<%BANNER%>

Inheritance and Mapping of Resistance to Bacterial Spot Race T4 (Xanthomonas perforans) in Tomato, and its Relationship ...

Permanent Link: http://ufdc.ufl.edu/UFE0022671/00001

Material Information

Title: Inheritance and Mapping of Resistance to Bacterial Spot Race T4 (Xanthomonas perforans) in Tomato, and its Relationship to Race T3 Hypersensitivity, and Inheritance of Race T3 Hypersensitivity from PI 126932
Physical Description: 1 online resource (132 p.)
Language: english
Creator: Hutton, Samuel
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: additive, bacteria, bacterial, campestris, dominance, epistasis, esculentum, euvesicatoria, hr, hypersensitivity, inheritance, linkage, lycopersicon, lycopersicum, mapping, markers, molecular, perforans, pimpinellifolium, qtl, quantitative, recessive, recombination, resistance, solanum, spot, suppression, tomato, vesicatoria, xanthomonas
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Resistance to bacterial spot of tomato (Solanum lycopersicum), race T4 (Xanthomonas perforans) was characterized in three advanced breeding lines: Fla. 8326, Fla. 8233, and Fla. 8517; by generation means analysis (GMA). GMA of Fla. 8326 for two of three seasons (fall 2006 and summer 2007) indicated resistance is mostly dominant with significant additive and epistatic effects. GMA of Fla. 8233 in the spring of 2007 and of Fla. 8517 in the summer of 2007 also showed dominance to be the main effect in addition to additive and epistatic effects. Duplicate dominance or recessive suppressor type epistasis was indicated in each breeding line. Resistant (R) and susceptible (S) F2 plants were selected from each of the three populations and the F3 and F4 progeny of these selections were evaluated to confirm resistance or susceptibility prior to including them for selective genotyping. Approximately 500 PCR-based markers, located primarily near areas of the genome where bacterial resistance genes have previously been identified, were screened to identify 269 polymorphic markers between S. lycopersicum and resistance sources. Polymorphic markers representing possible regions of introgression in each breeding line were analyzed for Transmission Disequilibrium (TD) across R and S selections from each family to identify markers linked to resistance QTL. TD analysis indicated the following significant QTL: a PI 114490 resistance locus on chromosome 3 in Fla. 8517; a PI 128216 resistance locus on chromosome 9 in Fla. 8233 and Fla. 8517; a H7998/PI 128216 resistance locus on chromosome 11 in Fla. 8326, Fla. 8233, and Fla. 8517; and an OH9242 susceptibility locus on chromosome 12 in Fla. 8517. Non-significant but plausible QTL were indicated on chromosomes 1, 5 and 10. Race T3 hypersensitivity (HR) was controlled by a common locus in PI 126932 and PI 128216, which was different from the race T3 HR locus in H7981. Race T3 HR was inherited from PI 126932 as a single, dominant gene. Race T4 field resistance was not associated with race T3 HR in Fla. 8326, Fla. 8233 or Fla. 8517.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Samuel Hutton.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Scott, John W.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022671:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022671/00001

Material Information

Title: Inheritance and Mapping of Resistance to Bacterial Spot Race T4 (Xanthomonas perforans) in Tomato, and its Relationship to Race T3 Hypersensitivity, and Inheritance of Race T3 Hypersensitivity from PI 126932
Physical Description: 1 online resource (132 p.)
Language: english
Creator: Hutton, Samuel
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: additive, bacteria, bacterial, campestris, dominance, epistasis, esculentum, euvesicatoria, hr, hypersensitivity, inheritance, linkage, lycopersicon, lycopersicum, mapping, markers, molecular, perforans, pimpinellifolium, qtl, quantitative, recessive, recombination, resistance, solanum, spot, suppression, tomato, vesicatoria, xanthomonas
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Resistance to bacterial spot of tomato (Solanum lycopersicum), race T4 (Xanthomonas perforans) was characterized in three advanced breeding lines: Fla. 8326, Fla. 8233, and Fla. 8517; by generation means analysis (GMA). GMA of Fla. 8326 for two of three seasons (fall 2006 and summer 2007) indicated resistance is mostly dominant with significant additive and epistatic effects. GMA of Fla. 8233 in the spring of 2007 and of Fla. 8517 in the summer of 2007 also showed dominance to be the main effect in addition to additive and epistatic effects. Duplicate dominance or recessive suppressor type epistasis was indicated in each breeding line. Resistant (R) and susceptible (S) F2 plants were selected from each of the three populations and the F3 and F4 progeny of these selections were evaluated to confirm resistance or susceptibility prior to including them for selective genotyping. Approximately 500 PCR-based markers, located primarily near areas of the genome where bacterial resistance genes have previously been identified, were screened to identify 269 polymorphic markers between S. lycopersicum and resistance sources. Polymorphic markers representing possible regions of introgression in each breeding line were analyzed for Transmission Disequilibrium (TD) across R and S selections from each family to identify markers linked to resistance QTL. TD analysis indicated the following significant QTL: a PI 114490 resistance locus on chromosome 3 in Fla. 8517; a PI 128216 resistance locus on chromosome 9 in Fla. 8233 and Fla. 8517; a H7998/PI 128216 resistance locus on chromosome 11 in Fla. 8326, Fla. 8233, and Fla. 8517; and an OH9242 susceptibility locus on chromosome 12 in Fla. 8517. Non-significant but plausible QTL were indicated on chromosomes 1, 5 and 10. Race T3 hypersensitivity (HR) was controlled by a common locus in PI 126932 and PI 128216, which was different from the race T3 HR locus in H7981. Race T3 HR was inherited from PI 126932 as a single, dominant gene. Race T4 field resistance was not associated with race T3 HR in Fla. 8326, Fla. 8233 or Fla. 8517.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Samuel Hutton.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Scott, John W.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022671:00001


This item has the following downloads:


Full Text

PAGE 1

1 INHERITANCE AND MAPPING OF RESISTANCE TO BACTERIAL SPOT RACE T4 ( Xanthomonas perforans ) IN TOMATO, AND ITS RELATIONSHIP TO RACE T3 HYPERSENSITIVITY, AND INHERITANCE OF RACE T3 HYPERSENSITIVITY FROM PI 126932 By SAMUEL FORREST HUTTON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

PAGE 2

2 2008 Samuel Forrest Hutton

PAGE 3

3 To my sister, Catherine

PAGE 4

4 ACKNOWLEDGMENTS I would first like to thank the chair of my supervisory committee, Dr. J.W. Scott, for the opportunity to pursue this Ph.D. in his tomato breeding program. His wealth of knowledge, sense of humor, and ordering of priorities made him an excellent mentor, and I cannot imagine a better advisor. I am very appreciative to the other members of my supervisory committee, Drs. E.A. Kabelka, J.B. Jones and H.J. Klee, for their advice and support. They were always open t o help, and I only regret I did not take advantage of their assistance more than I did I owe a great deal of thanks to Cathy Provenzano, Rosa Ayala, Sarah Smith, Jose Diaz and Dolly Cummings as members of the tomato breeding project who provided assistan ce in the field, greenhouse and lab. Drs. Yuanfu Ji and Aliya Momotaz are greatly appreciated for their assistance with molecular techniques. I am also indebted to Dr. Jeremy Edwards for being a continual source of advice and for helping improve the effici ency of my labwork. I would not have accomplished nearly so much with this research if it had not been for the help I received in specific areas from a number of professors and researchers. Dr. J.B. Jones, Dr. R.E. Stall and Jerry Minsavage provided tremen dous assistance with my greenhouse experiment s in Gainesvi lle. Drs. D.M. Francis, Matt Robbins and Sung Chur Sim at Ohio State University were more than generous in their contributions to marker development and screening, data analysis, and hypothesis form ation. I thank my wife, Emily, for her love and support during the past four years. I also thank our daughter, Anna Christine, for the joy she has brought us both for the past year and a half Soli Deo Gloria!

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGEMENTS 4 6 LIST OF FIGURES 8 CHAPTER 1 INTRODUCTI 1 2 2 INHERITANCE OF RESISTANCE TO XANTHOMONAS PERFORANS RACE T4 IN FLORIDA BREEDING LIN 1 9 3 ANALYSIS OF MOLECULA R MARKERS FOR LINKAG E TO RESISTANCE LOCI IN F LORIDA BREEDING LINE S 8233, 8517 AND 832 4 6 4 GENETIC CONTROL OF RACE T3 HYPERSENSITIVITY FROM PI 126932 AN D THE RELATIONSHIP B ETWEEN RACE T3 H YPERSENSITIVITY AND RACE T4 RESISTANCE APPENDIX A 9 8 B ADDITIONAL MOLECULAR MARKER 4 C DNA SOURCES FOR SELE CTIVE GENOTYPING OF RESISTANT AND SUSCEPTIBLE SELECTIO NS 5 LIST OF REFERENCES 7 BIOGR ..13 2

PAGE 6

6 LIST OF TABLES Table Page 2 1 Bacterial sp ot race T4 disease severity for Florida 8326 (P 1 ), Florida 7946 (P 2 ), F 1 F 2 and backcross generations, and joint scaling tests for goodness of fit to an additive dominan 2 2 2 Estimates of ad ditive, dominance, and interaction parameters for the Florida 8326 x Florida 7946 family 3 3 2 3 Bacterial spot race T4 disease severity for Florida 8233 (P 1 ), Florida 7776 (P 2 ), F 1 F 2 and backcross generations in spring 2007, and joint scaling test for goodness of fit to an additive 4 2 4 Estimates of additive, dominance, and interaction parameters for the Florida 8233 x Florida 7776 fa mily in spring 2007 5 2 5 Bacterial spot race T4 disease severity for Florida 8517 (P 1 ), Florida 7776 (P 2 ), F 1 F 2 and backcross generations in spring 2007, and joint scaling test for goodness of fit to a n additive 6 2 6 Estimates of additive, dominance, and interaction parameters for the Florida 8517 x Florida 7776 family in summer 2007 7 3 1 Markers 6 7 3 2 Disease severity on resistant and susceptible selections from the Florida 7776 x Florida 8233 F 2 generation, and subsequent prog eny in later seas ... 80 3 3 Genotypic data on resistant and susceptible progeny selections (see Table 3 2) f or m arkers polymorphic between Florida 8 233 and Florida 81 3 4 Disease severity on r esistant and susceptible selections from the Florida 7776 x Florida 8517 F 2 generation, and subsequent progeny in later ...8 2 3 5 Genotypic data on resistant and susceptible progeny selections (see Table 3 4) for m arkers polymorphic between Florida 8517 ...8 3 3 6 Disease severity on resistant and susceptible selections from the Florida 7946 x Florida 8326 F 2 generation, and subsequent progeny in later seasons ..8 5 3 7 Genotypic data on resistant and susceptible progeny selections (see Table 3 6) for markers polymorphic between Florida 8 326 and Florida 7 94 6

PAGE 7

7 4 1 Hypersensitivity as measured b y time to confluent necrosis in tomato plants infiltrated with Xanthomonas perforans 5 4 2 Hypersensitivity to race T3 of Xanthomonas perforans on rooted tomato cuttings in fall 2005 a nd fall 200 6 6 4 3 Segregation of plants for bacterial spot race T3 hypersensitivity and race T4 field resistance in three F 2 7 B 1 Markers screened by a mo dified EcoTILLING approach to identify polymorphisms between PI 114490 5 B 2 Technical information for markers polymorphic among genotypes resistant and susceptible to bacte 10 B 3 Non polymorphic m 3 C 1 DNA sources for selective genotyping of resistant and susceptible selections from Fla. 8233, Fla. 8517, and Fla. 8326 familie s 6

PAGE 8

8 LIST OF FIGURES Figure Page 2 1 Bacterial spot race T4 disease sev erity frequency distributions for tomato parents Fla. 8326, Fla. 7946, and generations derived from them at Citra, FL, during fall 2005. BC = backcross. Plants were rated on the Horsfall Barratt (1945) scale, where higher numbers indicate more disease 3 8 2 2 Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8326, Fla. 7946, and generations derived from them at Balm, FL, during spring 2006. BC = backcross. Plants were r ated on the Horsfall Barratt (1945) scale, where higher numbers indicate more disease (see text). 9 2 3 Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8326, Fla. 7946, and gener ations derived from them at Citra, FL, during summer 2007. BC = backcross. Plants were rated on the Horsfall 40 2 4 Bacterial spot race T4 di sease severity frequency distributions for tomato parents Fla. 8233, Fla. 7776, and generations derived from them at Balm, FL, during spring 2007. BC = backcross. Plants were rated on the Horsfall Barratt (1945) scale, where higher numbers indicate more disease (see text). 41 2 5 Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8517, Fla. 7776 and generations derived from them at Citra, FL, during summer 2007. BC = backcross. Plan ts were rated on the Horsfall Barratt (1945) scale, where higher numbers indicate more disease (see text). 2 2 6 Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8233, Fla. 8326, and the F 2 generation derived from them at Citra, FL, during fall 2005 and spring 2006. Plants were rated on the Horsfall Barratt (1945) scale, where higher numbers indicate more disease (see text). 3 2 7 Bacter ial spot race T4 disease severity frequency distribution for tomato parents Fla. 8517, Fla. 8233, and the F 2 generation derived from them at Citra, FL, during summer 2007. Plants were rated on the Horsfall Barratt (1945) scale, where higher numbers indi cate more disease (see text 4 2 8 Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8326, Fla. 8517, and the F 2 generation derived from them at Citra, FL, during summer 20 07. Plants were rated on the Horsfall Barratt (1945) scale, where higher numbers indicate more disease (see text 5 A 1. Pedigree of Fla. 8517. (Both Fla. 7655B and Fla. 7600 contain H7998 in their pedigrees.)

PAGE 9

9 A 2. Putative pedigree of Fla. 8233. [However, PI 114490 is now thought to be incorrectly recorded in this pedigree. It appears that PI 128216 was actually crossed to Fla. 7655 (see Ch. 3).] A 3. A 4. Pedigree of Fla. 7776 A 5. Pedigree of Fla. 7946

PAGE 10

10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INHERITANCE AND MAPPING OF RESISTANCE TO BACTERIAL SPOT RACE T4 ( Xanthomonas perforans ) IN TOMATO, AND ITS RELATIONSHIP TO RACE T3 HYPERSENSITIVITY, AND INHERITANCE OF RACE T3 HYPERSENSITIVITY FROM PI 126932 By Samuel Forrest Hutt on December 2008 Chair: John W. Scott Major: Horticultural Science Resistance to bacterial spot of tomato ( Solanum lycopersicum) race T4 ( Xanthomonas perforans ) was characterized i n three advanced breeding lines: Fla. 8326, Fla. 8233, and Fla. 8517 ; b y generation means analysis (GMA). GMA of Fla. 8326 for two of three seasons (fall 2006 and summer 2007) indicated resistance is mostly dominant with significant additive and epistatic effects. GMA of Fla. 8233 in the spring of 2007 and of Fla. 8517 in the summer of 2007 also showed dominance to be the main effect in addition to additive and epistatic effects. Duplicate dominance or recessive suppressor type epistasis was indicated in each breeding line. Resistant (R) and susceptible (S) F 2 plants were sele cted from each of the three populations and t he F 3 and F 4 progeny of these selectio ns were evaluated to confirm resistance or susceptibility prior to including them for selective genotyping Approximately 500 PCR based markers located primarily near areas of the genome where bacterial resistance genes have previously been identified, were screened to identify 269 polymorphic markers between S. lycopersicum and resistance sources. Polymorphic markers representing possible regions of introgression in each br eeding line were analyzed for Transmission Disequilibrium (TD) across R and S selections

PAGE 11

11 from each family to identify markers linked to resistance QTL. TD analysis indicated the following signifi cant QTL: a PI 114490 resistance locus on chromosome 3 in Fla 8517 ; a PI 128216 resistance locus on chromosome 9 in Fla. 8233 and Fla. 8517 ; a H7998/PI 128216 resistance locus on chromosome 11 in Fla. 8326, Fla. 8233 and Fla. 8517; and an OH9242 susceptibility locus on chromosome 12 in Fla. 8517 Non significant b ut plausible QTL were indicated on chromosomes 1, 5 and 10. Race T3 hypersensitivity (HR) was controlled by a common locus in PI 126932 and PI 128216, which was different from the race T3 HR locus in H7981. Race T3 HR was inherited from PI 126932 as a sing le, dominant gene. Race T4 field resistance was not associated with race T3 HR in Fla. 8326, Fla. 8233 or Fla. 8517.

PAGE 12

12 CHAPTER 1 INTRODUCTION Fresh market tomato ( Solanum lycopersic um L. ) is the most valuable vegetable cr op produced in Florida. The 2007 2 00 8 tomato crop was harvested from 37 8 00 acres and was worth slightly over 464 million dollars (FASS, 2008 ). The number of harvested acres, although still very high, has declined over the past 15 years. Florida growers today face challenges from exotic di seases, encroaching urbanization, higher production costs, increased global competition, and the phase out of methyl bromide. In response, growers are continually seeking ways to maximize efficiency by cutting costs and increasing yields. Yield reduction o f tomatoes results from a number of factors, including adverse weather conditions, cultural problems, disease, insects, nematodes, and weeds. Disease is of particular significance in Florida because of the The most pervasive di sease that faces Florida tomato production is bacterial spot. Bacterial spot of tomato is caused by three species of Xanthomonas: Xanthomonas euvesicatoria, X vesicatoria, and X perforans (Jones, et al., 2000; Jones, et al., 2005); these species were fo rmerly named X campestris pv. vesicatoria and X vesicatoria (Jones, et al., 1923), and secondary spread of the disease within plant beds and production fields oc curs primarily by wind driven rain and surface drainage of water that contains the bacterium (Sherf and Macnab, 1986). Thus, disease prevalence is greatest when temperatures are high and rainfall is frequent. Symptoms of this disease occur on leaves, stem s, and fruit. Necrotic lesions generally appear as small well defined spots. Leaves, however, may also take on a blighted or scorched appearance under conditions of heavy moisture, such as is experienced in Florida during the fall (Sherf and Macnab, 1986). Severe losses can occur due to leaf infections that

PAGE 13

13 cause defoliation, which may result in both yield losses and reduced quality from cracking, sunscald, or black shoulder (Scott and Jones, 1986). Pohronezny and Volin (1983) documented that total marketa ble yield losses rang ed from 17 to 52% under late and early bacterial spot infections, respectively. Bacterial spot especially reduced the number of USDA large size fruit (the fruit bringing the highest return to the producer). Control of bacterial spot i s based primarily on the use of bactericides and has become more difficult over the years. Antibiotic sprays, particularly streptomycin, were once very efficacious in controlling the disease, but are no longer used due to the ability of the bacterium to d evelop resistance (Lai et al., 1977; Stall and Thayer, 1962). Copper formulations proved effective in reducing bacterial spot development (Stall, 1959). However, after years of application, many strains of the bacterium have developed copper resistance, an d these resistant strains are now dominant in Florida (Marco and Stall, 1983). Copper applied in combination with mancozeb has been demonstrated to more effectively control bacterial spot than copper applied alone (Conover and Gerhold, 1981; Marco and Stal l, 1983). Current control practices utilize this latter tank mix, but control is often poor during periods of high disease pressure (Jones and Jones, 1985; Jones et al. 1991a b). Because disease control is poor and there is concern about excessive use of pesticides, alternative methods for controlling bacterial spot have been researched Compounds, such as Actigard, that induce systemic acquired resistance (SAR) in the plant have been reported as effective control alternatives (Louws et al., 2001), and Ac igard is routinely used in north Florida against bacterial spot on field grown fresh market tomato (Obradovic et al., 2005). The use of bacteriophages can also help control bacterial spot of tomato (Balogh et al., 2003; Flaherty et al.,

PAGE 14

14 2000), and integrat ed use of SAR inducers and phages may complement each other as an alternative management strategy (Obradovic et al., 2005). M uch effort has been directed to ward breeding for resistant varieties, but development has been difficult due to the limited availa bility of resistance sources, multigenic control of resistance, and the emergence of new races of the pathogen. Prior to 1989, all known strains from various areas of the world were of a single race (T1) of X euvesicatoria This race induced a hypersensit ive response (HR) on the resistant genotype Hawaii 7998 (H7998) a small fruited inbred bred for bacterial wilt resistance in Hawaii (Jones and Scott, 1986; Scott and Jones, 1986). Efforts were underway to incorporate this resistance into improved germplas m (Scott and Jones, 1989; Scott et al., 1991). Field resistance was largely additive and controlled by three to five effective factors (Scott and Jones, 1989). Whereas hypersensitive genes are generally thought to be single dominant genes, race T1 hypersen sitivity from H7998 was initially determined to be associated with either two (Whalen et al., 1993) or three genes (Wang et al., 1994). Yu et al. (1995) subsequently identified three regions of the genome: Rx 1 and Rx 2 located on the short and long arm s of chromosome 1 respectively ; and Rx 3 located on chr omosome 5, confirming the multi genic control of race T1 hypersensitivity in H7998. F ield resistance however, was not explained by the hypersensitive response alone. Wang (1992) found correlation coeff icients of only 0.39 to 0.41 between hypersensitivity and field resistance in two field F 2 populations. Likewise, Somodi et al. (1996) reported correlation coefficients of 0.31 to 0.52 between hypersensitivity and field resistance in two F 2 populations. R ace 2 (T2) ( X vesicatoria ) first was identified from Brazil in 1989 (Wang et al., 1990), and later, it was found in several other locations around the world (Bouzar et al., 1994b; Stall et al., 1994). This new race did not induce a hypersensitive reaction on H7998 as did T1. In

PAGE 15

15 addition, T2 strains were phenotypically different from T1 strains (Bouzar et al., 1994a b; Jones et al., 1993) and were amylolytic and pectolytic, whereas T1 strains were not (Bouzar et al., 1994a; Stall et al., 1994). Strains of a third race (T3), X perforans which were classified originially as T2, were isolated in Florida beginning in 1991 and were described by Jones et al. (1995). The incorrect identification was due to the fact that these strains were also amylolytic and pec tolytic, and they produced a compatible reaction on the tomato genotype H7998. However, unlike T1 and T2 strains, T3 strains induced a rapid hypersensitive response on the tomato genotype Hawaii 7981 (H7981) and S pimpinellifolium accessions PI 126932 and PI 128216. In vitro studies found that T3 was antagonistic to T1 (El Morsy et al., 1994), and before T1 resistant cultivars could be developed in Florida, the T3 strain largely replaced T1 (Jones et al. 1998). Scott et al. (1995) reported on the hypers ensitivity and/or resistance of a number of lines to race T3 in 2 years of testing, including Hawaii 7981 (H7981), PI 128216, PI 126932, H7998, PI 114490, PI 155372, PI 340905 S and PI 126428. The former three lines all produced a hypersensitive reaction w hen infiltrated with X. perforans race T3, whereas the latter five did not. Of those lines producing race T3 hypersensitivity, H7981 showed the highest level of r esistance, and selections of PI 126932 and PI 128216 had partial resistance. PI 114490, PI 155 372, PI 340905 S and PI 126428 also displayed partial resistance, and H7998 exhibited a low level of tolerance. The hypersensitive response in H7981 was determined to be controlled by an incompletely dominant gene, Xv3 (Scott et al., 1996), but field resis tance was later determined to be quantitatively conferred by Xv3 and other resistance genes (Scott et al., 2001). It is not known whether T3 hypersensitivity in PI 128216 and PI 126932 is conferred by Xv3 or by a different

PAGE 16

16 gene with similar function One objective of this research is to determine the allelism of the T3 Hr genes in each of these sources A novel source of resistance to T3 was found in the wild species S pennellii LA716 and described by Astua Monge, et al. (2000 a ). This resistance, which c auses a hypersensitive response with T3 strains, was determined to be different from Xv3 in H7981, as previously Xv4 a resistance gene corresponding to the avirulence gene avrXv4 in the pathogen (Astua Monge, et al., 2000 a ). Because of the potential for X vesicatoria race T2 to emerge in Florida, resistance to this race, as well as to X euvesicatoria race T1 and to X perforans race T3 was desired. Scott et al. (1997) screened a number of tomato genotypes for resistance to race T2 and compared these data to published results for races T1 and T3. H7981 was highly resistant to race T3 b ut susceptible to races T1 and T2. PI 126932, PI 128216 and PI 126428 were also resistant to race T3 but susceptible to races T1 and T2. H7998, which was resistant to race T1, showed the same low level of tolerance to race T2 as it did to race T3 PI 155372 and PI 114490 were the only lines with desirable levels of resistance to all three races, with the lat ter having the highest and most consistent levels of resistance. Scott et al. (2003) reported on the inheritance of resistance from PI 114490 to race T2. This resistance was determined to be additive and controlled by two genes, where all four alleles we re required for maximum resistance. A strong relationship between resistance genes for races T1 and T2 was observed, allowing for selection for resistance to either of these races to result in resistance to both races. Resistance to race T2 and race T3 segre gated independently, indicating that race T3 resistance is not controlled by the same genes as race T2 resistance in PI 114490.

PAGE 17

17 Recently, races T4 and T5 of X perforans have emerged and appear to be associated with mutagenesis of tomato race 3 strains (M insavage et al. 2003 ). The r ace T4 strain s arose from mutation s in the avrXv3 gene while the race T5 strain s contain mutations in both the avrXv3 and avrXv4 gene s Race T4 strains overcame the hypersensitive resistance in H7981, PI 128216 and PI 126932 ( Minsavage et al. 2003 ; Astua Monge et al., 2000b ) but have incompatible interactions when inoculated on LA716 ; whereas race T5 strains are compatible on genotypes containing Xv3 and/or Xv4 to that would be effective across races would be desira races T3 and T4 are problems in Florida, resistance to both of these races is needed to prevent bacterial spot. Additionally, since race T1 might re emerge i f resistance to races T3, T4 and T5 was achieved, and race T2 could potentially become a problem in Florida resistance to races T1 and T2 is also desireable. PI 114490 is resistant to race T4 (Scott, et al. 2006) as well as to the first three races of the pathogen, but l ittle is known about the genetics resistance to race T4 Although race T4 overcame the T3 Hr resistance of PI 128216, th is line ha s non hypersensitive resistance to race T4 (Scott et al., 2006) Another goal of this research is to determine if the T3 hyper sensitive resistance of th is line plays a role in its T4 resistance. This study will also investigate T4 resistance from PI 114490, PI 128216 and PI 126932, and seek to determine whether any of these sources have common T4 resistance genes. One approach to answer this question will be to identify molecular markers linked to important resistant genes ; these markers will also be very useful in efforts to pyramid resistance genes. Three advanced breeding lines have been developed that carry resistance to race T4. Florida 8233 is a large fruited fresh market tomato with PI 114490 and H7998 recorded in its

PAGE 18

18 pedigree. It has a moderate to high level of resistance to race T4. Florida 8517, with PI 114490, PI 128216 and H7998 in its pedigree, is a plum tomato with mo derate to high resistance. Florida 8326 has PI 126932 and H7998 in its pedigree. It is a large fruited fresh market tomato with only a moderate level of resistance. T o summarize, t he objectives of this research were to 1) determine the inheritance of race T4 resistance from each of the three above resistant breeding line s 2) identify molecular markers linked to resistance genes in each breeding line 3) elucidate the inheritance of the race T3 hypersensitivity from PI 126932 and determine if it is conferre d by the same locus as Xv3 in H7981, and 4) investigate the relationship between race T3 HR and race T4 field resistance from Fla. 8233 and Fla. 8517.

PAGE 19

19 CHAPTER 2 INHERITANCE OF RESISTANCE TO XANTHOMONAS PERFORANS RACE T4 IN FLORIDA BREEDING LINES 8326, 823 3 AND 8517 Introduction Bacterial spot of tomato ( Solanum l ycopersicum L.) is the most pervasive disease that faces Florida tomato production Race T1, caused by Xanthomonas euvesicatoria was the endemic race in Florida until 1991 when race T3 ( X. perfo rans ) emerged. The latter was antagonistic to (El Morsy et al ., 1994) and largely replaced race T1 (Jones et al., 1998) Race T4 came about as a result of a mutation in the X. perforans avrXv3 gene (Jones, unpublished), and has recently become prevalent ( J ones, unpublished ). Five races of the bacterial spot pathogen hae been identified without any selection pressure from monocultures of resistant cultivars. Despite this, host resistance still seems an attractive control strategy because bacterial spot is ex tremely difficult to control by chemical means, especially during hot, rainy weather common in Florida early in the fall production season. Resistance to race T1 was identified in Hawaii 7998 (H7998) (Scott and Jones, 1986), and this resistance was based in part on hypersensitivity (Jones and Scott, 1986) Field resistance was not explained by hypersensitivity alone, but was largely additive and controlled by 3 to 5 effective factors (Scott and Jones, 1989). The race T1 hypersensitive response of H7998 was controlled by three regions of the genome (Wang et al., 1994; Yu et al., 1995), while field resistance was conferred by hypersensitivity and other genes (Wang, 1992; Somodi et al., 1996). Resistance to race T3 was identified in a number of sources, includ ing hypersensitive resistance in H7981, PI 128216 and PI 126932 (Scott et al., 1995; Jones et al., 1995) and non hypersensitive resistance in PI 1144 90 (Scott et al., 1995). Hawaii 7981 displayed the highest level of resistance to race T3, and field resist ance was conferred by the incompletely dominant

PAGE 20

20 resistance gene, Xv3 (Scott et al., 1996) and other resistance genes (Scott et al., 2001). Race T4 overcame the Xv3 based hypersensitiv ity of H7981, PI 126932 and PI 128216 ( Minsavage, et al., 2003; Astua Mo nge et al., 2000b ), but not the field resistance of PI 114490 or PI 128216 (Scott et al., 2006). Ideally, r esistance is needed that is effective across multiple races of bacterial spot. Particularly, since races T3 and T4 are problems in Florida, resista nce to both of these races is needed to minimize damage associated with bacterial spot. However, little is known about the genetics of race T4 resistance. Three advanced breeding lines with resistance to bacterial spot races T3 and T4 have been developed ( Scott et al., 2006). Florida 8326 is a large fruited fresh market tomato with PI 126932 and H7998 in its pedigree; Florida 8233 is a large fruited fresh market tomato with PI 128216 and H7998 in its pedigree; Florida 8517 is a plum tomato with PI 114490, P I 128216 and H7998 in its pedigree. Both Fla. 8233 and Fla. 8517 have moderate to high levels of resistance to race T4, while Fla. 8326 has only a moderate level of resistance to this race. The primary objective of this research was to determine the inheri tance of resistance to race T4 in Florida breeding lines 8326, 8233 and 8517. A secondary objective was to evaluate the potential for comb in ing resistance genes from each of these sources for a higher level of resistance to race T4. Materials and Methods E xperimental Design, Inoculation and Disease Evaluation Within each experiment a randomized complete block de sign was used with four blocks, each with ten plant s per plot for the parent, F 1 and reciprocal F 1 (RF 1 ) lines and two plots of 25 plant s for the F 2 generation s. The R F 1 was included in each experiment to test for maternal inheritance of resistance For all experiments, seed were sown in growth rooms in Black Beauty

PAGE 21

21 spent coal (Reed Minerals Div., Highland, IN) and transplanted approximately 7 to 1 0 days later to Speedling trays (3.8 cm 3 cell size) (Speedling, Sun City, FL) in the greenhouse where seedlings were grown for four weeks Plants were transplanted to field beds that were 20 cm high and 81 cm wide that had been fumigated with 67% methyl bromide : 33% chloropicrin at 197 kg ha 1 (175 lbs per acre) and covered with reflective plastic mulch. Plants were spaced 46 cm apart in rows, with 152 cm between rows, staked and tied, and irrigated by drip tape beneath the plastic mulch of each bed. A r ecommended fertilizer program was followed and plants were sprayed with pesticides (excluding copper) as needed throughout the season ( Olsen et al., 2007 2008 ). I noculum was produced by growing the bacterial strains on Difco nutrient agar (Becton Dickinso n and Company, Sparks, Md.) for 24 the agar plates and suspended in 10 m M MgSO 4 7 H 2 O, and the suspensions were standardized to A 600 = 0. 30 (a concentration of approximately 2 to 5 x 10 8 colony forming units ( cfu)/mL). Inoculum was applied either at this concentration without surfactant, or was diluted to approximately 10 6 cfu/mL subsequent to standardization and applied along with Silwet L77 at 0.025% (v/v) as indicated below Inoculum was applied by misting t he foliage with a backpack sprayer Plants were rated for disease severity in the field using the Horsfall and Barratt scale (1945), where 1 = 0%, 2 = 0% 3%, 3 = 3% 6%, 4 = 6% 12%, 5 = 12% 25%, 6 = 25% 50%, 7 = 50% 75%, 8 = 75% 87%, 9 = 87% 94%, 10 = 94% 9 7%, 11 = 97% 100%, and 12 = 100% diseased tissue. Data were subjected to generation means analysis (Mather and Jinks, 1982) using a spreadsheet program (Ng, 1990). Plant Materials Florida 8326

PAGE 22

22 The race T4 susceptible inbred Fla. 7946 was crossed to Fl a. 8 326 and subsequently the F 1 was self pollinated to produce F 2 seed and crossed to each parent to produce backcrosses. These generations w ere used for inheritance stud ies in fall 2005 spring 2006 and summer 2007. For the fall 2005 experiment seed were so wn on 29 July and transplanted to the field in Citra, FL on 9 September. R ace T4 inoculum (at a concentration of approximately 2 to 5 x 10 8 cfu ml 1 ) was applied to the plants early in the morning on 16 September, and each plant was rated for disease on 19 October. For the spring 2006 experiment seed were sown on 17 February and transplanted to the field in Balm, FL on 29 March; race T3 inoculum (at a concentration of approximately 2 to 5 x 10 8 cfu mL 1 ) was applied to the plants early in the mornings on 5 May and again on 24 May because conditions were not favorable for disease during the first inocula tion. However, subsequent race identification of plant lesions, and disease severity of control lines, each indicated that race T4 was responsible for field i nfection. Each plant was rated for disease the week of 26 June. For the summer 2007 experiment seed were sown on 8 June, and race T4 inoculum (at a concentration of approximately 2 to 5 x 10 6 cfu mL 1 ) was applied to plants on 20 July, prior to trans plant ing to the field in Citra, FL on 26 July. Each plant was rated for disease on 3 October Florida 8233 The race T4 susceptible inbred Fla. 7776 was crossed to Fla. 8233, and subsequently the F 1 was self pollinated to produce F 2 seed and crossed to each pare nt to produce backcrosses. These generations were used for inheritance studies in fall 2006, spring 2007 and summer 2007. Extremely poor field conditions in fall 2006 and summer 2007 made disease severity rating s very difficult and resulted in unreliable d ata that will not be presented here. For the spring 2007 experiment seed were sown on 1 February and transplanted to the field in Balm, FL on 13

PAGE 23

23 March. Race T4 inoculum (at a concentration of approximately 2 to 5 x 10 8 cfu mL 1 ) was applied early in the m orning on 25 April, and each plant was rated for disease on 1 May. Florida 8517 The race T4 susceptible inbred Fla. 7776 was crossed to Fla. 8517, and subsequently the F 1 was self pollinated to produce F 2 seed and crossed to each parent to produce backcro sses. These generations were used for inheritance studies in fall 2006, spring 2007 and summer 2007. Extremely poor field conditions in fall 2006 and low disease levels in spring 2007 made disease severity ratings very difficult and resulted in unreliable data that will not be presented here. For the summer 2007 experiment seed were sown on 8 June, and race T4 inoculum (at a concentration of approximately 2 to 5 x 10 6 cfu m L 1 ) was applied to plants on 20 July, prior to trans planting to the field in Citra, FL on 26 July. Each plant was rated for disease on 3 October Combined resistance Florida 8233 was crossed to Fla. 8326, and subsequently the F 1 was self pollinated to produce F 2 seed. These generations were included in the Fla.8326 inheritance studies i n Citra, FL in fall 2005 and in Balm FL in spring 2006 Dates for seed sowing, transplanting, inoculation and disease evaluation were the same as those stated above for the Fla. 8326 fall 2005 and spring 2006 inheritance studies Florida 8326 was crossed to Fla. 8517 Fla. 8517 was crossed to Fla. 8233, and each F 1 w as subsequently self pollinated to produce F 2 seed. These generations were used for an inheritance study in summer 2007 in Citra, FL. Seed were sown on 8 June, and race T4 inoculum (at a concen tration of approximately 2 to 5 x 10 6 cfu mL 1 ) was applied to plants on 20 July, prior to transplanting to the field on 26 July. Each plant was rated for disease on 4 October.

PAGE 24

24 Analysis of variance using the GLM procedure of the Statistical Analysis System (SAS Institute, Cary, N.C.) was used to test for differences between F 1 and RF 1 generations, and for differences between resistant parents Results In two of the three seasons that Fla. 8326 was tested, the F 1 and R F 1 were not significantly different, ind icative of nuclear inheritance; likewise, maternal inheritance was not indicated in Fla. 8233 or Fla. 8517 (data not shown). Thus within each family, the F 1 and R F 1 generations were combined for generation means analysis. Florida 8326 Disease pressure was lower in fall 2005 than in either the spring 2006 or summer 2007 experiments as is evidenced by the higher mean disease severity of Fla. 7946 in each of the latte r seasons (Table 2 1). In 2005, disease severities of the F 1 and F 2 were intermediate between the resistant and susceptible parents and skewed toward the susceptible parent (Figure 2 1). The mean of the BCP 1 was approximately equal to the midparent, and the BCP 2 mean was approximately equal to the susceptible parent (Tables 2 1 and 2 2). The fall 2005 data had an acceptable fit to an additive dominance genetic model using the joint scaling test (Mather and Jinks, 1971) (Table 2 1), with only the additive effect having significan ce (Table 2 2 ). Broad sense heritability was estimated to be 0.66 by th e method of Allard (1960) ; narrow sense heritability was estimated at 1.23 by the method of Warner (1952); and the method of Wright (1934) implicated one effective factor contributing to resistance (data not shown). In spring 2006, disease severities of the F 1 and F 2 were intermediate between the resistant and susceptible parents and skewed toward the resistant parent (Figure 2 2). The BCP 1 was skewed toward the resistant parent as expected but the BCP 2 segregated in a continuous pattern

PAGE 25

25 (Figure 2 2) and had a lower mean than the midparent (Tables 2 1 and 2 2). In summer 2007, the F 1 and F 2 disease severities were both intermediate between the resistant and susceptible parents, but the F 2 mean disease severity was higher than would be expected under an a dditive dominance model (Figure 2 3 Table 2 1 ). The BCP 1 mean was approximately equal to that of the F 1 while the BCP 2 had a lower mean than expected (Table 2 1). Neither the spring 2006 nor the summer 2007 data fit an additive dominance model due primar ily to deviations in the BCP 2 generation for both seasons and deviations in the F 2 generation in 2007 (Mather and Jinks, 1971) (Table 2 1). Thus, an interaction analysis was performed that revealed significant homozygous x homozygous ([i]) interactions in summer 2007, and significant homozygous x heterozygous ([j]) and heterozygous x heterozygous ([l]) interactions in spring 2006 and summer 2007 (Table 2 2). Because the [h] and [l] parameters had opposite signs, duplicate dominance or recessive suppression type of epistasis was indicated (Mather and Jinks, 1982). Dominance and additivity were significant each season, and dominance had a greater effect. The presence of epistasis and dominance prevented the estimates of effective factors and heritabilities. In fall 2005, 36 of 113 F 2 41 were as susceptible as Fla. 7 94 2 plants were resistant while only 10 were susceptible. In summer 2007, 21 of 152 F 2 plants were as 94 6 (disease approximately 27% of F 2 plants were resistant and 30% were susceptible. Florida 8233

PAGE 26

26 Disease pressure in spring 2007 was moderate. The disease severities of Fla. 8233 and Fla. 7776 were distinguishable from one another but there was a low percentage of overlap between the two distributions (Figure 2 4). The F 1 was intermediate between re sistant and susceptible parents and skewed toward resistance (Table 2 3, Figure 2 4) The BCP 1 had an excess of resistant plants, resulting in a lower mean disease severity than was expected. The BCP 2 was distributed between the two parents and was skewed slightly toward susceptibility. The F 2 showed continuous variation between the two parents. An additive dominance model was not sufficient to explain the data primarily due to deviations in the backcross generations (Table 2 3). An interaction analysis r evealed significant homozygous x homozygous ([i]) and heterozygous x heterozygous ([l]) interactions (Table 2 4).The opposite signs of the [h] and [l] parameters indicated duplicate dominance or reces sive suppression type epistasis. Additive and dominance effects were also significant, and dominance had the greatest contribution to resistance. The presence of dominance and epistasis prevented the estimates of effective factors and heritabilities. Of the 198 F 2 plants evaluated, 49 ( 25%) were as resistant a s Fla. 8233 with disease Florida 8517 Under moderate to high disease pressure in summer 2007, parents separated with a small overlap Mean disease severity of the F 1 and F 2 were intermediate between the resistant and susceptible parents, and each was skewed slightly toward susceptible (Table 2 5, Figure 2 5). The backcross to the resistant parent was more susceptible than expected, with a mean disease severity appro ximately equal to the F 1 and some very susceptible plants; and the BCP 2 was

PAGE 27

27 slightly less susceptible than the susceptible parent. Deviations in the BCP 1 generation and, to a lesser extent, in the F 2 generation resulted in the inadequacy of an additive dom inance model to explain the data. An i nteraction analysis identified significant, homozygous x homozygous ([i]), homozygous x heterozygous ([j]), and heterozygous x heterozygous ([l]) interactions, and duplicate dominance or recessive suppression type epi stasis was again indicated (Table 2 6). D ominance and additive effects were significant, and dominance was the primary contributor to resistance. One hundred sixty three F 2 plants were evaluated in summer 2007. Of these, 37 (23%) susceptible as Fla. 7776. Combined Resistance In fall 2005, Fla. 8326 had a lower mean disease severity than Fla. 8233 ( P = 0.0024) and in spring 2006, Fla. 8233 was more resistant than Fla. 8326 ( P < 0.0001) (Figure 2 6). The F 2 generation had a mean disease severity intermediate between the two parents in both seasons F 2 progeny disease severities were s lightly skewed toward resistance in 2005 and 2006 and were distributed mainly within the range of the two parents. The F 2 distribution included o ne plant that was rated more resistant than either parent in 2005 and s everal F 2 plants in 2006 with slightly h igher disease severity ratings than either parent In summer 2007, the parents Fla. 8517 and Fla. 8233 were approximately equal and a fair number of individual plants were rated as susceptible (disease severity > 5) (Figure 2 7). T he mean disease severi ty of the F 2 generation was similar to that of Fla. 8517 F 2 p rogeny did not

PAGE 28

28 appear to segregate for individuals that were more susceptible than the parents, but the F 2 distribution included several plants with less disease than either parent. Fla. 8517 w as more resistant than Fla. 8326 in summer 2007 ( P = 0.0002 ) (Figure 2 8) and both parents included individuals rated as susceptible (disease severity > 5) The F 2 progeny from the cross between these two lines had disease severities that w ere distributed primarily within the ranges of the two parents and slightly skewed toward Fla. 8326 Only 3 F 2 plants were identified with less disease than Fla. 8517, and none were more susceptible than either parent Discussion The University of Florid has bred for bacterial spot The main source of r esistance to race T 1 from H7998 was overcome in Florida by the emergence of race T3 ; H7981, PI 126932 and PI 128216 were each identified as resis tant to the third race and all three had race T3 hypersensitivity as well (Jones et al., 1995). Most of the breeding efforts for race T3 resistance focused on H7981 because this line had the highest level of resistance to race T3 (Scott et al., 1995), but PI 126932 and PI 128216 were also included in the breeding program to a lesser extent Race T3 resistant breeding lines with these latter sources in their pedigrees include Fla. 8326, Fla. 8233 and one of the parents of Fla. 8517 Each of these three bree ding lines also has non hypersensitive race T4 resistan ce indicating that they may have durable resistance. Results from inheritance studies presented in this research indicate that resistance in all three breeding lines is primarily dominant, but that ep istasis is important and additive effects contribute significantly. Although b oth Fla. 7776 and Fla. 7946 are susceptible to bacterial spot, Fla. 7946 is much more susceptible than Fla. 7776. Both were used as susceptible parents to study

PAGE 29

29 inheritance. Res istant and susceptible individuals were easily distinguished in the Fla. 8326 x Fla. 7946 cross, even in fall 2005 when disease pressure was low. This is in contrast to crosses involving Fla. 7776, where resistant and susceptible parents still separated, b ut with a small percentage of overlap. This emphasizes the importance of choosing parents with adequate separation, especially when working with a quantitative trait. In fall 2005, under low bacterial spot disease pressure, Florida 8326 inheritance data f it an additive dominance model However, an erroneous narrow sense heritability was calculated. absence of dominance and the absence of epistasis. The presence of th ese effects in Fla. 8326, although undetected in fall 2005, is possibly the reason for the overestimation of narrow sense heritability, since both dominance and epistasis were significant in the Fla. 8326 spring 2006 and summer 2007 inheritance studies. Ad ditionally, one effective factor was identified in fall 2005. It may be that under low disease pressure, a single gene in Fla. 8326 can provide acceptable resistance, but that under higher pressure, a second, interacting gene is needed. This concept will b e discussed further in Chapter 3. Comb in ing resistance from various sources is an approach for developing durable resistance, as well as for increasing the level of achievable resistance. Each of the resistant breedi ng lines used in this study was crossed with one another to determine the potential for pyramiding their resistance genes and to estimate whether these lines had QTL in common In general, susceptible plants did not segregate from the F 2 generation s of the two crosses involving Fla. 8326; nor we re segregates identified that were more resistant than either parent This suggests that resistance in Fla. 8326 is based on QTL in common with Fla. 8233 and Fla. 8517, and that additional resistance genes contribute to the higher level of resistance in th e latter two

PAGE 30

30 lines thus limiting the potential for pyramiding genes from Fla. 8326. By this hypothesis it follows that susceptible segregates were not identified in the F 2 between Fla. 8517 and Fla. 8233. However, the identification of a 6 F 2 plants from this cross with lower disease severity ratings than either parent supports the possibility that these two lines may not have all QTL in common. This is not certain, since the identification of these individuals may simply be the result of the large popula tion size of the F 2 relative to the parents. These plants were selected and are being tested to determine if they are, in fact, more resistant than either parent. The multi genic control of the partial resistance to race T4 from Fla. 8326, Fla. 8233 and Fla 8517 presents significant difficulties for developing resistant cultivars. Although the F 1 w as an improvement over the susceptible parent in each inheritance study, this would probably not be a commercially acceptable level of resistance with the partial ly resistant parent s used Thus it will be necessary to incorporate this resistance into both parents of a hybrid. Further more i ncorporation of this resistance into lines that are not highly susceptible (such as Fla. 7776) will be particularly difficult, since progeny with moderate and higher levels of resistance are not easily distinguish able This, combined with the complicating effects of dominance and epistasis, necessitates that s e lections be evaluated in subsequent trials to obtain plants homozygous for resistant alleles. The potential for pyramiding resistance QTL from Fla. 8326, Fla. 8233 and Fla. 8517 for durable resistance depends, in part, on the source of each QTL. Evidence from complementation tests suggests that Fla. 8326 has limited usefuln ess in pyramiding, but that it may be possible to pyramid resistance genes from Fla. 8233 and Fla. 8517. Each of these resistant breeding lines has two or more potential donors of resistance in its pedigree, but it is uncertain which donors are contributin g to resistance Chapter 3 discusses the identification of molecular markers linked to

PAGE 31

31 resistance QTL in each line and addresses the source of each QTL. The use of molecular markers linked to resistance QTL will also be extremely use ful in applied breeding work to overcome the aforementioned difficulties in obtain ing p lants homozygous for resistance alleles.

PAGE 32

32

PAGE 33

33

PAGE 34

34

PAGE 35

35

PAGE 36

36

PAGE 37

37

PAGE 38

38 Figure 2 1. Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8326, Fla. 7946, and generations derived from them at Citra, FL, during fall 2005. BC = backcross. Plants were r ated on the Hor sfall Barratt (1945) scale, where h igher number s indicate more disease ( see text ) 0 5 10 15 20 25 30 2 3 4 5 6 7 Frequency (No. Plants) Disease severity Fla. 8326 (P 1 ) 0 5 10 15 20 25 30 2 3 4 5 6 7 Frequency (No. Plants) Disease severity Fla. 7946 (P 2 ) 0 10 20 30 40 2 3 4 5 6 7 Frequency (No. Plants) Disease severity F 1 0 10 20 30 40 2 3 4 5 6 7 Frequency (No. Plants) Disease severity F 2 0 5 10 15 20 2 3 4 5 6 7 Frequency (No. Plants) Disease severity BCP 1 0 10 20 30 40 2 3 4 5 6 7 Frequency (No. Plants) Disease severity BCP 2

PAGE 39

39 Figure 2 2. Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8326 Fla. 7946 and generations derived from them at Balm, FL, during spring 2006. BC = backcross. Plants were r ated on the Horsfall Barratt (1945) scale, where h igher number s indicate more disease ( see text ) 0 5 10 15 20 25 30 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity Fla. 8326 (P 1 ) 0 5 10 15 20 25 30 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity Fla. 7946 (P 2 ) 0 5 10 15 20 25 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity F 1 0 10 20 30 40 50 60 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity F 2 0 10 20 30 40 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity BCP 1 0 5 10 15 20 25 30 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity BCP 2

PAGE 40

40 Figure 2 3. Bacterial spot race T4 disease severity frequency distributions for tomato paren ts Fla. 8326, Fla. 7946 and generations derived from them at Citra, FL, during summer 2007. BC = backcross. Plants were r ated on the Horsfall Barratt (1945) scale, where h igher number s indicate more disease ( see text ) 0 5 10 15 20 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity Fla. 8326 (P 1 ) 0 5 10 15 20 25 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity Fla. 7946 (P 2 ) 0 10 20 30 40 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity F 1 0 20 40 60 80 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity F 2 0 5 10 15 20 25 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity BCP 1 0 10 20 30 40 50 2 3 4 5 6 7 8 9 Frequency (No. Plants) Disease severity BCP 2

PAGE 41

41 Figure 2 4. Bacterial s pot race T4 disease severity frequency distributions for tomato parents Fla. 8233, Fla. 7776 and generations derived from them at Balm, FL, during spring 2007. BC = backcross. Plants were r ated on the Horsfall Barratt (1945) scale, where h igher number s in dicate more disease ( see text ) 0 5 10 15 20 2 3 4 5 6 7 Frequency (No. Plants) Disease severity Fla. 8233 (P 1 ) 0 5 10 15 20 25 2 3 4 5 6 7 Frequency (No. Plants) Disease severity Fla. 7776 (P 2 ) 0 5 10 15 20 25 30 2 3 4 5 6 7 Frequency (No. Plants) Disease severity F 1 0 20 40 60 80 2 3 4 5 6 7 Frequency (No. Plants) Disease severity F 2 0 10 20 30 40 2 3 4 5 6 7 Frequency (No. Plants) Disease severity BCP 1 0 5 10 15 20 25 30 2 3 4 5 6 7 Frequency (No. Plants) Disease severity BCP 2

PAGE 42

42 Figure 2 5. Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8517, Fla. 7776 and generations derived from them at Citra, FL, during summer 2007. BC = backcross. Plants were r ated on the Horsfall Barratt (1945) scale, where h igher number s indicate more disease ( see text ) 0 5 10 15 20 25 30 3 4 5 6 7 Frequency (No. Plants) Disease severity Fla. 8517 (P 1 ) 0 5 10 15 20 3 4 5 6 7 Frequency (No. Plants) Disease severity Fla. 7776 (P 2 ) 0 10 20 30 40 50 60 3 4 5 6 7 Frequency (No. Plants) Disease severity F 1 0 20 40 60 80 100 3 4 5 6 7 Frequency (No. Plants) Disease severity F 2 0 10 20 30 40 3 4 5 6 7 Frequency (No. Plants) Disease severity BCP 1 0 10 20 30 40 50 60 3 4 5 6 7 Frequency (No. Plants) Disease severity BCP 2

PAGE 43

43 Figure 2 6. Bacterial spot race T4 disease severity frequency distributions for tomato parents Fla. 8233 Fla. 8326 and the F 2 generation derive d from them at Citra, FL, during fall 2005 and spring 2006 Plants were r ated on the Horsfall Barratt (1945) scale, where h igher number s indicate more disease ( see text ) 0 5 10 15 20 2 3 4 5 Frequency (No. Plants) Disease severity FALL 2005 Fla. 8233 = 4.04 0 5 10 15 20 2 3 4 5 Frequency (No. Plants) Disease severity SPRING 2006 Fla. 8233 = 2.89 0 5 10 15 2 3 4 5 Frequency (No. Plants) Disease severity Fla. 8326 = 3.65 0 10 20 30 2 3 4 5 Frequency (No. Plants) Disease severity Fla. 8326 = 3.65 0 20 40 60 80 2 3 4 5 Frequency (No. Plants) Disease severity F 2 = 3.76 0 20 40 60 80 2 3 4 5 Frequency (No. Plants) Disease severity F 2 = 3.46

PAGE 44

44 Figure 2 7 Bacterial spot race T4 disease severity frequency distributions fo r tomato parents Fla. 8517 Fla. 8233 and the F 2 generation derived from them at Citra, FL, during summer 2007. Plants were r ated on the Horsfall Barratt (1945) scale, where h igher number s indicate more disease ( see text ) 0 2 4 6 8 10 12 3 4 5 6 Frequency (No. Plants) Disease severity Fla. 8517 = 4.64 0 2 4 6 8 10 3 4 5 6 Frequency (No. Plants) Disease severity Fla. 8233 = 4.98 0 10 20 30 40 50 3 4 5 6 Frequency (No. Plants) Disease severity F 2 = 4.62

PAGE 45

45 Figure 2 8 Bacterial sp ot race T4 disease severity frequency distributions for tomato parents Fla. 8326 Fla. 8517 and the F 2 generation derived from them at Citra, FL, during summer 2007. Plants were r ated on the Horsfall Barratt (1945) scale, where h igher number s indicate mor e disease ( see text ) 0 2 4 6 8 10 12 3 4 5 6 Frequency (No. Plants) Disease severity Fla. 8326 = 5.35 0 2 4 6 8 10 12 3 4 5 6 Frequency (No. Plants) Disease severity Fla. 8517 = 4.61 0 10 20 30 40 3 4 5 6 Frequency (No. Plants) Disease severity F 2 = 5.03

PAGE 46

46 CHAPTER 3 ANALYSIS OF MOLECULA R MARKERS FOR LINKAG E TO RESISTANCE LOCI IN FLORIDA BREEDING LIN ES 8233, 8517 AND 83 26 Introduction Bacterial spot of tomato is the most pervasive disease that faces Florida tomato ( Solanum lycopersic um L. ) production. It is caused by three species of Xanthomonas: Xanthomonas euvesicatoria, X vesicatoria, and X perforans (Jones et a l., 2000; Jones et al., 2006). Disease prevalence is greatest when temperatures are high and rainfall is frequent condi tions typically experienced in Florida during the early fall Severe losses can occur due to leaf infections that cause defoliation, which may result in both yield losses and reduced quality from cracking, sunscald, or black shoulder (Scott and Jones, 1986 ). Bacterial spot is primarily controlled by the use of bactericides which provide less than adequate control during periods of high disease pressure (Jones and Jon es, 1985; Jones et al. 1991a, b). There has been much effort directed to breeding for resis tant varieties, but development has been difficult due to the multigenic control of resistance and the emergence of new races of the pathogen. Prior to 1989, all strains that had been collected from various areas of the world were of a single race (T1) of X euvesicatoria This strain induced a hypersensitive response (HR) on the resistant genotype Hawaii 7998 (H7998) (Jones and Scott, 1986; Scott and Jones, 1986), and e fforts were underway to incorporate this resistance into improved germplasm (Scott and J ones, 1989; Scott et al. 1991). Field resistance was reported to be largely additive and controlled by three to five effective factors, but was not explained by hypersensitivity alone (Scott and Jones, 1989; Wang, 1992; Somodi et al., 1996). Genetic contro l of the HR was initially determined to be associated with three regions of the genome: Rx 1 located on the short arm of chromosome 1; Rx 2 located on the long arm of chromosome 1; and Rx 3 located on

PAGE 47

47 chromosome 5 (Yu et al., 1995). Subsequent work by Y ang et al. (2005) using molecular markers linked to Rx 1 and Rx 3 determined that Rx 3 was the primary contributor to race T1 HR, and that this locus explained 41% of field resistance to race T1 in an inbred backcross population. In 1989, race 2 (T2) ( X v esicatoria ) was determined to be quite prevalent in Brazil (Wang et al., 1990), and other areas of the world (Bouzar et al., 1994b; Stall et al., 1994). Strains of a third race (T3), X perforans which were classified originially as race T2, were isolated in Florida beginning in 1991 and were described by Jones et al. (1995). Race T3 strains induced a rapid hypersensitive response on the tomato genotype Hawaii 7981 (H7981) and S pimpinellifolium accessions PI 126932 and PI 128216. In vitro studies found t hat race T3 was antagonistic to race T1 (El Morsy et al., 1994), and before race T1 resistant cultivars could be developed in Florida, the race T3 strain largely replaced T1 (Jones et al. 1998). Scott et al. (1995) reported on the hypersensitivity and/or resistance of a number of lines to race T3 in 2 years of testing, including Hawaii 7981 (H7981), PI 128216, PI 126932, H7998 and PI 114490. The former three lines all produced a hypersensitive reaction when infiltrated with X. perforans race T3, whereas t he latter two did not. Of the former three lines, H7981 showed the highest level of r esistance, and selections of PI 126932 and PI 128216 had partial resistance. PI 114490 displayed partial resistance, and H7998 exhibited a low level of resistance The hyp ersensitive response in H7981 was determined to be controlled by an incompletely dominant gene, Xv3 (Scott et al., 1996), but field resistance was later determined to be quantitatively conferred by Xv3 and other resistance genes (Scott et al., 2001). Beca use of the potential for X vesicatoria race T2 to emerge in Florida, resistance to this race, as well as to X euvesicatoria race T1 and to X perforans race T3 was desired. Scott et al.

PAGE 48

48 (1997) screened a number of tomato genotypes for resistance to race T2 and compared these data to published results for races T1 and T3. H7981 was highly resistant to race T3 b ut susceptible to races T1 and T2. PI 126932 and PI 128216 were also resistant to race T3 but susceptible to races T1 and T2. H7998, resistant to ra ce T1, had a low level of resistance to races T2 and T3. PI 114490 was one of only two lines with desirable levels of resistance to all three races, and had the highest and most consistent levels Scott et al. (2003) determined that race T2 resistance in P I 114490 is additive and controlled by two genes; it was also reported that a strong relationship existed between resistance genes in this PI for races T1 and T2, but that race T3 resistance is likely controlled by different genes. Recently a fourth race, X perforans race T4, has emerged that overcame the Xv3 based hypersensitive resistance in H7981, PI 128216 and PI 126932 ( Minsavage et al., 2003 ; Astua Monge et al., 2000b ). Scott et al. (2003) pointed out that a durable resistance that is effective acro ss races is needed In particular, since races T3 and T4 are the prevalent races in Florida, resistance to both of these races is needed to prevent bacterial spot. PI 114490 is resistant to race T4 as well as to the first three races of the pathogen (Scott et al. 2006 ), but little is known about the genetics of this resistance; furthermore, although race T4 overcame the T3 H R resistance of PI 128216 and PI 126932, the former has non hypersensitive resistance to race T4 and breeding lines with race T4 resi stance presumably derived from these PIs have been developed (Scott et al., 2006) The identification of molecular markers linked to resistance genes from each of these sources would be very useful in incorporating resistance into improved germplasm, as we ll as in efforts to pyramid resistance genes. Three advanced breeding lines have been developed that have resistance to race T4. Florida 8233 is a large fruited fresh market tomato with PI 114490 and H7998 recorded in its

PAGE 49

49 pedigree. It has a moderate to hig h level of resistance to race T4. Florida 8517, with PI 114490, PI 128216 and H7998 in its pedigree, is a plum tomato with moderate to high resistance. Florida 8326 has PI 126932 and H7998 in its pedigree. It is a large fruited fresh market tomato with onl y a moderate level of resistance. The primary objective of this research was to identify molecular markers linked to T4 resistance genes in each of these resistant breeding lines. A secondary objective was to develop and compile an inventory of markers pol ymorphic among resistant and susceptible genotypes used in this study. Materials and Methods Plant Materials Three advanced breeding lines with resistance to bacterial spot race T4 of tomato were used as donor parents to develop three separate F 2 populatio ns. Florida 8233, Fla. 8517 and Fla.8326 were selected for field resistance over multiple seasons, without using molecular markers. Fla. 8233 and Fla. 8517 were each crossed to Fla. 7776, a susceptible inbred line. Fla. 8326 was crossed to Fla. 7946, a hig hly susceptible inbred line. Individual F 2 plants, selected from each population on the basis of highest or lowest level of disease severity were re evaluated as F 3 (and in some cases, F 4 ) families to confirm resistance or susceptibility. Selections with consistently high or low levels of disease severity were genotyped with markers polymorphic between the two parents of their respective family. In screens to identif y these polymorphic markers, PI 114490, PI 128216, PI 126932, H7981 and H7998 were included as potential resistance donors, and Fla. 7776 and Fla. 7946 were included as susceptible genotypes. Molecular Markers Approximately 500 PCR based markers were screened t o detect polymorphisms among PI 114490, PI 128216 PI 126932, H7981, H7998, Fla. 7776 and Fla. 7946. Many of the markers

PAGE 50

50 were obtained from the Tomato Mapping Resource Database ( http://www.tomatomap.net ) or were recently developed ( M. Robbins and S. Sim, personal communication), and had accompanying po lymorphism information for genotypes used in this study. All other markers, unless otherwise indicated, were obtained from the Solanaceae genome network (SGN) ( http://www.sgn.cornell.edu ) or were developed in house from RFLP probe, unigene or BAC sequences found on SGN. For markers that did not have accompanying polymorphism information, screening was initially done to identify CAPS (cleaved amplified polymorphic sequence) markers. Here, PCR product of each marker w as digested with 15 to 20 different frequent cutting restriction enzymes to identify polymorphic restriction sites among the genotypes screened. Additional markers polymorphic between PI 114490 and Fla. 7776 were identified using an EcoTILLING approach on agarose gels, as described by Raghavan et al. (2007), with two exceptions: W hen making an enzyme mixture, 10x NEBuffer 1 (New England Biolabs, Inc.) was added to the celery juice extract (CJE) in CJE buffer and nanopure water, instead of the additional cel ery juice extract buffer; also digestions were carried out for one hour rather than for 15 minutes. Polymorphic markers identified by this approach were then sequenced and, where possible, converted to CAPS for screening of other genotypes. The EcoTILLING approach was also used to screen several polymorphi c markers that could not be converted to CAPS. Experimental Design, Inoculation and Disease Evaluation Within each inheritance study a randomized complete block de sign was used with four blocks, each with ten plant s per plot for the parent lines and two plots of 25 plant s for the F 2 generation Progeny of F 2 selections were evaluated in confirming experiments where a randomized complete block design was used with 3 blocks and 6 or 8 plant s per plot For all

PAGE 51

51 experiments, seed were sown in growth rooms in Black Beauty spent coal (Reed Minerals Div., Highland, IN) and transplanted approximately 7 to 10 days later to Speedling trays (3.8 cm 3 cell size) (Speedling, Sun City, FL) in the greenhouse, where seedlings were grown for four weeks Plants were transpl anted to field beds that were 20 cm high and 81 cm wide that had been fumigated with 67% methyl bromide : 33% chloropicrin at 197 kg ha 1 (175 lbs per acre) and covered with reflective plastic mulch. Plants were spaced 46 cm apart in rows, with 152 cm betw een rows, staked and tied, and irrigated by drip tape beneath the plastic mulch of each bed. A recommended fertilizer program was followed, and plants were sprayed with pesticides (excluding copper) as needed throughout the season ( Olsen et al., 2007 2008 ) I noculum was produced by growing the bacteria on Difco nutrient agar (Becton Dickinson and Company, Sparks, Md.) for 24 the agar plates and suspended in 10 mM MgSO 4 7 H 2 O, and the suspensions were standardized to A 600 = 0. 30 (a con centration of approximately 2 to 5 x 10 8 colony forming units (cfu)/mL). Inoculum was applied either at this concentration without surfactant, or was diluted to approximately 10 6 cfu/mL subsequent to standardization and applied along with Silwet L77 at 0.0 25% (v/v) as indicated below Inoculum was applied by misting the foliage with a backpack sprayer Plants were rated for disease severity in the field using the Horsfall and Barratt scale (1945), where 1 = 0%, 2 = 0% 3%, 3 = 3% 6%, 4 = 6% 12%, 5 = 12% 25 %, 6 = 25% 50%, 7 = 50% 75%, 8 = 75% 87%, 9 = 87% 94%, 10 = 94% 97%, 11 = 97% 100%, and 12 = 100% diseased tissue. Florida 8233 Florida 8233 was crossed to Fla. 7776 and subsequently the F 1 was self pollinated to produce F 2 seed. B oth parents and the F 2 generation were included in an inheritance study in fall

PAGE 52

52 2006 and repeated in spring 2007 and summer 2007. In fall 2006, seed were sown on 17 July and transplanted to the field in Balm, FL on 25 August. Race T4 inoculum (at a concentration of approximatel y 2 to 5 x10 8 cfu mL 1 ) was applied early in the morning of 20 September, and plants were rated for disease on 17 October. For the spring 2007 inheritance study seed were sown on 1 February and transplanted to the field in Balm, FL on 13 March. Race T4 in oculum ( same concentration as above ) was applied on 25 April, and individual plants were rated for disease severity on 2 May. For the summer 2007 inheritance study, seed were sown on 8 June, and race T4 inoculum (at a concentration of approximately 2 to 5 x10 6 cfu mL 1 ) was applied to plants on 20 July, prior to transplanting to the field in Citra, FL on 26 July. Individual F 2 plants were selected for resistance or susceptibility within each experiment. Progeny of F 2 selections made in fall 2006 were evalua ted in experiments in spring 2007 and summer 2007 to confirm resistance or susceptibility ; progeny of spring 2007 selections were evaluated in a confirming experiment in summer 2007; and progeny of F 2 selections made in summer 2007 were evaluated in spring 2008. For the spring 2007 confirmation experiment in Balm, FL plants were rated for disease severity on 1 May, and all other procedures were the same as described for the spring 2007 inheritance study, above. Likewise for the summer 2007 confirmation exp eriment in Citra, FL disease severity was rated on 26 September, and all other procedures were the same as for the summer 2007 inheritance study. For the spring 2008 experiment in Balm, FL l ow disease levels r esulted in ambiguous data that will not be pr esented Florida 8517 Florida 8 517 was crossed to Fla. 7776 and subsequently the F 1 was self pollinated to produce F 2 seed. B oth parents and the F 2 generation were included in an inheritance study in fall 2006 at Balm, FL and repeated in summer 2007 at Ci tra, FL In fall 2006 plants were rated for

PAGE 53

53 disease on 18 October and in summer 2007 plants were rated on 3 October. All other procedures were the same as described for the Fla. 8233 inheritance stud ies. Progeny of F 2 selections made in fall 2006 were ev aluated in experiments in spring 2007 and summer 2007 to confirm resistance or susceptibility ; and progeny of F 2 selections made in summer 2007 were evaluated in a confirming experiment in spring 2008. For the spring 2007 experiment all procedures were th e same as for the spring 2007 Fla. 8233 confirmation experiment above. Likewise for the summer 2007 confirmation experiment in Citra, FL, disease severity was rated on 26 September, and all other procedures were the same as for the Fla. 8233 summer 2007 i nheritance study. In the spring 2008 experiment in Balm, FL, l ow disease levels resulted in ambiguous data that will not be presented Florida 8326 Florida 8326 was crossed to Fla. 7946 and subsequently the F 1 was self pollinated to produce F 2 seed. B oth parents and the F 2 generation were included in an inheritance study in spring 2006 a nd repeated in summer 2007. For the spring 2006 experiment, seed were sown on 17 February and transplanted to the field in Balm, FL on 29 March. Race T3 inoculum (at a conc entration of approximately 2 to 5 x 10 8 cfu mL 1 ) was applied to the plants early in the mornings on 5 May and again on 24 May because conditions were not favorable for disease during the first inoculation However, subsequent race identification of plant lesions, and disease severity of control lines, indicated that race T4 caused the resultant field infection. Each plant was rated for race T4 disease severity the week of 26 June. For the summer 2007 inheritance study, each plant was rated for disease on 3 October; all other procedures were the same as described for the summer 2007 Fla. 8233 inheritance study.

PAGE 54

54 Progeny of F 2 selections made in spring 2006 were evaluated in confirming experiments in spring 2007 and summer 2007, and progeny of F 2 selections ma de in summer 2007 were evaluated in spring 2008. For the spring 2007 experiment, all procedures were the same as for the spring 2007 Fla. 8233 confirmation experiment, above. Likewise for the summer 2007 confirmation experiment in Citra, FL, disease sever ity was rated on 26 September, and all other procedures were the same as for the Fla. 8233 summer 2007 inheritance study. In spring 2008, seed were sown on 5 February and transplanted to the field in Balm, FL on 13 March. Fields were naturally infected wit h race T4, and each plant was rated for disease on 2 May. Marker Analysis Within each family, markers polymorphic between the two parents were analyzed across selected progeny based on a modification of the Transmission Disequillibrium (TD) Test (George, et al., 1999; Zhu and Elston, 2001). For this approach, selections were grouped as resistant or susceptible, and marker data w ere scored on the basis of the probability of a resistant allele (++, +/ -) for co dominant markers, or on the basis of the pre sence of a resistant allele (+, ) for dominant markers. A regression analysis was used for all markers using the Reg procedure of the Statistical Analysis System (SAS Institute, Cary, N.C.) Results Two hundred five molecular markers without polymorphism information were initially screened by restriction digestions to identify polymorphisms among resistant and susceptible genotypes. Compared with Fla. 7776; 92 of these markers were polymorphic with PI 128216, 80 were polymorphic with PI 126932, and 19 wer e polymorphic with PI 114490. Because this latter PI was considered to be an important source of resistance in two of the resistant breeding lines, 227 markers (some of them previously screened by restriction digestions) were screened for

PAGE 55

55 polymorphisms bet ween PI 114490 and Fla. 7776 by the modified EcoTILLING approach (Table B 1 ). Thirty polymorphic markers were identified by this approach, 24 of which were included in the screening of resistant and susceptible genotypes. One hundred forty two additional p olymorphic markers were included on the basis of accompanying polymorphism information for a total of 269 markers polymorphic among resistant and susceptible genotypes (Table 3 1 ). Florida 8233 Eleven resistant and five susceptible selections were made fr om the Fla. 8233 x Fla. 7776 F 2 generation over t hree seasons (Table 3 2 ). In fall 2006, E507 225 was selected as a resistant F 2 plant. Segregation for resistance was observed among its F 3 progeny in spring 2007, and two divergent selections were made, res ulting in resistant selection 4 and susceptible selection 2. summer 2007 was the best season for distinguishing resistant and susceptible selections. Resistant selections 7 through 11 and susceptible selections 3 through 5 correspond to F 2 plants select ed in summer 2007 Progeny of these later selections were included in a confirmation experiment in spring 2008, but differences between resistant and susceptible selections w ere not clear due to low disease pressure. Thus, i nclusion of these selections in mar ker analysis is based solely on their F 2 ratings in summer 2007. Twenty one polymorphic markers were identified between Fla. 8233 and Fla. 7776 (Table 3 3 ), representing as many as 10 introgression regions. Three introgressions on chromosomes 4 (CT20145 t o CT10184 ), 5 (CT20210I to Rx3 L1) and 9 (SSR383) appear to have originated from PI 128216, while no introgression in Fla.8233 necessarily originated from PI 114490. H7998 appears to be the source of an introgression on chromosome 11 (C2_At3g54470 to CT201 81). The source of an introgression on chromosome 7 (CT20052 to

PAGE 56

56 C2_At5g20180) could not be determined by its three representative markers. Five individual markers indentified possible introgression regions on chromosomes 1, 3, 7 and 10. Analysis of marker s for TD did not identify any marker as significant at the P = 0.05 level. One marker, TG403, was marginally non significant ( P = 0.059). The markers LEOH316 and Rx3 L1 on chromosome 5, although not significant, together indicate the possibility that they could be flanking a resistance locus because of the pattern of recombination between them. Chromosome 11 markers TG286 3 and CT20181 also show a pattern of resistance associated with the H7998 allele at this locus, with this allele present in several of th e resistant selections and absent in all of the susceptible selections. Likewise, the chromosome 9 marker, SSR383, seems to show a pattern of resistance being associated with the Fla. 8233 allele, as several of the resistant selections we re homozygous for this allele, but s usceptible selections we re all heterozygous or homozygous for the Fla. 7776 allele. Fl orida 8517 Nine resistant and 8 susceptible selections were made from the Fla. 8517 x Fla. 7776 F 2 generation over two seasons (Table 3 4 ). In fall 20 06, E514 246 was selected as a resistant F 2 plant. Segregation for resistance was observed among its F 3 progeny in spring 2007, and two divergent selections were made, resulting in resistant selection 1 and susceptible selection 3. Resistant selections 5 t hrough 9 and susceptible selections 5 through 8 correspond to F 2 plants selected in summer 2007 Progeny of these selections were included in a confirmation experiment in spring 2008, but low disease pressure in the latter season ma de it difficult to disc ern differences in resistant and susceptible selections Thus, inclusion of these later selections is based solely on their F 2 ratings in summer 2007.

PAGE 57

57 Forty eight markers were polymorphic between Fla. 8517 and Fla. 7776, representing as many as 15 introgr ession regions in Fla. 8517 (Table 3 5 ). Introgressions on each of chromosomes 2, 3, 4 and 11 were initially found to have originated from either PI 114490 or PI 128216. Upon analysis of Fla. 8349 and Fla. 8350 the parents of Fla. 8517 (Figure A 1 ) the int rogressions on chromosomes 2 and 3 were determined to have descended from PI 114490 via Fla. 8350, the chromosome 4 introgression descended through Fla. 8350 and appears to have come from Fla. 7600, and one of the introgressions on chromosome 11 was determ ined to have originated from PI 128216 and descended through Fla. 8349 (data not shown). A second chromosome 11 introgression originated from either H7998 or PI 114490. The chromosome 5 introgression, originally thought to have come from PI 128216, appears to have resulted from a recombination event between markers Rx3 L1 and CosOH73. The upper portion of this introgression apparently originated from the processing line OH9242 in the pedigree of Fla. 8350 while the lower portion came from PI 128216 in the pedigree of Fla. 8349 (data not shown). The introgression on chromosome 12 also appears to have resulted from a recombination event within two separate introgressed regions in each of Fla. 8349 and Fla. 8350. The upper portion of this Fla. 8517 introgressi on (CT100 to C2_At5g42740) descended through Fla. 8349, likely from PI 128216, while the lower portion (SSR20) corresponds to the Fla. 7600/OH9242 allele from Fla. 8350 (data not shown). Six individual markers indentified possible introgressions on chromos omes 1, 7, 8, 9 and 10. Three introgression regions were significant for T D in the Fla. 8517 family at the P = 0.05 level (Table 3 5 ). The chromosome 3 introgression from PI 114490 was a highly significant resistance locus for all four markers representin g that region. The PI 128216 introgression on chromosome 11 was also a significant resistance locus for all three corresponding markers.

PAGE 58

58 SSR20 on chromosome 12 was also highly significant, indicating that the Fla. 7600/OH9242 allele at this locus is associ ated with susceptibility. Although not significant, SSR383 on chromosome 9 showed a pattern of resistance associated with the Fla. 8517, as several of the resistant selections are homozygous for this allele, but susceptible selections are all heterozygous or homozygous for the Fla.7776 allele. Likewise, the pattern of recombination between markers CT10050 and CT10649, representing the chromosome 2 PI 114490 introgression in Fla. 8517, indicate the possibility that they could be flanking a resistance locus. Fl orida 8326 Seven resistant and 8 susceptible selections were made from the Fla. 8326 x Fla. 7946 F 2 generation over two seasons (Table 3 6 ). In spring 2006, E707 166 was selected as a resistant F 2 plant. Segregation for resistance was observed among its F 3 progeny in spring 2007, and two divergent selections were made, resulting in resistant selection 7 and susceptible selection 1. Susceptible selection 4 segregated for resistance in summer 2007 and spring 2008. Despite the low disease pressure in spring 2008, a clear disease screen was achieved confirming selecti ons made the previous season; this was attributed to the high susceptibility of Fla. 7946, which resulted in sufficient contrast between resistant and susceptible selections. Nineteen markers w ere polymorphic between Fla. 8326 and Fla. 7946, representing up to 9 regions of introgression in Fla. 8326 (Table 3 8). Three markers on chromosome 3 as well as two individual markers on chromosome one and TG403 on chromosome 10 indicated introgressions t hat likely descended from PI 126932. Eight markers on chromosome 11 represent an introgression from H7998. Five additional markers indicated possible introgressions on chromosomes 2, 3, 7 and 9.

PAGE 59

59 All chromosome 11 markers were significant for TD at the P = 0.05 level in the Fla. 8326 family, while markers TOM196, SSR637, TOM144 and LEOH57 were highly significant ( P < 0.0001). No other regions of introgression were significant, although marker Cf9 on chromosome 1 was marginally non signficant ( P = 0.063). Di scussion The rapid rate at which resistance to bacterial spot of tomato has been overcome is quite alarming, especially when considering that this has occurred without the deployment of resistant cultivars For resistance to be successful, it must be effe ctive against all present races of the pathogen and durable against the emergence of new races. Our approach has been to utilize molecular markers to identify resistance genes from a number of different sources conferring both race specific and broad spect rum resistance, and then to pyramid these genes to possibly attain a higher level of resistance PI 114490, PI 128216, PI 126932 and H7998 each have resistance or partial resistance to multiple races of bacterial spot. Florida breeding lines 8233, 8517 and 8326 also have resistance to multiple races of bacterial spot ( Scott et al., 2006 ), presumably derived from one or more of these sources. Despite the pervasiveness of bacterial spot in Florida, periods of lower disease pressure often occur especially in the spring production season when rainfall is less frequent. When disease pressure and secondary spread are low, the ability to distinguish resistant and susceptible genotypes depends on the level of contrast between the two. This was evidenced in the spr ing 2008 confirmation experiments: Florida 8326 has only moderate resistance to race T4, but progeny of F 2 selections were clearly distinguishable because of the high susceptibility of Fla. 7946; alternatively, because Fla. 7776 is not as susceptible as Fl a. 7946 it was not possible to

PAGE 60

60 clearly distinguish selections from the Fla. 8233 or Fla. 8517 families, even though each of these lines are more resistant than Fla. 8326. Florida 8233 has PI 114490 recorded in its pedigree, but none of its introgressions identified by markers from this research were clearly descended from this PI. However, three (on chromosomes 4, 5 and 9) appear to have descended from PI 128216. It was already known that an error existed in the pedigree of Fla. 8233, because this line ha s T3 hypersensitivity, while none of the parents in its recorded pedigree are T3 hypersensitive (J. Scott, personal communication). These results suggest that the mistake most likely occurred when crossing Fla. 7655 with PI 114490 (see Figure A 2 ), and pol len from PI 128216 was used instead. By this explanation, PI 128216 would also be the source of the T3 hypersensitivity. The lack of significance for markers in the Fla. 8233 family is likely due to the low number of individuals represented in the suscept ible pool. Still, four regions of introgression showed a pattern of resistance associated with the Fla. 8233 allele. A PI 128216 introgression on chromosome 9 is present in both Fla. 8233 and Fla. 8517. While analysis of this marker for either family was n on significant, a combined analysis on selections from both families was significant ( P = 0.0217). Although this PI 128216 allele was distributed among both resistant and susceptible selections, selections homozygous for this allele were only present withi n the resistant groups, suggesting that this locus may confer the additive resistance identified in each of these breeding lines by the generation means analysis (see Ch. 2). Similarly marker TG403 on chromosome 10 failed to show significance in the Fla. 8233 or Fla. 8517 families. In this case, resistance appears to be associated with th e PI 128216 allele in Fla. 8233 ( P = 0.059), where only one susceptible s election is heterozygous and all others are homozygous for the Fla. 7776 allele, but resistance do es not appear to be associated with the Fla. 8517 allele ( P = 0.488). A

PAGE 61

61 combined analysis on selections from both families was not significant ( P = 0.7841). However, it could not be determined whether the Fla. 8517 allele descended from PI 128216 or from PI 114490 Thus, the possibility of a PI 128216 resistance QTL on chromosome 10 is not precluded Because the map positions for the markers used in this study were not all determined from a common mapping population, markers representing each introgressio n were arranged in the most logical order, considering the recombination patterns among selected progeny. Provided the orders are correct, the chromosome 5 region between markers LEOH316 and Rx3 L1 displayed a surprisingly high amount of recombination, wit h 20 of the 33 selections from Fla. 8233 and Fla. 8517 showing cross overs in this region. In the Fla. 8233 family, 8 of the resistant selections are homozygous for the resistant allele for marker LEOH316, and no resistant alleles are present in susceptibl e progeny for marker Rx3 L1, suggesting that a resistance gene could be located between the two markers. Moreover, resistant selection 4 has at least one copy of the resistant allele at this locus, but susceptible selection 2 (selected divergently from the same F 3 plant as resistant selection 4) only has the susceptible allele. Such a pattern of association between observed resistance and the presence of the resistant allele was not observed in the Fla. 8517 family. However, the PI 128216 introgression in F la. 8517 does not span the region between LEOH 316 and Rx3 L1, and thus does not include this plausible resistance locus. Having a resistance gene from PI 128216 at this locus seems possible, given that resistance genes are often organized in clusters, and this region contains a gene important for T1 hypersensitivity in H7998 (Yang, et al., 2005; Yu et al., 1995). A gene cluster at this locus could also explain the high rate of recombination within selected progeny, as varying levels of recombination are ob served between component genes of a cluster (Michelmore and Meyers, 1998). Alternatively, a T4 resistance gene at this locus could simply be an alternative allele from that of H7998.

PAGE 62

62 Both Fla. 8233 and Fla. 8326 have a chromosome 11 introgression from H799 8. While the size of the introgression in Fla. 8326 appears to span a larger region, this is not necessarily the case. Instead, t his could be due to the lack of polymorphic markers in the region that distinguish H7998 alleles from Fla. 7776 alleles. This i s rather unfortunate, since the significance levels of chromosome 11 markers in the Fla. 8326 family indicate the QTL may be near the upper portion of this introgression, precisely where markers are lacking in the Fla. 8233 family. An obvious question with regard to this introgression is why such a clear effect is observed in the Fla. 8326 family, where all resistant selections possess the H7998 allele, while not all resistant selections in the Fla. 8233 family carry it. One possible explanation is that the upper part of the H7998 introgression containing the QTL is not present in Fla. 8233, and resistance in this line is not associated with this locus. The present research cannot rule out this possibility, especially considering the lack of significance for TD at this locus. On the other hand, those resistant selections in the Fla. 8233 family that appear to lack the introgression could be recombinants that only lost the lower portion of the introgression, while retaining the portion of the introgression for which there are no polymorphic markers. Another, very likely explanation is that resistance in Fla. 8233 may be quantitative while Fla. 8326 may have only one major resistance gene. Thus, Fla. 8233 selections lacking a gene on chromosome 11 but possessin g one or more genes from other loci, still show resistance relative to the moderately susceptible Fla. 7776; whereas Fla. 8326 selections must all carry the chromosome 11 QTL to express resistance relative to the highly susceptible Fla. 7946 This seems po ssible, as p lausible QTL on chromosomes 5, 9 and 10 explain the resistance observed in Fla. 8233 resistant selections 1, 2, 4, 5, 6 and 9, which all lack the chromosome 11 introgression. Together with the chromosome 11 locus, these four QTL account for the resistance or susceptibility observed in 15 of the 16 Fla. 8233 selections. The lack

PAGE 63

63 of resistance in susceptible selection 5, which has the resistant allele for marker TG403, could be explained by a possible recombination between the QTL and the marker, but more markers in this region would be needed to confirm this. A problem with attributing T4 resistance in Fla. 8326 to a single H7998 gene is that Fla. 8326 exhibits a higher level of resistance to T4 than does H7998 (Scott et al., 2006). This is sugge stive of epistasis where some non H7998 gene in Fla. 8326 is effecting this higher level of resista nce from the chromosome 11 QTL. Indeed, only one factor was indicated in Fla. 8326 by the fall 2005 inheritance study (see Ch. 2); under the low disease pre ssure that season the H7998 gene evidently provided sufficient resistance t o explain the variation that was observed. However, under the higher disease pressure in spring 2006 and summer 2007, epistatic effects were significant, indicating that a second g ene was required for full resistance. Th e epistatic gene of Fla. 8326 would theoretically be present in resistant selections and absent in the susceptible selections that carried the chromosome 11 introgression. Marker Cf9 maps to a region on the short arm of chromosome 1 that was identified as a contributor to T1 hypersensitivity from H7998 (Yu et al., 1995). This marker indicates a PI 126932 introgression in Fla. 8326 that was marginally non significant for T D analysis, but is a likely candidate for such a QTL. Resistant selection 4 is the only resistant individual that lacks this allele, but it also had one of the highest disease severity ratings of all resistant selections in summer 2007 (selection 7 was rated higher, but was also heterozygous for the ch romosome 11 introgression). Of the susceptible selections that have the Cf9 resistant allele, only selection 4 carried the chromosome 11 introgression, and this selection also segregated for resistance Moreover, susceptible selection 1 and resistant selec tion 7 were divergent selection s from the same F 3 family but only the latter carries the chromosome 1 resistant allele. Thus this chromosome 1

PAGE 64

64 locus seems to account for the recessive suppression type epistatic effect identified by the genera tion means a nalysis in Fla. 8326 (see Ch. 2). The same effect was likewise identified in Fla. 8233 and Fla. 8517 by the ir inheritance studies (see Ch. 2) and the chromosome 1 resistant allele is also present in m ost of the resistant selections from each of these fami lies ; however, the lack of susceptible selections missing this allele while hav ing the chromosome 11 QTL limits the confidence with which the recessive suppression interaction can be inferred regarding Fla. 8233 and Fla. 8517 The development of one or mor e codominant markers in this region, together with the screening of larger resistant and susceptible pools, would help to confirm or rule out this possibility. Hawaii 7997 is a source of resistance to bacterial wilt ( Ralstonia solanacearum ) in the Universi ty of Florida tomato breeding program and it is susceptible t o race T4 bacterial spot (J.W. Scott, personal comm.) T his resistance has been incorporated into several breeding lines that are also susceptible to bacterial spot, yet resistance to bacterial spot race T4 has been observed in a number of advanced breeding lines developed from this material (Scott, unpublished) This is suggestive of an interaction either between a bacterial spot resistance gene in H7997 and an effecting gene in some of the susc eptible breeding lines, or between a bacterial spot resistance gene in some of the susceptible breeding lines and an effecting gene in H7997. Both possibilities lend support to the present theory that epistasis is contributing to bacterial spot resistance and that the native background of the resistance gene does not always contain the secondary gene. The chromosome 12 introgression in Fla. 8517 apparently came about by a recombination event between a PI 128216 introgression in Fla. 8349 and a n OH9242 intr ogression in Fla. 8350, as both introgressions were present in early selections of this breeding

PAGE 65

65 line. The latter introgression, represented by SSR20, was a significant susceptibility QTL in the Fla. 8517 family, emphasizing that it is often an invalid ass umption that resistant alleles are only present in the resistant parent. The association of this locus with susceptibility from OH9242 is supported by the fact that this allele was segregating in the early Fla. 8517 selection used in this study, but is abs ent in later selections of the same line (data not shown) QTL for resistance in Fla. 8517 are located on chromosomes 3, 9 and 11. The QTL on chromosomes 3 and 11 appear to exhibit dominant gene action and may be the primary contributors to the dominant ef fect identified by the generation means analysis (see Ch. 2). PI 114490 is the donor of the resistant allele on chromosome 3 ; th is region has also been associated with the Xv4 based resistance to race T4 from S. penn e llii LA 716 (Astua Monge et al., 2000a ) When Fla. 8517 was selected from the Fla. 8349 x Fla. 8350 F 2 plot, two additional selections were also made. Evaluation of the F 3 families from each of these selections indicated that the other two lines were less resistant than Fla. 8517. Interestingl y, neither of these two additional selections carried the chromosome 3 introgression from PI 114490 or the chromosome 9 introgression from PI 128216. None of the other resistance loci demonstrated this pattern among the three selections (data not shown). P I 128216 is also the donor of the resistant allele on chromosome 11. If the PI 128216 chromosome 11 QTL is an allele at the same locus as in H7998, this research would suggest an allelic series where H7998 and PI 128216 > Fla. 7776 > Fla. 7946. The plausib le Fla. 8517 resistance QTL on chromosomes 3, 9 and 11, and the susceptibility QTL on chromosome 12 account for the resistance or susceptibility observed in 16 of the 17 selections Only r esistant selection 1 also lacks all of the resistance QTL markers, but it also does not have the OH9242 suscept ibility allele on chromosome 12

PAGE 66

66 The identification of multiple QTL conferring bacterial spot race T4 resistance from four separate sources raises hopes that an acceptable level of durable resistance can be achi eved. The potential for successfully pyramiding these QTL to result in higher and more durable levels of resistance depends in part on whether the resistance genes have different mechanisms of resistance. Further research is necessary to determine whether these QTL contain unique resistance genes, or if they are essentially mimic genes. Efforts are underway both to pyramid these resistance QTL for evaluation of their combined effect, and to develop additional molecular markers for finer mapping and individu al confirmation of each plausible resistance locus. Upon confirmation of all QTL, each locus should be tested to determine the level of resistance provided agai nst each race of bacterial spot as well as its mechanism of resistance. To aid in the evaluatio n of individual QTL crosses have been made between Fla. 7946 and both Fla. 8233 and Fla. 8517 for QTL evaluation in a more susceptible background. Plausible resistance QTL are also being incorporated into a number of susceptible breeding lines by MAS usin g the markers identified by this research.

PAGE 67

67

PAGE 68

68

PAGE 69

69

PAGE 70

70

PAGE 71

71

PAGE 72

72

PAGE 73

73

PAGE 74

74

PAGE 75

75

PAGE 76

76

PAGE 77

77

PAGE 78

78

PAGE 79

79

PAGE 80

80

PAGE 81

81

PAGE 82

82

PAGE 83

83

PAGE 84

84

PAGE 85

85

PAGE 86

86

PAGE 87

87 CHAPTER 4 GENETIC CONTROL OF RACE T3 HYPERSENSITIVITY FROM PI 126932 AND T HE RELATIONSHIP BETWEEN T3 HYPERSENSITIVITY AND RACE T4 RESISTANCE Introduction Bacterial spot of tomato ( Solanum lycopersicum L. ) is one of the most serious diseases that f aces Florida tomato production. Four r aces of the bacterial spot pathogen in tomato have been isolated under field conditions (Jones et al., 2005) and one strain of a fifth race has been identified (Minsavage et al., 2003; Jones, unpublished) In Florida, Xanthomonas euvesicatoria race T1 was prevalent until 1991 when X perforans race T3 emerged (Jones et al., 1995). Race T3 was antagonistic to race T1 (Jones et al., 1998) and largely replaced the latter race. X perforans race T4 recently emerged and has been isolated in several locations in Florida since 199 8 (Minsavage et al., 2003) Clearly, resistance to races T3 and T4 is needed in Florida. One of the most r esistant sources of race T3 resistance is Hawaii 7981 ( H7981 ) (Scott et al., 1995) This resistance is based largely on hypersensitivity (HR) conferred by the incompletely dominant gene Xv3 but field resistance is also based on other genes (Scott et al., 2001). Race T3 HR and field resistance ha ve also been identified in PI 128216 and PI 126932 (Scott et al., 1995). Advanced breeding line Fla. 8326 is a large fruited fresh market tomato with resistance to bacterial spot race T3 (Scott, unpublished) and t olerance to other races (Scott, et al., 2006). Resistance sources in its pedigree include PI 126932 and Hawaii 7998 ( H7998 ) T3 resistance is provided, at least in part, by HR from PI 126932, and partial resistance to race T4 is derived from H7998 a nd poss ibly from PI 126932 (see Ch. 3). Race T4 overcame the Xv3 based hypersensitive resistance in H7981, as well as the HR in PI 128216 and PI 126932 (Jones, unpublished) It was not surprising that race T4 overcame this resistance in all three sources since t he HR in each of these three lines is based upon recognition of a common avirulence gene,

PAGE 88

88 avrXv3 (Astua Monge, et al., 2000b), and race 4 came about by a mutation s in the avrXv 3 gene (Minsavage et al., 2003 ). It is not known, however, whether the HR in PI 126932 and PI 128216 is conferred by Xv3 or by a different gene operating by the s ame mechanism, nor how this trait is inherited in the two PI s. Advanced breeding line Fla. 8517 is a plum tomato with resistance to race T4 (Scott et al., 2006) from PI 1144 90 and PI 128216 (see Ch. 3) and HR to race T3 from PI 128216. Florida 8233 is a large fruited fresh market tomato with resistance to races T3 (Scott, unpublished) and T4 (Scott et al., 2006) and it is race T3 hypersensitive Race T4 resistance is derived from H7998 and PI 128216, and race T3 HR comes from PI 128216 (see Ch. 3) While multiple genes appear to confer T4 resistance in Fla. 8517 and Fla. 8233 it is not known whether any of these genes are involved in T3 HR from PI 128216. The objectives of t his research were to: 1) determine the genetic control of HR from PI 126932, 2) determine the allelism of the T3 HR gene s in H7981, PI 126932 and PI 128216 and 3 ) test for an association between the T3 hypersensitivity in Fla. 8326, Fla. 8233 and Fla. 85 17 and T4 field resistance in each of these lines. Materials and Methods Inoculum Preparation, Plant Inoculations and Disease Evaluations X perforans races T3 and T4 inoculum were each produced by growing the bacteria on Difco nutrient agar (Becton Dickin son and Company, Sparks, Md.) for 24 cells were removed from the agar plates and suspended in either sterile tap water (for fall 2005 experiments) or in 10 mM MgSO 4 7 H 2 O (for fall 2006 experiments) and the suspensions were standard ized to A 600 =0. 30 (a concentration of approximately 2 to 5 x 10 8 colony forming units (cfu)/mL). For race T3, leaves were infiltrated with the bacterial suspension as described by

PAGE 89

89 Hibberd et al. (1987). Infiltrated plants were subsequently moved to a growt h room that was kept at a constant temperature of 24C with a 16 h light period. Assessments for HR were carried out at 12 to 24 hour intervals for 24 to 72 hours after infiltration Infiltrated areas exhibiting confluent necrosis within 36 hours were scor ed as hypersensitive. For race T4 field inoculations, the bacterial suspension was applied by misting the foliage with a backpack sprayer early in the morning before sunrise. Field p lants were rated for disease severity in the field using the Horsfall and Barratt scale (1945), where 1 = 0%, 2 = 0% 3%, 3 = 3% 6%, 4 = 6% 12%, 5 = 12% 25%, 6 = 25% 50%, 7 = 50% 75%, 8 = 75% 87%, 9 = 87% 94%, 10 = 94% 97%, 11 = 97% 100%, and 12 = 100% diseased tissue. Plant Materials Florida 8326 (T3 hypersensitivity from PI 126932) was crossed to Fla. 8021 and Fla. 7946 both susceptible breeding line s to race s T3 and T4 of X perforans ; to Fla. 8000, a breeding line with T3 HR from H7981; and to Fla. 8233 (T3 HR from PI 128216) The F 1 s were subsequently self pollinated to p roduce F 2 seed. In f all 2005, F 1 and F 2 generations of crosses involving Fla. 8021 and Fla. 8000, parents of those crosses, and H7981 were grown in the greenhouse at temper atures ranging from 25 to 35C After 4 to 5 weeks, the main stem was removed above the fully expanded third true leaf. Approximately 3 days after top p ing, plants were inoculated with race T3 for testing for an HR Also in f all 2005, parents, F 1 and F 2 generation s of the cross es involving Fla. 7946 and Fla. 8233 were included as part of a field experiment in Citra, FL. Seed were sown on 29 July, and plants were transplanted to the field on 9 September. P lants were inoculated with race T4 inoculum on 16 September and rated for disease severity on 19 October. Cuttings we re taken from field plants on 22 November and labeled according to family and plant number rooted

PAGE 90

90 under misters, and grown in the greenhouse at temper atures ranging from 25 to 35C. After 4 to 5 weeks plants were topped and tested for T3 HR as described above. Thus, each pl ant was rated in the field for race T4 disease severity and in the greenhouse for race T3 HR Florida 8233 and Fla. 8517 were each crossed to Fla. 7776, a susceptible breeding line to race s T3 and T4 of X. perforans and each F 1 w as subsequently self polli nated to produce F 2 seed. Parents, F 1 and F 2 generations of these crosses were included as part of field experiments in Citra and Balm, FL in f all 2006. Seed were sown on 17 July and trans planted to the field in Balm, FL on 25 August, and to the field in Citra, FL on 30 August. Race T4 inoculum was applied on 19 and 20 September to the plants in Citra and Balm respectively. Plants at Citra were rated for d isease severity on 11, 12 October, and plants at Balm were rated on 17, 18 October. Cuttings were tak en from plants at both locations the week of 1 October and labeled according to family and plant number rooted under misters and grown in the greenhouse at temper atures ranging from 25 to 35C. After 4 to 5 weeks, plants were topped and tested for T3 HR as described above. Thus, each plant was rated in the field for race T4 disease severity and in the greenhouse for race T3 HR For all field experiments, seed were sown in growth rooms in Black Beauty spent coal (Reed Minerals Div., Highland, IN) and tran splanted approximately 7 to 10 days later to Speedling trays (3.8 cm 3 cell size) (Speedling, Sun City, FL) in the greenhouse, where seedlings were grown for four weeks. Plants were then transplanted to field beds that were 20 cm high and 81 cm wide that h ad been fumigated with 67% methyl bromide : 33% chloropicrin at 197 kg ha 1 (175 lbs per acre) and covered with reflective plastic mulch. Plants were spaced 46 cm apart in rows, with 152 cm between rows, staked and tied, and irrigated by drip tape beneath the plastic mulch of each bed. A recommended fertilizer program was followed, and plants were

PAGE 91

91 sprayed with pesticides (excluding copper) as needed throughout the season ( Olsen et al., 2007 2008 ). Results Inheritance of the gene for HR to X perforans race T3 was studied in the cross between Fla. 8326 (resistant parent) and Fla. 8021 (susceptible parent). Their F 1 displayed a resistant phenotype (Table 4 1), and the corresponding F 2 segregat ion fit a 3:1 ratio into resistant and susceptible individuals, ind icating HR was conferred by a single, dominant gene. In the cross between Fla. 8326 and Fla. 8000, the F 1 displayed the expected HR phenotype, and the F 2 segregation of resistant and susceptible plant s fit a 15:1 ratio (Table 4 1) indicating HR was confer red by two dominant genes. I n the cross between Fla. 8233 and Fla. 8326, the F 2 did not segregate any susceptible individuals (Table 4 2) indicating they both have the same gene conferring HR. Thus, results suggest that PI 126932 and PI 128216 share a com mon T3 HR gene, but that this gene is at a different locus than the T3 HR gene in H7981. In f all 2005, race T4 disease severity was rated in the field, and cuttings from parents and 41 (Fla. 7946 x Fla. 8326) F 2 plants and were tested for race T3 HR (Tabl e 4 2 ). Disease severity ratings for race T4 in f all 2006 were very difficult to assess due to extremely poor field conditions at Citra, FL and low disease pressure at Citra and Balm, FL. In spite of this, race T4 field disease severity was rated and race T3 HR was measured on cuttings of parents 312 (Fla. 7776 x Fla. 8233) F 2 plants and 289 (Fla. 7776 x Fla. 8517) F 2 plants (Table 4 2 ) Within each of the three crosses, Chi square contingency tests were used to evaluate the segregation of F 2 plants for r ace T4 field resistance and race T3 hypersensitivity to test for a relationship between the two traits In each F 2 family, the two traits segregated idependently, indicating that there is n o relationship between race T3 HR and race T4 field resistance (Tab le 4 3 )

PAGE 92

92 Discussion In breeding for X perforans race T3 resistance, several sources of resistance are available, some of which are race T3 HR and i nclude H7981, PI 126932 and PI 128216 (Jones et al., 1995) The highest level of resistance in the field wa s identified in H7981 (Scott et al., 1995) and b reeding efforts for race T3 resistance t hus focused primarily on H7981 while inheritance of resistance from PI 126932 and PI 128216 was never studied. Interest in the two PIs as resistance donors has increas ed since the emergence of race T4 which overcame the HR of all three resistance sources but not the field resistance of PI 128216 (Scott et al., 2006) ; additionally, race T4 resistant breeding lines with resistance presumably derived from these PIs have b een developed, providing supportive evidence for the presence of non HR resistance genes in these backgrounds. Herein we present evidence that genetic control of T3 HR in PI 128216 and PI 126932 is provided by a different locus than in H7981. I t is general ly believed that hypersensitive responses are controlled by single dominant genes, but this is not always the case. X. euvesicatori a race T1 HR in H7998 appears to be controlled by three factors ( Wang et al., 1994; Yu et al., 1995), and T3 HR in H7981 is c ontrolled by a single incompletely dominant gene (Scott et al., 1996). Understanding the genetic control of HR from PI 126932 is important in determining whether this resistance would be needed in one or both parents to obtain a full level of hypersensitiv e resistance in a hybrid. Because the T3 H R gene in PI 126932 appears to be controlled by a single dominant gene, use of this gene is probably more suitable for hybrid development than Xv3 from H7981. However, control plants heterozygous for Xv3 from H7981 were not included in the study; it is therefore not clear whether the T3 HR gene in PI 126932 is truly dominant, or if the screen was not precise enough to distinguish a rapid HR from an intermediate response.

PAGE 93

93 One approach to obtain resistance to multipl e races of bacterial spot involves the pyramiding of major resistance genes to each race. Hawaii 7981, PI 1269 32 and PI 128216 all produce an HR in response to race T3, and all have race T3 field resistance (Scott et al., 1995; Scott et al., 2001). R ace T4 overcame the HR of each line ( Astua Monge et al., 2000b), as well as the field resistance of H7981 (Scott, unpublished) and PI 126932 (Scott et al., 2006) Thus, t he race T3 hypersensitive genes appear to be the primary contributor to race T3 field resist ance in H7981 and PI 126932 and this may be the case in PI 128216 as well In developing advanced breeding lines with race T3 field resistance, it may be desirable to make crosses among lines that have resistance derived from one or another of these sourc es. Because PI 126932 and PI 128216 appear to share a common race T3 HR locus crosses between lines with PI derived HR would be expected to maintain this trait in all progeny. However, because the PI race T3 HR locus is different from the H7981 Xv3 locus, selected progeny of crosses between lines with Hawaiian derived HR and lines with PI derived HR would need to be screene d to be certain that race T3 hypersensitivity was maintained The race T3 HR genes in H7981, PI 126932 and PI 128216 all act by recogni zing the same avirulence factor, avrXv3 (Astua Monge et al. 2000b). R ace T4 appears to be associated with mutagenesis of the avrXv3 gene in tomato race 3 strains ( Minsavage et al., 2003 ). Considering this, the lack of a relationship between race T3 HR and race T4 field resistance is not surprising. The hypothesis that such a relationship might have exist ed is based on the ideas that either: 1) the race T3 H R gene could contribute a level of non hypersensitive resistance which, in combination with other res istance genes, is effective against race T4, or 2) the race T3 H R gene is linked to another race T4 resistance QTL as disease resistance genes often cluster in

PAGE 94

94 the genome (Michelmore and Meyers, 1988) Neither appears to be the case, and race T4 resistanc e appears independent of race T3 HR

PAGE 95

95

PAGE 96

96

PAGE 97

97

PAGE 98

98 APPENDIX A PEDIGREES

PAGE 99

99 Figure A 1 Pedigree of Fla 8517 (Both Fla. 7655B and Fla. 7600 contain H7998 in their pedigrees.)

PAGE 100

100 Figure A 2. Putative pedigree of Fl a 8233 [ However, PI 114490 is now thought to be i ncorrectly recorded in th is pedigree. It appears that PI 128216 was actually crossed to Fla. 7655 (see Ch. 3) .] (Fla 7655 contains H7998 in its pedigree.)

PAGE 101

101 Figure A 3. Pedigree of Fla 8326 ( Fla. 7708 contains H7998 in its pedigree )

PAGE 102

102 Figure A 4. Pedigree of Fla 7776

PAGE 103

103 Figure A 5 Pedigree of Fla 7946

PAGE 104

104 APPENDIX B ADDITIONAL MOLECULAR MARKER INFORMATION

PAGE 105

105

PAGE 106

106

PAGE 107

107

PAGE 108

108

PAGE 109

109

PAGE 110

110

PAGE 111

111

PAGE 112

112

PAGE 113

113

PAGE 114

114

PAGE 115

115

PAGE 116

116

PAGE 117

117

PAGE 118

118

PAGE 119

119

PAGE 120

120

PAGE 121

121

PAGE 122

122

PAGE 123

123

PAGE 124

124

PAGE 125

125 APPENDIX C DNA SOURCES FOR SELECTIVE GENOTY PING OF RESISTANT AND SUSCEP TIBLE SELECTIONS

PAGE 126

126

PAGE 127

127 LIST OF REFERENCES Allard, R. W. 1960. Principles of plant b reeding. John Wiley & Sons. New York Astua Monge, G., G.V. Minsavage, R.E. Stall, C.E. Vallejos, M.J. Davis and J.B. Jones 2000 a Xv4 vrxv4 : A new gene for gene interaction identified between Xanthomonas campestris pv. vesicatoria race T3 and the wild tomato relative Lycopersicon pennellii Mol. Plant Micro be Interact. 13: 1346 1355. Astua Monge, G., G.V. Minsavage, R.E. Stall, M.J. Davis, U. Bonas and J.B. Jones. 2000b. Resistance of tomato and pepper to T3 strains of Xanthomonas campestris pv. Vesicatoria is specified by a plant inducible avirulence gene. Mol. Plant Microbe Interact. 13:911 921. Balogh, B., J.B. Jones, M.T. Momol, S.M. Olson, A. Obradovic, P. King and L.E. Jackson. 2003. Improved efficacy of newly formulated bacteriophages for management of bacterial spot on tomato. Plant Dis. 87:949 954. Bouzar, H., J.B. Jones, G.V. Minsavage, R.E. Stall, and J.W. Scott. 1994a. Proteins unique to phenotypically dist inct groups of Xanthomonas campestris pv. vesicatoria revealed by sil v er staining. Phytopathology 84:39 44. Bouzar, H., J.B. Jones, R.E. St all, N.C. Hodge, G.V. Minsavage, A.A. Benedict, and A.M. Alvarez. 1994b. Physiological, chemical, serological, and pathogenic analyses of a world wide collection of Xanthomonas campestris pv. vesicatoria strains. Phytopathology 84:663 671. Conover, R.A. and N.R. Gerhold. 1981. Mixtures of copper and maneb or mancozeb for control of bacterial spot of tomato and their compatibility for control of fungus diseases. Proc. Fla. State Hort. Soc. 94:154 156. El Morsy, G.A., G.C. Somodi, J.W. Scott, R.E. Stall, and J.B. Jones. 1994. Aggressiveness of Xanthomonas campestris pv. vesicatoria tomato race 3 (T3) strains over to mato race 1 (T1) strains: Evidence for antagonism. Phytopathology 84:1094. (Abstr.) FASS. 200 8 Vegetable acreage, production and value; 200 7 200 8 Florida agricultural statistics service, Orlando, FL. http://ww w.nass.usda.gov/fl/ (last accessed 8/1 /0 8 ). Flaherty, J.E., J.B. Jones, B.K. Harbaugh, G.C. Somodi and L.E. Jackson. 2000. Control of bacterial spot on tomato in the greenhouse and field with H mutant bacteriophages. HortScience 35:882 884. Gardner, M .W. and Kendrick, J.B. 1923. Bacterial spot of tomato and pepper. Phytopathology 13: 307 315.

PAGE 128

128 George, V., H.K. Tiwari, X. Zhu and R.C. Elston. 1999. A test of transmission/disequilibrium for quantitative traits in pedigree data, by multiple regression Am. J. Hum. Genet. 65:236 245. Getz, S., C.T. Stevens, and D.W. Fulbright. 1983. Influence of developmental stage on susceptibility of tomato fruit to Pseudomonas syringae pv. tomato Phytopathology 73:36 38. Horsfall, J.G. and R.W. Barratt. 1945. An improved grading system for measuring plant diseases. Phytopathology 35:655. Jones, J.B. and J.P. Jones. 1985. The effect of bactericides, tank mixing time and spray schedule on bacterial leaf spot of tomato. Proc. Fla. State Hortic. Soc. 98: 244 247. Jones, J.B. and J.W. Scott. 1986. Hypersensitive response in tomato to Xanthomonas campestris pv. vesicatoria Plant Dis. 70:337 339. Jones, J.B., G.H. Lacy, and H. Bouzar. 2006. Reclassification of the xanthomonads associated with bacterial spot disea se of tomato and pepper. Syst. Appl. Microbiol. 29:85 86. Jones, J.B., G.H. Lacy H. Bouzar, G.V. Minsavage, R.E. Stall and N.W. Schaad. 2005. Bacterial spot Worldwide distribution, importance and review. Acta Horticulturae 695:27 33. Jones, J.B., G.V. Minsavage, R.E. Stall, R.O. Kelly, and H. Bouzar. 1993. Genetic analysis of a DNA region involved in expression of two epitopes associated with lipopolysaccharide in Xanthomonas campestris pv. vesicatoria Phytopathology 83:551 556. Jones, J.B., H. Bouz ar, R.E. Stall, E.C. Almira, P.D. Roberts, B.W. Bowen, J. Sudberry, P.M. Strickler, and J. Chun. 2000. Systematic analysis of xanthomonads ( Xanthomonas spp.) associated with pepper and tomato lesions. Intl. J. Syst. Evol. Microbiol. 50:1211 1219. Jones, J.B., R.E. Stall and H. Bouzar 1998. Diversity among Xanthomonads pathogenic on pepper and tomato. Annu. Rev. Phytopathol. 36: 41 58. Jones, J.B., R.E. Stall, J.W. Scott, G.C. Somodi, H. Bouzar and N.C. Hodge. 1995. A third tomato race of Xanthomonas campestris pv. Vesicatoria. Plant Disease 79: 395 398. Jones, J.B., S.S Woltz, J.P. Jones and K.L. Portier, K.L. 1991a. Population dynamics of Xanthomonas campestris pv. vesicatoria on tomato leaflets treated with copper bactericides. Phytopathology 81 : 714 719. Jones, J.B., S.S Woltz, R.O. Kelly and G. Harris. 1991b. The role of ionic copper, total copper, and select bactericides on control of bacterial spot of tomato. Proc. Fla. State Hortic. Soc. 104: 257 259.

PAGE 129

129 Lai, M., N.J. Panopoulos, and S Sha ffer. 1977. Transmission of R plasmids among Xanthomonas spp. And other plant pathogenic bacteria. Phytopathology 67:1044 1050. Louws, F.J., M. Wilson, H.L. Campbell, D.A. Cuppels, J.B. Jones, P.B. Showemaker, F. Sahin and S.A. Miller. 2001. Field con trol of bacterial spot and bacterial speck of tomato using a plant activator. Plant Dis. 85:481 488. Mather, K. and J.L. Jinks. 1971. Biometrical genetics. Co rnell Univ. Press, Ithaca. Mather, K. and J.L. Jinks. 1982. Biometrical genetics: the study of c ontinuous variation. 3 rd ed. Chapman and Hall, London. Marco, G.M., and R.E. Stall. 1983. Control of bacterial spot of pepper initiated by strains of Xanthomonas campestris pv. vesicatoria that differ in sensitivity to copper. Plant Disease 67:779 891. Michelmore, R.W. and B.C. Meyers. 1998. Clusters of resistance genes in plants evolve by divergent selection and a birth and death process. Genome Res. 8:1113 1130. Minsavage, G.V., B. Balogh, R.E. Stall and J.B. Jones. 2003. New tomato races of Xant homonas campestris pv. vesicatoria assoiated with mutagenesis of tomato race 3 strains. Phytopathology 93:S62. (Abstr.) Ng, T.J. 1990. Generation means analysis by microcomputer. Hort Science 25:363. Obradovic, A., J.B. Jones, M.T. Momol, S.M. Olson, L.E. J ackson, B. Balogh, K. Guven and F.B. Iriarte. 2005. Integration of biological control agents and systemic acquired resistance inducers against bacterial spot of tomato. Plant Dis. 89:712 716. Olson, S.M., W.M. Stall, M.T. Momol, S.E. Webb, T.G. Taylor, S .A. Smith and E.H. Simonne and E. McAvoy. 2007 2008. Tomato Production in Florida p. 409 428 In: S.M. Olson and E. Simonne (eds.) Vegetable Production Handbook for Florida Food 360, Lincolnshire Pilowsky, M. a nd D. Zutra. 1986. Reaction of differe nt tomato genotypes to the bacterial speck pathogen ( Pseudomonas syringae pv. tomato ). Phytoparasitica 14:39 42. Pohronezny, K. and R.B. Volin. 1983. The effect of bacterial spot on yield and quality of fresh market tomatoes. HortScience 18:68 70. Ragha van, C., Ma.E.B. Naredo, H. Wang, G. Atienza, B. Liu, F. Qiu, K.L. McNally, and H.Leung. 2007. Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping. Mol Breeding 19:87 101. Scott, J.W. 2004. Fla. 7946 tomato breed ing line resistant to Fusarium oxysporum f.sp. lycopersici races 1, 2, and 3. HortScience 39:440 441.

PAGE 130

130 Scott, J.W., D.M Francis, S.A. Miller, G.C. Somodi, and J.B. Jones. 2003. Tomato bacterial spot resistance derived from PI 114490; inheritance of resis tance to race T2 and relationship across three pathogen races. J. Amer. Soc. Hort. Sci. 128: 698 703. Scott, J.W. and J.B. Jones 1989. Inheritance of resistance to foliar bacterial spot of tomato incited by Xanthomonas campestris pv. vesicatoria J. Am er. Soc. Hort. Sci. 114: 111 114. Scott, J.W. and J.B. Jones. 1986. Sources of resistance to bacterial spot [ Xanthomonas campestris pv. vesicatoria (Doidge) Dye] in tomato. HortScience 21: 304 306. Scott, J.W., J.B. Jones, and G.C. Somodi. 1991. Diseas e severity of tomato hybrids heterozygous or homozygous for resistance to bacterial spot: commercial outlook. Proc. Fla. State hortic. Soc. 104: 259 262. Scott, J.W., J.B. Jones, G.C. Somodi 2001. Inheritance of resistance in tomato to race T3 of the bacterial spot pathogen. J. Amer. Soc. Hort. Sci. 126: 436 441. Scott, J.W., J.B. Jones, G.C. Somodi, and R.E. Stall. 1995. Screening tomato accessions for resistance to Xanthomonas campestris pv. vesicatoria, race T3. HortScience 30 : 579 581. Scott, J .W., S.A. Miller, R.E. Stall, J.B. Jones, G.C. Somodi, V. Barbosa, D.L. Francis, and F. Sahin. 1997. Resistance to race T2 of the bacterial spot pathogen in tomato. HortScience 32: 724 727. Scott, J.W., S.M. Olson, H.H. Bryan, J.A. Bartz, D.N. Maynard, P .J. Stofella. 2006. Solar Fire hybrid tomato: Fla. 7776 tomato breeding line. HortScience 41:1504 1505. Scott, J.W., R.E. Stall, J.B. Jones, and G.C. Somodi. 1996. A single gene controls the hypersensitive response of Hawaii 7981 to race 3 (T3) of the b acterial spot pathogen. Rpt. Tomato Genet. Coop. 46: 23. Scott, J.W., S.F. Hutton, J.B. Jones, D.M. Francis, and S.A. Miller. 2006. Resistance to bacterial spot race T4 and breeding for durable, broad spectrum resistance to other races. Rpt. Tomato Gene t. Coop. 56:33 36. Sherf, A.F. and A.A. Macnab. 1986. Tomato. p. 599 696. In : Vegetable diseases and their control. 2 nd ed. John Wiley & Sons, New York. Somodi, G.C., J.B. Jones, J.W. Scott, J.F. Wang, and R.E. Stall. 1996. Relationship between the hyp ersensitive reaction and field resistance to tomato race 1 of Xanthomonas campestris pv. vesicatoria Plant Disease 80: 1151 1154. Stall, R.E. 1959. An evaluation of spray materials for control of bacterial spot on field seeded tomatoes. Plant Dis. Rptr 43:725 728.

PAGE 131

131 Stall, R.E. and P.L. Thayer. 1962. Streptomycin resistance of the bacterial spot pathogen and control with streptomycin. Plant Dis. Reptr. 46:389 392. Stall, R.E., C. Beaulieu, D. Egel, N.C. Hodge, R.P. Leite, G.V. Minsavage, H. Bouzar, J. B. Jones, A.M. Alvarez and A.A. Benedict. 1994. Two genetically diverse strains are included in Xanthomonas campestris pv. vesicatoria Int. J. Syst. Bacteriol. 44:47 53. Wang, J.F. 1992. Resistance to Xanthomonas campestris pv. vesicatoria in tomato. PhD diss. Univ. Florida, Gainesville. Wang, J.F., J.B. Jones, J.W. Scott and R.E. Stall. 1994. Several genes in Lycopersicon esculentum control hypersensitivity to Xanthomonas campestris pv. vesicatoria Phytopathology 84:702 706. Wang, J.F., J.B. Jo nes J.W. Scott, and R.E. Stall. 1990. A new race of the tomato group of strains of Xanthomonas campestris pv. vesicatoria Phytopathology 80:1070 Warner, J.N. 1952. A method for estimating heritability. Agron. J. 44:427 430. Whalen, M.C., J.F. Wang, F.M. Carland, M.E. Heiskell, D. Dahlbeck, G.V. Minsavage, J.B. Jones, J.W. Scott, R.E. Stall, and B.J. Staskawicz. 1993. Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998. Mol. Plant Microbe Interact. 6:616 627. Wright, S. 1934. The results of crosses between inbred strains of guinea pigs, differing in number of digits. Genetics 19:537 551. Yang, W.C., E.J. Sacks, M.L. Ivey, S.A. Miller, and D.M. Francis. 2005. Res istance in Lycopersicon esculentum intraspecific crosses to race T1 strains of Xanthomonas campestris pv. vesicatoria causing bacterial spot of tomato. Phytopathology 95:519 527. Yu, Z.H., J.F. Wang R.E. Stall, and C.E. Vallejos. 1995. Genomic localizat ion of genes that control a hypersensitive reaction to Xanthomonas campestris pv. vesicatoria (Doidge)Dye. Genetics 141: 675 682. Yunis, H., Y. Bashan, Y. Okon, and Y. Henis. 1980. Weather dependence, yield losses, and control of bacterial speck of tom ato caused by Pseudomonas tomato Plant Dis. 64:937 939. Zhu, X. and R.C. Elston. 2001. Transmission/disequilibrium test for quantitative traits. Genet. Epidemiol. 20:57 74.

PAGE 132

132 BIOGRAPHICAL SKETCH Samuel Forrest Hutton was born on November 14, 1977 in G reenwood, MS. He grew up with an older and two younger sisters in Tchula, MS, where his father farmed cotton, rice, soybean and corn. He worked for his father each summer until graduating from Cruger Tchula Academy in 1996. Sam then attended Delta State Un iversity for one year before transferring to Mississippi State University where he received his B.S. in agronomy in December 2000 He began graduate school at the University of Minnesota in August of the following year and married Emily D. Jones one year later The two of them remained in MN until June 2004 when Sam graduated with a M.S. in soybean breeding. In August 2004, Sam began his doctorate in tomato breeding at the University of Florida under Dr. J.W. Scott He lived in Gainesville for nearly two y ears and then mov ed to Tampa for completion of his research at the GCREC. His first child, Anna Christine, was born in December, 2006 and his second arrived soon after his dissertation defense. Upon completion of his Ph.D., Sam continued working in J.W. S doctoral researcher.