249449_GPA2_exons_1-3 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AF266747_RGC1 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAGC------AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAGC------EU352874_SH-RGH6_gene_complete ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AC237866_RH137K16__2833_ch_12 ATGCATTGCTTCCACCGTGAAACAGTGGATGGCAGC------AC237866_RH137K16_36092-ch_12 ATACATTGATTCCACCGTGAAACAGTGGATGGCAGC------AC237866_RH137K16_46342-ch_12 ATGCATTGATTCCTTCATGATGCAGTGGTTGGCAAT------AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 ATACATTGATTCCACCGTGAAACAGTGGATGGCAGC------AC238225_RH125I04_29334 ATGCATTGCTTCCACCGTGAAACAGTGGATGGCAGC------AC238225_RH125I04_65982_ch_5 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAGC------AC238225_RH125I04_89012 ATACATTGATTCCACCGTGAAACAGTGGATGGCAAC------AC238291_RH153N17_34642_ch_12 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AC238291_RH153N17_96301_ch_12 ATACATTGATTCTACCGTGAAACAGTGGATGGCAGC------AC238387_RH192P22_23420_ch_12 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAAC------AC238387_RH192P22_66050_ch_12 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAGC------PGSC0003DMB000000063:400004..4 ACGCATTGATTCCCTCATGATGCAGTGGTTGGCAAT------PGSC0003DMB000000116:773867..7 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAGC------PGSC0003DMB000000116:833883..8 ATGCATTGCTTCCACCGTGAAACAGTGGATGGCAGC------PGSC0003DMT400007570-Gpa2 ACGCATTGATTCCCTCATGATGCAGTGGTTGGCAAT------PGSC0003DMT400010966-Gpa2 GAAGAACATGTACATGACAAAAAACAAAGATAATAT------PGSC0003DMT400010970-Gpa2 ACACATTGATTCCATCATGAAGGAGTGGATGGCAATCCGGGACGGGTGCAGCAACATCAA PGSC0003DMT400010987-NBS-codin ACGCGTTGATTCCATGCTCAATGAGTGGATGGAAAT------PGSC0003DMT400020342-Gpa2 ATGCATTGATTCCACCGTGAAACAGTGGATGGCAGC------PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 ATGTATTGATTCCTTCATGATGCAGTGGTTGGCAAT------PGSC0003DMT400020360- Gpa2 ATGCATTGATTCCTTCATGATGCAGTGGTTGGCAAT------PGSC0003DMT400036058-Gpa2 ACGCATTGATTCCACAATGAACAAGTGGATGGAAAT------PGSC0003DMT400036104-NBS-LRR GCGCGTTGATTCCATGGTGAACAAGTGGAAAGATGT------PGSC0003DMT400062366-Gpa2 ACACATTGGTTGCACAATGAACAAGTGGAAGGAAAT------PGSC0003DMT400071638- PSH-RGH7 AGACATTGATTCCAGAGTCAACAAGTGGATGAAAAT------PGSC0003DMT400020353-Gpa2 ATGCATTGCTTCCACCGTGAAACAGTGGATGGCAGC------

160

730 740 750 760 770 780 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 ------ATCGGAC------Lorret -amplified by 106Rx2 ------ATCGGAC------Courage- amplified by 106Rx2 ------ATCGGAC------Luca -amplified by 106Rx2 ------ATCGGAC------Nativ -amplified by 106Rx2 ------ATCGGAC------White Lady -amplified by 106Rx ------ATCGGAC------Bzura-amplified by 106Rx2 ------ATCGGAC------AJ249448_S.acaule_Rx2 ------ATCGGAC------AJ011801_Rx_gene_complete_cds ------ATCGGAC------AJ011801_Rx_gene_join_(11849-1 ------ATCGGAC------AF195939_Gpa2_gene_complete_cd ------ATCGGAC------AJ249449_GPA2_exons_1-3 ------ATCGGAC------AF266747_RGC1 ------ATCGGAC------AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 ------ATCGGAC------EU352874_SH-RGH6_gene_complete ------ATCGGAC------AC237866_RH137K16__2833_ch_12 ------ATCGGAC------AC237866_RH137K16_36092-ch_12 ------ATCGGAC------AC237866_RH137K16_46342-ch_12 ------ATGGGAC------AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 ------ATCGGAC------AC238225_RH125I04_29334 ------ATCGGAC------AC238225_RH125I04_65982_ch_5 ------ATCGGAC------AC238225_RH125I04_89012 ------ATCGGAC------AC238291_RH153N17_34642_ch_12 ------ATCGGAC------AC238291_RH153N17_96301_ch_12 ------ATCGGAC------AC238387_RH192P22_23420_ch_12 ------ATCGGAC------AC238387_RH192P22_66050_ch_12 ------ATCGGAC------PGSC0003DMB000000063:400004..4 ------ATGGAACTG------PGSC0003DMB000000116:773867..7 ------ATCGGAC------PGSC0003DMB000000116:833883..8 ------ATCGGAC------PGSC0003DMT400007570-Gpa2 ------ATGGAACTG------PGSC0003DMT400010966-Gpa2 ------PGSC0003DMT400010970-Gpa2 AGAGTATATGATTGTCTCAGAATCGAGCAAAAGTTTTTTGGCAAAATCTAAAATGAGACA PGSC0003DMT400010987-NBS-codin ------ACACAACAT------PGSC0003DMT400020342-Gpa2 ------ATCGGAC------PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 ------ATGGGACTG------PGSC0003DMT400020360- Gpa2 ------ATGGGACTG------PGSC0003DMT400036058-Gpa2 ------GCAGAACAT------PGSC0003DMT400036104-NBS-LRR ------PGSC0003DMT400062366-Gpa2 ------GCAGAACAA------PGSC0003DMT400071638- PSH-RGH7 ------GCAAAAATT------PGSC0003DMT400020353-Gpa2 ------ATCGGAC------

161

790 800 810 820 830 840 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 ------Lorret -amplified by 106Rx2 ------Courage- amplified by 106Rx2 ------Luca -amplified by 106Rx2 ------Nativ -amplified by 106Rx2 ------White Lady -amplified by 106Rx ------Bzura-amplified by 106Rx2 ------AJ249448_S.acaule_Rx2 ------AJ011801_Rx_gene_complete_cds ------AJ011801_Rx_gene_join_(11849-1 ------AF195939_Gpa2_gene_complete_cd ------AJ249449_GPA2_exons_1-3 ------AF266747_RGC1 ------AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 ------EU352874_SH-RGH6_gene_complete ------AC237866_RH137K16__2833_ch_12 ------AC237866_RH137K16_36092-ch_12 ------AC237866_RH137K16_46342-ch_12 ------AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 ------AC238225_RH125I04_29334 ------AC238225_RH125I04_65982_ch_5 ------AC238225_RH125I04_89012 ------AC238291_RH153N17_34642_ch_12 ------AC238291_RH153N17_96301_ch_12 ------AC238387_RH192P22_23420_ch_12 ------AC238387_RH192P22_66050_ch_12 ------PGSC0003DMB000000063:400004..4 ------PGSC0003DMB000000116:773867..7 ------PGSC0003DMB000000116:833883..8 ------PGSC0003DMT400007570-Gpa2 ------PGSC0003DMT400010966-Gpa2 ------PGSC0003DMT400010970-Gpa2 AGGACTAGCGTTTTGGAAACGAGCAGTAGGACACATTGATTCTATGATAAACAACAGGAT PGSC0003DMT400010987-NBS-codin ------PGSC0003DMT400020342-Gpa2 ------PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 ------PGSC0003DMT400020360- Gpa2 ------PGSC0003DMT400036058-Gpa2 ------PGSC0003DMT400036104-NBS-LRR ------PGSC0003DMT400062366-Gpa2 ------PGSC0003DMT400071638- PSH-RGH7 ------PGSC0003DMT400020353-Gpa2 ------

162

850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT Lorret -amplified by 106Rx2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT Courage- amplified by 106Rx2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT Luca -amplified by 106Rx2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT Nativ -amplified by 106Rx2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT White Lady -amplified by 106Rx ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT Bzura-amplified by 106Rx2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AJ249448_S.acaule_Rx2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AJ011801_Rx_gene_complete_cds ------AGCATGAAAGATCTAAAACCACAAACTAGCTCGCTTGT AJ011801_Rx_gene_join_(11849-1 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCGCTTGT AF195939_Gpa2_gene_complete_cd ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AJ249449_GPA2_exons_1-3 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AF266747_RGC1 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 ------AGCATGAAAGATCTAAAACAACAAACTAGCTCACTTGT EU352874_SH-RGH6_gene_complete ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC237866_RH137K16__2833_ch_12 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC237866_RH137K16_36092-ch_12 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC237866_RH137K16_46342-ch_12 ------TGCTTTAAAGATTTCAAAGCACAAAATTTTTGTCTATC AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC238225_RH125I04_29334 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC238225_RH125I04_65982_ch_5 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC238225_RH125I04_89012 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC238291_RH153N17_34642_ch_12 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC238291_RH153N17_96301_ch_12 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC238387_RH192P22_23420_ch_12 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT AC238387_RH192P22_66050_ch_12 ------AGCATGAAAGATCTAAAACAACAAACTAGCTCACTTGT PGSC0003DMB000000063:400004..4 ------GTACAGTAAAATCAAAGATTTGAAAGCACGAAAGTTTTCTCTAGC PGSC0003DMB000000116:773867..7 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT PGSC0003DMB000000116:833883..8 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT PGSC0003DMT400007570-Gpa2 ------GTACAGTAAAATCAAAGATTTGAAAGCACGAAAGTTTTCTCTAGC PGSC0003DMT400010966-Gpa2 ------TATTGCCAATACATCGTCATCTCA PGSC0003DMT400010970-Gpa2 GATGAAGCAGAGCATGTACACCAAAAGCAAAGATGTAGAAGGACAGAATTTGACTCTTGC PGSC0003DMT400010987-NBS-codin ------GTACACCAAAAGCAAAGATGAAGAAGCACAT------CTTGC PGSC0003DMT400020342-Gpa2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 ------GTACAACAACATTAAAGATTTCAAAGCACAAAATTTCTGTCTATC PGSC0003DMT400020360- Gpa2 ------GTACAACAACTTTAAAGATTTCAAAGCACAAAATTTTTGTCTATC PGSC0003DMT400036058-Gpa2 ------GTACACCAAAAGGAAAGATGAAGAAGCACATAACTTGGATCTTGC PGSC0003DMT400036104-NBS-LRR ------AGAAGCACATAACTTGGCTTTCAC PGSC0003DMT400062366-Gpa2 ------GTACAACAATATCAAAGACCTCAAAGTACAAAGCATGTCTCTTGG PGSC0003DMT400071638- PSH-RGH7 ------GTACACCAGAACCAAAGATGTACAAGGGAATAACTCGGCTCTCGC PGSC0003DMT400020353-Gpa2 ------AGCATGAAAGATCTAAAACCACAAACTAGCTCACTTGT

163

910 920 930 940 950 960 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 C---AGTTTACCTGAC---CATGCTTTTGAGCAGCCTGGGAAT---ATAATGGTTGGCCG Lorret -amplified by 106Rx2 C---AGTTTACCTGAC---CATGCTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCG Courage- amplified by 106Rx2 C---AGTTTACCTGAC---CATGCTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCG Luca -amplified by 106Rx2 C---AGTTTACCTGAC---CATGCTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCG Nativ -amplified by 106Rx2 C---AGTTTACCTGAC---CATGCTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCG White Lady -amplified by 106Rx C---AGTTTACCTGAC---CATGCTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCG Bzura-amplified by 106Rx2 C---AGTTTACCTGAC---CATGCTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCG AJ249448_S.acaule_Rx2 C---AGTTTACCTGAC---CATGCTTTTGAGCAGCCTGAGAAT---ATAATGGTTGGCCG AJ011801_Rx_gene_complete_cds C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AJ011801_Rx_gene_join_(11849-1 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AF195939_Gpa2_gene_complete_cd C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AJ249449_GPA2_exons_1-3 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AF266747_RGC1 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGATAAT---ATAATGGTTGGCCG AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG EU352874_SH-RGH6_gene_complete C---AGTTTACCTGAA---CATGCTTTTGAGCAGCCCGAGAAT---ATAATGGTTGGCTA AC237866_RH137K16__2833_ch_12 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AC237866_RH137K16_36092-ch_12 C---AGTTTACCTGAA---CATGCTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AC237866_RH137K16_46342-ch_12 C---AAGATACCTGAA---CGTGCTGTTGAGCGGTCCGAGGAT---ATAATGGTTGGCTA AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 C---AGTTTACCTGAA---CATGCTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AC238225_RH125I04_29334 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AC238225_RH125I04_65982_ch_5 C---AGTTTACCTGAA---CATGCTGTTGAACAGCCCGAGAAT---ATAATGGTTGGCCG AC238225_RH125I04_89012 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AC238291_RH153N17_34642_ch_12 C---AGTTTACCTGAA---CATGCTTTTGAGCAGCCCGAGAAT---ATAATGGTTGGCTA AC238291_RH153N17_96301_ch_12 C---AGTTTACCTGAA---CATGCTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AC238387_RH192P22_23420_ch_12 C---AGTTTACCTGAA---CATGCTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG AC238387_RH192P22_66050_ch_12 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG PGSC0003DMB000000063:400004..4 C------GAA---CATGCTGTAGGGAAGCCTGAGAAT---ATAATGGTTGGCCA PGSC0003DMB000000116:773867..7 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG PGSC0003DMB000000116:833883..8 C---AGTTTACCTGAA---CATGCTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG PGSC0003DMT400007570-Gpa2 CCCTAGTATACCTGAA---CATGCTGTAGGGAAGCCTGAGAAT---ATAATGGTTGGCCA PGSC0003DMT400010966-Gpa2 A------CATGCCTTA---TTAGAGCCTGATGAGAACATGAAT---ATG---GTTGGTGA PGSC0003DMT400010970-Gpa2 C---AGTACATCTCGA---CATTCCTTGGAGCATGAGAATA------TGATGGTTGGCCA PGSC0003DMT400010987-NBS-codin TAGTACTACATCGATATCTCAACATGTTGTGGAGCCTCAAGAT---ATGATGGTTGGACA PGSC0003DMT400020342-Gpa2 C---AGTTTACCTGAA---CATGATGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 C---AAGATACCTGAA---CTTGCTGTTGAGCGGTCCGAGGTT---ATAATGGTTGGCTA PGSC0003DMT400020360- Gpa2 C---AAGATACCTGAA---CGTGCTGTTGAGCGGTCCGAGGAT---ATAATGGTTGGCTA PGSC0003DMT400036058-Gpa2 TAGTACTACATCGATGTCTCAACATGTTGTGGAGCCTCAGGAT---ATGATGGTTGGACA PGSC0003DMT400036104-NBS-LRR C------AGTACTACA---TCTCAACATGTTGTGGAGCCGATT------GTTGGCCA PGSC0003DMT400062366-Gpa2 C------GATGTATCT---CGACTTGCTGTAGAGACTGAGAAT---GTGATGGTTGGCCA PGSC0003DMT400071638- PSH-RGH7 C---AGTACATCTCAA---CATGTTGTGGAGCCTGAGGATAATTATATGATGGTTGGTCA PGSC0003DMT400020353-Gpa2 C---AGTTTACCTGAA---CATGCTGTTGAGCAGCCCGAGAAT---ATAATGGTTGGCCG

164

970 980 990 1000 1010 1020 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA Lorret -amplified by 106Rx2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA Courage- amplified by 106Rx2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA Luca -amplified by 106Rx2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA Nativ -amplified by 106Rx2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA White Lady -amplified by 106Rx TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA Bzura-amplified by 106Rx2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA AJ249448_S.acaule_Rx2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA AJ011801_Rx_gene_complete_cds TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AJ011801_Rx_gene_join_(11849-1 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AF195939_Gpa2_gene_complete_cd TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AJ249449_GPA2_exons_1-3 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AF266747_RGC1 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCAAGAGGAGGAAGGGAACTAGA EU352874_SH-RGH6_gene_complete TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA AC237866_RH137K16__2833_ch_12 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA AC237866_RH137K16_36092-ch_12 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AC237866_RH137K16_46342-ch_12 TGAAAATGAATTCAAGATGAT---GCTGGATCAACTTGCTAGAGGAGAAAGAGAACTAGC AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AC238225_RH125I04_29334 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA AC238225_RH125I04_65982_ch_5 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAGCTAGA AC238225_RH125I04_89012 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AC238291_RH153N17_34642_ch_12 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA AC238291_RH153N17_96301_ch_12 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA AC238387_RH192P22_23420_ch_12 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGTTAGAGGAGGAAGGGAACTAGA AC238387_RH192P22_66050_ch_12 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCAAGAGGAGGAAGGGAACTAGA PGSC0003DMB000000063:400004..4 TGAAAATGAATTCGAGATGAT---GCTCAATCAAGTTGCTGGAGGAGAAAGGGAACTAGA PGSC0003DMB000000116:773867..7 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA PGSC0003DMB000000116:833883..8 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA PGSC0003DMT400007570-Gpa2 TGAAAATGAATTCGAGATGAT---GCTCAATCAAGTTGCTGGAGGAGAAAGGGAACTAGA PGSC0003DMT400010966-Gpa2 TGAAAATGAATTCGAGATGAT---GCAGGGGAGACTTACAAGAGGAGGAAGGGAACTAAA PGSC0003DMT400010970-Gpa2 TGAAAATGAATTCGAGATGAT---GCAGGACCAACTTACTAGAGGTGCCAGTGATCTAGA PGSC0003DMT400010987-NBS-codin TGAAAATGAACTCGAGATGATCATGCAGGATCAGCTTGCTAGAGGAGCAAGTGAACTTGA PGSC0003DMT400020342-Gpa2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 TGAAAATGAATTCAAGATGAT---GCTGGATCAACTTGCTAGAGGAGAAAGAGAACTAGA PGSC0003DMT400020360- Gpa2 TGAAAATGAATTCAAGATGAT---GCTGGATCAACTTGCTAGAGGAGAAAGAGAACTAGC PGSC0003DMT400036058-Gpa2 TGAAAATGAATTCGAGATAATCATGGAGGATCAGCTTGCTAGAGGAGCAAGTGAACTTGA PGSC0003DMT400036104-NBS-LRR TAAAAATGAATTCGAGATGAT---GCAGGATCAACTTGTCAGAGGAGCAAGTGAACTAGA PGSC0003DMT400062366-Gpa2 TGAAAATGAATTTGAGATGAT---GCAGGATCAAATTGCTAGAGGATCAAATGAACTAGA PGSC0003DMT400071638- PSH-RGH7 TGAAAATGAACTCGAGATGAT---GCAGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA PGSC0003DMT400020353-Gpa2 TGAAAATGAATTTGAGATGAT---GCTGGATCAACTTGCTAGAGGAGGAAGGGAACTAGA

165

1030 1040 1050 1060 1070 1080 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA Lorret -amplified by 106Rx2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA Courage- amplified by 106Rx2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA Luca -amplified by 106Rx2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA Nativ -amplified by 106Rx2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTG White Lady -amplified by 106Rx AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA Bzura-amplified by 106Rx2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA AJ249448_S.acaule_Rx2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA AJ011801_Rx_gene_complete_cds AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTA AJ011801_Rx_gene_join_(11849-1 AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTA AF195939_Gpa2_gene_complete_cd AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTA AJ249449_GPA2_exons_1-3 AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTA AF266747_RGC1 AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTA AF266746_RGC3_pseudogene ------EU352875_SH-RGH7 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTATA EU352874_SH-RGH6_gene_complete AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTA AC237866_RH137K16__2833_ch_12 AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTA AC237866_RH137K16_36092-ch_12 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA AC237866_RH137K16_46342-ch_12 AGTTGTATCAATTGTAGGTATGGGAGGCATTGGCAAGACAACTTTGGCTACGAAACTCTA AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA AC238225_RH125I04_29334 AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTGCAAAACTCTA AC238225_RH125I04_65982_ch_5 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA AC238225_RH125I04_89012 AGTTGTCTCAATCGTAGGGATGGGAGGCATTGGGAAAACAACTTTGGCTACAAAACTCTA AC238291_RH153N17_34642_ch_12 AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTA AC238291_RH153N17_96301_ch_12 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA AC238387_RH192P22_23420_ch_12 AGTTGTCTCAATCGTAGGGATGGGAGGCATCGGGAAAACAACTTTGGCTACAAAACTCTA AC238387_RH192P22_66050_ch_12 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTATA PGSC0003DMB000000063:400004..4 AGTTGTCTCAATTGTAGGTATGGGAGGCATTGGCAAAACAACTTTGGCTACAAAACTCTA PGSC0003DMB000000116:773867..7 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA PGSC0003DMB000000116:833883..8 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA PGSC0003DMT400007570-Gpa2 AGTTGTCTCAATTGTAGGTATGGGAGGCATTGGCAAAACAACTTTGGCTACAAAACTCTA PGSC0003DMT400010966-Gpa2 AGTTGTCTCGATTGTCGGTATGGGTGGCATTGGCAAGACAACTTTGGCTAACAAAATCTA PGSC0003DMT400010970-Gpa2 AATTGTCTCAATCGTTGGGATGGGGGGCTTAGGCAAGACAACTTTGGCTAACAAAATTTT PGSC0003DMT400010987-NBS-codin AGTTGTCTCCATTGTAGGTATTGGGGGCATCGGTAAGACAACTTTGGCTGACAAAATTTA PGSC0003DMT400020342-Gpa2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 AGTTGTATCAATTGTAGGTATGGGAGGCATTGGCAAGACAACTTTGGCAACAAAACTCTA PGSC0003DMT400020360- Gpa2 AGTTGTATCAATTGTAGGTATGGGAGGCATTGGCAAGACAACTTTGGCTACGAAACTCTA PGSC0003DMT400036058-Gpa2 AGTTGTCTCCATTGTAGGTATGGGGGGCATCGGTAAGACAACTTTGGCTGACAAAATTTA PGSC0003DMT400036104-NBS-LRR AGTTGTCTCAATTGTAGGTATGGGTGGCATCGGTAAAACAACTTTGGCTAACAAAATTTA PGSC0003DMT400062366-Gpa2 AGTTGTCTCAATTGTCGGGATGGGAGGCATTGGCAAGACAACTTTGGCTAACAAAGTTTA PGSC0003DMT400071638- PSH-RGH7 AGTTGTCTCAATTGT------AGGCATCGGTAAGACAACTTTGGCGAACAAAATCTA PGSC0003DMT400020353-Gpa2 AGTTGTCTCAATCGTAGGGATGGGAGGTATCGGGAAAACAACTTTGGCTACAAAACTCTA

166

1090 1100 1110 1120 1130 1140 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA Lorret -amplified by 106Rx2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA Courage- amplified by 106Rx2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA Luca -amplified by 106Rx2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA Nativ -amplified by 106Rx2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA White Lady -amplified by 106Rx TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA Bzura-amplified by 106Rx2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AJ249448_S.acaule_Rx2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AJ011801_Rx_gene_complete_cds TAGTGATCCGTGCATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AJ011801_Rx_gene_join_(11849-1 TAGTGATCCGTGCATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AF195939_Gpa2_gene_complete_cd TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AJ249449_GPA2_exons_1-3 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AF266747_RGC1 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AF266746_RGC3_pseudogene -AAAACAACTTTGGCTACAAAACTCTATACTGATCCTTACATTATGTCTCGATTTGATAT EU352875_SH-RGH7 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA EU352874_SH-RGH6_gene_complete TAGTGATCCTTGCATTATGCCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AC237866_RH137K16__2833_ch_12 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGTAACTGTTTCGCAGGA AC237866_RH137K16_36092-ch_12 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AC237866_RH137K16_46342-ch_12 CAACGATCCATGCATGATGTATCGTTTTGACATTCGTGCTAAAGCTACTGTTTCACAAGA AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AC238225_RH125I04_29334 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGTAACTGTTTCGCAGGA AC238225_RH125I04_65982_ch_5 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTACAAAAGTAACTGTTTCACAGGA AC238225_RH125I04_89012 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AC238291_RH153N17_34642_ch_12 TAGTGATCCTTGCATTATGCCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AC238291_RH153N17_96301_ch_12 TAGTGAACCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA AC238387_RH192P22_23420_ch_12 TAGTGATCCTTGCATTATGTCTCGATTTGATATTCATGCAAAAGCAACTGTTTCACAAGA AC238387_RH192P22_66050_ch_12 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA PGSC0003DMB000000063:400004..4 TAGAGATCCACGCATTATGTCTCACTTTGACATTCTTGCAAAAGCTACTGTTTCGCAAGA PGSC0003DMB000000116:773867..7 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA PGSC0003DMB000000116:833883..8 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA PGSC0003DMT400007570-Gpa2 TAGAGATCCACGCATTATGTCTCACTTTGACATTCTTGCAAAAGCTACTGTTTCGCAAGA PGSC0003DMT400010966-Gpa2 TAGTGATCCATTCATTATGTCTCACTTTGACATTCGTGGAAACGTAACTGTTTCACAAGA PGSC0003DMT400010970-Gpa2 CTGTGACCCATTCGTTATGTCTTGTTTTGATATACGTGCAAAAGTCACCATCTCACAAGA PGSC0003DMT400010987-NBS-codin TAATGATCCATTCATAATGTCACACTTTGACATTCGTGCAAAAGCTACTGTTTCACAAGA PGSC0003DMT400020342-Gpa2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 CAACGATCCATGCATGATGTATCGCTTTGACATACGTGCAAAAGCTACTGTTTCACAAGA PGSC0003DMT400020360- Gpa2 CAACGATCCATGCATGATGTATCGTTTTGACATTCGTGCTAAAGCTACTGTTTCACAAGA PGSC0003DMT400036058-Gpa2 TAATGATCCATTCATAATGTCACACTTTGACATTCGTGCAAAAGCTACTGTTTTACAAGA PGSC0003DMT400036104-NBS-LRR CAATGATTCATTCATTATGTCTCACTTTGACGTTCGTGCAAAAGCTACTGTTTCACAAGA PGSC0003DMT400062366-Gpa2 CAGTGATCCATTCATTATGTCTCGCTTTGACATCCGTGCAAAAATTACTGTCTCACAAGA PGSC0003DMT400071638- PSH-RGH7 CAATGATCCATTCATAATGTCACATTTTGACATTCGTGCAAAAGCTACTGTTTCACAAGA PGSC0003DMT400020353-Gpa2 TAGTGATCCTTACATTATGTCTCGATTTGATATTCGTGCAAAAGCAACTGTTTCACAAGA

167

1150 1160 1170 1180 1190 1200 ....|....|....|....|....|....|....|....|....|....|....|....| Hopehely -amplified by 106Rx2 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAG------Lorret -amplified by 106Rx2 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAG------Courage- amplified by 106Rx2 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAG------Luca -amplified by 106Rx2 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAG------Nativ -amplified by 106Rx2 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAG------White Lady -amplified by 106Rx GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAG------Bzura-amplified by 106Rx2 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAG------AJ249448_S.acaule_Rx2 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGA AJ011801_Rx_gene_complete_cds GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGA AJ011801_Rx_gene_join_(11849-1 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGA AF195939_Gpa2_gene_complete_cd GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTA AJ249449_GPA2_exons_1-3 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTA AF266747_RGC1 GTATTGTGTGAGAAATGTATTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATGA AF266746_RGC3_pseudogene TCGTGCAAAAGCAACTGTTTCACAAGAGTATTGTGTGAGAAATGTACTCCTAGGCCTTCT EU352875_SH-RGH7 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTA EU352874_SH-RGH6_gene_complete GTATTGTGTGAGAAATGTACTACAAGGCCTTCTTTCTTCGATAAGTGATGAACCTGATGA AC237866_RH137K16__2833_ch_12 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTTCTTCGATAAGTGATGAACCTGAGAA AC237866_RH137K16_36092-ch_12 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGA AC237866_RH137K16_46342-ch_12 GTATTGTGTGAGAAATGTTTTCCTAGACCTTCTTTCTTGTATAAGTGATAAACCTTATGA AC237866_RH137K16_50554_ch_12 ------Primers_used_to_amplify gene_A ------AC238225_RH125I04_13358_ch_5 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGA AC238225_RH125I04_29334 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTTCTTCGATAAGTGATGAACCTGAGAA AC238225_RH125I04_65982_ch_5 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTTCTTCGATAAGTAATGAACCTGATGA AC238225_RH125I04_89012 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGA AC238291_RH153N17_34642_ch_12 GTATTGTGTGAGAAATGTACTACAAGGCCTTCTTTCTTCGATAAGTGATGAACCTGATGA AC238291_RH153N17_96301_ch_12 GTATTGTGTGAGAAATGTACTTCAAGGCCTTCTTTCTTCGATAAGTGATGAACCTGATGA AC238387_RH192P22_23420_ch_12 GTATTGTGTGAGAAATGTACTCCAAGGCCTTCTTTCTTCGATAAGTGATGAACCTGATGA AC238387_RH192P22_66050_ch_12 GTATTGTGTGAGAAATGTACTCCTAGGCCTTCTTTCTTTGACAAGTGATGAACCTGATTA PGSC0003DMB000000063:400004..4 GTACTGTGTGAGAAATGTACTCCTTGCCCTTCTTGCTTCGACAAGTGAGGAACCTGATGA PGSC0003DMB000000116:773867..7 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTTCTTCGATAAGTAATGAACCTGATGA PGSC0003DMB000000116:833883..8 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGA PGSC0003DMT400007570-Gpa2 GTACTGTGTGAGAAATGTACTCCTTGCCCTTCTTGCTTCGACAAGTGAGGAACCTGATGA PGSC0003DMT400010966-Gpa2 GTATTGTAGGGAATATGTACTCCTAGGTCTTCTTTCTTCTGTAAGTGGAATGAGTAGTCA PGSC0003DMT400010970-Gpa2 GTATTGTGTGAGAAATGTACTCTTATACCTTCTTTATTCCGTACGTGGAAAGACTGATGC PGSC0003DMT400010987-NBS-codin GTATTGTGCGAAAAAAGTACTCCTAAGTCTTCTTTCTTCGACTAGTGGAAAGATCGATGA PGSC0003DMT400020342-Gpa2 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTTCTTCGATAAGTAATGAACCTGATGA PGSC0003DMT400020346-NBS-LRR ------PGSC0003DMT400020349-Gpa2 GTATTGTGTGAGAAATGTTTTCCTAGACCTTCTTTCTTGTATAAGTGATAAACCTTATGA PGSC0003DMT400020360- Gpa2 GTATTGTGTGAGAAATGTTTTCCTAGACCTTCTTTCTTGTATAAGTGATAAACCTTATGA PGSC0003DMT400036058-Gpa2 GTATTGTGCGAAAAAAGTACTCCTAAGTCTTCTTTCTTCGACTAATGGAAAGATCGATGA PGSC0003DMT400036104-NBS-LRR GCATTGTGTGAGAAATGTACTCTTAACCCTTCTTTCTTGTATTAGTGTAAAGACTGATGA PGSC0003DMT400062366-Gpa2 GTATTGTGCAAGAAATGTACTTCTAGGCCTTCTTTCTTCTGTAAGTGGAAAGGCTGATGA PGSC0003DMT400071638- PSH-RGH7 GTATTGCGAGAAAAATGTA------TGC PGSC0003DMT400020353-Gpa2 GTATTGTGTGAGAAATGTAATCCTAGGCCTTCTTCCTTCGATAAGTGATGAACCTGATGA

168

Appendix 8 Annotations and BLAST scores of differentially expressed sequences induced with PVYNTN in White Lady

SGN BLASTX NCBI BLAST X Size Clone ID Accession no. Putative function Coverage E-Value Identity Putative function Coverage E-Value Identity (bp) Accession no. (%) (%) (%) (%)

Cont1 831 PGSC0003DMG402019042 Cytochrome P450 like_TBP 26.71 3.6e-31 86.67 BAA10929.1 Cytochrome P450 like_TBP 60 3e-70 80

Cont2 558 PGSC0003DMG400031495 Senescence-associated protein 5.0e-44 29.57 100 BAB33421 Putative senescence-associated 85 2e-58 81 protein

F2-164 398 PGSC0003DMG400026665 Zinc knuckle (CcHc-type) family protein 24.12 1.6e-14 100 EOX97864 Zinc knuckle family protein isoform 2 22 8e-10 90

175f2 390 PGSC0003DMG400023055 F-box protein 99.50 4.8e-68 100 AAU93580 Putative F-box protein 98 3e-82 92

79r2 245 PGSC0003DMG400031277 F-box domain-containing protein 99.18 5.9e-24 62.96 ABO92989 F-box domain-containing protein 97 2e-26 65

161r2 453 PGSC0003DMG400043984 Pectinesterase inhibitor 58.71 5.0e-80 100 XP_004248923 Probable pectinesterase/pectinesterase 98 2e-57 54 inhibitor 12-like

6r2 809 PGSC0003DMG400008384 CTR1 kinase 11.52% 9.7e-22 96.2 NP_001234457 CTR1-like protein kinase 28 6e-18 64

11r,2 386 PGSC0003DMG400027904 Relative to APETALA2 1 21.09 1.0e-10 100 CAR92295 Relative to APETALA2 1 19 8e-08 100

218-2 384 PGSC0003DMG400034988 SKP1 1 100 2.5e-64 100 XP_004236226 PREDICTED: SKP1-like protein 4- 99 8e-65 80 like

283 522 PGSC0003DMG400028887 Lipoxygenase 25.29 6.2e-18 100 XP_004247024 PREDICTED: linoleate 9S- 25 2e-18 98 lipoxygenase 5, chloroplastic-like

290 262 PGSC0003DMG400009512 Kunitz-type proteinase inhibitor 79.01 9.2e-41 100 SPI2_SOLTU Serine protease inhibitor 2 81 6e-41 97

327 290 PGSC0003DMG400033335 Calcium-dependent protein kinase 8 99.31 3.6e-47 100 XP_004250931 PREDICTED: calcium-dependent 99 3e-58 100 protein kinase 7-like

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SGN BLASTX NCBI BLAST X Size Clone ID Accession no. Putative function Coverage E-Value Identity Putative function Coverage E-Value Identity (bp) Accession no. (%) (%) (%) (%)

395-2 1153 PGSC0003DMG400040601 Gene of unknown function 23.70 7.4e-47 100 - No significant similarity found - - -

389 595 PGSC0003DMG400043824 Gene of unknown function 53.03 3.5e-49 100 XP_004244403 PREDICTED: dolichol phosphate- 21 3e-18 98 mannose biosynthesis regulatory protein-like isoform 1

228 341 PGSC0003DMG400030199 Conserved gene of unknown function 94.69 1.7e-53 100 - No significant similarity found - - -

94-2 203 PGSC0003DMG400043130 Conserved gene of unknown function 99.01 6.4e-34 100 XP_003635721 Hypothetical protein MTR_004s0025 81 6e-14 58

428f 624 PGSC0003DMG400026589 Conserved gene of unknown function 86.68 1.3e-108 100 - No significant - - -

410-r2 356 PGSC0003DMG400044769 Gene of unknown function 32.87 1.7e-14 100 - No significant similarity found - - -

212-f3 257 PGSC0003DMG400037253 Gene of unknown function 46.69 5.7e-14 67.50 - No significant similarity found - - -

400-f2 248 PGSC0003DMG400034976 Gene of unknown function 98.78 8.4e-36 90.12 - No significant similarity found - - -

115-f3 846 PGSC0003DMG400037998 Gene of unknown function 47.87 3.9e-63 84.44 - No significant similarity found - - -

130r2 181 PGSC0003DMG400022158 Gene of unknown function 97.79 5.1e-29 100 - No significant similarity found - - -

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SGN BLASTX NCBI BLAST X Size Clone ID Accession no. Putative function Coverage E-Value Identity Putative function Coverage E-Value Identity (bp) Accession no. (%) (%) (%) (%)

125f 277 - Nosignificant similarity found - - - - No significant similarity found - - -

Cont5 110 - No significant similarity found - - - - No significant similarity found - - -

11 192 - No significant similarity found - - - - No significant similarity found - - -

63 325 - No significant similarity found - - - - No significant similarity found - - -

75f 330 - No significant similarity found - - - - No significant similarity found - - -

236r2 221 - No significant similarity found - - - - No significant similarity found - - -

236f2 310 - No significant similarity found - - - - No significant similarity found - - -

252-2 121 - No significant similarity found - - - - No significant similarity found - - -

175r2 310 - No significant similarity found - - - - No significant similarity found - - -

23r2 354 - No significant similarity found - - - - No significant similarity found - - -

161f2 289 - No significant similarity found - - - - No significant similarity found - - -

48r2 235 - No significant similarity found - - - - No significant similarity found - - -

3f2 235 - No significant similarity found - - - - No significant similarity found - - -

171

Appendix 9 Annotations and BLAST scores of differentially expressed sequences induced by PVX in White Lady

SGN BLASTX NCBI BLAST X Size Clone ID Accession no. Putative function Coverage E-Value Identity Putative function Coverage E-Value Identity (bp) Accession no. (%) (%) (%) (%)

232-2 755 PGSC0003DMG400009830 Ureide permease 1 54.58 1.0e-77 79.56 XP_004237058 PREDICTED: ureide permease 2-like 66 8e-81 96

256 609 PGSC0003DMG400008828 Mitochondrial respiratory chain 96.55 2.5e-62 61.62 EGW00718.1 AFG3-like protein 2 99 2e-140 98 complexes assembly protein AFG3

209 258 PGSC0003DMG401019042 Cytochrome P450 like_TBP 60.47 3.2e-23 98.08 XP_003614380 Cytochrome P450 likeTBP 53 2e-16 87

245 564 PGSC0003DMG400007381 Zinc finger (Ran-binding) family 40.43 5.1e-41 100 XP_004245200 Zinc finger Ran-binding domain- 58.7 7e-08 72 protein containing protein 2-like

57-2vi 453 PGSC0003DMG400031495 Senescence-associated protein 66.23 1.3e-49 98.00 AGB85033 Senescence-associated protein 80 6e-71 89

54-2vi 293 PGSC0003DMG400023055 F-box protein 99.32 5.0e-50 100 AAU93580 Putative F-box protein 99 2e-58 93

50-2 453 PGSC0003DMG400031277 F-box domain-containing protein 41.72 3.8e-36 73.02 ABO92991 F-box domain-containing protein 73 2e-30 70

62-2v 520 PGSC0003DMG401010480 Peroxidase 44 45.35 8.3e-72 97.44 XP_004249800 Peroxidase 44-like 86 3e-64 90

59-2v 378 PGSC0003DMG400012318 WRKY transcription factor 100 2.7 e-65 98.2 XP_004245564 WRKY transcription factor 22-like 100 9e-56 86

110-2v 302 PGSC0003DMG400016502 Transcription factor 64.57 7.5e-24 80.56 XP_004241742 Transcription factor bHLH68-like 61 2e-22 72

143-2v 520 PGSC0003DMG400002191 Ethylene-responsive transcription 99.81 3.0e-95 100 XP_004250840 Ethylene-responsive transcription 99 3e-89 89 factor factor LEP-like

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(Continued)

SGN BLASTX NCBI BLAST X Size Clone ID Accession no. Putative function Coverage E-Value Identity Putative function Coverage E-Value Identity (bp) Accession no. (%) (%) (%) (%)

388-2v 699 PGSC0003DMG400005910 Cellular nucleic acid binding protein 17.94 2.0e-16 100 XP_004241367 Uncharacterized protein 17 9e-14 95

440-2v 381 PGSC0003DMG400037625 Flavin-containing monooxygenase 32.28 1.4e-17 100 XP_004247957 Flavin-containing monooxygenase 32 7e-17 93 family protein YUCCA10-like

361-v2 754 PGSC0003DMG400019605 Pyruvate kinase family protein 99.47 4.9e-130 100 XP_004240954 Plastidial pyruvate kinase 4, 99 3e-167 96 chloroplastic-like

18-v2 300 PGSC0003DMG400012943 Gene of unknown function 53.00 1.2e-23 100 - No significant similarity found - - -

11-v2 367 PGSC0003DMG400017213 Conserved gene of unknown function 30.19 2.6e-33 100 XP_004247744 PREDICTED: glycolipid transfer 30 2e-24 98 protein-like

133-v2 431 PGSC0003DMG400029434 Gene of unknown function 38.28 1.1e-24 100 - No significant similarity found - - -

46-v2 656 PGSC0003DMG402022530 Conserved gene of unknown function 20.61 2.7e-12 66.67 XP_004234531 PREDICTED: calmodulin-lysine N- 13 1e-08 90 methyltransferase-like

231 230 PGSC0003DMG400041407 Gene of unknown function 26.09 2.7e-05 95.00 - No significant similarity found - - -

266f 1127 PGSC0003DMG400042313 Conserved gene of unknown function 29.34 4.1e-56 96.43 XP_003614394 Hypothetical protein MTR_5g051130 64 2e-68 93

78-2v 290 PGSC0003DMG400024298 Gene of unknown function 42.41 4.1e-07 68.29 - No significant similarity found - - -

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(Continued)

SGN BLASTX NCBI BLAST X Size Clone ID Accession no. Putative function Coverage E-Value Identity Putative function Coverage E-Value Identity (bp) Accession no. (%) (%) (%) (%)

152 467 - No significant similarity found - - - ABB45495v NADH dehydrogenase subunit 2 55 2e-25 93

478 164 - No significant similarity found - - - XP_003544026 PREDICTED: uncharacterized protein 98 1e-25 93

423 492 - No significant similarity found - - - ADZ45521 GREBP, a cGMP-response 68 1e-68 99 element-binding protein

150 418 - No significant similarity found - - - EDM17840 RCG40545, isoform CRA_b 99 3e-59 100

111 257 - No significant similarity found - - - XP_003544026 Uncharacterized protein 70 4e-28 90

376 968 - No significant similarity found - - - XP_0036143 Hypothetical protein 66 9e-80 90 94 MTR_5g051130

323-2 196 - No significant similarity found - - - - No significant similarity found - - -

174

Appendix 10

Genetic variability statistics for 17 intron primers within F1 population and potato cultivars

Marker Genotype Size range (bp) Scored ATETRA analysis POPGENE analysis Hc H′c h I bands

PKF11 F1 150-600 3 0.41 0.75 0.17 0.27 cultivar s 4 0.56 0.99 0.20 0.31

LBR57 F1 500-1100 5 0.75 1.07 0.35 0.53 cultivars 4 0.71 1.01 0.40 0.59

AVTPSH F1 180-1200 4 0.69 1.15 0.29 0.43 cultivars 4 0.70 1.30 0.05 0.09

PGRSH F1 180-890 4 0.79 0.80 0.43 0.62 cultivars 4 0.77 1.22 0.37 0.56

RP3a35 F1 130-360 5 0.69 1.26 0.23 0.37 cultivars 5 0.78 1.20 0.40 0.59

NB89 F1 150-300 3 0.40 0.69 0.20 0.31 cultivars 3 0.59 0.91 0.28 0.40

PTA-83 F1 150-1200 4 0.61 1.05 0.32 0.47 cultivars 4 0.69 1.08 0.33 0.48

R1L333 F1 150-390 4 0.45 0.94 0.14 0.24 cultivars 4 0.58 1.03 0.20 0.29

ATP-218 F1 100-400 4 0.61 1.19 0.35 0.51 cultivars 3 0.51 0.83 0.22 0.25

Cin F1 170-380 5 0.72 1.06 0.36 0.50 cultivars 4 0.57 1.29 0.07 0.18

TREPSH F1 180-520 3 0.65 1.04 0.15 0.22 cultivars 3 0.67 0.98 0.15 0.22

TSWVP F1 120-350 4 0.66 1.09 0.33 0.47 cultivars 3 0.50 0.82 0.15 0.23

TRP77 F1 150-500 3 0.46 0.79 0.14 0.21 cultivars 4 0.53 1.02 0.02 0.05

Nitsh F1 250-530 3 0.37 0.70 0.20 0.32 cultivars 3 0.44 0.77 0.17 0.27

RPB36 F1 150-800 2 0.42 0.60 0.38 0.57 cultivars 2 0.42 0.60 0.23 0.32

Cat F1 220-250 2 0.08 0.24 0.04 0.09 cultivars 2 0.12 0.27 0.06 0.12

Antiv1 F1 780-1000 5 0.76 1.14 0.35 0.55 cultivars 5 0.73 0.96 0.30 0.44

Mean F1 3.71 0.56 0.92 0.26 0.39 cultivars 3.59 0.58 0.96 0.21 0.32 Hc: Heterozygosity corrected for sample size; Hc′: Shannon index corrected for sample size; h: Expected heterozygosity or Nei’s gene diversity; I: Shannon’s information index of phenotypic diversity.

175

Appendix 11

Genetic variability statistics for 23 IT primers in wild Solanum species

Marker Genotype Size range Scored ATETRA analysis POPGENE analysis

(bp) bands Hc H′c h I PKF11 sect. Archaesolanum 210-700 8 0.61 3.13 0.26 0.42 sect. Solanum 300-700 6 0.81 2.30 0.34 0.51 S. nigrum 310-780 6 0.76 1.23 0.10 0.15 section AVTPSH sect. Archaesolanum 170-600 6 0.77 2.78 0.27 0.43 sect. Solanum 170-650 6 0.77 Invalida 0.27 0.43 S. nigrum 170-300 5 0.67 1.22 0.22 0.36

PGRSH sect. Archaesolanum 180-290 2 0.34 0.59 0.34 0.52 sect. Solanum 180-350 6 0.77 2.70 0.32 0.50 S. nigrum 250 1 -b - - -

NB89 sect. Archaesolanum 100-550 4 0.60 0.93 0.37 0.56 sect. Solanum 100-400 4 0.70 1.28 0.36 0.54 S. nigrum 100-600 6 0.70 1.96 0.18 3.00

PTA-83 sect. Archaesolanum 110-1000 5 0.73 2.88 0.28 0.44 sect. Solanum 110-300 3 0.68 1.38 0.37 0.55 S. nigrum 110-300 3 - - - -

R1L333 sect. Archaesolanum 160 1 - - - - sect. Solanum 160-250 4 0.37 1.69 0.23 0.39 S. nigrum 100-400 2 0.09 0.39 0.04 0.10

ATPb-218 sect. Archaesolanum 210-400 2 0.40 0.62 0.34 0.52 sect. Solanum 180-400 3 0.42 1.03 0.27 0.43 Archaesolanum S. nigrum 180-400 2 0.10 0.42 0.04 0.10

Cin sectArchaesolanum. Archaesolanum 150-700 5 0.70 2.28 0.15 0.24 sect. Solanum 150-400 4 0.66 1.58 0.33 0.47 Archaesolanum S. nigrum 150-700 4 - - - - Archaesolanum TREPSH sect. Archaesolanum 100-200 2 0.33 0.57 0.18 0.27 rchaesolanumsect. Solanum 100-200 2 0.38 0.54 0.20 0.30 S. nigrum 100-550 4 0.50 1.09 0.15 0.27

TSWVP sect. Archaesolanum 140-900 2 0.33 0.57 0.18 0.27 sect. Solanum 140-200 2 0.39 0.54 0.20 0.30 S. nigrum 140-250 2 - - - -

TRP77 sect. Archaesolanum 350-2000 5 0.45 3.14 0.28 0.50 sect. Solanum 100-1500 5 0.57 Invalid 0.30 0.50 S. nigrum 500-2000 5 0.75 2.18 0.34 0.51

Nitsh sect. Archaesolanum 100-1100 8 0.79 Invalid 0.28 0.44 sect. Solanum 100-300 5 0.57 1.93 0.16 0.28 S. nigrum 100-1200 8 0.85 Invalid 0.21 0.36

RPB36 sect. Archaesolanum 110-900 3 0.65 Invalid 0.43 0.63 sect. Solanum 300 1 - - - - S. nigrum 300 1 - - - -

Cat sect. Archaesolanum 250-1500 4 0.75 2.76 0.29 0.43 sect. Solanum 250-1700 4 0.64 1.29 0.31 0.49 S. nigrum 250-1500 6 0.45 1.60 0.16 0.23

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Appendix 11

(Continued)

Marker Genotype Size range Scored ATETRA analysis POPGENE analysis

(bp) bands Hc H′c h I Antiv1 sect. Archaesolanum 900-1100 7 0.78 2.77 0.30 0.47 sect. Solanum 200-1100 7 0.81 2.83 0.29 0.45 S. nigrum 200-1100 6 0.85 2.05 0.41 0.60

Mresis sect. Archaesolanum 170-200 2 0.19 0.63 0.09 0.17 sect. Solanum 170-200 2 0.45 0.57 0.25 0.34 S. nigrum 170-200 2 - - - -

Cad sect. Archaesolanum 100-1600 4 0.73 1.81 0.37 0.54 sect. Solanum 1300-1600 3 0.62 0.79 0.50 0.69 S. nigrum 1300-1600 1 - - - -

Winsh sect. Archaesolanum 100-250 2 - - - - sect. Solanum 100-250 2 0.69 1.05 0.25 0.34 S. nigrum 100-250 2 0.44 0.84 0.12 0.21

Str sect. Archaesolanum 100-200 2 0.19 0.63 0.00 0.00 sect. Solanum 100-400 3 0.41 1.01 0.15 0.26 S. nigrum 100-400 4 0.61 1.22 0.27 0.40

PT11 sect. Archaesolanum 150-320 3 0.54 1.03 0.27 0.40 sect. Solanum 150-320 3 0.64 0.92 0.14 0.20 S. nigrum 150-320 2 - - - -

Pe54 sect. Archaesolanum 150-1200 5 0.74 Invalid 0.34 0.52 sect. Solanum 150-800 7 0.82 Invalid 0.28 0.45 S. nigrum 150-1200 7 0.62 2.27 0.20 0.34

Cop12 sect. Archaesolanum 210-1200 6 0.68 Invalid 0.21 0.35 sect. Solanum 160-900 6 0.66 1.85 0.24 0.39 S. nigrum 160-1200 8 0.70 2.25 0.20 0.34

Cunf34 sect. Archaesolanum 150-800 6 0.73 Invalid 0.31 0.48 sect. Solanum 150-800 10 0.85 Invalid 0.25 0.40 S. nigrum 100-800 8 0.76 2.27 0.19 0.33

Mean sect. Archaesolanum 4.23 0.54 1.70 0.28 0.43 sect. Solanum 4.41 0.62 1.40 0.27 0.42 Archaesolanum S. nigrum 4.50 0.59 1.50 0.19 0.49 Hc: Heterozygosity corrected for sample size;4.50 Hc′: Shannon index corrected for sample size; h: Expected heterozygosity or Nei’s gene diversity; I: Shannon’s information index of phenotypic diversity; a: ATETRA software applies the number of singletons (genotypes that only occur once) to estimate the number of unsampled genotypes. This correction works well for sample sizes over 50, but for smaller sample sizes, a small bias is still present. The correction in loci that was not possible the program generated “Invalid”. (Van Puyvelde et al., 2010); b: Monomorphic.

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Appendix 12 Characteristics of the monomorphic loci and their intron-targeting primer sequences

1 1 Locus Putative function Primer sequence (5′-3′) Ta Transcript ID Ch

LRRsh1 LRR receptor-like F: TCATAGATGGAAATTTGGTTGAA 52 PGSC0003DMG4000312 X kinaseserine/threonine -protein R:TCATAGATGGAAATTTGGTTGAA AATTGCAGGGCAAAGTTCAG 28 TCATAGATGGAAATTTGGTTGAA Cunsh Conserved gene of unknown F: CCCAAATGTGCGTCATGAAT 54 PGSC0003DMG4000290 IX function R: CACTAAGGGAAAGCTTATGGTATG 10

Gufsh Gene of unknown function F: CCTTCACTGGGACCCACAT 55 PGSC0003DMG4000422 IX R: TTGGGGGTAGGACTGAATATG 98

Th91 Thioredoxin F: CGGACTCTCATTCGAGATCTTT 55 PGSC0003DMG4000038 IX R: TGATGAAAGTTACAAGTCTGAAAGG 91

Cuf32 Conserved Gene of unknown F: GTTGACCAAGCATCATGGAG 55 PGSC0003DMG4000145 II function R: GCATGGTTTATGTTAAGGGCTTACT 32

CYT73 Cytochrome P450 71A4 F: CAACAAACGCATCCTGTGTA 52 PGSC0003DMG4000142 III R: CATAATCAACAGCACCATTTGTAA 73

Cht83 Chitinase F: TTCATTTTGGTTTCTGCTTGG 58 PGSC0003DMG4000198 - R: AAGTGTAGTGTCCGGGGCTA 83

Gul94 Gulonolactone oxidase F: AAATTATTGAGATTAAGCGGGTCA 52 PGSC0003DMG4000054 VIII R: TTACAATGGATTGAATAGAATTTGG 94

Armsh Armadillo/beta-catenin repeat F: TTGTGACTAATGGAAAGCCTCT 55 PGSC0003DMG4020201 VI familyprotein / BTB/POZ domain R: TGAAAAAGATTGTTGGGCAGA 26

Ar/Ser45 Arginine/serine-rich splicing F: TTCCAAACTTCTGCCTTAGCA 56 PGSC0003DMG4000146 VI factor R: CACAGAGGCTGAGTGTCCAA 45

TRASH Tospovirus resistance protein A F: GGAGAATCTTACCGAGCTTCAA 56 PGSC0003DMG4000065 IX R: TCTCCAAGGCACCACTCTTC 71

R1L380 late blight resistance protein R1 F: TCAAAGCAAAGATTCAGGAAAA 55 PGSC0003DMG4000033 V A4 R: TCATTCATCCTCGGAGTCCT 80

AC701 Acyl-CoA binding protein F: CGGTTACCGTCAGTCATGG 58 PGSC0003DMG4000247 I R: CATCAGTTTGACCAACAATGG 01

R2L18 R2 late blight resistance protein F: CGCTGAAAGAGCAGACAACA 55 PGSC0003DMG4000115 IV R: CTTTTCATTTCTGGAACCATTG 18

GUF98 Gene of unknown function F: ACCTTCACTGGGACCCACAT 56 PGSC0003DMG4000422 VIIII R: GGGGGTAGGACTGAATATGTAGA 98 º 1 Ta: annealing temperature in C; Ch: location on potato chromosome; Available in SOL Genomics Network (http://sgn.cornell.edu/).

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