CHAPTER 2 Molecular Tagging of Resistance Genes to Septoria Leaf Spot in Tomato (Solanum Lycopersicum L.)

ABSTRACT

JOSHI, BAL KRISHNA. Molecular Tagging of Resistance Genes to Septoria Leaf Spot and Late Blight in Tomato (Solanum lycopersicum L.). (Under the direction of Dr. Dilip R. Panthee.)

Septoria leaf spot caused by Septoria lycopersici Speg. and late blight caused by Phytopthora infestans (Mont.) de Bary are two important foliar diseases of tomato (Solanum lycopersicum L.). Identification of DNA markers linked to these diseases is a prerequisite for accelerating resistance breeding through marker-assisted selection (MAS). The objectives of the present study were to investigate the inheritance pattern of septoria leaf spot resistance and identify molecular markers linked to septoria leaf spot and late blight resistance in tomato. Two tomato inbred lines, NC 85L-1W(2007), resistant to late blight and susceptible to septoria leaf spot, and NC 839-2(2007)-1, resistant to septoria leaf spot and susceptible to late blight were crossed to produce an

F1 progeny. A total of 250 F2 plants, and 10 plants of each parents and F1 were grown in field plots at the Mountain Horticultural Crops Research and Extension Center (MHCREC), Mills River, NC in the summer of 2009. Severity of these two diseases was scored from the same population in different time intervals under natural inoculums using a scale of 0 to 5, where 0 = no disease and 5 = complete development of disease on a plant. Different levels of infestation of both pathogens were observed in the plant population which indicated that, inoculum pressure was enough to screen the population. DNA was extracted from 2-3 week old plants. Both parental lines were screened with a total of 379 molecular markers that included 157 simple sequence repeats (SSR), two conserved ortholog sets (COS), 23 M-13 tailing SSRs, and 197 randomly amplified polymorphic DNA (RAPD) markers. Two DNA bulks, identified as the resistant bulk (RB) and susceptible bulk (SB) were prepared from eight resistant and eight susceptible F2 individuals. Transgressive-segregants among the F2 population

were found only for susceptibility of septoria leaf spot. The segregation ratio of resistant and susceptible plants for resistance to septoria leaf spot fit the expected ratio of 3:1 (2 = 3.014, P = 0.083) indicating that the inheritance of resistance was based on a single dominant gene. While none of the SSR and COS were found to be polymorphic between parents, a total of 34 RAPD primers (17.26%) were polymorphic, of which 11 primers (32.35%) were polymorphic between resistant and susceptible bulks of septoria leaf spot, however, six were found unlinked. Five RAPD primers were identified linked to septoria disease reaction, of which two were linked to the susceptible and three were linked to the resistance loci.

Transgressive segregation for resistance and susceptibility to late blight was also observed in this study. Out of the 34 RAPD markers screened, 16 (47%) produced polymorphic bands between resistant and susceptible bulks of late blight lines. Four RAPD primers were identified as linked markers, two with susceptible and two with resistance loci. Because of the low reproducibility of RAPD, these markers will be converted to sequence characterized amplified region (SCAR) markers. These markers will be evaluated for their utility in a marker-assisted selection (MAS) program in tomato for septoria leaf spot and late blight. A total of 9 RAPD markers have been identified linked to these two traits using the F2 population through a BSA technique.

Molecular Tagging of Resistance Genes to Septoria Leaf Spot and Late Blight in Tomato (Solanum lycopersicum L.)

by Bal Krishna Joshi

A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science

Horticultural Science

Raleigh, North Carolina

2011

APPROVED BY:

______Bryon Sosinski Frank J. Louws

______G. Craig Yencho Consuelo Arellano

______Dilip R. Panthee Committee Chair BIOGRAPHY

I was born in 26 June 1967 at Samibhanjyang, Lamjung, Nepal. After completion of School Leaving Certificate, I joined the Institute of Agriculture and Animal Science (IAAS) at Rampur, Chitwan and earned a Bachelor in Science (Ag) with Plant Breeding elective in 1992.

After graduation, I worked for the Unitarian Service Committee-Canada-Nepal (USCCN) for 5 years as an Agriculture and Livestock Development Coordinator until I joined the IAAS for studies toward a Master degree with a major in plant breeding. After my Master degree in 2000, I joined the Nepal Agricultural Research Council (NARC), Kathmandu. I also served as an editorial member for the Nepal Agriculture Research Journal and some proceedings. During this time, I also taught plant breeding to undergraduate students of the Himalayan College of Agricultural Sciences and Technology, Purbanchal University in 2007 and 2008.

I received the national level scientific award in 2007, and I have published more than 30 research papers, the details of which are available at http://sites.google.com/site/jafgaubase). In 2009, I enrolled at North Carolina State University, USA, to pursue a Master of Science in Horticulture under the guidance of Dr. Dilip R. Panthee.

ii ACKNOWLEDGMENTS

I would like to express my sincere thanks to the following who encouraged me in my scientific pursuits.

To Dr. Dilip R. Panthee, advisor, for the invaluable guidance, encouragement and fruitful discussions. Thank you for giving me the opportunity to gain knowledge in the field of plant breeding and the experience gathered from your lab.

To other advisory committee members, Dr. C. Arellano, Dr. G.C. Yencho, Dr. F.J. Louws and Dr. B. Sosinski, for their guidance and suggestions gained during the progress of my academic and research work.

To Candice Anderson for her support during field work and Dr. Mahfuzur Rahman (Department of Plant Pathology) for his support in pathological works.

To all my family members, relatives and friends that have helped, supported and always encouraged me.

Finally, to Ganga (my wife) and Benjika (daughter), for providing me moral support, for their continuous love and encouragement, and for their patience and understanding.

iii TABLE OF CONTENTS

LIST OF TABLES ...... vii LIST OF FIGURES ...... viii LIST OF APPENDICES ...... x ABBREVIATIONS...... xi

CHAPTER 1 General Introduction ...... 1 Tomato Origin and Taxonomy ...... 1 Tomato Production ...... 5 Foliar Fungal Diseases ...... 11 Tomato Genetics and Breeding ...... 12 Genome Mapping ...... 13 Molecular Markers ...... 14 Random Amplified Polymorphic DNA (RAPD) ...... 15 Linkage and QTL Maps ...... 16 Mapping Population ...... 18 Artificial vs. Natural Inoculums for Mapping Resistance Genes ...... 19 Experimental Design for Mapping Resistance Genes ...... 21 Bulked Segregant Analysis ...... 22 Objectives...... 26 References ...... 27

CHAPTER 2 Molecular Tagging of Resistance Genes to Septoria Leaf Spot in Tomato (Solanum lycopersicum L.) ...... 39 Abstract ...... 39 Introduction ...... 40 Material and Methods ...... 43 Plant Materials ...... 43 Field Evaluation ...... 43 DNA Extraction, Quantification and Dilution ...... 45 PCR-based Molecular Markers Screening ...... 46 Simple Sequence Repeat (SSR) ...... 46 M-13 Tailing SSR ...... 46 Random Amplified Polymorphic DNA (RAPD) ...... 47 Disease Scoring ...... 47 Bulked Segregant Analysis ...... 48 Gel Electrophoresis ...... 48 DNA Extractions from the Gel for Sequencing ...... 48 Data Analysis ...... 49

iv Results ...... 49 Segregation of Resistance ...... 49 RAPD Markers ...... 50 RAPD Markers and Bulked Segregant Analysis ...... 52 Discussion...... 62 References ...... 65

CHAPTER 3 Molecular Tagging of Resistance Genes to Late Blight in Tomato ...... 68 Abstract ...... 68 Introduction ...... 69 Material and Methods ...... 72 Plant Materials ...... 72 Field Evaluation ...... 74 DNA Extraction, Quantification and Dilution ...... 74 PCR-based Markers Screening ...... 75 SSR ...... 75 Bulked Segergant Analysis ...... 76 Gel Electrophoresis ...... 77 DNA Extractions from the Gel for Sequencing ...... 77 Data Scoring and Analysis ...... 77 Results ...... 78 RAPD Markers ...... 79 RAPD Markers and Bulked Segregant Analysis ...... 79 Discussion...... 89 References ...... 92

CHAPTER 4 Isolation and Culture of Septoria lycopersici Speg. from Septoria Leaf Spot affected Tomato Leaves...... 95 Introduction ...... 95 Symptoms ...... 96 Host Pathogen Interaction ...... 96 Resistance Breeding ...... 97 Material and Methods ...... 98 Plant Materials ...... 98 Clinical Test and Spore Identification ...... 98 Incubation, Isolation and Culture ...... 98 Sterilization and Aseptic Technique...... 99 Culture Media ...... 99 Subculture and Storage ...... 101 Results and Discussion ...... 101 Clinical Test and Spore Identification ...... 101

v Isolation and Culture Techniques ...... 103 Colony Growth and Characters ...... 103 Subculture and Storage ...... 103 Culture Media ...... 105 Conclusion ...... 105 References ...... 106

APPENDICES ...... 109

vi LIST OF TABLES

Table 1. Wild relatives of tomato (http://tgrc.ucdavis.edu/key.aspx) ...... 4 Table 2. Disease resistance genes and their linkage group in 12 chromosomes of tomato ...... 17 Table 3. Mapping population, marker types and number of QTLs detected for disease resistance and fruit quality traits in tomato ...... 20 Table 4. Use of bulked segregant analysis (BSA) to identify traits of interest in different crop species...... 24 Table 5. Parental description along with their pedigrees and coefficient of parentage ..... 44 Table 6. Segregation ratio of resistant and susceptible progenies to septoria leaf spot observed in the F2 population of tomato derived from NC 085 x NC 839, 2009 ...... 53 Table 7. RAPD primers polymorphic between resistant and susceptible parents of tomato to septoria leaf spot ...... 54 Table 8. Polymorphic RAPD markers and their size between resistance and susceptible bulks of tomato to septoria leaf spot ...... 55 Table 9. Polymorphic bands of RAPD markers linked to either resistance or susceptible genes of tomato for septoria leaf spot ...... 56 Table 10. Specific marker bands linked to either resistance or susceptible genes to septoria leaf spot (SLS) of tomato ...... 57 Table 11. Informative RAPD markers associated to tomato genes in relation to reaction with septoria leaf spot ...... 58 Table 12. Parental description along with their pedigrees and coefficient of parentage ..... 73 Table 13. RAPD primers polymorphic between resistant and susceptible parents of the NC 08135 F2 population of tomato screened for late blight resistance ...... 81 Table 14. Polymorphic RAPD markers and band size between late blight resistant and susceptible bulks of tomato ...... 82 Table 15. Polymorphic bands of RAPD markers linked to genes in tomato conferring resistance or susceptibility to late blight ...... 83 Table 16. Specific marker bands linked to genes for resistance or susceptibility to late blight of tomato ...... 84 Table 17. Informative RAPD markers associated with susceptible or resistance genes to late blight of tomato ...... 85

vii LIST OF FIGURES

Figure 1. Top 15 countries in term of total harvested area for tomato production in the world, 2009...... 6 Figure 2. Total tomato production in million mt of top 15 countries in the world, 2009...... 7 Figure 3. Top 22 countries in term of tomato productivity (ton/ha) in the world, 2009...... 8 Figure 4. Total harvested areas (ha), productivity and farm value of tomato in USA over two decades...... 9 Figure 5. Tomato export and import volume (ton) in USA from 2000 to 2007...... 10 Figure 6. General description of bulked segregant analysis (BSA) technique...... 23

Figure 7. Frequency distribution of 234 F2 individuals derived from NC-085L- 1W(2007) x NC-839-2(2007)-1 based on the score of severity of septoria leaf spot in tomato, Mills River, 2009...... 51 Figure 8. Electrophoresis pattern of DNA fragments generated by RAPD markers (A. MRTOMR-031, B. MRTOMR-121). Polymorphic band (i.e., linked to susceptible) between parents, and between resistant and susceptible bulks are indicated by arrow...... 59 Figure 9. Electrophoresis pattern of DNA fragments generated by RAPD marker (A. MRTOMR-022, B. MRTOMR-117 and C. MRTOMR-118). Polymorphic band (i.e., linked to resistance) between parents and between resistant and susceptible bulks are indicated by arrow...... 60 Figure 10. RAPD marker (A. MRTOMR-130) showing polymorphic band (indicated by arrow) only to parents, i.e., band with unlinked loci and RAPD marker (MRTOMR-146) showing band (indicated by arrow) only in two bulks...... 61 Figure 11. Electrophoretic pattern of DNA fragments generated by RAPD marker (A. MRTOMR-026, B. MRTOMR-046). Polymorphic band (i.e., linked to susceptible) between parents and between resistant and susceptible bulks are indicated by arrow...... 86 Figure 12. Electrophoretic pattern of DNA fragments generated by RAPD markers (A. MRTOMR-031, B. MRTOMR-038). Polymorphic band (i.e., linked to resistance) between parents and between resistant and susceptible bulks are indicated by arrow...... 87

viii Figure 13. A. RAPD marker (MRTOMR-040) showing polymorphic band (indicated by arrow) only to parents, i.e., band with unlinked loci. B. RAPD marker (MRTOMR-022) showing band (indicated by arrow) only in two bulks...... 88 Figure 14. Tomato leaves infected with septoria leaf spot (Septoria lycopersici) (A), extrusion of white mass of spore from pycnidia (B), and enlarged extrusion of spore (C)...... 100 Figure 15. Blue stained thread like spore with septa of septoria (A) and enlarged spore (B)...... 102 Figure 16. White button like initial growth of septoria (A), fully grown colony (B) and colony in slant media for storage at 4oC (C)...... 104

ix LIST OF APPENDICES

Appendix 1. Monthly weather conditions during tomato growing period (shaded area) in Mills River, North Carolina, 2009 ...... 109

Appendix 2. Parents [NC 85L-1W(2007) x NC 839-2(2007)] and their F1 (NC 08135) ...... 110 Appendix 3. Samples of tomato plant infected by two foliar diseases, late blight and septoria leaf spot ...... 111 Appendix 4. List of SSR, RAPD and other markers used to screen parents...... 112 Appendix 5. DNA extraction protocol from tomato fresh leaves ...... 115 Appendix 6. Extraction of DNA bands from agarose gels ...... 116 Appendix 7. SSR profiles of parental lines showing monomorphic based on agarose gel, polyacryalamide gel and capillary electrophoresis ...... 118 Appendix 8. Chemicals and reagents for DNA extraction and PCR ...... 119

x ABBREVIATIONS

AFLP Amplified Fragment Length Polymorphism BC Back Cross BSA Bulked Segregant Analysis CAPS Cleaved Amplified Polymorphic Sequence CIM Composite Interval Mapping COP Coefficient of Parentage COS Conserved Ortholog Set CTAB Cetyl TrimethylAmmonium Bromide DH Doubled Haploid dNTP DeoxyriboNucleotide TriPhosphate EDTA Ethylene Diamine Tetra Acetic acid EST Expressed Sequence Tag EtBr Ethidium Bromide IM Interval Mapping ISSR Inter Simple Sequence Repeat MAS Marker Assisted Selection ML Maximum Likelihood NIL Near Isogenic Lines PCR Polymerase Chain Reaction PDA Potato Dextrose Agar QTL Quantitative Trait Loci RAPD Random Amplified Polymorphic DNA RB Resistant Bulk RBC Reciprocal Back Cross RFLP Restriction Fragment Length Polymorphism RGA Resistance Gene Analog RH Relative Humidity RIL Recombinant Inbred Lines RP Resistant Parent SB Susceptible Bulk SCAR Sequence Characterized Amplified Region SDW Sterile Distilled Water SIM Simple Interval Mapping SLS Septoria Leaf Spot SNP Single Nucleotide Polymorphism SP Susceptible Parent SSC Soluble Solids Content SSR Simple Sequence Repeat

xi Ta Annealing Temperature TAE Tris-Acetate-EDTA TBE Tris-Boric-EDTA TE Tris-EDTA Tm Melting Temperature ToMV Tomato Mosaic Virus TSWV Tomato Spotted Wilt Virus TYLCV Tomato Yellow Leaf Curl Virus

xii

CHAPTER 1 General Introduction

Tomato (Solanum lycopersicum L., 2n = 24) is an herbaceous, usually sprawling plant in the Solanaceae or nightshade family that is typically cultivated for its fruits for human consumption. It is a perishable vegetable occupying a large volume in the international trade. Literature shows that tomato was first used for culinary purposes in Southern Europe (Ray, 1673; Miller, 1752) and it is an important crop rich in vitamins, minerals and antioxidants (Rick, 1980; Vinson et al., 1998; Nguyen and Schwartz, 1999; Willcox et al., 2003). High levels of dietary lycopene available in tomato have been found useful in reducing cancer risk (Giovannucci, 2002).

TOMATO ORIGIN AND TAXONOMY Linnaeus (1753) reported Solanum lycopersicum in America calidiore (a citation of the type). The wild relatives of Solanum lycopersicum are distributed in South America, along the coast and high Andes from central Ecuador, through Peru to northern Chile and in the Galapagos Islands (Muller, 1940; Luckwill, 1943; Rick, 1973; Taylor, 1986; Jorgensen and Léon-Yánez, 1999; Darwin et al., 2003; Peralta et al., 2005).

Early botanists recognized tomato as Solanum pomiferum (Sabine, 1820; Luckwill, 1943). Anguillara (1561, cited by Peralta and Spooner, 2007) named tomato as Lycopersicon, which means ‘wolf peach’. Tournefort (1694) classified cultivated tomato in the genus Lycopersicon, which was further described by Miller (1754). Linnaeus (1753) described the species as Solanum lycopersicum. Miller (1754) reconsidered Tournefort’s classification and grouped tomato into the genus Lycopersicon. Classical and modern authors (Muller, 1940; Luckwill, 1943; Hawkes, 1990; Rick et al., 1990) have treated tomato as Lycopersicon. Now, tomato is assigned to the genus Solanum based on the phylogenetic relationship using molecular data (Spooner et al., 1993; Peralta and Spooner, 2001; Peralta et al., 2005).

Rick et al. (1990), and Peralta and Spooner (2001) reported that the closest species of cultivated tomato (Solanum lycopersicum, formerly Lycopersicon esculentum) is Solanum pimpinellifolium (Jusl.) Mill. (formerly Lycopersicon pimpinellifolium). Key distinguishing characters between these two species are fruit size and leaf margin. The fruit diameter of Solanum lycopersicum is more than 1.5 cm with serrated leaf margin; whereas, fruit diameter of Solanum pimpinellifolium is less than 1.5 cm with undulated or entire leaf margin.

There are many subspecies of Solanum lycopersicum and they are typically characterized by the shape, size and color of the fruit. For example, Solanum lycopersicum var. cerasiforme (Dunal) Alef. which has typically small fruit (fruit diameter 1.5-2.5 cm with 2-loculed) is called Cherry tomato (Rick et al., 1990; Rick and Holle, 1990; Spooner et al., 1993). S. lycopersicum var. pyriforme (Dunal) Alef. has pear shaped fruit usually <3.8 cm in length and is called Pear tomato. S. lycopersicum var. grandifolium is called potato-leaved tomato and it has large and entire with few secondary leaves. Tomato with upright and erect, very compact plants is the characteristics of S. lycopersicum var. validium. S. lycopersicum var. lycopersicum are the common garden tomatoes, and they vary in their overall growth habit, fruit quality and fruit characteristics.

Taxonomical classification

Order: Solanales

Family: Solanaceae

Subfamily: Solanoideae

Tribe: Solaneae

Genus: Solanum L.

Subgenus: Lycopersicon (Mill.) Seithe

Species: Solanum lycopersicum L.

Synonyms

Lycopersicon galenii Mill.

Lycopersicon esculentum Mill.

Solanum spurium J.F.Gmel.

Solanum luridum Salisb.

Solanum pomiferum Cav.

Solanum humboldtii Willd.

Lycopersicon pyruforme Dunal

Lycopersicon humboldtii (Willd.) Dunal

Lycopersicon cerasiforme Dunal.

Lycopersicon lycopersicum (L.) H. Karst.

(Sources: http://www.uniprot.org/taxonomy/4081, http://www.ncbi.nlm.nih.gov/guide/taxonomy/, http://plants.usda.gov/classification.html, Jan. 2011)

Table 1. Wild relatives of tomato (http://tgrc.ucdavis.edu/key.aspx) SN Old nomenclature New nomenclature

1. L. pimpinellifolium Solanum pimpinellifolium

2. L. cheesmanii Solanum cheesmaniae

3. L. cheesmanii f. minor Solanum galapagense

4. L. parviflorum Solanum neorickii

5. L. chmielewskii Solanum chmielewskii

6. L. chilense Solanum chilense

7. L. peruvianum Solanum peruvianum

8. L. pennellii Solanum pennellii

9. L. hirsutum Solanum habrochaites

10. S. lycopersicoides Solanum lycopersicoides

11. S. sitiens Solanum sitiens

12. S. juglandifolium Solanum juglandifolium

13. S. ochranthum Solanum ochranthum

SN = Serial number.

TOMATO PRODUCTION Tomato is a tropical vegetable and used both as fresh and processed form worldwide. Tomato is second among vegetables in the world after potato in term of production with a total production of 136.2 million mt in 2008 (http://faostat.fao.org). Among the countries, China is the top producer growing in 35% of the total world’s tomato harvested area. Other top countries in descending order are USA, India, Turkey, Egypt, Italy and Iran (Figure 1). The leading countries in terms of total production are China (34.1 million mt), USA (14.1 million mt), India (11.1 million mt), Turkey (10.7 million mt) and Egypt (10 million mt) (Figure 2). The productivity of the Netherlands is the highest followed by Belgium, Ukraine, Denmark, Ireland, Iceland, Norway and Sweden (Figure 3). USA ranked 21st for tomato productivity in the world.

Among the leading vegetables in USA, tomato ranks second with a production of 14.1 million tons in 2009. Economically, tomato is the third most important vegetable (with a total farm value of $2.1 B) in US after potato ($2.6 B) and lettuce ($2.16 B) (http://www.nass.usda.gov/Publications/index.asp). As a source of vitamins and minerals in the US diet, it ranks first (Rick, 1980) among vegetables and fruits. The trend of tomato harvested areas, productivity and farm value over decades are given in Figure 4. The productivity is increasing over the years, but total harvested areas have remained almost same. The farm value of tomato has fluctuated over the years. The import volume of tomato in USA is almost four times more than export volume (Figure 5).

Cameroon 1% Iraq Others (76) 1% 14% Uzbekistan 1% Spain China 1% Brazil 35% 2% Cuba 2% Ukraine 2% Russian Fed. 3% Italy 3% Iran 4% USA 4% Egypt India 6% Turkey 14% 7%

Figure 1. Top 15 countries in term of total harvested area for tomato production in the world, 2009. (Source: FAOSTAT, 2010).

Portugal, 1.3

Morocco, 1.3 Others (76), Greece, 1.4 18.3 Ukraine, 2.0 China, 34.1 Uzbekistan, 2.1 Russian Federation, Brazil, 4.2 2.2 Spain, 4.7

Iran, 5.9 USA, 14.1

Italy, 6.4

Egypt, 10.0 India, 11.1

Turkey, 10.7

Figure 2. Total tomato production in million mt of top 15 countries in the world, 2009. (Source: FAOSTAT, 2010).

600.0

500.0 480.0

425.5

410.6

400.0 400.0

400.0 370.3

341.3

338.0 336.7

300.0

241.4

223.3 Yield, t/ha Yield,

200.0 167.2

161.1

123.5

104.9

88.3

84.2

83.3

83.0

82.3 80.6

100.0 80.2 50.6

0.0

USA

UAE

Israel

World

France

Cyprus

Ireland

Austria

Iceland

Finland

Norway

Ukraine

Sweden

Belgium

Portugal

Hungary

Palestine

Germany

Denmark

Switzerland

Netherlands

Luxembourg New Zealand New

Figure 3. Top 22 countries in term of tomato productivity (ton/ha) in the world, 2009. (Source: FAOSTAT, 2010.)

250 0.25 Farm value, $/kg (Y = 0.15 + 0.002X) Area, x1000 ha (Y = 187.16 - 1200x) 200 0.20

150 0.15

Yield, t/ha (Y = 55.71 + 1.03x)

100 0.10 Farm value, $/kg Farmvalue,

50 0.05 Total area, x1000 ha; Yield, t/ha Yield, ha; area, x1000 Total

0 0.00

Figure 4. Total harvested areas (ha), productivity and farm value of tomato in USA over two decades. (Source: http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1210)

1,200,000 Export volume, t Import volume, t 1,000,000

800,000

600,000

Export/ import, t import, Export/ 400,000

200,000

0 2000 2001 2002 2003 2004 2005 2006 2007

Figure 5. Tomato export and import volume (ton) in USA from 2000 to 2007. (Source: http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do?documentID=1210)

FOLIAR FUNGAL DISEASES Over 200 diseases, caused by pathogenic fungi, bacteria, viruses and nematodes are reported in tomato (Lukyanenko, 1991); hence, a major concern in tomato production is disease management. Two approaches have been adopted for disease management - non- breeding (e.g., use of fungicides) and breeding (use of resistance genes). The contribution of plant breeding toward the development of resistance is considered the best in tomato crop improvement. Resistance (R) genes have been identified for more than 30 tomato pathogens especially in wild species of tomato (Foolad et al., 2008). Among the long list of the diseases in tomato, two foliar diseases, septoria leaf spot (SLS) and late blight (LB), are economically important and attention to the development of the durable resistance to these pathogens is needed.

Septoria leaf spot (Septoria lycopersici Speg.) is one of the most devastating foliar diseases in humid regions, particularly during the periods of rainfall, frequent dew or overhead irrigation (Andrus and Reynard, 1945; Delahaut and Stevenson, 2004). It may cause complete defoliation leading to a significant crop loss under favorable environment conditions for disease development. Tomatoes may often be infected with pathogens of septoria leaf spot and early blight (Altenaria solani) simultaneously. The fungus overwinters in plant debris, on seed, or on weeds such as nightshade, jimsonweed, ground cherry and horse-nettle (Delahaut and Stevenson, 2004). Spores of the fungi may be splashed or blown onto tomato leaves. Spores of septoria can survive up to 3 years in infested debris.

Late blight (Phytopthora infestans (Mont.) de Bary) is generally recognized from the leaflet margins having purple, dark brown or black water-soaked lesions with a pale yellowish-green border. It can infect leaves, stems and fruits. Spores overwinter in the field in plant debris and can survive even under harsh conditions, becoming inoculum for next year’s crop (Gavino et al., 2000). The structure called sporangia produces the spores and the optimum temperature for sporangia germination is above 21oC. The rapid asexual reproduction of this pathogen can damage a tomato field quickly and sexual reproduction

enables it to develop new races through recombination or mutation. Therefore, a resistance mechanism in the host plant can quickly be overcome by late blight.

TOMATO GENETICS AND BREEDING Tomato is a diploid crop species with 2n=2x=24 chromosomes. It is autogamous in nature with a genome size of 950 Mbp. Reshuffling of genes through recombination is the principal way of developing improved genotypes in most breeding programs. Genetic diversity is necessary to develop new varieties through hybridization. Diversity in terms of its use and production environments of tomato is high. However, the genetic base of cultivated tomato is narrow, which is the foundation of many breeding programs (Bai and Lindhout 2007). It is estimated that the genomes of tomato cultivars contain 5% of the genetic variation of their wild relatives (Miller and Tanksley 1990). This has been further supported as less polymorphisms are typically detected with molecular markers within the cultivated tomato gene pool (Garcia-Martinez et al. 2006; Park et al. 2004; Villand et al. 1998). A high level of polymorphism was found among the Solanum genus using SSR markers (Alvarez et al., 2001). However, there is limited information on diversity at the varietal level using DNA markers. Major factors in reducing variation in tomato are self- pollination, founder effects, bottlenecks, and both artificial and natural selection (Rick 1958; Rick and Fobes 1975).

Wild Cherry tomato (Solanum lycopersicum var. cerasiforme) is the ancestor of cultivated tomato (Peralta and Spooner, 2007). Use of wild species in developing tomato breeding lines has increased the genetic base to some extent. Rick (1973), and Williams and St. Clair (1993) reported that germplasm base of cultivated tomato has been broadened using other Solanum species. Existence of 12 wild species of tomato (Bai and Lindhout, 2007) has provided great scope to broaden the genetic base by gene introgression, which can be exploited to widen the genetic base of tomato. However, the effect of recombination

on levels of polymorphism in Solanum is much weaker than in other well-studied species (Baudry et al., 2001).

Around the world, more than 75,000 accessions of tomato are conserved in gene banks of more than 120 countries (Robertson and Labate, 2007). The largest collections are managed by: The United States Department of Agriculture (USDA), Geneva, NY; The Asian Vegetable Research and Development Center (AVRDC), Tainan, Taiwan, and the Tomato Genetic Resources Center (TGRC) located in the Department of Vegetable Center at the University of California. These are the important sources of diversity in tomato breeding programs and many mutants are available. Several varieties of tomato have been developed through conventional breeding, even though it takes many years, especially to develop the homozygous lines. The main steps in conventional breeding are selection of parents, hybridization, selection and generation advancement, and evaluation. Selection based on the phenotypic markers depends on the environment and genotypes. In case of a self- pollinated crop, selection is effective only after getting complete homozygosity, which takes almost 6 years. On the other hand, molecular markers if available can be applied at any crop stage of early generation. It is also effective to identify the homozygote as well as heterozygote plants. Molecular marker-assisted breeding in tomato, however, has still not been widely used, either due to limited number of useful markers or availability of allele- specificity.

GENOME MAPPING The International Solanaceae Genome Project (SOL) has started sequencing the genome of tomato (http://solgenomics.net/). Ten countries (China, France, India, Italy, Japan, Korea, the Netherlands, Spain, United Kingdom, and the United States) are involved in the project (Mueller et al., 2005). Tomato is a model plant for the study of a number of economically important traits including fruit development and plant defense (Li et al., 2001; Tanksley, 2004). In 2009, a whole genome shotgun approach was initiated for the tomato

genome and draft sequences are already available (http://solgenomics.net/genomes/ Solanum_lycopersicum/index.pl). This sequence data will be very useful for further understanding the genetics of different traits.

Molecular Markers Availability of polymorphic molecular markers is a pre-requisite for developing genetic maps and molecular breeding. The number of markers is also very important for the construction of a saturated molecular linkage map. After the invention of molecular markers, which are many in numbers, it is now possible to develop saturated maps and to select the genotype directly. Limitations of association with morphological and biochemical markers are overcome by employing molecular markers. Molecular markers are useful for accelerating breeding process by improving precision and making selection more efficient. Markers linked to the gene(s) of interest help to select plants genotypically. Identification of marker along with the development of linkage map is the prerequisite for initiating MAS in a breeding program. Even quantitative traits can be dissected using molecular markers. Individual quantitative trait loci (QTL) can be characterized and transferred with the help of molecular markers.

The genetic marker should be polymorphic, co-dominant, non-epistatic and insensitive to environment to be used in MAS. There are a number of marker types used in plant breeding. Morphological markers are mostly few and depend on genotype- environment interaction. Isozyme is a protein based marker that has also limitation in use due to low level of polymorphism. The first DNA based marker is restriction fragment length polymorphism (RFLP) (Botstein et al., 1980). RFLP involves fragmenting a sample of DNA by restriction enzyme and then hybridization with a probe. It has been widely used on genome mapping. It needs a large sample of DNA and the process is cumbersome. Alternatively, PCR based markers, e.g., random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), single nucleotide polymorphism (SNP) are now considered more useful and simple. RAPD markers are usually decamer-based primers that amplify the random segments of

genomic DNA (Williams et al., 1990). SSR are repeating sequences of DNA and generally they are neutral and co-dominant (Hearne et al., 1992). SNP is the variation in DNA due to single nucleotide polymorphism in the genome (Wang et al., 1998) and abundant. RAPD is dominant whereas SSR, RFLP, sequence characterized amplified region (SCAR), cleaved amplified polymorphic sequence (CAPS) and SNP are co-dominant markers.

There are a number of linkage maps developed on tomato genome and different markers have been identified (Table 2) suitable to select genotype with the traits of interest. For example, RAPD markers linked to disease resistance genes have been identified by screening nearly-isogenic lines (Klein-Lankhorst et al., 1991; Martin et al., 1991). De Giovanni et al. (2004) identified RAPD marker linked to the ol-2 gene, which is resistance to powdery mildew, using bulked segregant analysis (BSA) to an F2. The distance between marker and ol-2 gene was also estimated through linkage analysis. Stevens et al. (1995) and Chagué et al. (1996) have identified RAPD markers linked to the Sw-5 gene. Czech et al. (2003) have used MAS for developing tomato spotted wilt virus (TSWV) resistant tomato using polymerase chain reaction (PCR) based markers, SCAR 421.

Random Amplified Polymorphic DNA (RAPD) Selection of markers depends on the types of objectives and target crop species. SSR and SNP are generally crop specific and these are not available for many crop species. On the other hand, without prior knowledge on the sequences of target genome, RAPD can be used. RAPD is a PCR based multi-locus molecular marker system and is dominant in nature. This type of marker was first described by Williams et al. (1990). Usually decamer primers are used to amplify the homologous sites of the target genome. Polymorphisms are detected as presence or absence of bands. RAPD markers, although old, are considered still useful for developing genetic maps and analyzing populations (Michelmore et al., 1991). These markers have been used widely in many species because of their application without prior sequence information. However, RAPD is very sensitive to reaction conditions and dominant in nature.

RAPD markers have been used in tomato for developing linkage maps (Grandillo and Tanksley, 1996; Saliba-Colombani et al., 2000), tagging resistant genes (Table 4) and diversity analysis (Villand et al 1998). They were also found very useful to discriminate potato cultivars (Sosinski and Douches, 1996) and can be used in tomato for the same purpose. Villand et al. (1998) used 41 RAPD primers which generated 98 polymorphic RAPD markers to estimate relationships among 96 accessions of tomato collected from a wide geographic range.

Linkage and QTL Maps A linkage map is a map of markers as well as genes on chromosome based on recombination. A linkage map is the prerequisite for MAS and gene cloning. A linkage map of tomato with 153 morphological and physiological markers was developed in 1968 (cited in Foolad 2007). Tanksley and Mutschler (1990) developed and added isozyme markers in an already available morphological map. A dense map was developed in 1991 using 1030 RFLP markers and some morphological markers (Tanksley et al., 1992). Haanstra et al. (1999) constructed an integrated map from 1078 amplified fragment length polymorphism

(AFLP) and 67 RFLP using an F2 population of S. lycopersicun x S. pennellii. After the invention of PCR based markers, many quantitative traits have been mapped in tomato genome (Table 3). The availability of complete DNA sequences and SNP markers will enhance the molecular based breeding of tomato.

QTLs are responsible for the majority of important crop characteristics, including regulation of fruit development and ripening. Advanced backcross breeding was initially used to genetically isolate a QTL that plays a major role in fruit mass variation between cultivated tomato and small fruited wild species S. pennellii (Alpert et al., 1995). QTLs were detected for a number of traits, e.g., resistance to tomato yellow leaf curl virus (TYLCV), early blight in inter-specific populations (Zamir et al., 1994; Foolad et al., 2002) of tomato.

Table 2. Disease resistance genes and their linkage group in 12 chromosomes of tomato SN Trait Gene Linked markers Gene Reference location 1 Bacterial Rcm1.0 TG059 1 Sandbrink et al., 1995 canker Rcm7.0 TG061, TG174, 7 Sandbrink et al., 1995 TG210A 2 Bacterial wilt Rrs4.0/Bw-4 TG268 4 Thoquet et al., 1996 Rrs6.0/Bw-1 TG118, CP18 6 Thoquet et al 1996 3 Fusarium wilt I, I-1, I-2, I-2C, I- CT226, TG572, P7- 7, 11 Bournival et al., 1990; 3 43DF3/R1 Sarfatti et al., 1991; Ori et al., 1997; Simons et al., 1998; Hemming et al., 2004 4 Late blight Ph-1, Ph-2, Ph-3 CP105, TG233, 7, 9, 10 Pierce, 1971; Moreau et TG591 al., 1998; Chunwongse et al., 2002

5 Powdery Ol-1, Ol-2 TG20, SCAU31500, 4, 6 Huang et al., 2000; De mildew SCAF10, SCAG11 Giovanni et al., 2004 6 Verticillium Ve GP39 9 Diwan et al., 1999 wilt 7 ToMV Tm-1, Tm-2 TG101, TG79 2, 9 Young and Tanksley, 1989; Levesque et al., 1990 8 TSWV Sw-5 SCAR-421, SCR-2 9 Chagué et al., 1996; Stevens et al., 1996 9 TYLCV Ty-3, Ty-3a FLUW25, P6-25 6 Ji et al., 2008 SN = Serial number. TSWV = Tomato spotted wilt virus. TYLCV = Tomato yellow leaf curl virus. ToMV = Tomato mosaic virus.

Mapping Population

Commonly used mapping populations are F2, backcross (BC), recombinant inbred lines (RIL), near isogenic lines (NIL) and doubled haploids (DH) lines. In case of asexual crops an F1 population is used. DH is developed by chromosome doubling of haploid cell, e.g., microspores or macrospores. BC progeny is generated by crossing F1 with one of its parents.

RILs are obtained by successive self-pollination of F2 individuals. NILs are the lines that have the same genotypes except in one locus and it is generally developed through backcross. An

F2 population is the earliest segregating generation available for mapping genes from the crosses of self-pollinated crop species and F1 population for non-fixed parents and provides the greatest genetic window around the locus (Michelmore et al., 1991). All the possible genotypes can be deduced from the parental genotypes. Findings from the F2 population can be further verified using advanced generations. The heterozygous genotypes along with two homozygous in F2 population allow estimates of the degree of dominance for loci linked to markers. The F2 populations of tomato have been commonly used to map QTL for a number of traits in tomato (Table 3), for example, acylsugars (Mutschler et al., 1996), eleven quantitative traits (days to first true leaf, days to first flower, plant height, total number of flower buds, number of internodes on the primary stem, total number of internodes, number of well-developed branches, total fresh weight and total dry weight of the aerial portion of the plant, diameter of the stem at the first internode and leaflet width/length ratio) (DeVicente and Tanksley, 1993), fruit quality traits (Paterson et al., 1991), soluble solid (Osborn et al., 1987; Tanksley and Hewitt, 1988), salt tolerance (Breto et al., 1994; Foolad et al., 1997), fruit morphology (Van der Knaap and Tanksley, 2003) and fruit size (Lippman and Tanksley, 2001).

Genotypic variation within cultivated tomato is reported to be low. Linkage map and QTL detection have been based on inter-specific crosses in tomato (Paterson et al., 1991; DeVicente and Tanksley, 1993; Mutschler et al., 1996; Fulton et al., 1997; Bai et al., 2003; Van der Knaap and Tanksley, 2003). Advancement of molecular markers systems such as

SNP is abundant and might be useful to study the intra-specific population of tomato. Polymorphism is generally high and easy to detect in the population generated from interspecific crosses. However, several limitations are associated with such a population. For example, limitations include low fertility, segregation distortions, linkage drag between desirable and undesirable traits, and non-direct use for the creation of new crop varieties (Tanksley and Nelson, 1996). Linkage maps have also been constructed based on intraspecific crosses in tomato using morphological, RFLP, RAPD and AFLP markers (Saliba- Colombani et al., 2000). Causse et al. (2001) used intra-specific populations of tomato to map QTLs for fruit quality traits and detected one to five QTLs per trait. Choice of parental lines, segregating population and markers are determining factors for construction of genetic map. If polymorphic markers are available, intra specific crosses offer valuable and useful information to further advance the molecular breeding.

Artificial vs. Natural Inoculums for Mapping Resistance Genes Artificial inoculation is a common method for studying host-pathogen interactions. Genes as well as QTLs are mapped in a host by creating artificial disease pressure. However, because of the cost and time involved in the artificial inoculation, natural infestation might equally be considered suitable for mapping QTLs. Use of hot spots for specific disease and spreader rows help to increase the disease pressure and are cost effective ways of resistance breeding. Additionally, predictability of the favorable environment for a certain disease can be utilized to study the host-pathogen interaction in natural condition. For example, two QTLs for glume botch resistance in wheat were identified using composite interval mapping from naturally inoculated populations (Schnurbusch et al., 2003). One of these QTLs explained 31.2% of the observed phenotypic variation for the resistance within the population. Aphid resistance in barley under natural infestation was studied and detected a single QTL consistently over two years explaining at least 22% of total phenotypic variation (Moharramipour et al., 1997). Natural infestation was also used by Spaner et al. (1998) in barley and by Frei et al. (2005) in common bean.

Table 3. Mapping population, marker types and number of QTLs detected for disease resistance and fruit quality traits in tomato Population Traits Marker QTL Variation, % References 2 2 Source Type Size analyzed Number Types Detection Detected, R t R i method n

S. esculentum/ S. F2 104 Powdery 318 AFLP IM 3 68 Bai et al., 2003 parrifolium mildew S. esculentum/ S. F2, F3 350 Fruit wt, SSC, 71 RFLP ML 7-13 44-72 4-42 Paterson et al., 1991 cheesmanii pH S. esculentum/ S. F2 160 Fruit size and 93 RFLP Regression 3, 7 30, 46 13-17, Van der Knaap and pimpinellifolium shape 7-19 Tanksley, 2003 S. esculentum/ S. F2 234 Acylsugar 150 RFLP IM 5 Mutschler et al., 1996 pennellii S. esculentum/S. RIL 144 Organoleptic 103 RFLP, SIM, CIM 1-5 9-45 Causse et al., 2001; Saliba- esculentum var. traits RAPD, Colombani et al., 2001 cerasiforme AFLP S. esc/ S. BC, 213, Late blight 104, 98 RFLP IM 2-6 >20, 22 19-27 Brouwer and St. Clair, hirsutum RBC 133 2004 S. esc/ S. BC1 145 Early blight 141, 23 RFLP, SIM, CIM 10 >57 8-26 Foolad et al., 2002 hirsutum RGAs S. esc/ S. chilense BC1S1 50 TYLCV 61 RFLP ANOVA 3 Zamir et al., 1994 S. esculentum/ S. F8 RIL 97 Fruit wt, SSC, 132 RFLP ANOVA 7-13 4-42 Goldman et al., 1995 cheesmanii seed wt, color S. esc./ S BC1F5 Fruit color ANOVA 13 15-89 Kabelka et al., 2004 hirsutum S. esc/ S. BC1 119 Lycopene 151 RFLP IM 1-7 39-75 4-33 Chen et al., 1999 pimpinellifolium content S. esc/ S. BC2F6 170 22 quality 126 RFLP Regression 71 4-17 Doganlar et al., 2002 pimpinellifolium traits QTL = Quantitative trait loci. BC = Backcross. RIL = Recombinant inbred lines. SSC = Soluble solids content. wt = weight. TYLCV = Tomato yellow curl leaf virus. IM = Interval mapping. ML = Maximum likelihood. SIM = Simple interval mapping. CIM = Composite interval mapping. RBC = Reciprocal 2 2 backcross. esc = esculentum. R t = Percentage of total variation for all the components of the trait. R i = Percentage of individual variation for all the components of the trait.

Experimental Design for Mapping Resistance Genes Genotypes of parents and their progenies for the traits of interest along with phenotypic measurement of quantitative traits are necessary to construct linkage map and to detect QTL. Randomized planting of male and female parents, F1, and F2 either in field or glasshouse have been used to map tomato QTLs (Osborn et al., 1987; Tanksley and Hewitt, 1988; Paterson et al., 1991; DeVicente and Tanksley, 1993; Breto et al., 1994; Alpert et al., 1995; Mutschler et al., 1996; Foolad et al., 1997; Saliba-Colombani et al., 2000; Causse et al., 2001; Lippman and Tanksley, 2001; Van der Knaap and Tanksley, 2003).

Replication is necessary and commonly used to estimate the error variance and to improve the significance of experimental results. However, for linkage and QTL analysis based on the F2 population, replication is not necessarily considered important (DeVienne and Causse, 2003). The number of different individuals in different genotypic classes serves the basis for such type of analysis. Here, power of QTL detection and precision of QTL location depends more on the population size rather than on marker density (Darvasi et al., 1993; Liu, 1998). For example, to determine whether two gene pairs are linked or assorting randomly, the conventional method is to compare the number of individuals observed in each phenotypic class with those expected on the basis of independent assortment, and then to test the deviation between these values by chi-square test. An F2 is the most conveniently illustrative population for QTL mapping on a marker by marker basis (DeVienne and Causse, 2003), because it is the only generation in which the three possible genotypes of one locus are present. We can construct a linkage map where there is an association between a marker and the trait. With the experiment of randomizing few individuals of male and female parents, F1 and F2 individuals, QTLs have been mapped in tomato for number of different traits, for examples powdery mildew resistance from 104 F2 individuals (Bai et al., 2003), salt tolerance from 206 F2 plants (Breto et al., 1994), different quantitative traits from 432 F2 plants, 32 female plants, 18 male plants and 33 F1 plants.

Furthermore, individual plants from BC1 population without replication have also been used for QTL analysis (Alpert et al., 1995).

Bulked Segregant Analysis Tagging of a particular segment of chromosome by molecular marker is the prerequisite of marker-assisted breeding. Earlier, use of near isogenic lines (NIL) was the common means of identifying the gene of interest. NIL are not available for most of the target regions and it takes a long time to develop. Alternatively, Michelmore et al. (1991) developed a very rapid and simple PCR-based method to identify the gene of interest called bulked segregant analysis (BSA) and identified RAPD markers linked to a disease resistance gene in lettuce. For BSA, any kinds of mapping populations, e.g., RIL, BC, F2 and DH that are segregating for a trait of interest, can be used (Table 4, Figure 6).

In BSA, two extreme phenotypes, i.e., low and high (or susceptible and resistant) of a particular trait from a segregating population, are compared using bulk DNA from these two extremes. DNA from individuals similar to trait of interest are bulked and assumed that the bulks are homozygous for the target interval and heterozygous for the rest of the interval as shown in Figure 6 (Giovannoni et al., 1991; Michelmore et al., 1991; Quarrie et al., 1999). DNA markers are then used to screen the parents and bulks. If polymorphism is found between bulks, this marker (polymorphic band) is expected to be associated with gene of interest.

RAPD marker is not considered suitable for MAS mainly due to its dominant nature, multiple loci amplification and low reproducibility, and sensitivity to reaction conditions. However, many disease resistance genes have been identified in tomato using RAPD (Table 4). Once RAPD markers linked to resistance genes are detected, they can be converted into co-dominant markers, for example, CAPS or SCAR, which are more reliable than RAPD. There may be potential to use BSA for identifying gene conferring multiple disease resistance (MDR) in a faster way. Zwonitzer et al. (2010) used RIL to see the evidence of MDR in maize following the conventional QTL mapping strategy.

P1 P2

Figure 6. General description of bulked segregant analysis (BSA) technique. After developing segregating population for trait of interest from crossing inbreds, two bulks are prepared from two extremes. These bulks and parents are then screened by markers to identify the polymorphic ones. P = Parent. B = Bulk. RIL = Recombinant inbred lines. BC = Back cross. DH = Double haploid. R = Resistance. S = Susceptible. Bar is a chromosome representation of each individual that contains a target region. The two lines across the bars indicate homozygote interval.

Table 4. Use of bulked segregant analysis (BSA) to identify traits of interest in different crop species SN Marker Bulked Crop Population Traits studied Reference Type Total Polymorphic between size used Parents Bulks 1. RFLP, 100 - 3 RAPD 14-20 Lettuce F2 Resistance to downy mildew Michelmore RAPD RAPD et al. 1991 2. RAPD 600 254 4 100, Tomato F4 QTL Resistance to TYLCV Chagué et al., 64 1997 3. RAPD 200 - 14 7-8 Tomato F7 (RIL) Flower number, fruit number, Lin et al., fruit setting percentage, yield, 2006 fruit weight 4. RAP D 382 117 6 67, 33 Tomato F2 Resistance to TSWV Chagué et al., 1996 5. RAPD 271 28 5 - Tomato F2 Resistance to TSWV Śmiech et al., 2000 6. RAPD 200 3 7-14 Tomato F2 Pedicel abscission and fruit Giovannoni ripening et al., 1991 7. SNP 1536 27 9, 8 Soybean F2 Resistance to soybean rust Hyten et al., 2009 8. SSR 540 - - 5 Wheat DH Resistance to stripe rust Lan et al., 2010 9. RAPD 222 63 4 14 Corn F2 Resistance to northern leaf Khampila et blight al., 2008 10. RAPD 200 7 3 8 Tomato F2 Male sterility Staniaszek et al., 2000 11. AFLP 256 13 2 10 Tomato F2 Resistance to bacterial wilt Miao et al., 2009 12. RAPD 10 and 2 and 1 10 Tomato F2 Heat tolerance Kamel et al., and 6 2010 ISSR

Table 4. Continued SN Marker Bulked Crop Population Traits studied Reference Type Total Polymorphic between size used Parents Bulks 13. RAPD 13 5-6 Apple F1 Scab and powdery mildew Sestras et al., resistance, ideotype 2009 14. RAPD 164 19 2 14, 3 Wheat F2 Stripe rust resistance Chague et al., 1999 15. RAPD 700 - 7 5 Barley DH Rhynchosporium secalis Barua et al., resistance 1993 16. SSR 48 1 8 Wheat NIL Male sterility Cao et al., 2009 17. SSR 109 4 10 Maize F2 Drought tolerance Gemenet et al., 2010 18. SSR 293 2 20 Rice RIL Drought tolerance Venuprasad et al., 2009 19. RAPD 213 47 1 and 1 10, 9 Tomato F2 Powdery mildew resistance De Giovanni and and 26 et al., 2004 AFLP 20. AFLP 128 1 5 Tomato F2 Late blight resistance Moreau et al., 1998 21. SSR 109 60 5 9 Wheat F3 Powdery mildew resistance Chantret et al., 2000 22. AFLP 42000 3 10 Tomato F2 Resistance to Cladosporium Thomas et al., fulvum 1995 SN = Serial number. DH = Doubled haploid. RIL = Recombinant inbred lines. NIL = Near isogenic lines. TYLCV = Tomato yellow leaf curl virus. TSWV = Tomato spotted wild virus. QTL = Quantitative trait loci. Bulked size means number of individuals used.

BSA has been an important technique to identify the DNA markers linked to trait of interest. Availability of markers is very important to accelerate the breeding process. To implement the MAS in tomato resistance breeding, it is necessary to tag the molecular markers linked to resistance genes. This study focused on the identification of markers through BSA technique with the following specific objectives.

OBJECTIVES The main objective of this study is the development of reliable molecular markers that are tightly linked to septoria leaf spot and late blight resistance genes in tomato. The specific objectives were to:

1. determine the inheritance pattern of septoria leaf spot in a tomato population derived from intra-species crosses; and

2. identify the molecular markers linked to septoria leaf spot and late blight resistance genes.

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CHAPTER 2 Molecular Tagging of Resistance Genes to Septoria Leaf Spot in Tomato (Solanum lycopersicum L.)

ABSTRACT Molecular marker linked to the resistance loci is necessary to accelerate the resistance breeding process through marker assisted selection (MAS). We studied the inheritance of resistance to Septoria lycopersici and identified RAPD markers linked to septoria leaf spot resistance in tomato using an F2 population and bulked segregant analysis. Two tomato inbred lines, NC 85L-1W(2007), susceptible to septoria leaf spot pathogen, and NC 839-2(2007)-1, resistant to septoria leaf spot pathogen, were crossed to produce an F1. A total of 250 F2 plants, and 10 plants each of P1, P2 and F1 were grown in research plots at the Mountain Horticultural Crops Research and Extension Center, Mills River, NC, in the summer of 2009. Disease severity was scored under natural inoculums using a scale of 0 to 5, where 0 = no disease and 5 = complete development of disease on the plant. DNA was extracted from 2-3 weeks old plants. The parental lines were screened with a total of 379 molecular markers including 157 SSR, two conserved ortholog set (COS), 23 M-13 tailing SSR and 197 RAPD primers. Two DNA bulk samples, identified as resistant bulk (RB) and susceptible bulk (SB), were prepared from the F2 individuals. The RB sample consisted of 8 individuals with a disease score of 0 and the SB sample contained 8 individuals with a score of 4 to 4.5. Transgressive segregants among the F2 population were found only for susceptibility. The segregation ratio of resistant and susceptible plants fit a 3:1 ratio (2 = 3.014, P = 0.083) indicating that the inheritance of resistance to septoria was based on a single dominant gene in the present study. Only 34 RAPD primers (17.26%) were found polymorphic between parents. Eleven RAPD primers (32.35%) showed polymorphism between resistant and susceptible bulks. Five RAPD primers were identified linked to septoria disease reactions. Among the five identified primers, two were linked to

susceptible and three were linked to resistance. Subject to verification in an independent population, these markers may be useful for MAS of septoria leaf spot resistance in tomato.

INTRODUCTION Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops worldwide. Among the foliar diseases of tomato, septoria leaf spot (Septoria lycopersici Speg.) is one of the most devastating diseases. It may cause complete defoliation leading to a significant crop loss under favorable environmental conditions, particularly in humid regions during periods of high rainfall, frequent dew, or over-head irrigation (Andrus and Reynard, 1945; Delahaut and Stevenson, 2004). Septoria occurs worldwide but it is particularly important in Canada and United States (Ferrandino and Elmer, 1992; Poysa and Tu, 1993).

Although fungicides are effective at controlling septoria, breeding for resistant varieties is a priority due to the cost involved in applying fungicides and environmental hazards associated with their use. However, with the use of fungicides, this disease has received less attention from breeders in the past (Locke, 1949; Barksdale and Stoner, 1978).

It has been reported that resistance to septoria is controlled by a single dominant gene (Barksdale and Stoner, 1978). The resistance in PI422397 is moderate and associated with small fruit size and late in maturity (Poysa and Tu, 1993). The majority of the resistant lines belong to the wild species, e.g., S. peruvianum, S. gladnulosum and S. pimpinellifolium. The highest degree of resistance was found in S. habrachaites (Andrus and Reynard, 1945; Locke, 1949).

Twenty-two accessions out of 700, mostly from S. habrachaites and S. peruvianum, had a score of 2.0 and 3.9 (when scored using a 0 to 9 scale, where 0 = no disease and 9 = severe disease) indicating that they were more resistant than PI422397 (Poysa and Tu, 1993). Useful levels of resistance have also been found in S. pennelli, S. pimpinellifolium, S. chilense, and S. lycopersicum var. cerasiforme (Poysa and Tu, 1993). Breeding lines of

interspecific crosses with S. habrachaites accessions had high levels of resistance. But, these interspecific lines had one or more undesirable horticultural traits such as indeterminate growth habit, late maturity or low yield.

Many resistant varieties of tomato have been developed through the introgression of resistant genes either from cultivated or from wild species of tomato. More than 8 years is necessary to develop resistant varieties through conventional breeding, i.e., using morphological markers or relying on phenotypic data. Molecular markers have emerged as a very useful tool for breeders to select the desirable genotypes at any crop stage. Molecular markers are useful for accelerating breeding process by improving precision and making selection more efficient. Markers linked to the gene(s) of interest can be used to select plants genotypically that are genetically similar to the recurrent parent possessing the desired horticultural traits. However, due to lack of molecular markers linked to septoria leaf spot of tomato, marker assisted selection (MAS) has not been initiated in septoria resistance breeding. Identification of markers is a prerequisite for MAS.

Earlier, use of near isogenic lines (NIL) has been the common means of identifying genes of interest. NILs are not available for most of the target regions and it takes a long time to develop NIL. Alternatively, Michelmore et al. (1991) developed a very rapid and simple PCR based method to identify the gene of interest called bulked segregant analysis (BSA) and identified random amplified polymorphic DNA (RAPD) markers linked to a disease resistance gene in lettuce. For BSA, any kind of mapping population e.g., recombinant inbred lines (RIL), backcross (BC), F2 or double haploid (DH) that are segregating for a trait of interest can be used.

In BSA, two extreme phenotypes, i.e., low and high (susceptible and resistant in case of resistance breeding) of a particular trait from a segregating population are compared using bulk DNA from these two extremes. DNA from individuals similar to the trait of interest are pooled and assumed that the bulks are homozygous for the target interval and heterozygote for the rest of the interval (Giovannoni et al., 1991; Michelmore et al., 1991;

Quarrie et al., 1999). DNA markers are then used to screen the parents and bulks. If polymorphisms are found between bulks, this marker (polymorphic band) is expected to be associated with the gene of interest.

Many disease resistance genes have been identified in tomato using RAPD following the BSA approach. Once identified, these markers can be converted into co-dominant markers, for example, cleaved amplified polymorphic sequence (CAPS) or sequence characterized amplified region (SCAR), which are more reliable than RAPDs. SCAR being a co-dominant marker can be applied in MAS. De Giovanni et al. (2004) identified a RAPD marker linked to the ol-2 gene conferring resistance to powdery mildew using BSA from an

F2 population. A single RAPD marker, OPU31500 with 1500 bp in size was detected in the susceptible bulk and converted to a CAPS marker. The distance between the marker and the ol-2 gene was also estimated through linkage analysis. Stevens et al. (1995) and Chagué et al. (1996) also identified RAPD markers linked to the Sw-5 gene that conferred resistance to tomato spotted wilt virus (TSWV). Linkage analysis mapped this marker within a distance of 10.5 cM from Sw-5. This RAPD marker was later converted into a SCAR marker. Śmiech et al.

(2000) used BSA in an F2 segregating population and found five primers that distinguished resistant and susceptible bulks.

DNA markers, though prerequisite for MAS, are not available for septoria leaf spot (SLS) resistance breeding in tomato. Information is also lacking on the linkage group of the resistance gene to septoria leaf spot. In such situations, BSA could be of valuable approach to tag the resistance gene without constructing linkage map.

In this study, we used the BSA technique to identify RAPD markers linked to septoria leaf spot resistance in tomato using an F2 population consisting of 8 resistant and 8 susceptible lines. An F2 population is the earliest segregating generation available for mapping genes from the crosses between inbreds and provides the greatest genetic window around the locus (Michelmore et al., 1991).

MATERIAL AND METHODS

Plant Materials Two tomato inbred lines, NC 85L-1W(2007) (referred onward as NC 85L) and NC

839-2(2007)-1 (referred here onward as NC 839) were crossed to produce F1 in greenhouse. NC 85L, used as a female, is susceptible to septoria leaf spot (susceptible parent, SP) and NC

839, used as male, is resistant to septoria leaf spot (resistant parent, RP). The F1 plants were selfed to produce F2 seeds. A total of 250 F2 plants, and 10 plants each of SP, RP and F1 were grown in this study. Among F2 plants, nine were blinds (seedlings without meristem) and data in six plants were missing. Therefore, we used 234 F2 plants in this study. The fruit of NC 85L was a mini roma type with dark red fruit color. It was developed by crossing a male sterile line, NC 051(x)-18, with a normal inbred line, NC 05108-2-166. The resistant line, NC 839 was a grape tomato with light red fruit color (Table 5). It was a product of two inbred lines, NC 0661 and NC 6109-1-196. The number of lines involved (i.e., crossing frequency) in developing NC 839 was higher than NC 85L.

Field Evaluation

A total of 280 plants consisting of 10 plants of each parent and F1 and 250 F2 plants were planted in the R16 Research Plot of the Mountain Horticultural Crops Research and Extension Center (MHCREC), Mills River, NC, during the summer of 2009. This research plot is a hotspot for septoria leaf spot and natural inoculum was abundant in the summer of 2009. Weather conditions of the tomato growing season are given in Appendix 1, and were favorable for disease development. Plants were evaluated for septoria leaf spot infection from natural inoculums using a scale of 0 to 5, where 0 = no disease and 5 = complete development of disease on the plant. Late blight infection was also scored from the same population. The experimental site was located at an altitude of 630 m (2067’) above sea level with latitude of 35.42721o and longitude of -82.55888o (http://www.nc- climate.ncsu.edu/cronos/?station=FLET).

Table 5. Parental description along with their pedigrees and coefficient of parentage Parent Maturity Fruit type, Disease Pedigree Common COP shape, reaction pedigree color NC 85L- Early Mini roma Late 051(x)- 0179(x)-1- 1W(2007) type, dark blight 18//0463/9722(x)- 18-4, 215E- red resistance 18 1(93), 0.227 NC 839- Average Grape Septoria 051(x)-18//CB25(x)- 9722(x)-18, 2(2007)-1 type, light leaf spot 18-3/9722(x)- 051, 03220, red resistance 18/0464 L3707 COP = Coefficient of parentage.

Seeding was done on June 1, 2009, in 30.5 x 45.5 cm (12 x 18”) trays containing peat moss and vermiculite. Trays were kept in the greenhouse with an average day temperature of 21.1oC (70oF). Twelve day old seedlings were transplanted into a 50-cell tray size of 12.7 x 24.4 cm (5 x 10”). Six week old seedlings were then transplanted in the field having loam soil with a row to row distance of 1.5 m (5’) and 45 cm (18”) plant to plant. The bed was raised and covered with black plastic. Other recommended cultural practices were followed as described in the Southeastern US 2010 Vegetable Crop Handbook (http://www.ncsu.edu/ enterprises/tomatoes/2010/04/07/2010-southeastern-us- vegetable-crop-handbook/).

DNA Extraction, Quantification and Dilution DNA was extracted from all samples following the method of Fulton et al. (1995). A detailed procedure of DNA extraction is given in Appendix 5. Briefly, approximately 100 mg of young leaves from 2-3 week old tomato seedlings were collected from the greenhouse in 1.5 ml Eppendorf tubes. The tubes were then dipped into liquid nitrogen and samples were ground using a glass rod. After adding 200 µl microprep buffer (see Appendix 5), samples were incubated in a 65oC water bath for about 60 min and filled with chloroform/isoamyl (24:1) solution. Samples were then centrifuged at 10,000 rpm for 5 minutes. The aqueous phase was pipetted off into a new micro-centrifuge tubes and 2/3rd to 1 times the volume of cold isopropanol was added to precipitate the DNA. After centrifuging this sample at 10,000 rpm for 5 minutes, the DNA pellet was separated and washed with 70% ethanol. The dry DNA pellet was resuspended in 100 µl of 1x TE buffer and stored at -20oC. Concentrations of DNA, which were estimated using spectrophotometer (NanoDrop 1000, Thermo Scientific, DE USA) ranged from 109 to 4621 ng/µl in all samples. Working solutions of DNA samples with a concentration of 20 ng/µl were prepared from original DNA samples in 1x TE buffer.

PCR-based Molecular Markers Screening

Simple Sequence Repeat (SSR) Two parental lines were screened with 157 SSR and two conserved ortholog set (COS) primers (Fulton et al., 2002) (Appendix 4). Amplification reactions were performed in a 10 l reaction volume containing PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 M of each primer and 0.625 U of Taq DNA polymerase with 20 ng template DNA. DNA amplifications were performed in a thermal cycler (Applied Biosystems, Life Technologies, USA) using the following cycling conditions: one cycle of 94oC for 3 min; 34 cycles of 94oC for 0.30 min, 52-58oC for 1min, 72oC for 1 min; one cycle of 72oC for 10 min followed by holding at 4oC.

M-13 Tailing SSR Conventional gel electrophoresis did not give any polymorphic SSR primers between parents. Therefore we used M-13 tailing SSR primers (Schuelke, 2000) to use the capillary electrophoresis system of fragment analysis. Twenty three M-13 tail SSR primers were screened. The M-13 sequence incorporated at the 5’-end of the forward primer was TGT AAA ACG ACG GCC AGT. Amplification was performed in a 25 l reaction volume consisting of 1x PCR buffer with 1.5 mM MgCl2, 0.2 mM dNTP, 0.08 µM forward primer with M-13 tail, 0.6 µM reverse primer, 0.6 µM IRD-labeled M-13 primer, 0.9 U Taq polymerase and 20 ng template DNA. The reaction was carried out in a thermal cycler (Applied Biosystems, Life Technologies, USA) using the following cycling conditions: one cycle of 94.0°C for 2:30 min, 15 cycles at 94.0°C for 0:30 min, 53-56oC for 0:30 min and 72.0°C for 1:00 min, 25 cycles of 94.0°C for 0:30 min, 50.0°C for 0:30 min and 72.0°C for 1:00 min and one cycle of 72.0°C for 7:00 min followed by holding at 4.0°C. This PCR profile is specific to the M-13 tailing protocol. The first 15 cycles amplify the specific region of interest at the annealing temperature (Ta) specific to the forward primer. The second round of amplification has 25 cycles where the Ta is specific to the M-13 primer and will allow the IRD-label to be incorporated.

An ABI plate was set up by adding 9.5 µl formamide /LIZ mix in 1 µl PCR product. Mix of LIZ standard and formamide was at the concentration of 1/200 by volume. The plate was denatured at 95oC for 5 min. DNA fragment analysis was done by capillary electrophoresis using an ABI 3730 DNA Analyzer (Applied Biosystems, Life Technologies, USA). Data were analyzed using GeneMarker Software V1.91 (SoftGenetics, USA). Electropherogram was then compared between parents to detect any polymorphisms.

Random Amplified Polymorphic DNA (RAPD) A total of 197 10-mer RAPD primers (Appendix 4) were screened using 20 ng DNA template of two parental lines. Primers polymorphic to parental lines were then used to screen resistant bulk and susceptible bulk samples. Amplification reactions were performed in 10 l reaction volumes containing 1x buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM

MgCl2), 200 M of each dNTP, 0.2 M primer and 1 U Taq polymerase. About 15 l mineral oil was overlaid on the reaction mixture. DNA amplifications were performed in a thermal cycler (Eppendorf, New York) using the following cycling condition: one cycle of 92oC for 3 min; 45 cycles of 92oC for 0.30 min, 42oC for 1 min and 72oC for 0.30 min; one cycle of 72oC for 8 min followed by holding at 4oC.

Disease Scoring Disease severity was scored at 77 days after seeding (August 17, 2009). Individual disease rating scores were based on visual assessment of symptom severity. The following scoring criteria was developed based on Winstead and Kelman (1952), Tu and Poysa (1990), Danesh et al. (1994) and used in this study. Severity of disease was scored at a scale of 0 to 5 with 0.5 increment, as 0 = no disease symptoms, 0.5 = Less than 10% leaf area with symptoms, 1 = 10-20% leaf area with symptoms, 1.5 = 20-30% leaf area with symptoms, 2 = 30-40% leaf area with symptoms, 2.5 = 40-50% leaf area with symptoms, 3= 50-60% leaf area with symptoms, 3.5 = 60-70% leaf area with symptoms, 4 = 70-80% leaf area with symptoms, 4.5 = 80-90% leaf area with symptoms, and 5 = 90-100% leaf area with symptoms.

For the inheritance study, we grouped all segregating plants into two, one resistance group with score from 0 to 2 and second, susceptible group with score from 2.5 to 5.

Bulked Segregant Analysis Bulked segregant analysis (BSA) was performed following the method of Michelmore et al. (1991). Two DNA bulks, called resistant bulk (RB) and susceptible bulk (SB) were prepared from F2 individuals. The RB consisted of 8 individuals with disease a score of 0 and SB contained 8 individuals with a score of 4 and 4.5. These two DNA bulks were prepared by pooling equal amounts of DNA (50 µl) from each individual. Non-correlation of disease score value of each bulk with total soluble solid indicated that, these bulks were from the two extremes of disease severity. PCR was run with polymorphic primers between parents on the bulks and parental DNA samples using the same reaction conditions as described above. PCR was repeated for at least two times for those primers that were polymorphic between bulks.

Gel Electrophoresis All RAPD and SSR PCR products were analyzed in 2% agarose gels containing 0.1 g/ml ethidium bromide in 1x TBE buffer (40 mM Tris-borate, pH 8.0, 1 mM EDTA) with a 100-bp ladder. Electrophoresis was run at 135 V for 2 hours. Gels were rinsed with water to enhance contrast and photographed under UV light. Polymorphism between parents by SSR primers was not found in agarose gel. Therefore, some PCR products with SSR primers were separated in 8% PAGE and stained with 0.5 µg/ml solution of ethidium bromide. Stained gels were photographed under UV light. RAPD fragments were scored as 1 for presence and 0 for absence. Band size was estimated based on a 100-bp DNA ladder.

DNA Extractions from the Gel for Sequencing Polymorphic RAPD bands between parents and bulks were extracted from the 2% agarose gel following the protocol of GenElute™ Gel Extraction Kit (Sigma) (Appendix 6). These DNA are being sequenced for converting useful RAPD primers into SCAR or CAPS markers.

Data Analysis

Scores of parental lines and F1 were averaged. Frequency of different score categories was estimated for F2 populations using SPSS v.17.0 (IBM, Corporation, New York, USA) for segregation analysis and frequency distribution. Skewness was estimated using SPSS v.17.0. Frequency data were analyzed by 2 to test the goodness of fit at an expected ratio of 3R: 1S lines using SAS v.9.1 (SAS Institute Inc., Cary, NC). Simple statistics based on the RAPD bands were calculated using MS Excel 2007.

The pedigrees of these two lines were traced back to estimate the coefficient of parentage (COP). The COP was estimated between parents on the assumption that all ancestors and parental lines are homozygous and a line derived from a cross obtains one- half of its genes from each parent. The computer software KIN (Tinker and Mather, 1993) was used to calculate the COP.

RESULTS

Segregation of Resistance Severity of septoria leaf spot (SLS) infestation was assessed on 234 individual plants at 77 days after planting, based on the percentage of total leaf area infected. The distribution of disease reaction was highly right-skewed (Figure 7). Only 234 plants out of 250 were scored for SLS either because some of the plants were blinds (with no meristem) or dead or mal-formed because of other factors. In tomato, blind plants are those seedlings that do not produce meristem and after few weeks such plants die. No symptoms were observed in the resistant parents, but the susceptible parents had intermediate levels of reaction (score from 1.5 to 3) against the septoria pathogen. Comparing the scores of F2 individuals with their parents, it was clear that transgressive segregants were found only towards susceptibility. This indicated that, the susceptible parent had also some contribution to resistance. Based on the distribution of F2 individuals, we found clearly two distinct groups, resistance and susceptible plants which allowed us to apply bulked

segregant analysis (BSA) to identify the linked markers. The distribution was right-skewed; however, there were enough individual plants for BSA using the two extremes.

About fifty percent of the F1 plants had a disease score of 0 indicating that resistance to the septoria pathogen was incomplete dominant. Among 234 F2 individuals, 164 were resistant and 70 (score greater than 2) were susceptible. The segregation ratio of resistant and susceptible plants fit the expected ratio of 3:1 (2 = 3.014, P = 0.083) for a single gene (Table 6), which indicates that the inheritance of resistance to septoria was based on a single dominant gene in the present study.

Two parents were phenotypically contrasting mainly for fruit type and color (Table 5). Fruit quality and shape of NC 839 are superior to NC 85L. Pedigree analysis of these parents showed that six parents were common. The coefficient of parentage between them was 0.23 indicating some dissimilarity between them. Variation was found between these parents in morphology and pedigree. However, we could not find any polymorphic SSR markers between them. We screened 180 SSR and two COS primers. Most of them were monomorphic and some of them did not amplify the genomic DNA of these parents (Appendix 7). Three systems of fragment analyses including agarose gel, polyacrylamide, and capillary gel electrophoresis did not detect any polymorphism, indicating lack of polymorphic SSR markers in these two lines.

RAPD Markers Out of 197 RAPD primers used to screen parental lines, 34 RAPD primers (17.26%) were polymorphic (Table 7). A total of 176 bands with a maximum fragment size of 1500 bp and minimum fragment size of 100 bp were amplified using 34 primers. Among these fragments, 84 were polymorphic between parents. The average number of bands per polymorphic RAPD primer was 4 and ranged from 2 to 8 bands. The number of polymorphic bands ranged from 1 to 4 with an average of 2. All 34 polymorphic RAPD primers were used to screen the resistant and susceptible bulks.

Figure 7. Frequency distribution of 234 F2 individuals derived from NC-085L-1W(2007) x NC- 839-2(2007)-1 based on the score of severity of septoria leaf spot in tomato, Mills River, NC, 2009.

The average phenotypic values of the parents and F1 are shown by the arrows. SP = Septoria leaf spot susceptible parent, NC 85L-1W(2007). RP = Septoria leaf spot resistance parent, NC 839-2(2007)-1.

RAPD Markers and Bulked Segregant Analysis Among the 34 polymorphic RAPD primers observed between parents, only 11 RAPD primers (32.35%) showed polymorphism between resistant and susceptible bulks (Table 8). A total of 87 bands were amplified by 34 RAPD primers. Among these bands, 34 and 20 bands were polymorphic between parents and between bulks, respectively. The size of bands ranged from 2000 to 150 bp. The average number of bands was seven per primer among total amplified bands and the average number of polymorphic bands between parents and between bulks was three and two, respectively. Five primers that distinguished parents as well as bulks were identified linked to septoria disease reaction (Table 9). One band of each of two primers, namely MRTOMR-121 and MRTOMR-031 (Figure 8) was found only in the susceptible parent NC 085L and susceptible bulk. Similarly, one band of each of 3 RAPD primers was amplified only in the resistant parent NC 839 and resistant bulk. Therefore, among five identified primers, two were linked to susceptible and three were linked to resistance. Amplified bands size linked to susceptible were 380 and 600 bp of RAPD primers MRTOMR-121 and MRTOMR-031, respectively (Table 10). Three RAPD primers linked to resistance were MRTOMR-022, MRTOMR-117 and MRTOMR-121 (Figure 9). The size of linked bands of these primers ranged from 1000 to 600 bp.

Useful RAPD primers related to disease reaction are listed in Table 11. Six primers were found to be unlinked to the loci (Figure 10). These primers distinguished only parents and not bulks, therefore, stated as unlinked markers. Some of the amplified bands were only found in bulks but not in either parent (Figure 10). This may be due to recombinants in the F2 population.

Table 6. Segregation ratio of resistant and susceptible progenies to septoria leaf spot observed in the F2 population of tomato derived from NC 085 x NC 839, 2009 Cross Resistant Susceptible Total Expected 2 P- (0-2 scale) (2.5-5 scale) ratio (R:S) value NC 085L-1W(2007)/ 164 70 234 3:1 3.014 0.083 NC 839-2(2007)-1

Table 7. RAPD primers polymorphic between resistant and susceptible parents of tomato to septoria leaf spot

Primer Sequence % GC Amplified Band size, Polymorphic % Tm bands bp band polymorphic Max Min MRTOMR-001 ATCGAGCACT 50 2 450 400 1 50 30 MRTOMR-003 AAGGGGCTGC 70 5 600 150 1 20 34 MRTOMR-014 CCGTCTCCAG 70 7 600 150 3 43 34 MRTOMR-022 CAGGGCCAGC 80 8 1000 100 6 75 36 MRTOMR-023 GGACCACGAA 60 3 650 100 2 67 32 MRTOMR-026 TAGGCACCGT 60 5 1000 300 3 60 32 MRTOMR-027 ACCTGATGCA 50 3 650 200 3 100 30 MRTOMR-029 CGCCATACGG 70 2 900 650 2 100 34 MRTOMR-031 GGGACGTCGC 80 5 600 200 2 40 36 MRTOMR-033 CGCTCGCGGC 90 5 1000 150 2 40 38 MRTOMR-038 TACCTTCGCC 60 6 1100 150 3 50 32 MRTOMR-039 CAGCACCACC 70 7 650 100 3 43 34 MRTOMR-040 ACAGTCCGCG 70 3 500 180 1 33 34 MRTOMR-046 CCATGCGCTA 60 5 600 100 4 80 32 MRTOMR-063 ACGAGTGACC 60 6 550 100 2 33 32 MRTOMR-078 GTGCCATGTG 60 4 700 200 1 25 32 MRTOMR-100 GAC GGC CCC A 80 7 1500 200 3 43 45 MRTOMR-110 ATG ACG ACC T 50 3 800 400 1 33 31 MRTOMR-112 CAT ACA CCT C 50 4 1500 650 2 50 25 MRTOMR-117 CCG AAC AAT C 50 6 950 300 5 83 28 MRTOMR-118 TGC TTG GGG G 70 4 1500 500 3 75 39 MRTOMR-121 GGC GTC GTA A 60 3 1100 550 2 67 36 MRTOMR-128 AGA CCC GGT C 70 3 600 300 1 33 38 MRTOMR-130 AGG TCT CTC G 60 3 700 250 1 33 32 MRTOMR-133 TTC AGC CAC A 50 3 900 300 2 67 32 MRTOMR-140 TGC CAA CGC C 70 5 800 250 1 20 42 MRTOMR-141 CAT TGG TGC T 50 2 900 450 1 50 31 MRTOMR-142 TTG CGC TTG T 50 2 800 400 1 50 37 MRTOMR-146 CGT TAC CGG G 70 5 2000 450 2 40 35 MRTOMR-161 TGT CTC CCT G 60 5 1500 700 1 20 32 MRTOMR-171 GAG GCCAGC G 80 4 700 200 1 25 43 MRTOMR-178 GCC ATC CGA A 60 2 800 650 1 50 35 MRTOMR-181 CAT GCG CTC C 70 3 800 400 1 33 39 OPK6 CACCTTTCCC 60 4 1000 600 1 25 31 P#72 GAGCACGGGA 70 4 600 250 1 25 39 bp = base pair. Tm = Melting temperature.

Table 8. Polymorphic RAPD markers and their size between resistance and susceptible bulks of tomato to septoria leaf spot Primer Amplified Size, bp Polymorphic % polymorphic

bands Max Min Between Between Between Between parents bulks parents bulks MRTOMR-022 8 1000 150 5 1 63 13 MRTOMR-031 6 1100 280 2 1 33 17 MRTOMR-038 5 800 500 3 1 60 20 MRTOMR-100 8 1500 350 4 1 50 13 MRTOMR-112 7 1500 300 1 3 14 43 MRTOMR-117 5 950 550 1 1 20 20 MRTOMR-118 9 1500 350 3 1 33 11 MRTOMR-121 8 1100 380 3 5 38 63 MRTOMR-130 4 700 250 1 25 0 MRTOMR-146 8 2000 450 4 1 50 13 MRTOMR-161 5 1500 700 1 2 20 40

Table 9. Polymorphic bands of RAPD markers linked to either resistance or susceptible genes of tomato for septoria leaf spot Marker PBN Size, bp SP RP RB SB Marker type MRTOMR-022 1 1000 0 1 1 0 P 2 800 0 1 0 0 - 3 600 1 0 1 1 - 7 250 1 0 1 1 - 8 150 1 0 1 1 - MRTOMR-031 1 1100 0 1 1 1 - 3 600 1 0 0 1 N MRTOMR-117 2 850 0 1 1 0 P MRTOMR-118 3 800 1 0 0 0 - 4 750 0 1 0 0 - 6 600 0 1 1 0 P MRTOMR-121 1 1100 0 0 0 1 - 3 900 1 1 0 0 - 4 850 1 1 0 1 - 6 650 1 1 1 0 - 7 420 1 0 0 0 - 8 380 1 0 0 1 N

PBN = Polymorphic band number. RP = Resistant parent. SP = Susceptible parent. RB = Resistant bulk. SB = Susceptible bulk. P = Positive. N = Negative.

Table 10. Specific marker bands linked to either resistance or susceptible genes to septoria leaf spot (SLS) of tomato Marker SLS SLS susceptible Genotype resistance P1 (NC 085L) P2 (NC 839) MRTOMR-022 1000 bp - - + MRTOMR-031 - 600 bp + - MRTOMR-117 850 bp - - + MRTOMR-118 600 bp - - + MRTOMR-121 - 380 bp + - + = Presence. - = Absence.

Table 11. Informative RAPD markers associated to tomato genes in relation to reaction with septoria leaf spot Linked to Linked to Unlinked to reaction With bands only susceptible resistant segment in bulks MRTOMR-031, MRTOMR-022, MRTOMR-022, MRTOMR- MRTOMR-022, MRTOMR-121 MRTOMR-117, 031, MRTOMR-038, MRTOMR-121, MRTOMR-142 MRTOMR-100, MRTOMR- MRTOMR-146, 112, MRTOMR-130 MRTOMR-161

Figure 8. Electrophoresis pattern of DNA fragments generated by RAPD markers (A. MRTOMR-031, B. MRTOMR-121). Polymorphic band (i.e., linked to susceptible) between parents, and between resistant and susceptible bulks are indicated by arrow. SP = Susceptible parent, NC 085L. RP = Resistant parent, NC 839. RB = Resistant bulk. SP = Susceptible bulk. M = Marker.

Figure 9. Electrophoresis pattern of DNA fragments generated by RAPD marker (A. MRTOMR-022, B. MRTOMR-117 and C. MRTOMR-118). Polymorphic band (i.e., linked to resistance) between parents and between resistant and susceptible bulks are indicated by arrow. SP = Susceptible parent, NC 085L. RP = Resistant parent, NC 839. RB = Resistant bulk. SP = Susceptible bulk. M = Marker.

A. B.

Figure 10. RAPD marker (A. MRTOMR-130) showing polymorphic band (indicated by arrow) only to parents, i.e., band with unlinked loci and RAPD marker (MRTOMR-146) showing band (indicated by arrow) only in two bulks. SP = Susceptible parent, NC 085L. RP = Resistant parent, NC 839. RB = Resistant bulk. SP = Susceptible bulk. M = Marker

DISCUSSION Resistance to septoria leaf spot in the present study was hypothesized to be controlled by a single incomplete dominant gene. Resistance has previously been reported to have conferred by dominant gene, denoted by Se (Andrus and Reynard, 1945) as well as recessive gene based on field observations in past studies (Wright and Lincoln, 1940). The differences found in the inheritance of resistance in this experiment might be due to use of different genotypes in different environment or different sources of resistance. The susceptible parent we used in this study did not appear completely susceptible suggesting that it might have some degree of resistance

Screening the F2 population with 197 RAPD primers, we identified three RAPD markers linked to the resistance and two RAPD markers linked to the susceptibility. Through the bulking of the extreme individuals from F2 population we were able to rapidly tag the markers associated with chromosomal segments or alleles that have a role in the reaction of tomato to septoria leaf spot. Using the BSA technique and 8 individuals in each bulk, five primers gave different band sizes that were found useful for considering as markers in septoria resistance breeding. Bands of two of these markers were only present in the susceptible parent and susceptible bulk, and bands of the other three markers were present only in the resistant parent and bulk. Therefore, these bands were considered associated with the susceptible alleles or resistant alleles.

The tagging of resistance genes using BSA is very fast and facilitates screening for new alleles of resistance for a particular disease. This is important because a resistant gene once tagged may not be effective for a long time either because of recombination in the host genome or mutation in pathogen.

The two parental lines used in this study were closely related to each other and SSR screening proved this findings. However, using RAPDs we were able to distinguish these parents at the molecular level. RAPDs are multilocus-based markers; therefore all primers

we identified here might be from the overlapping regions of a chromosome. For example, MRTOMR-022 produced a 1000 bp band and MRTOMR-118 a 600 bp band. The band produced by MRTOMR-118 might be the part of the band generated by MRTOMR-022. A number of disadvantages are associated with RAPDs, including the fact that they anneal in multiple sites, are dominant, and their sensitivity to reaction conditions may limit their use directly in MAS. Therefore, candidate RAPD markers are generally converted to SCAR or CAPS which produce the co-dominant banding pattern, and are much more useful for MAS.

BSA has been used in a number of crop species to tag a number of traits including quantitative traits. After identifying useful markers by BSA in tomato, MAS is now possible to apply during the selection of resistance to verticillium wilt, tomato spotted wilt virus, root knot nematode, powdery mildew, and fusarium wilt. De Giovanni et al. (2004) identified a RAPD marker linked to the ol-2 gene which confers resistance to powdery mildew. A single RAPD marker, OPU31500 with 1500 bp in size was detected in the susceptible bulk and converted to a CAPS marker. The distance between the marker and the ol-2 gene was also estimated through linkage analysis. Stevens et al. (1995) and Chagué et al. (1996) have identified RAPD markers linked to the Sw-5 gene for resistance to tomato spotted wilt virus (TSWV). Among the four RAPD markers linked to the gene of interest, two were tightly linked to the Sw-5 gene. Linkage analysis mapped this marker within a distance of 10.5 cM from Sw-5. This RAPD marker was later converted into a SCAR marker. Czech et al. (2003) have used MAS for developing TSWV resistant tomato using PCR markers. A single PCR band of 0.94 kb indicated the resistance genotype and the susceptible genotype showed an additional band of 0.9 kb. Smiech et al. (2000) found five primers that distinguished resistant and susceptible bulks. A PCR-based co-dominant marker, tightly linked to Mi has also been developed (Williamson et al., 1994).

We used disease scores that were based on natural infestation. Different levels of infestation were observed in the plant population, and positive and negative controls, which indicated that inoculum pressure was enough to screen the population. Spaner et al.

(1998) mapped loci affecting resistance to powdery mildew, leaf rust, stem rust, scald and net blotch in barley using field-scored data of disease severity under natural infestation. Natural infestation was also used by Frei et al. (2005) to identify QTLs resistance to thrips in common bean. Use of natural inoculum saves cost and time. Screening of target populations in conducive environments can be more successful in the long run to get the durable resistance genotype. However, pressure of natural inoculum might not always be enough to screen the lines. In such conditions, artificial inoculum is necessary and it can be considered useful to verify the field data information. The technique we used here to isolate and culture the septoria pathogen (Chapter 4) will be practically applicable in a septoria screening program.

Use of hot spot for specific disease help to increase the disease pressure and are a cost effective way of resistance breeding and is particularly useful in horizontal resistance breeding. Horizontal resistance system is generally considered suitable for long term cultivation of resistance variety. We tagged R gene for septoria leaf spot resistance based on the segregating population that were naturally infested in this study.

We identified seven potential RADP markers associated with resistance to the septoria infection. Four primers produced positive bands in resistant genotypes and these primers could be very useful in MAS. Because of low reproducibility of RAPD, we plan to convert these potential RAPD primers to SCAR and to find the tightly linked one, so that MAS can be applied using a single marker. MAS is cost effective and more precise, because it does not need to have a pathological evaluation and plants can be genotyped at any crop stage. Identified markers linked to resistance will also have a potential role for gene pyramiding (Hittalmani et al., 2000). SCAR markers may soon be available and facilitate the septoria resistance breeding program in tomato. To our knowledge, there are no other molecular markers reported associated with septoria leaf spot. RAPD identified in this study will be novel, which may be useful for speeding up the tomato breeding program aiming to improve septoria leaf spot resistance.

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Wright, V., and R.E. Lincoln. 1940. Resistance to defoliation diseases of tomato. Purdue Agric. Exp. Stn. Annu. Rep. 53:42-43.

CHAPTER 3 Molecular Tagging of Resistance Genes to Late Blight in Tomato

ABSTRACT A prerequisite for marker assisted selection (MAS) is the development of markers that is either tightly linked or in a known gene. We used the bulked segregant analysis (BSA) to identify RAPD markers linked to late blight resistance in tomato using an F2 population. Two tomato inbred lines, NC 85L-1W(2007) and NC 839-2(2007)-1 were crossed to produce an F1. NC 85L-1W(2007) was resistant to late blight (resistant parent, RP) and NC 839-

2(2007)-1 was susceptible to late blight (susceptible parent, SP). A total of 250 F2 plants, and

10 plants each of the RP, SP and F1 materials were grown at the Mountain Horticultural Crops Research and Extension Center (MHCREC), Mills River, NC, in 2009. Disease severity was scored on 85 day old plants using a scale of 0 to 5. DNA was extracted from 2-3 week old plants of all individual F2 plants, parents and F1. The parental lines were screened with 157 SSR and two conserved ortholog set (COS) primers, 23 M-13 tailing SSR primers and 197 10-mer RAPD primers. Two DNA bulk samples, called resistant bulk (RB) and susceptible bulk (SB) were prepared from 16 F2 individuals. We observed transgressive segregation for late blight resistance in this population. Only 34 RAPD primers (17.26%) were found to be polymorphic between parents. Sixteen RAPD primers (47%) out of 34 produced polymorphic bands between the resistant and susceptible bulks of late blight. Four RAPD primers, namely MRTOMR-026, MRTOMR-031, MRTOMR-038 and MRTOMR-046 were identified as linked markers to loci related to the late blight disease reaction. Two were linked to susceptibility and two to resistance. Due to the low reproducibility of RAPDs, it is necessary to convert these to SCARs and verify the tightly linked markers with late blight resistance loci, so that MAS can be applied using a single marker.

INTRODUCTION Tomato (Solanum lycopersicum L.) is a tropical vegetable used in fresh and processed forms. It is the second most consumed vegetables after potato in the world (FAOSTAT, 2010). Economically, tomato is the third most important vegetable in US after potato and lettuce. In terms of a source of vitamins and minerals in the US diet, it ranks first (Rick, 1980) among vegetables and fruits. Breeding efforts have been focused mainly on fruit yield and foliar diseases. The most important foliar disease of tomato is late blight (Panthee and Chen, 2010) caused by Phytopthora infestans (Mont.) de Bary, which can destroy a tomato field within a few days of occurrence (Fry and Goodwin, 1997) in a conducive environment for pathogen development. The most conducive environment for disease development is wet and cloudy conditions with cool temperatures. The airborne nature of late blight results in wide spread distribution in to a large area quickly. Davis et al. (2000) reported that late blight is the eighth most important disease of tomato in term of crop loss per acre in USA.

Advances have been made on resistance breeding to combat late blight in tomato. Foolad et al. (2008) extensively reviewed late blight resistance in tomato. Both vertical and horizontal resistances have been reported. A single dominant resistance gene to late blight is commonly found in wild tomato species. Four race specific R genes, Ph-1, Ph-2, Ph-3 and Ph-5 conferring resistance to late blight have been identified in S. pimpinellifolium. Ph-1 is a complete dominant gene and it has been mapped to the distal end of chromosome 7 (Foolad et al., 2008). LA-3707, a selection of S. pimpinellifolium from the Asian Vegetable Research and Development Center (AVRDC, Taiwan), contributed the Ph-3 gene, and Richter's wild tomato was the source of a partial dominant gene, Ph-2 (Gardner and Panthee, 2010b). The Ph-2 gene was mapped to chromosome 10. The Ph-3 gene, a partially dominant gene, is considered to be more durable than Ph-1 and Ph-2. The newly identified R gene, Ph-5, was found to be superior to all other R genes for late blight (Foolad et al., 2008). Quantitative trait loci (QTLs) have also been identified from backcross populations

developed by crossing cultivated tomato with wild tomatoes. QTLs for late blight are located in almost all 12 chromosomes (Brouwer et al., 2004), although the number of QTLs were not specified.

A few resistant varieties of tomato have been developed through the introgression of resistant genes, either from cultivated or from wild species of tomato (Panthee and Gardner, 2010; Gardner and Panthee, 2010a). However, these varieties may not be adaptable to all tomato growing regions and the resistance may not be long lasting. Therefore, working towards the development of new resistant varieties continuously is required. More than 8 years are required to develop a resistant variety through conventional breeding (e.g., using phenotypic selection). Molecular markers are useful for accelerating breeding work with more precise and efficient selection of desirable genotypes at any crop stage. Markers linked to the gene(s) of interest help to select plants genotypically that are genetically similar to the recurrent parent possessing the desired trait. However, due to the unavailability of PCR-based molecular markers tightly linked to late blight resistance, marker assisted selection (MAS) has not been adapted routinely breeding programs.

Earlier, use of near isogenic lines (NIL) was the common means of identifying genes of interest. NIL are not available for most of the target regions and takes a long time to develop. Alternatively, Michelmore et al. (1991) developed a very rapid and simple PCR- based method to identify the markers linked to gene of interest called bulked segregant analysis (BSA) and identified RAPD markers linked to a downy mildew disease resistance gene (Dm5/8) in lettuce. For BSA, any kind of mapping population e.g., recombinant inbred lines (RIL), backcross (BC), F2 or double haploid (DH) that are segregating for a trait of interest can be used. For dominant marker, e.g., RAPD, the F2 population is considered best, because of the doubled number of segregating loci in F2 than in the BC (Mackay and Caligari, 2000).

In BSA, two contrasting traits (e.g., resistance and susceptible) from a segregating population are compared using bulk DNA from these two extremes. DNA from individuals similar to traits of interest are bulked and assumed that the bulks are homozygous for the target interval and heterozygote for the rest of the interval (Giovannoni et al., 1991; Michelmore et al., 1991; Quarrie et al., 1999). DNA markers are then used to screen the parents and bulks. If polymorphism is found between bulks, this marker (polymorphic band) is expected to be associated with the gene of interest.

Different types of markers linked to disease resistance genes, e.g., Ph-3, ol-2 have been identified in tomato using random amplified polymorphic DNA (RAPD) following the BSA approach (Stevens et al., 1995; Chagué et al., 1996; De Giovanni et al., 2004). Once it is identified, this marker is then converted into co-dominant markers, for example cleaved amplified polymorphic sequence (CAPS), sequence characterized amplified region (SCAR) which are more reproducible than RAPDs. Chunwongse et al. (2002) identified the amplified fragment length polymorphism (AFLP) markers linked to Ph-3 using BSA. De Giovanni et al. (2004) identified a RAPD marker linked to the ol-2 gene conferring resistance to powdery mildew using BSA in an F2 population segregating for the ol-2 gene. A single RAPD marker, OPU31500 with 1500 bp in size was detected in the susceptible bulk and was converted to a CAPS marker. Stevens et al. (1995) and Chagué et al. (1996) also identified RAPD markers linked to the Sw-5 gene, resistance to tomato spotted wilt virus (TSWV). Linkage analysis mapped this marker within a distance of 10.5 cM from Sw-5. This RAPD marker was later converted into SCAR marker. Śmiech et al. (2000) used BSA in F2 segregating population and found five primers that distinguished resistant and susceptible bulks of TSWV.

In this study, we used the BSA method to identify RAPD markers linked to late blight resistance in tomato using an F2 population. An F2 population is the earliest segregating generation available for mapping genes from the crosses and provides the greatest genetic window around the locus (Michelmore et al., 1991). We tagged R gene based on a segregating population exposed to natural infestation. Uses of hot spot for specific disease

to screen the population are cost effective way of resistance breeding and particularly useful in horizontal resistance breeding. Horizontal resistance system is generally considered suitable for long term cultivation of resistance variety. Spaner et al. (1998) mapped loci affecting resistance to powdery mildew, leaf rust, stem rust, scald and net blotch in barley using field-scored data of disease severity under natural infestation conditions.

MATERIAL AND METHODS

Plant Materials Two tomato inbred lines, NC 85L-1W(2007) (referred onward as NC 85L) and NC

839-2(2007)-1 (referred onward as NC 839), were crossed to produce F1 in the greenhouse. The NC 85L, used as a female, is resistant to late blight (resistant parent, RP) and NC 839, used as male, is susceptible to late blight (susceptible parent, SP). The F1 plants were selfed to obtain F2 seeds. A total of 250 F2 plants, and 10 plants each of RP, SP and F1 were evaluated in this study. Nine F2 plants were blinds (seedling without meristem); therefore, we used 241 F2 plants. The fruit of NC 85L was a mini roma type with dark red fruit color (Table 12) whereas the fruit of NC 839 was a grape type with light red fruit color. The NC 85L selection was made for late blight and early blight resistance selected at Waynesville, NC, and the NC 839 selection was made at Mills River, NC, for outstanding fruit and plant type. The resistance source of late blight in NC 85L traces back to the L-3707 and Ritcher’s wild tomato (S. pimpinellifolium).

Table 12. Parental description along with their pedigrees and coefficient of parentage Parent Maturity Fruit type, Disease Pedigree Common COP shape, reaction pedigree color NC 85L- Early Mini roma Late blight 051(x)- 0179(x)-1- 1W(2007) type, dark resistance 18//0463/97 18-4, 215E- red 22(x)-18 1(93), NC 839- Average Grape Septoria 051(x)- 9722(x)-18, 0.227 2(2007)-1 type, light leaf spot 18//CB25(x) 051, 03220, red resistance -18- L3707 3/9722(x)-

18/0464 COP = Coefficient of parentage.

Field Evaluation

A total of 280 plants, consisting of 10 plants of each parent and F1, and 250 F2 plants were planted in R16 research plot of Mountain Horticultural Crops Research and Extension Center (MHCREC), Mills River, NC, in the summer of 2009. This research plot is a hot-spot for late blight and natural inoculum was observed at high pressure for screening the late blight segregating population in the summer of 2009. All these plants were evaluated for resistance to late blight using natural inoculum. The experimental site was in Mills River, Henderson, North Carolina, located at an altitude of 630 m above sea level and latitude of 35.42721o and longitude of -82.55888o (http://www.nc-climate.ncsu.edu/cronos/ ?station=FLET).

Seeding was done on June 1, 2009, in trays of size 30.5 x 45.7 cm containing peat moss and vermiculite. Trays were kept in the greenhouse with an average temperature of 21.1oC. Twelve day old seedlings were transplanted in a 50-cell tray size of 12.5 x 24.4 cm. Six week old seedlings were then transplanted in the field having loam type of soil at a row to row distance of 1.5 m and plant to plant distance of 45 cm. The bed was raised and covered with black plastic. Other recommended cultural practices were followed as described in Southeastern US 2010 Vegetable Crop Handbook (http://www.ncsu.edu/ enterprises/tomatoes/2010/04/07/2010-southeastern-us-vegetable-crop-handbook/).

DNA Extraction, Quantification and Dilution

DNA was extracted from all individual F2 plants, parents and F1 following the method of Fulton et al. (1995). Step by step procedure of DNA extraction is given in Appendix 5. Approximately 100 mg of young leaves from 2-3 week old tomato seedlings were collected from the greenhouse in 1.5 ml Eppendorf tube. This tube was then dipped into liquid nitrogen and samples were ground with a glass rod. After adding 200 µl microprep buffer, sample was incubated at 65oC water bath for about 60 min and filled with chloroform/ isoamyl (24:1) solution. Sample was then centrifuged at 10,000 rpm for 5 minutes. The aqueous phase was pipetted off into new micro-centrifuge tubes and 2/3rd to 1 times the

volume of cold isopropanol was added to precipitate DNA. After centrifuging this sample at 10,000 rpm for 5 minutes, the DNA pellet was separated and washed by 70% ethanol. The dry DNA pellet was resuspended in 100 µl of 1x TE buffer and stored at -20oC. Concentrations of DNA in all samples were estimated using spectrophotometer (NANO Drop 1000, Thermo Scientific, USA). Working solutions of DNA samples with a concentration of 20 ng/ µl were prepared from original DNA samples in 1x TE buffer.

PCR-based Markers Screening

SSR Two parental lines were screened with 157 SSR and two conserved ortholog set (COS) primers (Fulton et al., 2002) (Appendix 4). Amplification reactions were performed in a 10 l reaction volume containing PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 M of each primer and 0.625 U of Taq DNA polymerase with 20 ng template DNA. DNA amplifications were performed in a thermal cycler (Applied Biosystems, Life Technologies, USA) using the following cycling conditions: one cycle of 94oC for 3 min; 34 cycles of 94oC for 0.30 min, 52-58oC for 1 min, 72oC for 1 min; one cycle of 72oC for 10 min followed by holding at 4oC.

M-13 Tailing SSR

Conventional gel electrophoresis did not give any polymorphic SSR markers between parents. Therefore we used M-13 tailing SSR primers (Schuelke, 2000) to use the capillary electrophoresis system of fragment analysis. Twenty three M-13 tail SSR primers were screened (Appendix 4). M-13 sequence incorporated at beginning of forward primer was TGT AAA ACG ACG GCC AGT. Amplification was performed in a 25 l reaction volume consisting of 1x PCR buffer with 1.5 mM MgCl2, 0.2 mM dNTP, 0.08 µM forward primer with M-13 tail, 0.6 µM reverse primer, 0.6 µM IRD-labeled M-13 primer, 0.9 U Taq polymerase and 20 ng template DNA. The reaction was carried out in a thermal cycler (Life Technologies, USA) using the following cycling conditions: one cycle of 94.0°C for 2:30 min,

15 cycles of 94.0°C for 0:30 min, 53-56oC for 0:30 min and 72.0°C for 1:00 min, 25 cycles of 94.0°C for 0:30 min, 50.0°C for 0:30 min and 72.0°C for 1:00 min and one cycle of 72.0°C for 7:00 min followed by holding at 4.0°C. This PCR profile is specific to the M-13 tailing protocol. The first 15 cycles amplify the specific region of interest at the annealing temperature (Ta) specific to the forward primer. The second round of amplification has 25 cycles where the Ta is specific to the M-13 primer and will allow the IRD-label to be incorporated.

An ABI plate was set up by adding 9.5 µl formamide /LIZ mix in 1 µl PCR product. Mix of LIZ standard and formamide was at the concentration of 1/200 by volume. The plate was denatured at 95oC for 5 min. DNA fragment analysis was done by capillary electrophoresis using an ABI 3730 DNA Analyzer (Life Technologies, USA). Data were analyzed using GeneMarker Software V1.91 (SoftGenetics, USA). Electropherogram was then compared between parents to detect any polymorphisms.

RAPD

A total of 197 10-mer RAPD primers (Appendix 4) were screened using 20 ng DNA template of two parental lines. Primers polymorphic to parental lines were then used to screen resistant and susceptible bulk samples. Amplification reactions were performed in 10 l reaction volumes containing 1x buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM

MgCl2), 200 M of each dNTP, 0.2 M primer and 1 U Taq polymerase. About 15 l mineral oil was overlaid on the reaction mixture. DNA amplifications were performed in thermal cycler (Eppendorf) using the following cycling condition: one cycle of 92oC for 3 min; 45 cycles of 92oC for 0.30 min, 42oC for 1 min and 72oC for 0.30 min; one cycle of 72oC for 8 min followed by holding at 4oC.

Bulked Segergant Analysis Bulked segregant analysis was done following the method of Michelmore et al. (1991). Two DNA bulks, called resistant bulk (RB) and susceptible bulk (SB) were prepared

from F2 individuals. RB consisted of 8 individuals with a disease score of 0 and SB consisted of 8 individuals with a score of 4.5 and 5. The DNA was extracted separately from each individual of F2 population. Later, DNA bulk was prepared by pooling equal amounts of DNA

(50 l) of each of eight resistant and eight susceptible F2 plants. Non-correlation of disease score value of each bulk with total soluble solid indicated that these bulks were from the two extremes of disease severity. PCR was run with polymorphic primer between parents on the bulks and parental DNA samples using the same reaction conditions as described above. PCR was repeated for those primers that were polymorphic between bulks.

Gel Electrophoresis All RAPD and SSR PCR products were analyzed in 2% agarose gels containing 0.1 g/ml ethidium bromide in 1x TBE buffer (40 mM Tris-borate, pH 8.0, 1 mM EDTA) with a 100-bp ladder. Electrophoresis was run at 135 V for 2 hours. Gels were rinsed with water to enhance contrast and photographed under UV light. Polymorphism between parents by SSR primers was not found in agarose gel, therefore, some PCR products of SSR primers were separated on 8% PAGE and stained in ethidium bromide. Stained gels were photographed under UV light. RAPD fragments were scored as 1 for presence and 0 for absence. Band size was estimated based on a 100-bp ladder (Bioline USA Inc, MA, USA). Simple statistics based on the DNA bands were calculated using MS Excel 2007.

DNA Extractions from the Gel for Sequencing Polymorphic RAPD bands between parents and bulks were extracted from the 2% agarose gel following the Protocol of GenElute™ Gel Extraction Kit (Appendix 6). These DNA are being sequenced for converting useful RAPD primers to SCAR or CAPS.

Data Scoring and Analysis Disease severity was scored at 85 days after seeding (August 25, 2009). Individual disease rating scores were based on visual assessment of symptom severity. The following scoring criteria was developed based on Winstead and Kelman (1952), Tu and Poysa (1990), Danesh et al. (1994) and used in this study. Severity of disease was scored at a scale of 0 to

5 with 0.5 increment, as 0 = no disease symptoms, 0.5 = Less than 10% leaf area with symptoms, 1 = 10-20% leaf area with symptoms, 1.5 = 20-30% leaf area with symptoms, 2 = 30-40% leaf area with symptoms, 2.5 = 40-50% leaf area with symptoms, 3= 50-60% leaf area with symptoms, 3.5 = 60-70% leaf area with symptoms, 4 = 70-80% leaf area with symptoms, 4.5 = 80-90% leaf area with symptoms, and 5 = 90-100% leaf area with symptoms.

Scores of parental lines and F1 were averaged to see the transgressive segregants. The pedigrees of these two lines were traced back to estimate the coefficient of parentage (COP). COP was estimated between parents on the assumption that all ancestors and parental lines are homozygous and a line derived from a cross obtains one-half of its genes from each parent. The computer software KIN (Tinker and Mather, 1993) was used to calculate the coefficient of parentage (COP).

RESULTS The percentage of leaf area infected by the late blight pathogen was assessed 85 days after seeding. The average score of the resistant parent was 1.1 and that of the susceptible parent was 4.2. We observed transgressive segregation towards resistance as well as susceptible. The average score of F1 was 3.5, indicating that expression of resistance was incomplete. Some of F1 individuals had disease severity levels similar to the susceptible parent.

Pedigree analysis of the parents in the study showed that they had six parents in common. The coefficient of parentage between them was 0.23 indicating some dissimilarity between them. Variation was found between these lines for morphology and pedigree; however, we could not find any polymorphic SSR markers. We screened 180 SSR and two COS markers. Most of them exhibited monomorphic banding patterns and some of them did not amplify the genomic DNA of these parents (Appendix 7). Three systems of fragment analyses including agarose gel, polyacrylamide gel and capillary gel electrophoresis were

tried but none were able to detect any polymorphisms, indicating close similarity between these parents for SSR.

RAPD Markers Out of 197 RAPD primers used to screen parental lines, 34 RAPD primers (17.26%) were found polymorphic (Table 13). A total of 176 bands with maximum band size of 1500 bp and minimum band size of 100 bp were amplified using 34 primers. Among these bands, 84 were found polymorphic between parents. The average number of bands per polymorphic RAPD primer was 4, ranging from 2 to 8 bands. The number of polymorphic bands ranged from 1 to 4 with an average of 2. All these 34 polymorphic RAPD primers were used to screen the resistant and susceptible bulks.

RAPD Markers and Bulked Segregant Analysis Sixteen RAPD primers (47%) out of 34 gave polymorphic bands between late blight resistant and susceptible bulks of an F2 population (Table 14). A total of 105 RAPD bands were observed among four DNA samples consisting of two parents and two bulks samples. The total number of polymorphic bands observed between parents and between bulks was 35 and 23, respectively. On average, each primer amplified 7 bands. The average number of polymorphic bands per primer observed for the parental lines was 2 and for bulks 1. The largest band size was of 2000 bp and smallest was of 100 bp. The polymorphism shown by MRTOMR-046 was the largest among four samples.

The bands generated by MRTOMR-147 were mostly polymorphic between parents but none of these bands could distinguish the bulks. Four RAPD primers, namely MRTOMR- 026, MRTOMR-031, MRTOMR-038 and MRTOMR-046 were identified as linked markers to loci related to disease reaction (Table 15). Among those, two were linked to the susceptibility (Figure 11) and two to resistance (Figure 12). The band sizes of 1100 bp amplified by MRTOMR-026 and 800 bp amplified by MRTOMR-046 were found only in susceptible parent, i.e., NC 839 and susceptible bulk. Two other primers, MRTOMR-031 and

MRTOMR-038 produced band sizes of 550 bp and 1100 bp, respectively that were only in the resistant parent i.e., NC 085L and resistant bulk (Table 16).

The useful RAPD primers related to disease reaction are listed in Table 17. Eleven primers were found to have bands that were unlinked to the loci. These primers distinguished only parents and not bulks, and were therefore defined as unlinked markers (Figure 13A). The amplified bands of eight RAPD primers were only found in bulks but not in either parent (Figure 13B). This may be due to recombination in the F2 population.

Table 13. RAPD primers polymorphic between resistant and susceptible parents of the NC 08135 F2 population of tomato screened for late blight resistance

Primer Sequence % GC Amplified Band size, bp Polymorphic % Tm bands Max Min band polymorphic MRTOMR-001 ATCGAGCACT 50 2 450 400 1 50 30 MRTOMR-003 AAGGGGCTGC 70 5 600 150 1 20 34 MRTOMR-014 CCGTCTCCAG 70 7 600 150 3 43 34 MRTOMR-022 CAGGGCCAGC 80 8 1000 100 6 75 36 MRTOMR-023 GGACCACGAA 60 3 650 100 2 67 32 MRTOMR-026 TAGGCACCGT 60 5 1000 300 3 60 32 MRTOMR-027 ACCTGATGCA 50 3 650 200 3 100 30 MRTOMR-029 CGCCATACGG 70 2 900 650 2 100 34 MRTOMR-031 GGGACGTCGC 80 5 600 200 2 40 36 MRTOMR-033 CGCTCGCGGC 90 5 1000 150 2 40 38 MRTOMR-038 TACCTTCGCC 60 6 1100 150 3 50 32 MRTOMR-039 CAGCACCACC 70 7 650 100 3 43 34 MRTOMR-040 ACAGTCCGCG 70 3 500 180 1 33 34 MRTOMR-046 CCATGCGCTA 60 5 600 100 4 80 32 MRTOMR-063 ACGAGTGACC 60 6 550 100 2 33 32 MRTOMR-078 GTGCCATGTG 60 4 700 200 1 25 32 MRTOMR-100 GAC GGC CCC A 80 7 1500 200 3 43 45 MRTOMR-110 ATG ACG ACC T 50 3 800 400 1 33 31 MRTOMR-112 CAT ACA CCT C 50 4 1500 650 2 50 25 MRTOMR-117 CCG AAC AAT C 50 6 950 300 5 83 28 MRTOMR-118 TGC TTG GGG G 70 4 1500 500 3 75 39 MRTOMR-121 GGC GTC GTA A 60 3 1100 550 2 67 36 MRTOMR-128 AGA CCC GGT C 70 3 600 300 1 33 38 MRTOMR-130 AGG TCT CTC G 60 3 700 250 1 33 32 MRTOMR-133 TTC AGC CAC A 50 3 900 300 2 67 32 MRTOMR-140 TGC CAA CGC C 70 5 800 250 1 20 42 MRTOMR-141 CAT TGG TGC T 50 2 900 450 1 50 31 MRTOMR-142 TTG CGC TTG T 50 2 800 400 1 50 37 MRTOMR-146 CGT TAC CGG G 70 5 2000 450 2 40 35 MRTOMR-161 TGT CTC CCT G 60 5 1500 700 1 20 32 MRTOMR-171 GAG GCC AGC G 80 4 700 200 1 25 43 MRTOMR-178 GCC ATC CGA A 60 2 800 650 1 50 35 MRTOMR-181 CAT GCG CTC C 70 3 800 400 1 33 39 OPK6 CACCTTTCCC 60 4 1000 600 1 25 31 P#72 GAGCACGGGA 70 4 600 250 1 25 39 bp = base pair. Tm = Melting temperature.

Table 14. Polymorphic RAPD markers and band size between late blight resistant and susceptible bulks of tomato Primer Amplified Size, bp Polymorphic % polymorphic Max Min Between Between Between Between bands parents bulks parents bulks MRTOMR-014 7 1500 300 3 2 43 29 MRTOMR-022 7 1200 300 1 0 14 0 MRTOMR-026 7 1500 250 1 2 14 29 MRTOMR-031 7 2000 450 3 1 43 14 MRTOMR-038 4 1500 300 2 1 50 25 MRTOMR-040 10 1600 280 4 5 40 50 MRTOMR-046 9 2000 100 6 5 67 56 MRTOMR-050 4 1000 400 1 0 25 0 MRTOMR-063 11 1500 150 2 1 18 9 MRTOMR-076 6 1500 350 1 2 17 33 MRTOMR-110 6 1200 180 1 0 17 0 MRTOMR-112 5 1200 300 1 0 20 0 MRTOMR-121 6 100 380 2 0 33 0 MRTOMR-130 4 750 200 1 0 25 0 MRTOMR-146 9 1500 250 4 4 44 44 MRTOMR-147 3 900 500 2 0 67 0

Table 15. Polymorphic bands of RAPD markers linked to genes in tomato conferring resistance or susceptibility to late blight Name PBN Size, bp RP SP RB SB Marker type MRTOMR-026 2 1100 0 1 0 1 N 5 500 0 0 1 0 - MRTOMR-031 3 1200 1 0 0 0 - 6 550 1 0 1 0 P MRTOMR-038 2 1100 1 0 1 0 P 3 750 0 1 1 1 - MRTOMR-046 1 2000 0 1 0 0 - 2 1200 0 1 0 0 - 3 800 0 1 0 1 N 4 650 0 1 1 0 - 5 600 0 1 1 0 - 6 550 1 0 0 0 - 7 380 1 0 0 0 - 8 350 0 1 1 0 - PBN = Polymorphic band number. RP = Resistant parent. SP = Susceptible parent. RB = Resistant bulk. SB = Susceptible bulk. P = Positive. N = Negative.

Table 16. Specific marker bands linked to genes for resistance or susceptibility to late blight of tomato Marker LB LB Genotype resistance susceptible P1 (NC 085LW) P2 (NC 839-2) MRTOMR-026 - 1100 bp - + MRTOMR-031 550 bp - + - MRTOMR-038 1100 bp - + - MRTOMR-046 - 800 bp - + + = Presence. - = Absence.

Table 17. Informative RAPD markers associated with susceptible or resistance genes to late blight of tomato Linked to Linked to Unlinked With bands susceptible resistant only in bulks MRTOMR-026, MRTOMR-031, MRTOMR-014, MRTOMR- MRTOMR-014, MRTOMR-046, MRTOMR-038 022, MRTOMR-026 MRTOMR-022, MRTOMR-031, MRTOMR- MRTOMR-040, 040, MRTOMR-046, MRTOMR-046, MRTOMR-050, MRTOMR- MRTOMR-076, 112, MRTOMR-121, MRTOMR-112, MRTOMR-130, MRTOMR-147 MRTOMR-121, MRTOMR-146

Figure 11. Electrophoretic pattern of DNA fragments generated by RAPD marker (A. MRTOMR-026, B. MRTOMR-046). Polymorphic band (i.e., linked to susceptible) between parents and between resistant and susceptible bulks are indicated by arrow. RP = Resistant parent, NC 085L. SP = Susceptible parent, NC 839. RB = Resistant bulk. SP = Susceptible bulk. M = Marker.

Figure 12. Electrophoretic pattern of DNA fragments generated by RAPD markers (A. MRTOMR-031, B. MRTOMR-038). Polymorphic band (i.e., linked to resistance) between parents and between resistant and susceptible bulks are indicated by arrow. RP = Resistant parent, NC 085L. SP = Susceptible parent, NC 839. RB = Resistant bulk. SP = Susceptible bulk. M = Marker.

A. B.

Figure 13. A. RAPD marker (MRTOMR-040) showing polymorphic band (indicated by arrow) only to parents, i.e., band with unlinked loci. B. RAPD marker (MRTOMR-022) showing band (indicated by arrow) only in two bulks. RP = Resistant parent, NC 085L. SP = Susceptible parent, NC 839. RB = Resistant bulk. SP = Susceptible bulk. M = Marker.

DISCUSSION Among the Ph genes, resistant to late blight in tomato, Ph-2 and Ph-3 are partially dominant genes (Foolad et al., 2008). In our study, the resistant parent, NC 85L, also has Ph- 2 and Ph-3, derived from L-3707 and Ritcher’s wild tomato, respectively. The expressions of Ph-2 has been reported to be dependent on environmental conditions, crop physiological stage and pathogen isolate (Moreau et al., 1998). Ph-3, though superior to Ph-2, would not exhibit complete resistance to some isolates (Foolad et al., 2008).

Based on the screening of two bulks with 197 RAPD primers, we identified two RAPD markers linked to a putative resistance allele and two RAPD markers linked to the susceptible allele. Through the bulking of two extreme phenotypes of F2 populations we were able to rapidly tag the markers associated with the chromosomal segment that has a role on reaction to the late blight pathogen. With the BSA technique consisting of 8 individuals in each bulk, four primers gave different band sizes that were found to be linked to late blight resistance. There are some reports of markers identified in relation to late blight resistance in tomato (Chunwongse et al., 2002; Qiu et al., 2009). Chunwongse et al. (2002) identified AFLP markers linked to the Ph-3 gene using BSA and Qiu et al. (2009) identified one RAPD marker which was at distance of 5.8 cM from the target region of late blight resistance.

The probability of declaring an unlinked polymorphic marker linked to a gene is related to the size of the bulk and may be other factors. Both types of population and the markers should be considered during preparing the bulks (Michelmore et al., 1991). If there is small size of bulks, the frequency of false positives will increase. Michelmore et al. (1991) suggested that a few individuals per bulk are enough to identify the linked markers. The probability of finding an independent marker linked to the gene with bulk size of n is reported to be 2(1/4)n[1-(1/4)n]. We used 8 individuals and based on this formula, the proportion of false positives is about 3 x 10-5, which is very low.

In principle, BSA and NIL are effectively equivalent, and the many advantages of BSA over NIL are discussed by Michelmore et al. (1991). Tagging of resistance gene using BSA is very fast which facilitates the screening of new alleles for resistance to a particular pathogen. This is important because resistant genes once tagged may not be effective for a long time either because of recombination in the host genome or mutation in the pathogen. The BSA approach is also considered useful to fill the gaps in the maps.

The two parental lines used in this study are closely related to each other and SSR screening showed similar results. However, we found RAPD markers able to distinguish these parents at the molecular level. RAPD is a multilocus-marker, therefore; all primers we identified here might be from the overlap regions of the chromosome. For example, MRTOMR-026 produced polymorphic band size of 1100 bp between bulks and MRTOMR- 046 800 bp band. The band produced by MRTOMR-046 might be the part of the band generated by MRTOMR-026. These linkages should be verified by mapping the markers. Sequence of amplified region will help if they were from the same region and can blast to see the similarity and location in chromosome. A number of disadvantages associated with RAPD, for example, including annealing in multiple sites, dominant in nature, sensitive to reaction conditions may limit its use directly in MAS. Therefore, candidate RAPD markers are generally converted to SCAR or CAPS which produce the co-dominant banding pattern and are much more useful for MAS.

al. (1996) have identified RAPD markers linked to the Sw-5 gene for resistance to tomato spotted wilt virus (TSWV). Among the four RAPD markers linked to the gene of interest, two were tightly linked to the Sw-5 gene. Linkage analysis mapped this marker within a distance of 10.5 cM from Sw-5. This RAPD marker was later converted into a SCAR marker. Czech et al. (2003) have used MAS for developing TSWV resistant tomato using PCR markers. Smiech et al. (2000) used BSA in an F2 segregating population and found five primers that distinguished resistant and susceptible bulks.

We used disease scores that were based on natural infestation. Different levels of infestation rate were observed in an F2 population indicating that inoculum pressure was enough to screen the population. Use of natural inoculums saves cost and time. Screening of a target population in a target environment can be more successful in the long run to get the durable resistant genotype. Infestation from a natural population has been used in a number of crop species. For example, two QTLs for glume botch resistance in wheat were identified using composite interval mapping from naturally inoculated population (Schnurbusch et al., 2003). Spaner et al. (1998) mapped loci affecting resistance to powdery mildew, leaf rust, stem rust, scald and net blotch in barley using field-scored data of disease severity under natural infestation. Natural infestation was also used by Frei et al. (2005) to identify QTLs resistance to thrips in common bean.

We identified four potential RAPD primers associated with resistance to the late blight in tomato. Two primers yielded a DNA band in late blight resistant genotypes and these primers could be very useful in MAS. Because of low reproducibility of RAPD, these markers need to be converted to tightly-linked SCAR markers, so that MAS can be applied using a single marker. MAS is cost effective and more precise, because it does not need to have a pathological evaluation and can genotype at any growth stage. Further, linkage analysis is also necessary to identify the tightly linked marker.

REFERENCES Brouwer, D.J., E.S. Jones, and D.A.S. Clair. 2004. QTL analysis of quantitative resistance to Phytophthora infestans (late blight) in tomato and comparisons with potato. Genome 47:475-492.

Chunwongse, J., C. Chunwongse, L. Black, and P. Hanson. 2002. Molecular mapping of the Ph-3 gene for late blight resistance in tomato. J. Hort. Sci. Biotechnol. 77:281-286.

Danesh, D., S. Aarons, G.E. McGill, and N.D. Young. 1994. Genetic dissection of oligogenic resistance to bacterial wilt in tomato. Mol. Plant-Microbe Interact. 7:464-471.

Davis, R.M., G. Hamilton, W.T. Lanini, T.H. Spreen, and C. Osteen. 2000. The importance of pesticides and other pest management practices in U.S. tomato production. USDA, NAPIAR.

FAOSTAT. 2010. Food and Agriculture Organization of the United Nations. Available at http://faostat.fao.org/site/339/default.aspx (verified 25 June 2010).

Foolad, M., H. Merk, and H. Ashrafi. 2008. Genetics, genomics and breeding of late blight and early blight resistance in tomato. Crit. Rev. Plant Sci. 27:75-107.

Frei, A., M.W. Blair, C. Cardona, S.E. Beebe, H. Gu, and S. Dorn. 2005. QTL mapping of resistance to thrips palmi Karny in common bean. Crop Sci. 45:379-387.

Fry, W.E., and S.B. Goodwin. 1997. Resurgence of the Irish potato famine fungus. Bioscience 47:363-371.

Fulton, T.M., J. Chunwongse, and S.D. Tanksley. 1995. Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol. Biol. Rep. 13:207-209.

Gardner, R.G., and D.R. Panthee. 2010a. Plum Regal fresh-market plum tomato hybrid and its parents, NC 25P and NC 30P. HortScience 45:824-825.

Gardner, R.G., and D.R. Panthee. 2010b. NC 1 CELBR and NC 2 CELBR: Early blight and late blight-resistant fresh market tomato breeding lines. HortScience 45:975-976.

Mackay, I.J., and P.D.S. Caligari. 2000. Efficiencies of F2 and backcross generations for bulked segregant analysis using dominant markers. Crop Sci. 40:626-630.

Panthee, D.R., and F. Chen. 2010. Genomics of fungal disease resistance in tomato. Current Genomics 11:30-39.

Panthee, D.R., and R.G. Gardner. 2010. Mountain Merit: A late blight-resistant large-fruited tomato hybrid. HortScience 45:1547–1548.

Qiu, Y.P., H.T. Li, Z. Zhang, and Q.D. Zou. 2009. RAPD marker of the resistant gene Ph-3 for tomato late blight. Acta Horticulturae Sinica 36:1227-1232Available at (verified 29 March 2011).

Rick, C.M. 1980. Tomato. p. 669-680. In Hybridization of crop plants. Amer. Soc. Agron./Crop Sci. Soc. Amer., Madison, Wis.

Schuelke, M. 2000. An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology 18:233–234.

Stevens, M.R., E.M. Lamb, and D.D. Rhoads. 1995. Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses. Theor. Appl. Genet. 90:451-456.

Tinker, N.A., and D.E. Mather. 1993. KIN: Software for computing kinship coefficients. J. Hered. 84:238.

Tu, J.C., and V. Poysa. 1990. A brushing method of inoculation for screening tomato seedlings for resistance to Septoria lycopersici. Plant Dis. 74:294-297.

Winstead, N.N., and A. Kelman. 1952. Inoculation technique for evaluating resistance to Pseudomonas solanacearum. Phytopathology 42:628-634.

CHAPTER 4 Isolation and Culture of Septoria lycopersici Speg. from Septoria Leaf Spot affected Tomato Leaves

INTRODUCTION Septoria leaf spot (Septoria lycopersici Speg.) is one of the most devastating foliar diseases of tomato, particularly in humid regions during the periods of rainfall, frequent dew or over-head irrigation. It may cause complete defoliation leading to a significant crop loss under favorable environment conditions for disease development (Andrus and Reynard, 1945). The temperature range for sporulation varies from 15° to 27°C (59° to 80.5°F) with 25°C (77°F) being optimal. Free moisture is necessary for spore infection through stomata; therefore, a long-lasting dew and rainy days favor disease development (Delahaut and Stevenson, 2004); http://pubs.ext.vt.edu/450/450-711/450-711.html). Septoria occurs worldwide and effective fungicides are available to control septoria leaf spot. However, due to the cost involved in fungicides and environmental concern associated with fungicides, breeding for resistant variety is the priority in many breeding programs. With the use of fungicides this disease has got less attention from the breeder (Locke, 1949) in the past,

Tomatoes may often be infected with septoria leaf spot (SLS) and early blight (Altemaria solani) simultaneously (http://vegetablemdonline.ppath.cornell.edu/ factsheets/ Tomato_Septoria.htm). Septoria leaf spot is easily distinguished from early blight by the uniform, small size spots and the lack of concentric rings in the spots. Septoria overwinter in plant debris, on seed, or on weeds such as nightshade, jimsonweed, ground cherry and horse-nettle (Delahaut and Stevenson, 2004) and can survive up to 3 years. It is also reported as a soil borne fungal disease of tomato (Wyenandt et al., 2008). Wind and rain then spread spores to adjacent healthy leaves and plants. Spores of this fungus on debris

splash or blow on tomato leaves and start the disease cycle. Initial infection can be seen on lower leaves, and progresses upward.

SYMPTOMS Susceptible plant shows small, water-soaked round spots, about 3.2 mm in diameter, with a thin dark brown border and tan or a light center containing several small dark pycnidia (Figure 14). Yellow haloes often surround the spots, and severely infected leaves die and drop off. As the spots mature, they enlarge to diameter of about 6.4 mm and may coalesce. In the center of the spots are many dark brown, pimple-like structures called pycnidia, fruiting bodies of the fungus. The structures are large enough to be seen with the unaided eye or with the aid of a hand lens. Spots may also appear on stems, calyxes, and blossoms, but rarely on fruit. Heavily infected leaves will turn yellow, dry up, and drop off. This defoliation will result in sun-scalding of the fruit. Resistant type lesions are smaller and often resemble a dark rust colored fleck. Pycnidia, if present, are fewer, smaller and more difficult to detect than on a susceptible type lesion (Ferrandino and Elmer, 1992; Delahaut and Stevenson, 2004) .

HOST PATHOGEN INTERACTION Main infection route of S. lycopersici is through stomata but it may also occur by direct penetration of epidermal cells (Sohi and Sokhi, 1972). The enzyme tomatinase, produced by septoria pathogen, degrades -tomatine. The -tomatine is steroidal, glycoalkaloid saponin and has antimicrobial activity (Martin-Hernandez et al., 2000). Tomatinase-minus mutants are more sensitive to -tomatine than the wild-type strain. However, they can grow in the presence of 1 mM -tomatine, suggesting that non- degradative mechanisms of tolerance are also important. Some fungal pathogens of tomato are found to be more resistant to -tomatine (Melton et al., 1998). The necrotrophic tomato pathogen Septoria lycopersici produces an extracellular tomatinase enzyme that detoxifies -tomatine (Arneson and Durbin, 1967; 1968). The tomatine content which is

determined by the segregation of two codominant alleles of a single gene (Juvik and Stevens, 1982) is correlated with disease resistance in tomato. Variation is reported in - tomatine content in tomato species and accessions (Juvik et al., 1982). Cultivated tomato species produce lower amount of tomatine than wild species S. pimpinellifolium. The young leaves are rich in -tomatine (Roddick, 1974).

RESISTANCE BREEDING Development of host-plant resistance is considered the most effective and economical means to control diseases. Breeding efforts were not a priority due to effective control of the disease by fungicides (Barksdale and Stoner, 1978). However, with the severity of the problem, now it has been realized that breeding for septoria leaf spot resistance is important. Resistance has been reported to have single dominant gene action and is denoted by Se (Andrus and Reynard, 1945). However, recessive gene action has also been reported based on the field observation in a different study (Wright and Lincoln, 1940).

Due to cost involved in disease management, environmental hazards and likelihood of resistance development in the pathogen population from sole reliance on fungicides, breeding resistant varieties is a priority in tomato breeding programs including at NCSU. Screening germplasms for resistance under artificial inoculation is the most reliable option to create optimum disease pressure and maintaining consistency over several screening cycles. Artificial preparation of inoculums is also necessary to verify the markers identified using naturally infested population. Isolation and culture of inoculum is thus the most important first step to ensure the use of pathogen for artificial inoculation. However, an established protocol for isolation and maintenance of S. lycopersici inoculum is lacking at present. The objective of this experiment was to find a superior and reliable isolation and inoculum multiplication protocol for Septoria lycopersici.

MATERIAL AND METHODS

Plant materials Tomato leaves infected with septoria leaf spot were collected from research plots at Mountain Horticultural Crops Research and Extension Center (MHCREC), Mills River, and tomato growers’ field at Mills River (near MHCREC) in the summer of 2009.

Clinical Test and Spore Identification Infected leaf samples were taken to the Plant Pathology lab of NCSU and evaluated for disease symptoms visually and then using following three methods:

1. Using dissecting microscope: The lesions from an infected part of the leaf were observed directly under dissecting microscope. The symptoms of SLS, as described above, could be easily observed. Pycnidia, the fruiting bodies of SLS, were observed and characterized. 2. Using compound microscope: Slide mount of spores was prepared in a drop of water, first with a needle and then using blue staining solution (lactophenol cotton blue). Shape and length of spores were measured. Pycnidia mounted in a drop of water oozed conidia in large numbers. 3. Observing with naked eye after incubation: Infected leaves were incubated for 24 hours at room temperature using moist paper and observed with naked eye.

Incubation, Isolation and Culture After visual examination, leaves were kept in a moist chamber over night at room temperature to promote extrusion of spores from the pycnidia (Figure 14). After incubation of disease leaf sample for 24 hours, the following four different approaches were used to isolate the spores:

1. Sterile water was dropped on the spot of non-sterilized leaves and spores were streaked out in the media with the help of wire loop.

2. Under the dissecting microscope, spores were directly transferred with the help of needle by touching first infected leaf area and then the media.

3. A small piece of media was taken and touched to the infected leaf area and replaced in the petri dishes.

4. Leaf samples were sterilized and macerated. Then quadrant-streak technique (http://archive.microbelibrary.org/asmonly/details_print.asp?id=2808&lang=) was used to drop spore from macerated sample onto media with a platinum wire loop.

A single colony was selected and re-streaked on media. Pure culture was obtained by transferring either a hypha tip or streaked out the spore solution in the media following the bacterial streaking technique.

Sterilization and Aseptic Technique All operations including media transfer, isolation, and sub culture were performed in a laminar flow hood. Media was sterilized with a pressure-temperature sterilizing system (121oC, 15 psi for 20 min) and antibiotics were sterilized by filtration. Metal tools were sterilized in flame and working area with 75% ethyl alcohol. Surface sterilizing technique was followed to sterilize the leaf samples. Samples were dipped briefly in 70% alcohol and soaked in 10% bleach solution followed by rinsing in sterile distilled water.

Culture Media We used 3.9% Potato Dextrose Agar (PDA) media supplemented with Ampicillin @50 μg/mL. We dried the media by keeping in the laminar hood for a longer time, about one hour, before initiating culture to enhance sporulation of septoria (Pritchard and Porte, 1921). Media was stored in refrigerator for future use.

Figure 14. Tomato leaves infected with septoria leaf spot (Septoria lycopersici) (A), extrusion of white mass of spore from pycnidia (B), and enlarged extrusion of spore (C).

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Subculture and Storage Each colony was examined for the shape and length of spores to determine the right pathogen on a slide containing sterile distilled water (SDW) with cover slip under the microscope. Small pieces of colony were transferred to fresh media every three weeks. The colonies were maintained by routine transfer on fresh media. Colonies were transferred to tubes having slant PDA media and kept at room temperature for 3 weeks for long term storage. The fully grown colony was stored at 4oC.

RESULTS AND DISCUSSION

Clinical Test and Spore Identification Before isolation and culture, it is very important to identify the pathogen and associated pathogens with the target one. We used the following methods for diagnosis and spore identification of septoria leaf spot of tomato.

1. Using dissecting microscope: Pycnidia, the fruiting bodies of SLS were seen protruding from the center of leaf spot (Figure 14). Pycnidia were like pimple structures with dark on the bottom and white on the top. Pycnidia were black, globose to subglobose, ostiolate, 100-150 μm diameter. 2. Using compound microscope: Long and thread like shape of spores were observed. Spores were blue after staining with lactophenol cotton blue. Pycnidia mounted in a drop of water oozed conidia in large numbers. Spore (conidia) was hyaline and needle like, typically long thread (14-100 µm) with 2-6 septa and tapered on the both side (Figure 15). 3. Observing with naked eye after incubation: Several white dots were observed on the center of SLS after 24 hours incubation at room temperature (Figure 14). Incubation promoted extrusion of spores from the pycnidia.

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Figure 15. Blue stained thread like spore with septa of septoria (A) and enlarged spore (B).

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Isolation and Culture Techniques We used four methods for transferring isolates from the leaves. Among these methods, direct transfer of pycnidia with the help of a dissecting microscope was successful and found more effective. After 24 hours of incubation, infected leaf areas were observed under the dissecting microscope in the laminar hood. The pycnidia were seen clearly and these pycnidia were then transferred with the help of sterilized needle in PDA media. The other three methods, water aided spore transfer, media aided spore transfer and macerated sample-streaked out, were not found effective to isolate and culture septoria pathogens. After transferring, spores were incubated at room temperature for 1-2 weeks.

Colony Growth and Characters Spore growth was observed after 2 weeks. Spores were long threads with many septa and tapered both end. Growth of the spores was very slow, and its growth on the media look like small button white mass (Figure 16). At the initial growth stage, they were white but later turned black. The colony was very compact and hard (Figure 16). Few white spots were also observed in old colony.

Subculture and Storage Pure culture was obtained by transferring a very tip portion of the colony to media. We stored the single colony that can be used for further study. Pure culture is necessary to get the genetically similar population that is necessary to study the host-pathogen specific reaction. Colonies were verified observing long thread like spores under microscope and sub-cultured. Colony growth was maintained by routine transfer on PDA media. The colony of S. lycopersici was stored simply using slanted PDA media in tube at 4oC.

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Figure 16. White button like initial growth of septoria (A), fully grown colony (B) and colony in slant media for storage at 4oC (C).

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For inoculums preparation, the stored colony can be recovered by inoculating blocks of mycelium onto PDA and incubating in the dark at 22oC for about 14 days. Optimal conditions (Tu and Poysa, 1990) for inoculation are 1. Inoculums concentration of 106 spores/ ml, 2. Temperature at 24°C, 3. Moisture period of 48 hours and 4. A photoperiod of 14 hours. Literature showed that there are five different methods of inoculation, namely Spraying (Barksdale and Stoner, 1978), Spraying aided by spreader row (Poysa and Tu, 1993), Dipping (Andrus and Reynard, 1945), Brushing method (Tu and Poysa, 1990) and Detached leaf test (Martin-Hernandez et al., 2000).

Culture Media Spores were successfully cultured in PDA media. Use of cooked bean pod was also reported to be effective. Andrus and Reynard (1945) used cooked bean pod to culture, in which spores were sporulated copiously for screening and inheritance study of S. lycopersici resistance. The green bean pod was cooked and put into the tubes for S. lycopersici culture. Development of pycnidia is favored by dry air and hindered by moist air in tomato plants. Therefore, media was dried just by keeping it a longer time, about one hour on the laminar hood before culturing. Importance of dry media is supported by the findings of Pritchard and Porte (1921) who found variation in sporulation in culture media. Pycnidia were readily developed from the drier areas in the corn meal culture. Moist areas produced only after the medium becomes somewhat dry.

Conclusion A spore of SLS is thread-like, long with septa which can be directly isolated and cultured in selective PDA media. Colony growth is very slow and it is white at the initial growth stage and later it turns black in color and becomes compact and hard. Inoculums can be prepared from the stored colony to use in the tomato resistance breeding program. We hope this technique of isolation and culture of septoria will be useful in resistance breeding. We are planning to use the technique for artificial inoculation of septoria pathogen to verify the identified RAPD markers in Chapter 2.

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REFERENCES Andrus, C.F., and G.B. Reynard. 1945. Resistance to septoria leaf spot and its inheritance in tomatoes. Phytopathology 35:16-24.

Arneson, P.A., and R.D. Durbin. 1967. Hydrolysis of tomatine by Septoria lycopersici: A detoxification mechanism. Phytopathology 57:1358-1360.

Arneson, P.A., and R.D. Durbin. 1968. The sensitivity of fungi to alpha-tomatine. Phytopathology 58:536-537.

Barksdale, T.H., and A.K. Stoner. 1978. Resistance in tomato to Septoria lycopersici. Plant Dis. Rep. 62:844-847.

Delahaut, K., and W. Stevenson. 2004. Tomato disorders: Early blight and septoria leaf spot. The University of Wisconsin. A2606:R-0504.

Ferrandino, F.J., and W.H. Elmer. 1992. Reduction in tomato yield due to septoria leaf spot. Plant Disease 76:208-211.

Juvik, J.A., and M.A. Stevens. 1982. Inheritance of foliar alpha-tomatine content in tomatoes. J. Am. Soc. Hort. Sci. 107:1061-1065.

Juvik, J.A., M.A. Stevens, and C.M. Rick. 1982. Survey of the genus Lycopersicon for variability in alpha-tomatine content. HortScience 17:764-766.

Locke, S.B. 1949. Resistance to early blight and septoria leaf spot in the genus Lycopersicon. Phytopathology 39:829-836.

Martin-Hernandez, A.M., M. Dufresne, V. Hugouvieux, R. Melton, and A. Osbourn. 2000. Effects of targeted replacement of the tomatinase gene on the interaction of Septoria lycopersici with tomato plants. Mol. Plant-Microbe Interact. 13:1301-1311.

Melton, R.E., L.M. Flegg, J.K.M. Brown, R.P. Oliver, M.J. Daniels, and A.E. Osbourn. 1998. Heterologous expression of Septoria lycopersici tomatinase in Cladosporium fulvum: Effects on compatible and incompatible interactions with tomato seedlings. Mol. Plant-Microbe Interact. 11:228-236.

Poysa, V., and J.C. Tu. 1993. Response of cultivars and breeding lines of Lycopersicon spp. to Septoria lycopersici. Can. Plant Dis. Surv. 73:9-13.

Pritchard, F.J., and W.S. Porte. 1921. Relation of horse nettle (Solanum carolinese) to leaf spot of tomato (Septoria lycopersici). J. Agric. Res. XXI:501-506.

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Roddick, J.G. 1974. The steroidal glycoalkaloid alpha-tomatine. Phytochemistry 13:9-25.

Sohi, H.S., and S.S. Sokhi. 1972. Morphological, physiological and pathological studies in Septoria lycopersici. Indian Phytopathol. 26:666-673.

Tu, J.C., and V. Poysa. 1990. A brushing method of inoculation for screening tomato seedlings for resistance to Septoria lycopersici. Plant Dis. 74:294-297.

Wright, V., and R.E. Lincoln. 1940. Resistance to defoliation diseases of tomato. Purdue Agric. Exp. Stn. Annu. Rep. 53:42-43.

Wyenandt, C.A., L.H. Rhodes, R.M. Riedel, and M.A. Bennett. 2008. Cover crop mulch and fungicide program affect development of septoria leaf spot and marketable yield in processing tomato production. HortScience 43:807-810.

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APPENDICES

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APPENDICES

Appendix 1. Monthly weather conditions during tomato growing period (shaded area) in Mills River, North Carolina, 2009

Rainfall, mm Max Min RH, % 90 250

70 200

C 60 o 150 50

40 100

Temperature, Temperature, 30 RH, % and rainfall,and mm% RH, 20 50 10

0 0 Jan Feb Mar Apr May June Jul Aug Sep Oct Nov Dec

Seeding: 1 Jun Last harvest: 18 Sep

Min = Minimum temperature (°C). Max = Maximum temperature (°C). RH= Relative humidity (%).

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Appendix 2. Parents [NC 85L-1W(2007) x NC 839-2(2007)] and their F1 (NC 08135)

Late blight resistance Septoria leaf spot resistance

F1 08135)

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Appendix 3. Samples of tomato plant infected by two foliar diseases, late blight and septoria leaf spot

Symptom of septoria leaf spot Symptom of late blight

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Appendix 4. List of SSR, RAPD and other markers used to screen parents Marker type List SSR with M- SSR103-M13-F/SSR103-R, SSR104-M13-F/SSR104-R, SSR105-M13- 13 F primer F/SSR105-R, SSR134-M13-F/SSR134-R, SSR13-M13-F/SSR13-R, SSR146- (23) M13-F/SSR146-R, SSR14-M13-F/SSR14-R, SSR150-M13-F/SSR150-R, SSR155-M13-F/SSR155-R, SSR156-M13-F/SSR156-R, SSR15-M13- F/SSR15-R, SSR20-M13-F/SSR20-R, SSR218-M13-F/SSR218-R, SSR222- M13-F/SSR222-R, SSR223-M13-F/SSR223-R, SSR22-M13-F/SSR22-R, SSR308-M13-F/SSR308-R, SSR526-M13-F/SSR526-R, SSR589-M13- F/SSR589-R, SSR590-M13-F/SSR590-R, SSR593-M13-F/SSR593-R, SSR606-M13-F/SSR606-R, SSRB102358-M13-F/SSRB102358-R RAPD (197 K16, S12, P#72, OPK6, G5, MRTOMR-001, MRTOMR-021, MRTOMR-041, with 10-mer) MRTOMR-061, MRTOMR-081, MRTOMR-002, MRTOMR-022, MRTOMR- 042, MRTOMR-062, MRTOMR-082, MRTOMR-003, MRTOMR-023, MRTOMR-043, MRTOMR-063, MRTOMR-083, MRTOMR-004, MRTOMR- 024, MRTOMR-044, MRTOMR-064, MRTOMR-084, MRTOMR-005, MRTOMR-025, MRTOMR-045, MRTOMR-065, MRTOMR-085, MRTOMR- 006, MRTOMR-026, MRTOMR-046, MRTOMR-066, MRTOMR-086, MRTOMR-007, MRTOMR-027, MRTOMR-047, MRTOMR-067, MRTOMR- 087, MRTOMR-008, MRTOMR-028, MRTOMR-048, MRTOMR-068, MRTOMR-088, MRTOMR-009, MRTOMR-029, MRTOMR-049, MRTOMR- 069, MRTOMR-089, MRTOMR-010, MRTOMR-030, MRTOMR-050, MRTOMR-070, MRTOMR-090, MRTOMR-011, MRTOMR-031, MRTOMR- 051, MRTOMR-071, MRTOMR-091, MRTOMR-012, MRTOMR-032, MRTOMR-052, MRTOMR-072, MRTOMR-092, MRTOMR-013, MRTOMR- 033, MRTOMR-053, MRTOMR-073, MRTOMR-093, MRTOMR-014, MRTOMR-034, MRTOMR-054, MRTOMR-074, MRTOMR-094, MRTOMR- 015, MRTOMR-035, MRTOMR-055, MRTOMR-075, MRTOMR-095, MRTOMR-016, MRTOMR-036, MRTOMR-056, MRTOMR-076, MRTOMR- 096, MRTOMR-017, MRTOMR-037, MRTOMR-057, MRTOMR-077, MRTOMR-018, MRTOMR-038, MRTOMR-058, MRTOMR-078, MRTOMR- 019, MRTOMR-039, MRTOMR-059, MRTOMR-079, MRTOMR-020, MRTOMR-040, MRTOMR-060, MRTOMR-080, MRTOMR-097, MRTOMR- 109, MRTOMR-121, MRTOMR-133, MRTOMR-145, MRTOMR-157, MRTOMR-169, MRTOMR-181, MRTOMR-098, MRTOMR-110, MRTOMR- 122, MRTOMR-134, MRTOMR-146, MRTOMR-158, MRTOMR-170, MRTOMR-182, MRTOMR-099, MRTOMR-111, MRTOMR-123, MRTOMR- 135, MRTOMR-147, MRTOMR-159, MRTOMR-171, MRTOMR-183, MRTOMR-100, MRTOMR-112, MRTOMR-124, MRTOMR-136, MRTOMR- 148, MRTOMR-160, MRTOMR-172, MRTOMR-184, MRTOMR-101,

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Marker type List MRTOMR-113, MRTOMR-125, MRTOMR-137, MRTOMR-149, MRTOMR- 161, MRTOMR-173, MRTOMR-185, MRTOMR-102, MRTOMR-114, MRTOMR-126, MRTOMR-138, MRTOMR-150, MRTOMR-162, MRTOMR- 174, MRTOMR-186, MRTOMR-103, MRTOMR-115, MRTOMR-127, MRTOMR-139, MRTOMR-151, MRTOMR-163, MRTOMR-175, MRTOMR- 187, MRTOMR-104, MRTOMR-116, MRTOMR-128, MRTOMR-140, MRTOMR-152, MRTOMR-164, MRTOMR-176, MRTOMR-188, MRTOMR- 105, MRTOMR-117, MRTOMR-129, MRTOMR-141, MRTOMR-153, MRTOMR-165, MRTOMR-177, MRTOMR-189, MRTOMR-106, MRTOMR- 118, MRTOMR-130, MRTOMR-142, MRTOMR-154, MRTOMR-166, MRTOMR-178, MRTOMR-190, MRTOMR-107, MRTOMR-119, MRTOMR- 131, MRTOMR-143, MRTOMR-155, MRTOMR-167, MRTOMR-179, MRTOMR-191, MRTOMR-108, MRTOMR-120, MRTOMR-132, MRTOMR- 144, MRTOMR-156, MRTOMR-168, MRTOMR-180, MRTOMR-192 SSR and COS SSRB60800-F/SSRB60800-R, SSR150-F/SSR150-R, SSR155-F/SSR155-R, (159) SSR156-F/SSR156-R, SSR159-F/SSR159-R, SSR15-F/SSR15-R, SSR162- F/SSR162-R, SSR188-F/SSR188-R, SSR192-F/SSR192-R, SSR19-F/SSR19-R, SSR201-F/SSR201-R, SSR20-F/SSR20-R, SSR214-F/SSR214-R, SSR218- F/SSR218-R, SSR222-F/SSR222-R, SSR223-F/SSR223-R, SSR22-F/SSR22-R, SSR231-F/SSR231-R, SSR237-F/SSR237-R, SSR241-F/SSR241-R, SSR244- F/SSR244-R, SSR248-F/SSR248-R, SSR266-F/SSR266-R, SSR26-F/SSR26-R, SSR270-F/SSR270-R, SSR276-F/SSR276-R, SSR27-F/SSR27-R, SSR285- F/SSR285-R, SSR286-F/SSR286-R, SSR287-F/SSR287-R, SSR288-F/SSR288- R, SSR28-F/SSR28-R, SSR290-F/SSR290-R, SSR293-F/SSR293-R, SSR295- F/SSR295-R, SSR296-F/SSR296-R, SSR29-F/SSR29-R, SSR300-F/SSR300-R, SSR301-F/SSR301-R, SSR304-F/SSR304-R, SSR306-F/SSR306-R, SSR308- F/SSR308-R, SSR310-F/SSR310-R, SSR316-F/SSR316-R, SSR318-F/SSR318- R, SSR31-F/SSR31-R, SSR320-F/SSR320-R, SSR325-F/SSR325-R, SSR326- F/SSR326-R, SSR327-F/SSR327-R, SSR32-F/SSR32-R, SSR330-F/SSR330-R, SSR331-F/SSR331-R, SSR333-F/SSR333-R, SSR335-F/SSR335-R, SSR336- F/SSR336-R, SSR340-F/SSR340-R, SSR341-F/SSR341-R, SSR344-F/SSR344- R, SSR345-F/SSR345-R, SSR346-F/SSR346-R, SSR349-F/SSR349-R, SSR34- F/SSR34-R, SSR350-F/SSR350-R, SSR356-F/SSR356-R, SSR360-F/SSR360- R, SSR37-F/SSR37-R, SSR383-F/SSR383-R, SSR38-F/SSR38-R, SSR40- F/SSR40-R, SSR42-F/SSR42-R, SSR43-F/SSR43-R, SSR448-F/SSR448-R, SSR44-F/SSR44-R, SSR450-F/SSR450-R, SSR45-F/SSR45-R, SSR46- F/SSR46-R, SSR478-F/SSR478-R, SSR479-F/SSR479-R, SSR47-F/SSR47-R, SSR48-F/SSR48-R, SSR49-F/SSR49-R, SSR4-F/SSR4-R, SSR50-F/SSR50-R, SSR51-F/SSR51-R, SSR526-F/SSR526-R, SSR52-F/SSR52-R, SSR555- F/SSR555-R, SSR557-F/SSR557-R, SSR565-F/SSR565-R, SSR572-F/SSR572-

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Marker type List R, SSR578-F/SSR578-R, SSR57-F/SSR57-R, SSR580-F/SSR580-R, SSR582- F/SSR582-R, SSR586-F/SSR586-R, SSR589-F/SSR589-R, SSR590-F/SSR590- R, SSR593-F/SSR593-R, SSR594-F/SSR594-R, SSR595-F/SSR595-R, SSR596-F/SSR596-R, SSR598-F/SSR598-R, SSR599-F/SSR599-R, SSR5- F/SSR5-R, SSR601-F/SSR601-R, SSR602-F/SSR602-R, SSR603-F/SSR603-R, SSR605-F/SSR605-R, SSR606-F/SSR606-R, SSR61-F/SSR61-R, SSR62- F/SSR62-R, SSR63-F/SSR63-R, SSR65-F/SSR65-R, SSR66-F/SSR66-R, SSR67-F/SSR67-R, SSR69-F/SSR69-R, SSR70-F/SSR70-R, SSR72-F/SSR72- R, SSR73-F/SSR73-R, SSR74-F/SSR74-R, SSR75-F/SSR75-R, SSR76- F/SSR76-R, SSR80-F/SSR80-R, SSR85-F/SSR85-R, SSR86-F/SSR86-R, SSR92-F/SSR92-R, SSR94-F/SSR94-R, SSR96-F/SSR96-R, SSR98-F/SSR98- R, SSR99-F/SSR99-R, SSR9-F/SSR9-R, SSRB102358-F/SSRB102358-R, SSRB105694-F/SSRB105694-R, SSRB18031-F/SSRB18031-R, SSRB50753- F/SSRB50753-R, SSRB56555-F/SSRB56555-R, SSR103-F/SSR103-R, SSR104-F/SSR104-R, SSR105-F/SSR105-R, SSR108-F/SSR108-R, SSR110- F/SSR110-R, SSR111-F/SSR111-R, SSR112-F/SSR112-R, SSR115-F/SSR115- R, SSR117-F/SSR117-R, SSR11-F/SSR11-R, SSR122-F/SSR122-R, SSR124- F/SSR124-R, SSR125-F/SSR125-R, SSR128-F/SSR128-R, SSR134-F/SSR134- R, SSR135-F/SSR135-R, SSR136-F/SSR136-R, SSR13-F/SSR13-R, SSR146- F/SSR146-R, SSR14-F/SSR14-R, C2_At4g36530-F/C2_At4g36530-R, C2_At5g63840-F/C2_At5g63840-R

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Appendix 5. DNA extraction protocol from tomato fresh leaves (Fulton et al., 1995).

1. Collect 50-100 mg (approximately 4-8 new leaflets, up to 1.5 cm long) from a 1-3 week old tomato seedling(1) and nestle loosely in the bottom of a 1.5 ml Eppendorf tube (2). 2. Prepare fresh microprep buffer (see recipe below), keep at room temperature. 3. Add 200 µl of buffer and grind tissue with power drill and plastic bit (rinsing pestle with water between samples); add another 550 µl of buffer and either vortex lightly or shake entire rack by hand. 4. Incubate in 65oC water bath for 30-120 minutes. 5. Fill the tube with chloroform/ isoamyl (24:1). Mix well. This can be done by vortexing each tube or sandwiching tubes between two racks and vigorously inverting or shaking up and down 50-100 times. 6. Keep sample in ice for about 15-30’. 7. Centrifuge tubes at 10,000 rpm for 5 minutes. 8. Pipette off aqueous phase into new microfuge tubes (can repeat from step 5). Add 2/3-1 times the volume of cold isopropanol (or 70% ethyl alcohol) to each tube. Invert tubes until DNA precipitates. 9. Keep in fridge for 1 hour or overnight. 10. Immediately spin at 10,000 rpm for 5 minutes (no more), pour off isopropanol and wash pellet with 70% ethanol (3). 11. Can keep over night to dry. 12. Dry pellet by leaving tubes upside down on paper towels for approximately 1 hour or placing on sides in seed dryer for 15 minutes (longer if necessary). 13. Resuspend DNA in 50-150 µl of TE buffer.

Solutions for microprep buffer  DNA Extraction Buffer: 0.35 M Sorbitol, 0.1 M Tris-base, 5 mM EDTA; pH 7.5  Nuclei Lysis Buffer: 0.2 M Tris, 0.05 M EDTA, 2 M NaCl, 2% CTAB  Sarkosyl 5%

Microprep buffer: 2.5 parts DNA extraction buffer, 2.5 parts nuclei lysis buffer, 1.0 part 5% Sarkosyl. Add 0.3-0.5 g Sodium Bisulfite/100 ml buffer immediately before use (can be increased to avoid color in final product).

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Appendix 6. Extraction of DNA bands from agarose gels Protocol of GenElute™ Gel Extraction Kit (Product Code NA1111) provided by Sigma (USA)

Preparation Instructions

1. Wash Solution: Dilute the entire 12 ml of the Wash Solution Concentrate G with 48 ml of 95-100% ethanol prior to initial use. After each use, tightly cap the diluted Wash Solution to prevent the evaporation of ethanol. 2. Gel Solubilization Solution: The Gel Solubilization Solution will precipitate out of solution if stored at temperatures less than 18-25°C. Check to ensure that this solution is completely dissolved and that no crystals are present. If crystals are present, incubate at 37-50°C with periodic mixing until the crystals dissolve (approximately 5 minutes). 3. Agarose Gel Electrophoresis: Use fresh electrophoresis running buffer. Electrophoresis buffer, which has been used repeatedly, will reduce the DNA recovery efficiency. Minimize examination of ethidium bromide-stained gels with an UV trans-illuminator. If possible, use a trans-illuminator equipped with a long-wavelength (302 nm) UV light source, as this will minimize the damaging effects of UV light on nucleic acids. 4. Elution Solution: If purifying plasmid DNA or large linear DNA fragments (>3 Kb) preheat the Elution Solution to 65°C prior to use.

Spin Procedure for Agarose Gels

All centrifugations (spins) are performed at 12,000 to 16,000 x g.

1. Excise band. Excise the DNA fragment of interest from the agarose gel with a clean, sharp scalpel or razor blade. Trim away excess gel to minimize the amount of agarose. 2. Weigh gel. Weigh the gel slice in a tared colorless tube. 3. Solubilize gel. Add 3 gel volumes of the Gel Solubilization Solution to the gel slice. In other words, for every 100 mg of agarose gel, add 300 μl of Gel Solubilization Solution. Incubate the gel mixture at 50-60°C for 10 minutes, or until the gel slice is completely dissolved. Vortex briefly every 2-3 minutes during incubation to help dissolves the gel. 4. Prepare binding column. Preparation of the binding column can be completed while the agarose is being solubilized in step 3. Place the GenElute Binding Column G into one of the provided 2 ml collection tubes. Add 500 μl of the Column Preparation Solution to each binding column. Centrifuge for 1 minute. Discard flow-through liquid. 5. Check the color of the mixture. Once the gel slice is completely dissolved (step 3) make sure the color of the mixture is yellow (similar to fresh Gel Solubilization Solution with no gel slice) prior to proceeding to the following step. If the color of the mixture is red, add 10 μl of the 3 M Sodium Acetate Buffer, pH 5.2, and mix. The color should now be

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yellow. If not, add the 3 M Sodium Acetate Buffer, pH 5.2, in 10 μl increments until the mixture is yellow. 6. Add isopropanol. Add 1 gel volume of 100% isopropanol and mix until homogenous. For a gel with an agarose concentration greater than 2%, use 2 gel volumes of 100% isopropanol. 7. Bind DNA. Load the solubilized gel solution mixture from step 6 into the binding column that is assembled in a 2 ml collection tube. It is normal to see an occasional color change from yellow to red once the sample is applied to the binding column. If the volume of the gel mixture is >700 μl, load the sample onto the column in 700 μl portions. Centrifuge for 1 minute after loading the column each time. Discard the flow-through liquid. 8. Wash column. Add 700 μl of Wash Solution (diluted from Wash Solution Concentrate G as described under Preparation Instructions) to the binding column. Centrifuge for 1 minute. Remove the binding column from the collection tube and discard the flow- through liquid. Place the binding column back into the collection tube and centrifuge again for 1 minute without any additional wash solution to remove excess ethanol. Residual Wash Solution will not be completely removed unless the flow-through is discarded before the final centrifugation. 9. Elute DNA. Transfer the binding column to a fresh collection tube. Add 50 μl of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 minute. For efficient recovery of intact plasmid DNA, preheat the elution solution to 65°C prior to adding it to the membrane. Eluting at 65°C improves plasmid DNA recoveries by 2 to 3-fold. Yields of large linear DNA fragments (>3 Kb) can also be increased by up to 20% by preheating the elution solution to 65°C.

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Appendix 7. SSR profiles of parental lines showing monomorphic based on agarose gel, polyacryalamide gel and capillary electrophoresis

A. B. SSRB60800 SSR96 SSR85 SSR72 SSR65

SSR profiles of NC 85L-1W(2007) and NC 839-2(2007) on 2% agarose gel (A) and polyacrayalamide gel (B)

Electropherogram of SSR-606 in NC 85L-1W(207) and NC 839-2(2007)

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Appendix 8. Chemicals and reagents for DNA extraction and PCR

Liquid nitrogen Sarkosyl 5% Sodium bisulfate 0.35M Sorbitol: Dissolve 63.76 g (0.35 x 182.17) of Sorbitol in one liter of water 25mM MgCl2 MgCl2.6H2O 0.50825 g Extra pure water 100 ml Mix using hot stirrer and autoclave 0.5M EDTA pH7.5 EDTA (Na2EDTA.2H2O) 46.5 g Extra pure water 200 ml NaOH 5 g Adjust pH and add extra pure water to make 250 ml volume and autoclave 2M Tris HCl pH 7.5 Trizma base 24.22 g Extra pure water 70 ml HCl as per needed Add extra pure water to make final volume of 100 ml and autoclave 100x TE: 121 g of Tris and 37.2 g Na2EDTA.H2O in 800 ml water, adjust pH 8.0 with HCl, make up to 1 liter and autoclave 5M NaCl: 292 g of NaCl in 750 ml water and make up to 1 liter with water and autoclave 50x TAE buffer Trizma base (Tris) 242 g Acetic acid 57.1 ml 0.5M EDTA pH8.0 100 ml Extra pure water 700 ml Make a volume of 1 l and autoclave 50x TBE: 270 g of Tris (base), 138 g boric acid in 500 ml water. Add 100 ml 0.5M EDTA and make up to 1 liter. Store this solution in glass bottles at room temperature and discard any batches that develop a precipitate Loading buffer 0.5% Xylene cyanol 0.05 g 0.5% Bromo phenol blue 0.05 g Glycerol 40 ml Extra pure water 40 ml Mix using stirrer and autoclave 10 mg/ml Ethidium Bromide (EtBr) Add 1 g of ethidium bromide to 100 ml of ultrapure H2O Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved Wrap the container in aluminum foil or transfer the solution to a dark bottle and store at room temperature

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10 mM dNTP mix (dATP, dTTP, dCTP, dGTP) Mix equal amounts of dATP, dTTP, dCTP and dGTP (each with a concentration of 100 mM) in a 1.5-ml centrifuge tube. This solution has a concentration of 100 mM total dNTPs and 25 mM each dNTP. Take 100 µl of this solution and add 900 µl of sterile o ultrapure H2O. Store at –20 C 10x CTAB solution (10% CTAB, 0.7M NaCl) Mix 340 ml of autoclaved distilled water and add 40 g of CTAB. After dissolving completely add 16.46 g of NaCl and then fill it up to 400 ml with water. Autoclave Chl/Iso (Chloroform:Isoamyalcohol = 24:1 v/v) Do not autoclave. Avoid this chemical in contact with skin

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