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Inheritance and Identification of Molecular Markers Associated with Resistance to Squash Silverleaf Disorder in Summer S...

Permanent Link: http://ufdc.ufl.edu/UFE0041764/00001

Material Information

Title: Inheritance and Identification of Molecular Markers Associated with Resistance to Squash Silverleaf Disorder in Summer Squash (Cucurbita pepo)
Physical Description: 1 online resource (70 p.)
Language: english
Creator: Young, Kristen
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: caps, cucurbita, horticulture, molecular, plant, rapd, scar, squash, summer
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Squash silverleaf (SSL) disorder is a serious physiological disorder in summer squash (Cucurbita pepo L.) caused by the feeding nymphs of the silverleaf whitefly, Bemisia argentifolii (formerly known as Bemisia tabaci Gennadius, B strain). This disorder is characterized by a progressive silvering on the upper leaf surface and blanching of fruit that, in severe cases, renders them unmarketable. It has also been associated with a decrease in chlorophyll and carotenoid pigment production. A source of resistance to SSL disorder has been found in C. pepo breeding line, Zuc76, developed at the University of Florida. We characterized the inheritance of resistance to the leaf symptoms of SSL disorder within segregating progeny developed from crossing the Zuc76 with C. pepo Black Beauty, a cultivar susceptible to SSL disorder. Studies were conducted within a greenhouse using a completely randomized design. At the second to third true-leaf stage, each plant was infested with a total of 30 mating pairs of mixed-age adult silverleaf whiteflies. Twenty-one days after the initial infestation, the plants were evaluated for symptoms using a scale of 0 (no silvering) to 5 (95-100% silvering of upper leaf surface). Plants rated as 0 were considered resistant while those rated 1 through 5 were considered susceptible. Chi-square analysis of F2 and BC1 segregation data support a model in which a single recessive gene conferring resistance to the leaf symptoms of SSL disorder within the C. pepo breeding line Zuc76. Breeding squash with resistance to SSL disorder, derived from Zuc76, can be facilitated by using marker-assisted selection. Using F2 and BC1 progeny, segregating for resistance, seven random amplified polymorphic DNA (RAPD) markers were found associated with SSL disorder; two linked in coupling and five linked in repulsion. RAPD markers for plant breeding applications have the advantages of being simple, rapid and cost-efficient, however, they are sensitive to PCR conditions, resulting in poor reliability between runs. A failed attempt was made to convert four of the SSL disorder associated RAPD fragments to SCAR and CAPS markers for potential use in MAS. Further optimization of PCR parameters, primer redesign, sequencing of monomorphic bands and digestion by additional restriction enzymes may improve our chances of converting our SSL disorder associated RAPDs into SCAR and/or CAPS markers.
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 Kristen Young.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Kabelka, Eileen.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041764:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041764/00001

Material Information

Title: Inheritance and Identification of Molecular Markers Associated with Resistance to Squash Silverleaf Disorder in Summer Squash (Cucurbita pepo)
Physical Description: 1 online resource (70 p.)
Language: english
Creator: Young, Kristen
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: caps, cucurbita, horticulture, molecular, plant, rapd, scar, squash, summer
Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Squash silverleaf (SSL) disorder is a serious physiological disorder in summer squash (Cucurbita pepo L.) caused by the feeding nymphs of the silverleaf whitefly, Bemisia argentifolii (formerly known as Bemisia tabaci Gennadius, B strain). This disorder is characterized by a progressive silvering on the upper leaf surface and blanching of fruit that, in severe cases, renders them unmarketable. It has also been associated with a decrease in chlorophyll and carotenoid pigment production. A source of resistance to SSL disorder has been found in C. pepo breeding line, Zuc76, developed at the University of Florida. We characterized the inheritance of resistance to the leaf symptoms of SSL disorder within segregating progeny developed from crossing the Zuc76 with C. pepo Black Beauty, a cultivar susceptible to SSL disorder. Studies were conducted within a greenhouse using a completely randomized design. At the second to third true-leaf stage, each plant was infested with a total of 30 mating pairs of mixed-age adult silverleaf whiteflies. Twenty-one days after the initial infestation, the plants were evaluated for symptoms using a scale of 0 (no silvering) to 5 (95-100% silvering of upper leaf surface). Plants rated as 0 were considered resistant while those rated 1 through 5 were considered susceptible. Chi-square analysis of F2 and BC1 segregation data support a model in which a single recessive gene conferring resistance to the leaf symptoms of SSL disorder within the C. pepo breeding line Zuc76. Breeding squash with resistance to SSL disorder, derived from Zuc76, can be facilitated by using marker-assisted selection. Using F2 and BC1 progeny, segregating for resistance, seven random amplified polymorphic DNA (RAPD) markers were found associated with SSL disorder; two linked in coupling and five linked in repulsion. RAPD markers for plant breeding applications have the advantages of being simple, rapid and cost-efficient, however, they are sensitive to PCR conditions, resulting in poor reliability between runs. A failed attempt was made to convert four of the SSL disorder associated RAPD fragments to SCAR and CAPS markers for potential use in MAS. Further optimization of PCR parameters, primer redesign, sequencing of monomorphic bands and digestion by additional restriction enzymes may improve our chances of converting our SSL disorder associated RAPDs into SCAR and/or CAPS markers.
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 Kristen Young.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Kabelka, Eileen.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041764:00001


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1 INHERITANCE AND IDENTIFICATION OF MOLECULAR MARKERS ASSOCIATED WITH RESISTANCE TO SQUASH SILVERLEAF DISORDER IN SUMMER SQUASH ( Cucurbita pepo) By KRISTEN YOUNG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY O F FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Kristen Young

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3 To my parents, Ronnie and Pamela Young

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4 ACKNOWLEDGMENTS I foremost express my grati tude to Dr. Eileen Kabelka for her generous guidance, support and patience throughout this process. Her scholarship will always serve as a model. I am grateful to Dr. Don Huber and Dr. Steve Sargent for their valuable assistance, which allowed me to obtain this degree. Finally, I thank the many faculty and supporting staff members and fellow graduate students of the Horticultural Sciences Department who have aided me along the way to this degree.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 11 Introduction ............................................................................................................. 11 Taxonomy ......................................................................................................... 12 Production ........................................................................................................ 13 Cultivation ......................................................................................................... 14 Squash Silverleaf Disorder ..................................................................................... 15 Introduction ....................................................................................................... 15 Physiological and Cytological Characteristics .................................................. 16 Relationship to the Silverleaf Whitefly .............................................................. 16 Expression of Squash Genes SLW1 and SLW3 ............................................... 19 Silverleaf Whitefly ................................................................................................... 20 Taxonomy of the Silverleaf Whitefly ................................................................. 21 Management .................................................................................................... 21 Host Resistance ............................................................................................... 22 2 CHARACTERIZATION OF RESISTANCE TO SQUASH SILVERLEAF DISORDER CAUSED BY THE SILVERLEAF WHITELY ( Bemisia argentifolii ) IN SUMMER SQUASH ( Cucurbita pepo) .................................................................... 28 Introduction ............................................................................................................. 28 Materials and Methods ............................................................................................ 29 Plant Material ................................................................................................... 29 Greenhouse Experimental Design .................................................................... 29 Whitefly Culture and Plant Infestation .............................................................. 30 Scoring for Plant Response to Whitefly Infestation and Data Analysis ............. 30 Results and Discussion ........................................................................................... 31 3 INDENTIFICATION OF MOLECULAR MARKERS ASSOCIATED WITH RESISTANCE TO SQUASH SILVERLEAF DISO RDER IN SUMMER SQUASH ( Cucurbita pepo) ..................................................................................................... 37 Introduction ............................................................................................................. 37 Materials and Methods ............................................................................................ 39

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6 Plant Material ................................................................................................... 39 DNA Isolation ................................................................................................... 39 RAPD Analysis ................................................................................................. 40 Data Analysis ................................................................................................... 41 Results and Discussion ........................................................................................... 41 4 DEVELOPMENT OF SCAR AND CAPS MARKERS BASED ON RAPD MARKERS ASSOCIATED W ITH SSL DISORDER RESISTANCE ........................ 47 Introduction ............................................................................................................. 47 Materials and Methods ............................................................................................ 48 Plant Material and DNA Extracti on ................................................................... 48 Cloning and Sequencing of RAPD Fragments ................................................. 48 SCAR Primer Design and Amplification ........................................................... 49 CAPs ................................................................................................................ 50 Results and Discussion ........................................................................................... 50 LIST OF REFERENCES ............................................................................................... 62 BIOGRAPHICAL SKETCH ............................................................................................ 70

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7 LIST OF TABLES Table page 2 1 Segregation for resistance to squash silverleaf (SSL) disorder caused by the silverleaf whitefl y ( Bemisia argentifolii ) in Zuc76, Black Beauty and their F1, F2, and BC progeny. ........................................................................................... 36 3 1 Molecular markers associated with squash silverleaf (SSL) disorder resistance, derived from C. pepo breeding line Zuc76 ....................................... 46 4 1 C haracteristics of four RAPD fragments and their associated SCAR primers .... 61

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8 LIST O F FIGURES Figure page 1 1 SSL disorder symptoms initiate as venal leaf chlorosis, followed by silvering of the primary and secondary veins that progresses to interveinal areas, potentially covering the entire adaxial leaf surface. ............................................ 25 1 2 A pair of silverleaf whiteflies, Bemisia argentifolii ............................................... 26 1 3 Nymphs of silverleaf whitefly, Bemisia argentifolii .............................................. 26 1 4 Silverleaf whitefly ( Bemisia argentifolii ) life cycle ................................................ 27 2 1 Greenhouse studies to evaluate Zuc76, Black Beauty and their F1, F2, and BC progeny to squash silverleaf (SSL) disorder caused by the silverleaf whitefly ( Bemisia argentifolii ) .............................................................................. 33 2 2 Infestation of experimental plants with silverleaf whitefly, ( Bemisia argentifolii ) 34 2 3 Rating scale (0 5) for squash silverleaf disorder on Cucurbita pepo ............... 35 3 1 Relative position of markers OPC07, O PL07, OPBC16A and OPBC16B and the squash silverleaf disorder resistance locus ( sl ) in F2 progeny ..................... 45 4 2 Profiles of EcoRI digested plasmids containing RAPD fragments from primers OPBC16, OPC07 and OPL07. ............................................................... 56 4 3 PCR profile between the resistant and susceptible parents using sequence characterized amplified region (SCAR) marker ................................................. 57 4 4 PCR profile between the resistant and susceptible parents using sequence characterized amplified region (SCAR) marker .................................................. 58 4 5 Digestion patterns between the resistant and susceptible parents us ing the PCR products of C07S993 and L07S999 ........................................................... 59 4 6 Digestion patterns between the resistant and susceptible parents using the PC R products of BC16S554 and BC16S306. ..................................................... 60

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INHERITANCE AND IDENTIFICATION OF MOLECULAR MARKERS ASSOCIATED WITH RESISTANCE TO SQUASH SILVERLEAF DISORDER IN SUMMER SQUASH ( Cucurbita pepo) By Kristen Young May 2010 Chair: Eileen Kabelka Major: Horticultural Science Squash silverleaf (SSL) disorder is a serious physiological disorder in summer squash ( Cucurbita pepo L.) caused by the feeding nymphs of the silverleaf whitefly, Bemisia argentifolii (formerly known as Bemisia tabaci Gennadius, B strain). This disorder is characteriz ed by a progressive silvering on the upper leaf surface and blanching of fruit that, in severe cases, renders them unmarketable. It has also been associated with a decrease in chlorophyll and carotenoid pigment production. A source of resistance to SSL disorder has been found in C. pepo breeding line, Zuc76, developed at the University of Florida. We characterized the inheritance of resistance to the leaf symptoms of SSL disorder within segregating progeny developed from crossing the Zuc76 with C. pepo Black Beauty, a cultivar susceptible to SSL disorder. Studies were conducted within a greenhouse using a completely randomized design. At the second to third trueleaf stage, each plant was infested with a total of 30 mating pairs of mixedage adult sil verleaf whiteflies. Twenty one days after the initial infestation, the plants were evaluated for symptoms using a scale of 0 (no silvering) to 5 (95 100% silvering of upper leaf surface). Plants rated as 0 were considered resistant

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10 while those rated 1 thr ough 5 were considered susceptible. Chi square analysis of F2 and BC1 segregation data support a model in which a single recessive gene conferring resistance to the leaf symptoms of SSL disorder within the C. pepo breeding line Zuc76. Breeding squash wit h resistance to SSL disorder, derived from Zuc76, can be facilitated by using marker assisted selection. Using F2 and BC1 progeny, segregating for resistance, seven random amplified polymorphic DNA (RAPD) markers were found associated with SSL disorder ; tw o linked in coupling and five linked in repulsion. RAPD markers for plant breeding applications have the advantages of being simple, rapid and cost efficient, however, they are sensitive to PCR conditions, resulting in poor reliability between runs. A fail ed attempt was made to convert four of the SSL disorder associated RAPD fragments to SCAR and CAPS markers for potential use in MAS. Further optimization of PCR parameters, primer redesign, sequencing of monomorphic bands and digestion by additional restri ction enzymes may improve our chances of converting our SSL disorder associated RAPDs into SCAR and/or CAPS markers.

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11 CHAPTER 1 LITERATURE REVIEW Introduction The United States produced 303 thousand metric tons of squash, valuing $204 million, in 2008 (US DA, 2008). In the same year, Florida was the top squash producing state with a value of production of $53 million (USDA, 2008). Summer squash ( Cucurbita pepo L.) is the primary crop for Florida squash growers (Mossler and Nesheim, 2003). Squash silverleaf (SSL) is an economically important physiological disorder affecting summer squash. It is characterized by progressive silvering of veins and the adaxial leaf surface due to development of large air spaces between the mesophyll palisade cells and the adaxi al epidermis (Barro et al., 2007). Severely symptomatic plants can be stunted, leading to reduced fruit production. Fruit from severely symptomatic plants can appear blanched. Reduced fruit quality and yield has caused substantial economic loss to Florida squash growers (Maynard et al., 1989). The symptoms of SSL disorder are systemically induced by feeding by silverleaf whitefly ( Bemisia argentifolii ) nymphs (Jimenez et al., 1995). Infesting populations of silverleaf whitefly can be reduced through chemic al, cultural methods and biocontrol (McAuslane, 2009). Insecticide use is the primary method of whitefly control, but challenges of environmental impacts, economic costs and whitefly insecticide resistance has led to a renewed interest is developing squash cultivars with resistance to the disorder caused by whiteflies. A source of resistance in C. pepo to SSL disorder, designated Zuc76, was developed at the University of Florida.

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12 The objectives of this project include: (1) characterizing the inheritance of resistance to SSL disorder within Zuc76, and (2) identifying and developing molecular markers associated with SSL resistance derived from this breeding line. Characterization and the identification of molecular markers associated with SSL disorder resi stance will contribute to developing SSL disorder resistant C. pepo cultivars with superior horticultural potential. Squash ( Cucurbita pepo) Taxonomy The diverse genus Cucurbita includes some of the oldest domesticated plant species in the archeological r ecord (Decker, 1988). Five domesticated species and 22 wild species are found within the genus and are native to the Western hemisphere. C. pepo, C. moschata, C. maxima C. argyrosperma (formerly C. mixta ) and C. ficifolia are currently grown in a wide range of regions from cool temperate to tropical (Paris, 2005). C. pepo is the most widely grown species and includes summer squash and pumpkins. A number of cultigens of C. pepo were domesticated in the preColumbian Americas with the oldest record of t he species found in Valley of Oaxaca in Mexico and dated to nearly 10, 000 years old. C. pepo is considered the most polymorphic species within the plant kingdom (Zraidi et al., 2007). The fruit exhibit wide variation and are used in the infraspecific clas sification of cultivated members of the species into eleven cultivar groups (Paris, 2008). The three inedible, ornamental groups are the Round, Smooth Rinded Gourd group, the Oviform, SmoothRinded Gourd Group, and the Warted Gourd Group. The groups grown for their mature fruit are the Acorn Group and Pumpkin Group. The summer squash groups are vegetable marrow, cocozelle, scallop, crookneck, straightneck and zucchini.

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13 Summer squash are grown for their edible, immature fruit in temperate and subtropical regions (Paris, 2008). Shiny, intensely colored fruit of 100g to 200g are most desirable while dull, oversized fruit are unmarketable. During the midand late20th centuries, the value of summer squash exceeded pumpkins and winter squash leading to intensif ied breeding for increased crop quality (Paris, 2001). F1 hybrids were introduced commercially in the United States in the 1950s. The growth in popularity of summer squash into the new millennium has increased development of more disease resistant varieties. Records from the United Nations Food & Agriculture Organization show an increase in summer squash consumption greater than all other vegetables in Western Europe in the last decades of the millennium. Zucchini has become the most widely grown and economically important summer squash variety and has exceeded all other members of the Cucurbita genus (Paris, 1986; Paris, 2001). Some openpollinated zucchini cultivars, such as Black Beauty, are produced, but the majority of the market consists of over 100 hybrids (Paris, 1986). Production Squash, pumpkins and gourds are major vegetable crops with a combined worldwide production of 210 million tonnes in 2007 (FAOStat, 2009a). Summer squash represent a small portion of this production, but because summer sq uash production is concentrated in economically developed countries, the crop value may exceed the other Cucurbita varieties in this category (Paris, 2008). In 2007, the United States ranked 4th in production of squash, pumpkins and gourds with 864,180 me tric tonnes, of which 284, 220 tonnes were squash (FAOStat, 2009b; USDA, 2009). The value of United States squash production for 2008 was $204 million dollars, with Florida contributing $53 million dollars as the top squashproducing state (USDA, 2008). California is ranked

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14 second in terms of production value followed by New York, Georgia and Texas. In the United States, squash ranks in the top 15 vegetables in production, planting acreage and crop value (USDA, 2009). Squash production in the United States has changed greatly in the last two decades with nationwide production reports only becoming available in 2000 as squash production began to exceed other vegetables and melons (Cantliffe et al., 2007). Cultivation Summer squash are short season crops grown in warm seasons, typically fall and/or spring, when the threat of cold temperature to the herbaceous, frost sensitive plants is at a minimum ( Kemble et al., 2005; Paris, 2001). Summer squash are typically direct seeded or transplanted in flat ground or raised beds, with or without polyethelene mulch ( Kemble et al., 2005). Squash can be planted on plastic mulched beds following crops of other fruit or vegetables, but to avoid potential nematode and soil borne disease problems, summer squash should not follow other Cucurbitaceae crops. Well drained sand loam soils of pH 6.0 to 6.5 with high levels of organic matter are preferred ( Kemble et al. 2005; Peet, 2001b). Summer squash require relatively high fertilization rates, especially before flowering (Peet, 2001b). Fertilization rates are dependent upon soil testing results. Summer squash have palmate leaves and a bush habit (Paris, 1989; Paris, 2001). Because open growth and bush habits facilitate harvesting, vining varieties are rare. Foliage of C. pepo is t ypically rough and spiney, but smooth varieties of summer squash are desired for easier hand harvest and avoidance of scratches to the tender fruit (Paris, 1989). Squash plants are monoecious, producing conspicuous, nectar producing, orangeyellow, unisexual flowers (Paris, 2001). Squash plants produce male

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15 flowers 3 to 4 days before female flowers are formed and in a typical ratio of 3 males flowers per female flower (Peet, 2001a). Summer squash crops are highly dependent on effective bee pollination for c rop yield and quality ( Kemble et al., 2005; Stephens, 2003). Poor pollination results in small, misshapen or aborted squash fruit (Peet, 2001a). For adequate fruit set, one honeybee hive per acre of production is recommended. Squash require 40 to 50 days f rom seeding to reach marketable fruit size ( Kemble et al., 2005; Stephens, 2003). Market preference is for smaller, glossy fruit with intense color and less stippling (Paris, 2001). Yellow fruit are in demand in the United States, but not in other areas of production. Squash Silverleaf Disorder Introduction Squash silverleaf (SSL) disorder is a severe physiological disorder affecting economically important varieties of C. pepo C. maxima and C. moschata (Paris et al., 1993). It was first described in Israel in 1963 and was believed to associated with drought stress (Burger et al., 1983; McAuslane et al., 2004). The first widespread instance of SSL disorder affecting commercial plantings in Florida was in 1987 in Palm Beach County (Simons et al., 1988). Clas sic progressive leaf silvering was uniformly observed in a single planting of C. pepo in September and by December all commercial squash plantings in Palm Beach and Broward Counties. In addition to leaf symptoms, blossoms appeared frosted and fruit appeare d glazed or blanched. Dark green cultivars of zucchini appeared chlorotic to pale green in color in a uniform or mottled pattern. A reduction in yield in addition to the unmarketable conditions of the fruit contributed to severe economic losses during the 198788 growing season. SSL disorder has

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16 subsequently spread to many southern states, including Texas, Arizona and California and into Hawaii and the Caribbean region (Henneberry et al., 1999). Physiological and Cytological Characteristics SSL disorder symptoms initiate as venal leaf cholorosis, followed by silvering of the primary and secondary veins that progresses to interveinal areas, potentially covering the entire adaxial leaf surface (Fig. 1 1) (Jimenez et al., 1995; Simons et al., 1988). These sy mptoms are distinct from genetic silvering, which initiates in vein axils, usually appears in patches and does not expand to the entire laminar surface. Fruit and flowers are affected in severe cases of SSL disorder. Silvering related to SSL disorder is pe rmanent, though symptomatic plants can produce normal leaves. The silver appearance of the leaves is due to altered reflection of light as a result of the separation of the upper leaf epidermis from the palisade mesophyll (Schmalstig et al., 2001). Silvering symptoms are accompanied by a 1050% reduction in chlorophyll (Jimenez et al., 1995). Transmission electron micrographs of symptomatic leaf tissue revealed smaller chloroplasts with smaller starch grains in comparison to non affected control samples (Sc hmalstig et al., 2001). SSL disorder affected plants have reduced photosynthetic ability as evidenced by reduced net carbon rate exchanged and hypothesized to be a result of increased light reflectance, reduced chlorophyll levels, poorly developed chloropl ast and possible inhibition of the photosynthetic apparatus. Relationship to the Silverleaf Whitefly SSL disorder was first hypothesized to be associated with drought stress or other environmental factors such as air quality (Schuster et al., 1991; Simons et al., 1988). In the 19871988 Florida squash season, large populations of the silverleaf whitely (SLW), Bemisia argentifolii Bellows & Perring, (formally B. tabaci (Gennadius) strain B ) were

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17 observed in occurrences of severe silvering (Maynard, 1989). Subsequent greenhouse experiments showed that SSL disorder is associated with the feeding nymphs, not the adults, of the silverleaf whitefly (Costa et al., 1993; Schuster et al., 1991). As few as two immature whiteflies can induce silvering symptoms and the density of nymphal infestation is positively correlated with severity of symptoms (Costa et al., 1993). When the feeding nymphs are removed from the squash plant, new leaves appear free of symptoms (Schuster et al., 1991). Drought stress has been observ ed to intensify SSL disorder symptoms induced by the SLW (Carle et al., 1998; Paris, 1993). Jimenez et al. (1995) hypothesized that a translocated SLW borne toxin may be responsible for SSL disorder. The closely related sweetpotato whitefly ( Bemisia tabaci type A) does not induce SSL disorder. While plant defense responses differ in relation to physical damage associated with differential modes of insect feeding, silverleaf and sweetpotato whiteflies (SPW) are thought to have similar probing and feeding mec hanisms (van de Ven, 2000). Variations in composition of saliva or digestive symbiotes may be responsible for differential squash plant response to whitefly feeding (Jimenez et al., 1995; Ven et al., 2000). The components may be produced by the insect or by the endosymbiotic bacteria in the gut mycetomes of the SLW. Variations in species and frequency have been noted between B. tabaci and B. argentifolii endosymbionts (Chiel et al., 2007; Costa et al., 1995; Ruan et al., 2006). Endosymbionts affect their hosts ecology and evolution and have been shown to affect host plant range, susceptibility to environmental factors and resistance to pathogens and parasitoids in other species of agricultural insect pests.

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18 Bemisia argentifolii was first observed in the United States on poinsettia crops in South Florida in 1986 as it became a severe pest of economically important vegetable, field and ornamental crops (McAuslane, 2009). It quickly spread to other southern states with large areas of crop production, including Texas, Arizona and California. The SLW can over winter as far north as South Carolina and is a greenhouse pest in cooler latitudes. It has a broad host and geographical range, feeding on over 500 plant species in 74 families in mostly warm temperate and t ropical regions including the United States, Canada, Central and South America, the Caribbean, southern Europe, Africa, India and Australia (Brown et al., 1995; McAuslane, 2009). Crop damages attributed to SLW were estimated to exceed $500 million in the United States in 1991 (Perring et al., 1993). Damages to Florida crops due to direct feeding and virus transmission by the SLW were estimated to be $141.4 million. In Florida, SLW is a pest of a wide range of economically important crops including tomato, peppers, squash, cucumber, eggplant, watermelon, cabbage, potato, peanut, soybean, cotton and a number of ornamental crops such as poinsettia, hibiscus and chrysanthemum (McAuslane, 2009). SLW damage plants in a number of ways. Direct feeding on plant phl oem sap can cause loss of plant vigor, plant stunting, wilting, seedling death and reduced yield (McAuslane, 2009). Sooty mold grows on whitefly excreted honeydew, a sticky sugar rich waste. Sooty mold reduces fruit quality and leaf photosynthetic ability. Feeding by immature whiteflies is associated with physiological disorders in addition to SSL disorder. A physiological disorder of tomato, called irregular ripening, is characterized by streaked exterior fruit color and immature internal tissue that appears white

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19 (Schuster et al., 1990). It was first described in southwest Florida in 1987, coinciding with the appearance of SSL disorder. In 1989, Florida tomato growers lost $25 million due to the unmarketable fruit affected by tomato irregular ripening (Per ring et al., 1993). Other SLW associated physiological disorders include lettuce leaf yellowing and stem blanching, pepper streak, Brassica white stem and general leaf chlorosis in a number of crops (McAuslane, 2009). SLW vectors several plant pathogenic v iruses including tomato leaf curl virus, tomato mottle virus and bean golden mosaic virus. SLW serves as the vector for a number of viruses affecting Cucurbits. In 2006, Cucurbit leaf crumple virus (CLCV) was first reported in Florida on C. pepo (Akad et a l., 2008). The leaves of the symptomatic plants are curled, thickened crumpled and yellow. Fruit can become streaked, rendering them unmarketable (Webb et al., 2007). Squash vein yellowing virus (SqVYV) was detected on squash in Florida in 1983 and causes leaf veins to yellow (Baker et al., 2008). Cucurbit yellow stunting disorder virus causes initial chlorotic leaf spotting that progresses to the interveinal areas ( Davis et al., 2008). Leaves curl and become brittle. Fruit development is affected leading t o economic losses due to poor marketability. Expression of Squash Genes SLW1 and SLW3 Two genes, SLW1 and SLW3 are systematically induced in squash after feeding by the SLW, but not the SPW (van de Ven, 2000). SLW1 transcripts accumulated most abundantly in proximal SLW infested leaves, but were also detected in distal noninfested leaves not yet fully silvered. SLW1 RNAs were identified in proximal, SPW infested leaves, but were not present in distal leaves. Noninfested controls showed no SLW1 RNA accum ulation. SLW3 was expressed systemically only in SLW infested plants. Transcripts accumulated in distal, silvered leaves of SLW infested plants, but not

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20 in SPW infested plants. Similar expression levels were observed in both proximal SLW and SPW infested leaves, with low levels detected in the noninfested controls. SLW1 and SLW3 transcripts were only detected after whitefly nymphal feeding and not during adult feeding. Whitefly induction of SLW1 and SLW3 genes was compared with the noninfested plants res ponse to mechanical injury, methyl jasmonic acid (MeJA), and drought stress. Wounding and exogenously applied MeJa induced SLW1 expression. During a water deficit, SLW1 transcripts accumulated in low amounts in young and mature leaves while SLW3 RNAs were detected at high levels in young and mature leaves which suggest nymphal feeding may elicit similar signal responses by the squash plant as drought stress. SLW1 was identified to be a metallopeptidase like protein, which are involved in hydrolyzing peptic bonds. SLW3 is a betaGlucosidase like protein, which are involved in plant defense response to tissue damage. Silverleaf Whitefly Bemisia argentifolii n the family Aleyroidae, is similar in appearance to B. tabaci the sweetpotato whitefly (SPW), which has been in the United States since 1897 but not a significant agricultural pest (Brown et al., 1995; Perring et al., 1993). B. argentifolli was not distinguished from B. tabaci at the time of significant whitefly infestations in southwestern Florida begi nning in 1986. The expanded host range, increased populations and the appearance of plant physiological of unknown etiology was subsequently attributed to a new strain of B. tabaci designated strain B. Morphological, behavioral and pathological differenc es between the two strains of whiteflies have led scientists to classify the B strain as a new species, B. argentifolii Bellows & Perring ( Bellow et al., 1994) with the common designation of the silverleaf whitefly (SLW). The

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21 smaller SLW is more fecund an d has a broader host range and higher feeding rate than the SPW. The SPW does not cause plant physiological disorders nor transmit particular viruses that are only associated with the SLW. The SLW and SPW also show differences in esterase banding patterns and random amplified polymorphic DNA sequences. Taxonomy of the Silverleaf Whitefly Silverleaf whiteflies are about 0.8 to 1.0mm in length and have white wings with a yellow body (Fig. 12) (McAuslane, 2009). Piercing sucking mouthparts are used to feed on plant phloem sap at vein sites on the undersides of leaves (Brown et al., 1993; Hoddle, 1999; Johnson et al., 2005; Stansly, 2007). The SLW develops from an egg to an adult with four nymphal instar stages (Fig. 13). The adult female SLW deposit eggs on the underside of the host plant leaves. The first instar nymph emerges from the egg to find a minor veinfeeding site. The subsequent nymphal instars stages are immobile, feeding on plant phloem sap until the adult stage. Adult SLW usually emerge in the m orning. Male whiteflies are capable of mating upon emergence, while female whiteflies mate 2024 h after emergence. The lifecycle (Fig. 14) of the SLW is dependent on temperature (Hoddle, 1999). Adults emerge 29.9 days at 28 C and 49.9 at 22 C. The averag e number of days to egg hatch is 16.9 at 22 C and 10.4 at 28 C. Management Squash silverleaf disorder is currently managed through the control of SLW through chemical, cultural and biological methods. In most agricultural systems, chemical control using insecticides is the primary strategy of SLW management ( Henneberry et al., 1999). Single or mixtures of insecticides have provided reasonable control of whitefly in major crops. Research is shifting toward integrating alternative

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22 control methods due to co ncerns of environmental impacts and occurrences of whitefly insecticide resistance. SLW have been reported to have moderate to high resistance to a number of organophosphate, carbamate and pyrethroid insecticides (Brown et al., 1995). Insect growth regulat ors are considered environmentally safe, but also act on natural insect enemies ( Henneberry et al., 1999). Removal of crop residues to eliminate breeding sites during cropfree periods is an important cultural control method (McAuslane, 2009). Colored or reflective plastic mulches may be effective in deterring whiteflies. Biological control using natural enemies can potentially suppress SLW populations below economically injurious levels. Whiteflies have a number of natural enemies including some fungal pathogens, parasitoids and predatory insects. Incorporating natural enemies into management systems can be complex due to specific environmental requirements of each organism. Care must also be observed when using chemical control methods in conjunction wit h the use of natural enemies because insecticides have a negative effect on natural enemies. Host Resistance Host plant resistance (HPR) is an economically and environmentally sound alternative to chemical control methods to minimize the economical loss associated with SSL disorder (Teetes, 2009). HPR is the result of heritable plant traits that result in a plant being relatively less damaged in relation to a plant without the traits (Teetes, 2009). Differences in severity of silvering symptoms have been o bserved among and within the cultivar groups of C. pepo (Paris, 1993). Field and greenhouse experiments evaluating the six C. pepo cultivar groups showed cocozelle and vegetable marrow groups the least susceptible to silvering, while the crookneck and zucc hini groups showed the most severe symptoms of leaf silvering. The least susceptible cultivars were

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23 of Old World origin and current cultivation. The most susceptible cultivars are of Old World origin, but have been bred and cultivated in the New World for decades. While zucchini was shown as the most susceptible cultivar group, variation of symptom development has been observed among zucchini cultivars and breeding lines (McAuslane, 1996). Two University of Florida zucchini breeding lines, designated Zuc33 and Zuc76, found to be considerably less susceptible to SSL disorder than control varieties were evaluated in the field to determine relationships between whitefly population density and severity of symptoms. Zuc33 and Zuc76 supported similar white fly populations as the susceptible control but showed no silverleaf symptoms. The hypothesized mechanism of host plant resistance to SSL disorder is tolerance. Cordoza et al. (1999) ruled out antixenosis or insect colonization deterrence by the host plant, as the mechanism of resistance as resistant and susceptible zucchini genotypes did not significantly differ in SLW egg counts. Leaf characteristics such as trichome density and arrangement have been shown to influence whitefly oviposition on wild cucurbitaceous species (McAuslane, 1996). McAuslane (1996) observed more pubescent C. pepo cultigens were host to larger whitefly populations than less hairy cultigens. Cordoza et al. (1999) found no significant difference in trichome density between the zucchini breeding lines resistant to SSL disorder and the susceptible control. Resistant variety Zuc76 was found to be tolerant to SLW at initial infestation levels of 40, 80 and 160 SLW pairs, while the susceptible control exhibited maximum symptoms at the lowes t infestation. Development of squash genotypes with tolerance to whitefly feeding is an important tool in managing SSL disorder. Though the source of resistance, Zuc76,

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24 shows a number of undesirable horticultural traits, the gene(s) controlling SSL disorder resistance can be transferred to other cultivars and hybrids through traditional breeding methods. A previous study attempting to characterize the inheritance of resistance found in ZUC76 was not completely successful due to imperfect screening procedur es (Carle et al., 1998). Accurately identifying the mode of inheritance of SSL disorder resistance found in ZUC76 will contribute to future development a SSL disorder resistant C. pepo cultivar with superior horticultural potential.

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25 Figure 11. SSL di sorder symptoms initiate as venal leaf chlorosis followed by silvering of the primary and secondary veins that progresses to interveinal areas, potentially covering the entire adaxial leaf surface.

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26 Figure 12. A pair of silverleaf whiteflies, Bemisia argentifolii ( http://www.ars.usda.gov/is /AR/archive/nov07/vine1107.htm ) Figure 13. Nymphs of silver leaf whitefly, Bemisia argentifolii ( http://www.scienceimage. csiro.au/mediarelease/mr07227.html )

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27 Figure 14. Silverleaf whitefly ( Bemisia argentifolii ) life cycle ( http://www2.dpi.qld.gov.au/ horticulture/18512.html )

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28 CHAPTER 2 CHARACTERIZATION OF RESISTANCE TO SQUASH SILVERLEAF DISORDER CAUSED BY THE SILVER LEAF WHITELY ( Bemisia argentifolii ) IN SUMMER SQUASH ( Cucurbita p epo)1 Introduction Squash silverleaf (SSL) disorder is an economically important physiological disorder affecting squash, zucchini and pumpkin ( Cucurbita pepo ). It is characterized by a silver appearance of veins and the adaxial leaf surface caused by the progressive development of large air spaces between the mesophyll palisade cells and the adaxial epidermis due to increased or prolonged cell wall degradat ion (Barro et al., 2007; Jimenz, 1995; McAuslane, 2001). Silvered leaves exhibit reduced photosynthetic ability due a significant reduction in chlorophyll content, decreased chloroplast development and perhaps to increased light reflectance. Severely symptomatic plants can exhibit stunting and fruit yield reduction (Jimenez, 1995, McAuslane, 2001). In severe cases, squash fruit become blanched and unmarketable due to poor quality (Jimenez, 1995, Schmalstig et al., 2001). Reduced fruit quality and yield has caused substantial economic loss to Florida summer squash growers (Maynard et al, 1989). The value of Florida squash production was 53 million dollars in 2007 and summer squash is the primary crop for Florida squash growers ( Mossler and Nesheim, 2003; USDA, 2008). The symptoms of SSL disorder are systemically induced by feeding by immature nymphs of the silverleaf whitefly [ Bemisia argentifolii (formerly known as Bemisia tabaci Gennadius, B strain)] (Schuster et al. 1991). Silvering symptoms develop on new leaves upon maturation distal to the site of the nymph feeding. When the nymphs are 1 Reprinted with permission from Young, K.N. and E.A. Kabelka. 2009. Characterization of resistance to squash silverleaf disorder in summer squash. HortScience 44:12131214.

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29 removed, old leaves remain silver but new growth is free of symptoms (Jimenez, 1995). The expression of symptoms is dependent on density of nymphs present, level of env ironmental stress and cultivar (Paris, 1993). Infesting populations of silverleaf whitefly can be reduced through cultural methods and biocontrol however, control by chemical means is difficult due to the whiteflys resistance to some pesticides (McAuslane, 2007). Furthermore, the undersides of the leaves, the location of the nymphs and adult whiteflies are found on the plant, are difficult to reach by sprays. A source of resistance to SSL disorder within summer squash, designated Zuc76, was developed at t he University of Florida. The objective of this project is to characterize the inheritance of resistance to SSL disorder, caused by the silverleaf whitefly, within the summer squash breeding line Zuc76. Materials and Methods Plant Material The SSL disorder susceptible C. pepo cultivar, Black Beauty (BKB) and the SSL disorder resistant C. pepo breeding line, Zuc76, were used as parents to establish segregating populations. Black Beauty was obtained from commercial companies (Seed Savers, Decorah, IA and Reimer Seeds, Mount Holly, NC). Controlled pollinations were carried out in the greenhouse to generate F1 (BKB x Zuc76), F2 and reciprocal backcross (BKB x F1; F1 x BKB; F1 x Zuc76) progenies. Greenhouse Experimental Design Seed for all studies were sow n in individual 15.2 cm standard round plastic pots containing Fafard Growing Mix #3S ( Fafard Inc., Agawam, MA ). Studies to evaluate the F2 and BC1 progeny were performed using 61 cm cubed cages covered with fine mesh cloth to be insect proof (Fig. 2 1) E ach cage contained six randomly chosen plants.

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30 Plants were water regularly. As seedlings reached the first trueleaf stage, slow release fertilizer (Osmocote, 141414 N:P:K, Grace Sierra Horticulture Products, Milpitas, CA) was applied. Temperatures ranged from 2535 C throughout the year in the heated and evaporatively cooled greenhouse. Supplemental lighting was used, when needed, to maintain a 1 4 hour day length. Whitefly Culture and Plant Infestation Whitefly ( B. argentifolii ) adults were obtained f rom a colony reared on cotton ( Gossypium hirsutum ) and collard ( Brassica oleracea) in a laboratory maintained at 22 C and provided supplemental lighting for a 14 h day length. Starting at the second to third trueleaf stage, plants were infested with 30 pairs of mixed age adult whiteflies, aspirated into vials as aligned malefemale pairs from the surface a yellow foam card in the rearing cages (Fig. 2 2). Whiteflies were released by placing opened vials under the leaf canopy of each plant. Continuous feeding and ovipositing by the whiteflies were allowed for the duration of the experiments. Parental and F1 plants grown in noninfested cages served as negative controls. Parental and F1 plants grown among the progeny in infested cages served as positive controls. Scoring for Plant Response to Whitefly Infestation and Data Analysis Plants were visually rated for severity of SSL disorder symptoms 21 days after infestation The most silvered leaves of each plant showing sympt oms were scored u sing a scale of 0 t o 5 (Fig. 2 3), developed by Paris et al. (1987): 0 = green, completely nonsilvered leaves; 1 = leaves with silvering in and parallel to less than half of the veins ; 2 = leaves with s ilvering in and parallel to more than half of the veins ; 3 = leaves with silvering in and parallel to all of the veins ; 4 = leaves with silvering in all veins and some interveinal areas ; and 5 = enti re upper leave surface silvered. Plants rated as 0

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31 were considered resistant to SSL disorder while those rated 1 through 5 were considered susceptible. Chi square goodness of fit was used to test fit to the expected segregation ratios within segregating F2 and BC1 progeny to characterize resistance to SSL disorder. Results and Discussion Silvering of veins, interveinal areas and/ or the entire abaxial leaf surface was found in plants susceptible to the SSL disorder 21 days after whitefly infestation. These symptoms were absent in plants resistant to SSL disorder when grown in whitefly infested cages under the conditions of this study. Plants serving as negative controls remained free of symptoms. All experimental F1 progeny were susceptible to SSL disorder, exhibiting symptoms similar to the SSL disorder susceptible parent, Black Beauty (Table 21). F2 progeny segregated in a 1:3 [resistant (R):susceptible (S)] ratio. Progeny of the reciprocal backcrosses to the SSL disorder susceptible parent were all susceptible. Progeny of the backcross to the SSL disorder resistant parent segregated in a 1:1 (R:S) ratio. The segregation ratios support a model in which resistance to SSL disorder in Z uc 76 is conferred by a single recessive gene. SSL disorder resistance in a related species, C. moschata, was also found to be conferred by a single recessive gene ( sl ), but it is unknown if the gene is allelic to that found in C. pepo. In a study to determine the inheritance of the resistance to SSL disorder found within the C. pepo Zuc76 breeding line, Carle et al. (1998) established that inheritance of SSL disorder resistance was recessive, but wer e inconclusive in the number of genes involved, proposing one, two or four. The method for screening involved shaking whiteflies from host plants onto to the squash plants, which resulted in varied levels of the plants exposure to the whiteflies. Since in festation density of

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32 whitefly nymphs is directly correlated to the level of expression of SSL disorder symptoms (Costa et al., 1993, Yokomi et al. 1990), distributing a known number of adult mating pairs of whiteflies to each plant during the current study insured consistent levels of exposure. Environment also affects the expression of SSL symptoms. Paris et al. (1993) found drought stress increased the severity of silvering symptoms in C. pepo cultivars under controlled greenhouse conditions. Photoperiod and temperature may also contribute to differential symptom expression (Cardoza, 1999). An increase in temperature affects whiteflies, shortening the developmental time, including the time to emergence of the feeding nymphs (Butler et al., 1983, Cardoza et al., 1999). Conducting the inheritance study under greenhouse conditions limited the effects of temperature, photoperiod and moisture level on symptom expression. Identifying the mode of inheritance of SSL disorder resistance found in Zuc76 will contribute to future studies to develop a SSL disorder resistant C. pepo cultivar with superior horticultural potential.

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33 Figure 21. Greenhouse s tudies to evaluate Zuc76, B lack Beauty and their F1, F2, and BC progeny to squash silverleaf (SSL) disor der caused by the silverleaf whitefly ( Bemisia argentifolii ). A) Plants were placed in insect proof 61 cm cubed cages B) E ach cage contained six randomly chosen plants. A B

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34 Figure 22. Infestation of experimental plants with silverleaf whitefly, ( Be misia argentifolii ). A) Thirty pairs of m ixed age adult whiteflies were harvested from rearing cages by aspiration into vials as aligned malefemale pairs from the surface a yellow foam card. B) Whiteflies were released by placing opened vials under the leaf canopy of each plant. A B

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35 Figure 23. Rating scale (0 5) for squash silverleaf disorder on Cucurbita pepo. 0 = green, completely nonsilvered leaves 1 = leaves with silvering in and parallel to less than half of the veins 2 = leaves with silvering in and parallel to more than half of the veins 3 = leaves with silvering in and parallel to all of the 4 = leaves with silvering in all veins and some interveinal 5 = entire upper leave surface silvered

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36 Table 21. Segregation for resistance to squash silverleaf (SSL) disorder caused by the silverleaf whitefly ( Bemisia argentifolii ) in Zuc76, B lack Beauty and their F1, F2, and BC progeny. Genotype No. of plants z Expected Ratios (R:S) X 2 R S Zuc76 (Z76) 6 0 Black Beauty (BKB) 0 15 F 1 (BKB x Z76) 0 23 F 2 (BKB x Z76) 53 129 1:3 1.65 ns BC 1 (F 1 x Z76) 35 45 1:1 1.25 ns BC 1 (BKB x F 1 ) 0 53 0:1 BC 1 (F 1 x BKB) 0 23 0:1 zR= SSL disorder resistant, S = SSL disorder susceptible. ns, X2 value not significant ly different at P

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37 CHAPTER 3 INDENTIFICATION OF M OLECULAR MARKERS ASSOCIATED WITH RESIST ANCE TO SQUASH SILVERLEAF DISORDER IN SUMMER S QUASH ( Cucurbita pepo )2 Introduction Squash silverleaf (SSL) disorder is an important physiological disorder affecting economically important crops of Cucurbita pepo. It is characterized by permanent silvering of the leaf veins and the adaxial leaf surface. (Barro et al., 2007; Jimenz et al.,1995; McAuslane, 2001). Silvered leaves exhibit reduced photosynthetic ability. Severely affected plants can produce blanched fruit, which are unmarketable due to poor qual ity (Jimenez et al., 1995; Schmalstig et al., 2001). Reduced fruit quality and yield has caused substantial economic loss to Florida summer squash growers (Maynard et al., 1989). The value of Florida squash production was 53 million dollars in 2008 and sum mer squash is the primary crop for Florida squash growers ( Mossler et al., 2003; USDA, 2009 ). The symptoms of SSL disorder are induced by feeding immature nymphs of the silverleaf whitefly [ Bemisia argentifolii, (formerly known as Bemisia tabac i Gennadius, B strain)] (Costa et al., 1993; Schuster et al., 1991). Silvering symptoms are expressed on new leaves upon maturation distal to the site of the nymph feeding (Jimenez et al., 1995; Schuster et al., 1991). The expression of symptoms is dependent on densit y of nymphs present, level of environmental stress, and cultivar (Paris et al., 1993). An economically and ecologically sound form of control for SSL disorder is genetic resistance. A source of resistance to SSL disorder has been bred in a C. pepo 2 Reprinted in part with permission from Kabelka, E.A. and K.N. Young. 2010. Identif ication of molecular markers associated with resistance to squash silverleaf disorder in summer squash ( Cucurbita pepo). Euphytica 173(1):4954.

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38 breeding line, Zuc76, which has been characterized as being conferred by a single recessive gene (Chapter 2). Conventional breeding to incorporate SSL disorder resistance into squash cultivars requires time, labor and greenhouse space for generation of breeding populations and the phenotypic screening of adult plants for direct selection. Indirect selection using molecular markers linked to the SSL disorder resistance gene in Zuc76 can increase the efficiency of selection and reduce the time for cultivar development. The use of molecular markers based on polymerase chain reaction (PCR) can improve the efficiency of traditional plant breeding by indirectly selecting for the gene of interest. The PCR based random amplified polymorphic DNA protocol is simple, quick, a nd capable of high throughput. It requires small amounts of DNA template and no previous knowledge of DNA sequence. The RAPD procedure utilizes single oligonucleotide primers, usually consisting of ten bases, to amplify discrete random DNA fragments from a genomic DNA template (Mohler et al., 2005). At given temperatures, the random primers will anneal to multiple sites along the template. Sequences between the annealed primers may be amplified through PCR dependent upon distance between primers and extensi on time. RAPDs can be used as genetic markers due to the polymorphisms that are produced by sequence differences in the priming sites or from insertions deletions between the priming sites (Mohler et al., 2005). The polymorphisms are identified by electrophoresis as the presence or absence of individual amplified bands measured by molecular ladders on agarose gels. Random amplified polymorphic DNA analysis has been used to identify DNA markers associated with traits of economic importance for use in MAS in a number of

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39 important horticultural crops, such as fusarium wilt resistance in Cucumus melo, a member Cucurbitaceae (Zheng et al., 1999). For C. pepo, RAPD markers have been used successfully for assessing varietal genetic variation, for distinguishing cul tivars and for genetic mapping, (Brown, 2004; Paris et al., 2003; Zraidi et al., 2007). RAPD markers linked to morphological traits and disease resistance have been identified in C. pepo, but have yet to be applied to marker assisted selection (Brown, 2004). There are no reported molecular markers associated with SSL disorder. The objective of this project was to identify RAPD markers linked to SSL disorder resistance within the summer squash breeding line, Zuc76. Materials and Methods Plant Material Previ ously, a cross was made between Black Beauty (BKB), a C. pepo cultivar susceptible to SSL disorder and Z uc 76 a C. pepo breeding line resistant to SSL disorder under controlled greenhouse conditions. A single F1 plant was self pollinated to create F2 p rogeny and crossed with both parents to create reciprocal backcross (BC) progenies. T he parents, F1, F2 and BC1 progenies were evaluated for response to silverleaf whitefly infestation and resistance to SSL disorder in Zuc76 was determined to be conferred by a single recessive gene (Chapter 2) From this study, 93 F2 progeny, segregating 3:1 (susceptible: resistant), and 30 BC1 [(BKB x Zuc76) x Zuc76] progeny, segregating 1:1 (susceptible: resistant), were used to identify molecular markers associated with SSL disorder. DNA Isolation Genomic DNAs of previously harvested newly expanded true leaves of Zuc76, Black Beauty, F1 (BKB x Zuc76), 93 F2, and 30 BC1 [(BKB x Zuc76) x Zuc76] progeny

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40 (Chapter 2) were isolated using a modified CTAB method. Lyophilized le af tissue of each sample was ground to a fine powder by vortex using five 4 mm glass beads in 15 ml centrifuge tubes. Into each 15 ml centrifuge tube, 3 ml of an extraction buffer (0.35 M Sorbitol, 0.1 M Tris, 0.005 M EDTA, adjusted to pH 7.5), 3 ml of a n uclei lysis buffer (0.2 M Tris, 0.05 M EDTA, 2.0 M NaCl, 2% CTAB, adjusted to pH 7.5), and 1.5 ml 5% Sarkosyl were added. Samples were vortexed and incubated in a 65C water bath for 10 minutes. After cooling to room temperature, an equal volume of chlorof orm/isoamyl (24:1) was added, the samples mixed gently, and centrifuged at 800 g for 15 min. The aqueous phase was transferred to a new 15 ml centrifuge tube, 20 l of 10 mg/ml RNAse was added, and each sample incubated at 37C for 60 min. Samples were chi lled on ice for 15 min and one equal volume of isopropanol was added to each tube. Samples were gently inverted and then centrifuged at 3200 g for 45 min. The pelleted DNA was re suspended in 500 l Tris EDTA pH 7.5. RAPD Analysis The parents were screened with 1152 RAPD primers (Eurofins MWG Operon Technologies, USA). RAPD primers generating repeatable polymorphisms between the parental DNAs were used to genotype 93 segregating F2 progeny to identify putative markers associated with SSL disorder resista nce. Markers associated with SSL disorder were tested using 30 BC1 [(BKB x Zuc76) x Zuc76] progeny. 35 nmoles of plant genomic DNA, 2.0 mM MgCl2 DNA polymerase in buffer containing 200 mM Tris HcL, pH 8.4, and 500 mM KCl. Polymerase chain reaction (PCR) was performed as follows: denaturation at 94C for 5 min, followed by 40 cycles of 1 min at 94C, 1 min at 43C and 2 min at 72C, with a

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41 final extension at 72C for 5 min. The reactions were then cooled and held 4C. Twenty microliters of t he PCR products were separated on 1.5% agarose gels in 0.5% TBE buffer. The agarose gels were stained with 1 g ml1 ethidium bromide, visualized under UV light and photographed on a digital gel documentation system. The molecular weights of the RAPD products were estimated with a 1 Kb DNA ladder (Fisher Scientific, USA). Data Analysis Chisquare goodness of fit was used to test fit to the expected segregation ratios of RAPD fragments within the segregating F2 progeny derived from C. pepo Black Beauty and Zuc76. Single factor analysis of variance using PROC GLM of SAS ( Statistical Analysis System version 9.2, SAS Institute, Cary, NC) was performed to detect associations between each marker and SSL disorder in the segregating F2 and BC1 progeny. A signific ant association was declared if P < 0.05. Relative positions and distances between molecular marker loci and SSL disorder were estimated using JoinMap Version 3.0 (Van Ooijen and Voorrips 2001). Map distances in cM were estimated with the Kosambi mapping function. Results and Discussion Of the 1152 RAPD primers screened against the parental genotypes, C. pepo Zuc76 (SSL disorder resistant) and Black Beauty (SSL disorder susceptible), 55 (5%) produced reproducible banding profiles during amplification. T he low percentage of polymorphic RAPD loci between Zuc76 and Black Beauty may reflect their genetic similarity. Genotyping the F2 progeny derived from Zuc76 and Black Beauty revealed 12 of the 55 polymorphic RAPDs with distorted segregation ratios, based on chi square

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42 goodness of fit. These were eliminated from further analysis. Of the remaining 43 RAPD primers, six were found to be associated ( P < 0.05) with SSL disorder resistance based on singlefactor analysis of variance (Table 31). OPBC16 provided two PCR fragments both associated with SSL disorder resistance and labeled OPBC16A and OPBC16B. The seven RAPDs, OPC07, OPH01, OPL07, OPM01A, OPBA10, OPBC16A and OPBC16B, segregated in a 3:1 ratio in the F2 progeny indicating dominance of band when pres ent over its absence. OPC07 and OPBC16B were found coupled to SSL disorder resistance while OPH01, OPL07, OPM01, OPBA10, and OPBC16A were found linked in repulsion to SSL disorder resistance. Association of RAPD primers OPH01, OPL07, OPM01A, OPBA10, and O PBC16A with SSL disorder were examined in BC1 [(BKB x Zuc76) x Zuc76] progeny segregating 1:1 (resistant: susceptible). Because SSL resistance in Zuc76 is conferred by a single recessive gene, the markers OPC07 and OPBC16B, which are coupled with the resis tance allele, could not be tested in the BC1 progeny. Associations ( P < 0.05) between RAPD primer OPL07 and OBC16A with SSL disorder resistance were detected (Table 3 1). The relative position and distances of OPL07 and OPBC16A, including OPC07 and OPBC1 6B, to SSL disorder were estimated (Fig. 31). The RAPD marker, OPC07, is the closest marker to SSL disorder with its genetic distance estimated to be 20 cM. This is the first report describing molecular markers associated with resistance to SSL disorder resistance ( sl ) in summer squash ( C. pepo). In this study, the source of resistance to SSL disorder is derived from Zuc76, a breeding line with an observed number of undesirable characteristics including variable germination rates, heterophylly,

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43 and occasi onal dwarfism. The use of these markers has the potential of facilitating the introgression of the resistance gene from Zuc76 into C. pepo cultivars or parental breeding lines with superior horticultural potential. The use of RAPD markers in MAS is depend ent upon the breeding population and the orientation of the markers to the resistance allele (Johnson et al., 1995). To transfer a single recessive resistance gene into a susceptible but superior line, the donor (resistant) parent and the recurrent (susceptible) parent are crossed and the F1 self pollinated to generate the F2, which are screened for individuals homozygous recessive for the resistance gene (Staub et al., 2008). These individuals are backcrossed to the recurrent parent to produce a BC1. Homo zygous recessive individuals are self pollinated. The BC1S1 are screened for homozygous recessive plants, which are backcrossed to the recurrent parent. This process is repeated. Individuals in an advanced backcross generation showing the desired traits of the recurrent parent are self pollinated and screened for the homozygous recessive state for the resistance gene to produce the improved variety. Using backcross breeding to introgress the recessive resistance gene into superior lines would benefit from a RAPD marker linked in repulsion phase (Johnson et al., 1995). Selecting against the repulsionphase marker in the F2 and backcross populations will separate the homozygous resistant plants from plants with the dominant susceptible allele. In addition, two dominant markers in opposite linkage phase can be an efficient way to discriminate between heterozygous and homozygous individuals (reference). Fondevilla et al. (2008) showed that two dominant SCAR markers, one linked in coupling and the other in

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44 repulsi on to the powdery mildew resistance gene in pea ( Pisum sativa ), as highly efficient in distinguishing homozygous and heterozygous resistant plants. For a molecular marker to be effective in MAS, it must be an economic and simple method capable of rapid sc reening in breeding programs (Johnson et al., 1995; Mohler and Schwarz, 2004). RAPD markers satisfy these requirements, but suffer from the inherent problem of poor reproducibility due to sensitivity to PCR conditions. This problem can be overcome by devel oping RAPD markers into sequenced characterized amplified region (SCAR) markers (Dheng et al. 1997; Zhang et al. 2001). Converting markers into SCAR markers involves sequencing the RAPD fragment and designing primers specific to the polymorphic sequences. SCAR markers can potentially be developed into co dominant cleaved amplified polymorphic sequences (CAPS), which are produced by endonuclease digestion of the SCAR product. SCAR and CAPS markers have greater reproducibility and, therefore, greater potential in MAS. Future work from this study will include developing SCAR and/or CAPS markers based on the RAPD markers identified as linked to SSL disorder resistance.

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45 Figure 31. Relative position of markers OPC07, OPL07, OPBC16A and OPBC16B and the squash silverleaf disorder resistance locus ( sl ) in F2 progeny derived from crossing C. pepo Zuc76 (SSL disorder resistant) and Black Beauty (SSL disorder susceptible). Map distances on the left are expressed in centiMorgans. sl 0 OPC07 20 OPL07 23 OPBC16A 39 OPBC16B 42

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46 Table 31. Molecular markers associated with squash silverleaf (SSL) disorder resistance, derived from C. pepo breeding line Zuc76 Approx. fragment size (bp) P value z Primer Primer sequence (5 to 3) Ry S F2 progeny BC1 progeny OPC07 GTCCCGACGA 950 0.0124 N/A x OPH01 GGTCGGAGAA 1200 0.0470 ns OPL07 AGGCGGGAAC 850 <0.0001 0.0019 OPM01A GTTGGTGGCT 950 0.0437 ns OPBA10 GGACGTTGAG 800 0.0187 ns OPBC16A CTGGTGCTCA 600 0.00 10 0.001 OPBC16 B CTGGTGCTCA 300 0.0407 N/Ax zP value base d on singlefactor analysis of variance between each marker and SSL disorder in 93 F2 progeny, segregating 3:1 (susceptible: resistant), and 30 BC1 [(BKB x Zuc76) x Zuc76] progeny, segregating 1:1 (susceptible: resistant). ns= X2 value not significant ly dif ferent at P yR indicates SSL disorder resistant allele and S indicates SSL disorder susceptible allele xSSL resistance is conferred in Zuc76 by a single recessive gene, therefore, OPC07 and OPBC16B which are coupled with the resistance allele, could not be tested in the BC1 [(BKB x Zuc76) x Zuc76] progeny

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47 CHAPTER 4 DEVELOPMENT OF SCAR AND CAPS MARKERS BASED ON RAPD MARKERS ASSOCIATED WITH SSL DISORDER RESISTANCE Introduction The expression of the recessive gene, sl imparts resistance to squash silverleaf (SSL) disorder in zucchini squash ( Cucurbita pepo). The source of this resistance is breeding line Zuc76, which exhibits multiple undesirable horticultural characteristics. The resistance allele can be introgressed into cultivated varieties and lines that exhibit superior horticultural characteristics. The screening of resistance in segregating progenies is a resourceand timeconsuming process of evaluation of whitefly infested plants after weeks of symptom development in a climatecontroll ed greenhouse. Molecular markers linked to the trait of interest can be used in marker assisted selection (MAS) to identify desired genotypes at the seedling stage, bypassing tedious phenotype screening procedures (Zhang et al., 2001). Random amplified polymorphic DNA (RAPD) markers for plant breeding applications have the advantages of being simple, rapid and cost efficient (Johnson et al. 1995). The technique does not require large amounts of DNA template, radioactivity or foreknowledge of DNA sequences (Khampila et al., 2008; Zhang et al., 2001; Zraidi et al., 2007). However, RAPD markers are sensitive to PCR conditions, resulting in poor reliability between runs (Johnson et al., 1995). Because they are generally dominant, RAPD markers usually cannot di stinguish heterozygotes from one of the two homozygous genotypes (Zhang et al., 2001). The problems associated with RAPD markers can be overcome by converting RAPD markers to a sequence characterized amplified region (SCAR) markers (Deng et al., 1997; Zhang et al. 2001). The SCAR technique involves cloning and sequencing a RAPD fragment before designing primer

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48 pairs based on the terminal fragment sequences (Deng et al., 1997). SCAR markers are less sensitive to PCR conditions, generally allele specific and potentially co dominant (Zhang et al., 2001). SCAR marker development can be limited by the loss of polymorphism between genotypes. This is a result of the tolerance of mismatches by the priming sequences resulting in monomorphic SCAR fragments (Deng et al ., 1997; Zhang et al. 2001). Polymorphisms can be recovered by converting the SCAR to a cleaved amplified polymorphic sequence (CAPS), which is achieved by digesting the SCAR amplification product by restriction enzymes. RAPD markers were previously ident ified as linked to SSL disorder resistance in zucchini squash. In this study, the RAPD fragments were used to develop SCAR and CAPS markers for potential use in MAS and gene mapping. Materials and Methods Plant Material and DNA Extraction A SSL disorder resistant parent, Zuc76, and a SSL disorder susceptible parent, Black Beauty, were crossed to generate a segregating F2 generation. DNA was extracted from the parents and F2 generation following a modified CTAB protocol previously described in Chapter 3. As previously described in Chapter 3, the F2 generation was used to find RAPD markers associated with SSL resistance. Three of these RAPD markers (OPC07, OPL07 and OPBC16) were utilized for SCAR and CAPS marker development. Cloning and Sequencing of RAPD Fragments RAPD primers OPC07, OPL07 and OPBC16 were used to amplify genomic DNA from the SSL disorder resistant parent, Zuc76, and the SSL disorder susceptible parent, Black Beauty. Polymorphic PCR products were separated on 1.5% agarose gels

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49 before exc ision and purification with Wizard PCR preps DNA purification system (Promega Corporation, Madison, WI). The purified RAPD fragments were cloned into the pGem T Easy vector (Promega Corporation) following the manufacturers directions. Three to ten clones for each fragment were purified using Promega Wizard Plus SV Minipreps DNA System (Promega Corporation). Successful insertions of the DNA fragments into the vector were verified by plasmid digestion by EcoRI and electrophoresis. For each RAPD fragment, thr ee clones of expected size were sequenced by the method using M13 forward and reverse primers (Eurofins MWG Operon Technologies, USA). Sequenced data were compared with the known sequence data in the National Center for Biotechnology Information ( http://www.ncbi.nlm.nih.gov ) database using BLASTx program. SCAR Primer Design and Amplification Oligonucleotide primer pairs of 22 or 24 bases were designed based on the sequences of the RAPD fragments using Primer 3 software ( http://frodo.wi.mit.edu/primer3/ ). The primers consisted of the original RAPD primer decamer sequence plus an internal extension of 12 to 14 bases. Primers were designed to avoid primer dimer or sec ondary structure formation. Parental genomic DNAs were amplified with the new primers using the same reaction volumes and PCR conditions as the RAPD amplification excepting annealing temperature, which was set at the optimal priming temperature for each pr imer pair. When the polymorphisms were lost, annealing temperatures were modified to determine the effects on the PCR product. The annealing temperature was tested empirically over a gradient of 57.969C for OPC07 and OPL07, and 5161.9C for OPBC16A and OP BC16B. The amplified products were

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50 resolved on 1.5% agarose gel, stained with ethidium bromide and visualized under ultraviolet light. CAPs As the three SCAR primer pairs did not distinguish the parental genotypes, the SCAR products were screened for clea ved amplified p olymorphisms (CAPs) using eight restriction endonucleases (AluI, AvaII, BamHI, EcoRI, HaeIII, HindIII, HpaII, NcoI, NdeI, NotI, RsaI). Each 10 L digest reaction contained 2.0 L SCAR PCR product, 0.5 L of restriction enzyme and 1.0 L of 10x restriction enzyme buffer. The reaction was performed at 37C for 3 hrs. Digested products were visualized on ethidium bromidestained 1.5% agarose gel under ultraviolet light. Results and Discussion Four RAPD products produced from RAPD primers OPC07, OPL07 and OPBC16 (Fig. 4 1) were ligated into pGEM T Easy vectors. Clones containing the OPC07, OPBC16A and OPBC16B inserts exhibited more transformed E. coli colonies than OPL07. As a result, ten colonies containing the OPC07, OPBC16A and OPBC16B fragmen ts were cultured while only three were cultured for the OPL07 fragment. The appropriatesized fragments were recovered from each of the clones containing the OPBC16A, OPBC16B and OPC07 amplicons after digestion with EcoRI (Fig. 42). Only two of the three clones containing the OPL07 fragments produced appropriatesized bands after plasmid digestion. Three of the transformed vectors containing the OPBC16A, OPBC16B and OPC07 fragments were submitted for sequencing, while only two were submitted for OPL07. The sequences were bordered by the original RAPD decamer primers at the 3 and 5 ends. The complete sequences were determined by combining the reverse and forward sequences. The size of the fragments produced by

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51 the RAPD primer were found to be 952 bp for OP C07, 999 bp for OPL07, 554 bp for the OPBC16 A band and 306 bp for the OPBC16 B band (Table 41). The SCAR fragments were subsequently labeled C07S952, L07S999, BC16S554 and BC16S306. BLASTx search of the sequences yielded significant homology to sequence s in the database. The sequence C07S952 produced significant sequence alignment with lipoxygenase protein (Accesion: gb|AAB65767.1|). Products of the lipoxygenase pathway are used in a number of plant functions, including in defense to stress or feeding (P orta et al., 2002). Lipoxygenase transcripts are induced by jasmonic acid, a plant hormone that acts as a signaling molecule in response to wounding, such as insect damage. The sequences for L07S999, BC16S554 and BC16S306 matched hypothetical proteins with unknown functions. The SCAR primer pairs failed to differentiate the parental genotypes. Annealing temperature was modified to evaluate the effects on the SCAR products but changes in protocol did not recover the polymorphisms between the genotypes. In or der to differentiate between the two parents, the genotypes were screened for restriction site polymorphisms in the SCAR product using eight available restriction enzymes. Monomorphisms between the parental genotypes persisted and development of CAPS marke rs was unsuccessful. Of six RAPD markers found to be associated with SSL disorder resistance, four RAPD fragments were selected for isolation, cloning and sequencing for the development SCAR and CAPS markers. While the reproducibility of RAPD markers is so metimes unreliable due to sensitivity to PCR conditions, SCAR markers are more

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52 stable and reproducible between laboratories (Deng et al., 1997; Johnson et al., 1995). SCAR markers also have the advantage of being allelespecific. During the development of SCAR markers, the polymorphisms in the RAPD products differentiating genotypes can be lost. The RAPD polymorphisms are often a result of sequence differences of a single to a few nucleotides in the priming sites (Deng et al., 1997; Zhang et al., 2001). SC AR primers are designed to extend internally from the RAPD priming site. The loss of the original polymorphisms may occur when the extended SCAR primers tolerate the small mismatches in the RAPD priming site In our study, the four SCAR primer pairs were unable to reproduce the polymorphisms of the corresponding RAPD primers. The tolerance to priming mismatches can be reduced by increasing the annealing temperature. In this study, increasing annealing temperatures did eliminate nontarget fragments in BC1 6A and BC16B, but failed to recover polymorphisms. Polymorphisms may be recovered through the development of CAPS markers by digesting the SCAR products with restriction enzymes. Polymorphisms are a result of base mutations in restrictions sites (Deng et al., 1997). CAPS markers also have the advantage of being co dominant, distinguishing heterozygous from homozygous plants (Konieczny et al., 1993). A number of methods can be used to continue the development of the SSL disorder linked RAPD markers into S CARs or CAPS. Further optimization of PCR parameters, primer redesign, sequencing of monomorphic bands and digestion by additional restriction enzymes may recover the polymorphisms between the parental DNAs (Gutierrez et al., 2006; Zhang et al., 2001). Thi s study found that the

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53 modifications of annealing temperatures failed to differentiate between the parental genotypes. Another important component of PCR parameter optimization is MgCl2 concentration, which was not varied in this study (Deng et al., 1997; Zhang et al., 2001). The primers were designed to be 22 bp in length with one primer of 24 bp. Increasing the length of the SCAR primers up to 27 bp may make them more specific, stable and robust (Dheng et al., 1997; Wang et al., 2006). Six of nine RAPD p olymophisms were a result of mismatches at the primer annealing sites. Extension of the SCAR primers would not recover the polymorphism in this case. Future work could be focused on further development of the CAPs markers. In our study, the undifferentiat ed SCAR products were digested with eight endonucleases in effort to recover the polymorphisms. Moury et al. (2000) screened 25 enzymes for possible CAPS markers linked to tomato spotted wilt virus in pepper. Konieczny et al. (1993) used up to 50 restricti on enzymes in a screen to develop CAPS markers for Arabidopsis Screening additional endonucleases may reveal polymorphisms between the parental genotypes. Another approach to CAPS development involves recovering, cloning and sequencing the SCAR primer amplicons from both Zuc76 and Black Beauty. The sequences can be compared and screened for polymorphic restriction site mutations, which could be exploited for CAPS marker development (Zhang et al., 2001). Lastly, examining sequences of the parental genoty pes may yield divergences that can be used to recover polymorphisms using redesigned SCAR primers. Zhang et al. (2001) observed multiple base substitutions and small indels between the sequenced SCAR products of two parental lines of tomato. A forward prim er of 22 bp was designed to complement a segment containing three base substitutions. The redesigned forward

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54 and the original reverse primer produced the expected polymorphism, amplifying a single product in only one of the parental lines.

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55 Figure 41. PCR amplification products of parental genotypes using random amplified polymorphic DNA (RAPD) primers OPC07, OPL07 and OPBC16 showing banding pattern between resistant (R) and susceptible (S) plants with a 1 kb ladder (M).

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56 Figure 42. Profiles of EcoRI digested plasmids containing RAPD fragments from primers OPBC16, OPC07 and OPL07.

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57 Figure 43. PCR profile between the resistant and susceptible parents using sequence characterized amplified region (SCAR) marker (A) C07S993 and (B) L07S999 across a range of annealing temperatures from 57.9 C to 69 C. Arrow indicates band size of interest. Band sizes were measured against a 1 kb ladder (M).

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58 Figure 44. PCR profile between the resistant and susceptible parents using sequence characterized amplified region (SCAR) marker (A) BC16S554 and (B) BC16S306 across a range of annealing temperatures from 61.8 C to 72 C. Arrow indicates band size of interest. Band sizes were measured against a 1 kb ladder (M).

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59 Figure 45. Digestion patterns between the resistant and susceptible parents using the PCR products of C07S993 and L07S999. Enzymes used are AluI (Al), AvaII (Av), BamHI (Ba), EcoRI (Ec), HaeIII (Ha), HindIII (Hi), HpaII (Hp), NcoI (Nc), NdeI (Nd), NotI (No) and RsaI (Rs). Band sizes were measured against a 1 kb ladder (M).

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60 Figure 46. Digestion patterns between the resistant and susceptible parents using the PCR products of (a) BC16S554 and (b) BC16S306. Enzymes used are AluI (Al), AvaII (Av), BamHI (Ba), EcoRI (Ec), HaeIII (H a), HindIII (Hi), HpaII (Hp), NcoI (Nc), NdeI (Nd), NotI (No) and RsaI (Rs). Band sizes were measured against a 1 kb ladder (M).

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61 Table 41 Characteristics of four RAPD fragments and their associated SCAR primers RAPD Marker SCAR Characteristics Name Fragment Size (bp) SCAR Name Primer Pairs Sequence (5' to 3') Primer Length (bp) OPC07 953 C07S953 Forward GTCCCGACGA GAACGATCCG 20 Reverse GTCCCGACGA CTCTCAAGAAGC 22 OPL07 999 L07S999 Forward AGGCGGGAAC GGAGTGGAGA 20 Reverse AG GCGGGAAC ACTAGGGCTG 20 OPBC16A 554 BC16S554 Forward CTGGTGCTCA GGAATAAGAA 20 Reverse CTGGTGCTCA AACAAAAAGG 20 OPBC16B 306 BC16S306 Forward CTGGTGCTCA AACGAAAAGG 20 Reverse CTGGTGCTCA GGAACAAGAA 20

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62 LIST OF REFERENCES Akad, F., S. Webb, T.W. Nyoike, O.E. Liburd, W. Turechek, S. Adkins and J.E. Polston. Detection of Cucurbit leaf crumple virus in Florida Cucurbits. 2008. Plant Dis. 92(4):648. Baker, C., S. Webb and S. Adkins. 2008. Squash vein yellowing virus, causal agent of watermelon vine decline in Florida. Plant Pathol. Circ. 407. Bellows, T.S.J., T.M. Perring, R.J. Gill and D.H. Hendrick. 1994. Description of a species of Bemisia (Homoptera: Aleyroididade). Ann. Entomol. Soc. Am. 87:195206. Brown, J.K. 1995. The sweetpotato or silverleaf whiteflies: biotypes of Bemisia tabaci or a species complex? Ann. Entomol. Soc. Am. 40:51134. Brown, R.N. 2001. The use and development of molecular breeding tools in Cucurbita: a literature review. Cucurbit Genet. Coop. Rpt. 24:8790. Brown, R.N. and J.R Myers. 2001. RAPD markers linked to morphological and disease resistance traits in squash Cucurbit Genet. Coop. Rpt. 24:9193. Brown, R.N. and J.R. Myers. 2002. A genetic map of squash ( Cucurbita sp.) with randomly amplified polymorphic DNA markers and morphological markers. J. Am. Soc. Hortic. Sci. 127:568 575. Burger, Y., H.S. Paris, H. Nerson, Z. Karchi and M. Edelstein. 1983. Overcoming silvering disorder of Cucubita Cucurbit Genet. Coop. Rpt. 6:7071. Byrne, D.N. and T.S. Bellows, Jr. 1991. Whitef ly biology. Annu. Rev. Entomol. 36:431457. Cardoza, Y.J., H.J. McAuslane and S.E. Webb. 1999. Mechanisms of resistance to whitefly induced squash silverleaf disorder in zucchini. J. Econ. Entomol. 92:700707. Cantliffe, D.J., N.L. Shaw and P.J. Stoefella. 2007. Current trends in Cucurbit production in the U.S. Acta Hortic. 731:472478. Carle, R. B., S. E. Webb and A. Chandler. 1998. Genetic analysis of resistance to whitefly silvering in Cucurbita pepo L., p. 8489. In: J.D. McCreight (ed.). Cucurbitaceae 98: Evaluation and Enhancement of Cucurbit Germplasm. ASHS Press, Alexandria, Va. Chen, J., H.J. McAuslane, R.B. Carle and S.E. Webb. 2004. Impact of Bemisa argentifolii (Homoptera: Auchenorrhyncha: Aleyrodidae) infestation and squash silverleaf disorder on zucchini yield and quality. Plant Resist. 97(6):20832094.

PAGE 63

63 Chiel, E., Y. Gottlieb, E. Zchori Fein, N. Mozes Daube, N. Katzir, M. Inbar and M. Ghanim. 2007. Biotype dependent secondary symbiont communities in sympatric populations of Bemisia tabaci B. Entomol. Res. 97:407413. Cordoza, Y.J., H.J. McAuslane and S.E. Webb. 1999. Mechanisms of resistance to whitefly induced squash silverleaf disorder in zucchini. J. Econ. Entomol. 92(3):700707. Cordoza, Y.J., H.J. McAuslane and S.E. Webb. 2000. Effect of leaf age and silverleaf symptoms on oviposition site selection and development of Bemisia argentifolii (Homoptera: Aleyrodidae) on zucchini. Popul. Ecol. 29(2):220225. Costa, H.S. and J.K. Brown. 1991. Variation in biological characteristics and esterase patterns among populations of Bemisia tabaci and the association of one population with silverleaf symptom induction. Entom. Exp. Appl. 61:211219. Costa, H.S., D.E. Ullman, M.W. Johnson and B.E. Tabashnik. 1993. Squash silverleaf symptoms induced by immature, but not adult, Bemisia tabaci Phytopathology. 83:763766. Costa, H.S., D.M. Westcot, D.E. Ullman, J.K. Brown and M.W. Johnson. 1995. Morphological variation in Bemisia endosymbionts. Protoplasma 189:194202. Cuevas Marrero, H. and Wessel Beaver, L. 2008. Morphological and RAPD marker evidence of gene flow in openpollinated populations of Cucurbita moschata interplanted with C. argyrosperma, 347 351. In: M. Pitrat (ed.). Cucurbitacae 2008 Proceedings of the IXth EUCARPIA meeting on genetics and breeding of Cucurbitaceae. INRA, Avignon, France. Davis, R.M., T.A. Turini, B.J. Aegerter, and J.J. Stapleton. 2008. UC IPM Pest Management Guidelines: Cucurbits (UC ANR publication 3445). UC Davis IPM Online, Davis. July 2009. < http://www.ipm.ucdavis.edu/index.html >. Decker. D.S. 1988. Origin(s), evolution and systematics of Cucurbita pepo (Cucurbitaceae). Econ. Bot. 42(1):415. Dheng, Z., S. Huang, S. Xiao and F.G. Gmitter, Jr. 1997. Development and charac terization of SCAR markers linked to the citrus tristeza virus resistance gene from Poncirus trifoliata. Genome 40:697 704. FAOSTAT. 2009a. Food and Agriculture Organization of the United Nations. Chief, Electronic Publishing Poly and Support Brance, Communication Division, Rome. March 2008. < http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567>. FAOSTAT. 2009b. Food and Agriculture Organization of the United Nations. Chief, Electronic Publishing Poly and Support Brance, Communication Division, Rome. March 2008. < http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567>.

PAGE 64

64 Fazio, G., J.E. Staub and S.M. Chung. 2002. Development and characterization of PCR markers i n cucumber. J. Am. Soc. Hortic. Sci. 127(4):545557. Fondevilla, S., D. Rubiales, M.T. Moreno and A.M. Torres. 2008. Identification and validation of RAPD and SCAR markers linked to the gene Er3 conferring resistance to Erysiphe pisi DC in pea. Mol. Breedi ng 22:193 200. Gong L., G. Stift, R. Kofler, M. Pachner, T. Lelley. 2008. Microsatellites for the genus Cucurbita and an SSR based genetic linkage map of Cucurbita pepo L. Theor. Appl. Genet. 117:3748. Gutierrez, N., C.M. Avila, G. Duc, P. Marget, M.J. Su so, M.T. Moreno and A.M. Torres. 2006. CAPs markers to assist selection for low vicine and convicine contents in faba bean ( Vicia faba L.). Theor. Appl. Genet. 114:5966. Henneberry, T.J., and R.M Faust [eds.]. 1999. Silverleaf Whitefly: National Research, Action, and Technology Transfer Plan, 1997 2001 (Formerly Sweetpotato Whitefly, Strain B): Second Annual Review of the Second 5Year Plan, Held in Albuquerque, New Mexico, January 31 February 2, 1999. U.S. Department of Agriculture, Beltsville. July 2009. < http://www.ars.usda.gov/IS/np/silverleafwhitefly/slvrlfwhtfly99.htm > Hoddle, M.S. 1999. The Biology and Management of Silverleaf Whitefly, Bemisia argentifolii Bellows and Perring (Homopt era: Aleyrodidae) on Greenhouse Grown Ornamentals. University of California, Riverside. July 2009. < http://www.biocontrol.ucr.edu/bemisia.html >. Janila P., B. Sharma. 2004. RAPD and SCAR markers f or powdery mildew resistance gene er in pea. Plant Breeding 123:271284. Jiang, Y.X., N. Zareh, G.P. Walker and L.R. Teuber. 2003. Characterization of alfalfa germplasm expressing resistance to silverleaf whitefly, Bemisia argentifolii J. Appl. Entomol. 1 27:447457. Jimenez, D.R., R.K. Yokomi, RT Myer and JP Shapiro. 1995. Cytology and physiology of silverleaf whitefly induced squash silverleaf. Physiol. Mol. Plant P. 46:227242 Johnson, F.A., D.E. Short and J.L. Castner. 2005. Sweetpotato/Silverleaf White fly Life Stages and Damage. (SP90). University of Florida Institute of Food and Agricultural Sciences, Gainesville. July 2009. < http://edis.ifas.ufl.edu/IN004 >. Johnson, E., P.N. Miklas and J.R. Stavely. 1995. Coupling and repulsionphase RAPDs for markerassisted selection of PI 181996 rust resistance in common bean. Theor. Appl. Genet. 90:659664.

PAGE 65

65 Kemble, J.M., E.J. Sikora, M.G. Patterson, G.W. Zehnder and E. Bauske. 2005. Guide to Commercial Summer Squash Production. (ANR 1014). Alabama Cooperative Exte nsion System, Auburn. July 2009. < http://www.aces.edu/pubs/docs/A/ANR 1014/ >. Khampila, J. K. Lertrat, W. Saksirirat, J. Sanitchon, N. Muangsan and P. Theerakulpisut. 2008. Identification of RAPD and SCAR markers liked to northern leaf blight resistance in waxy corn ( Zea mays var. ceratina). Euphytica 164:615625 Konieczny, A. and F.M. Ausubel. 1993. A procedure for mapping Arabidopsis mutations using co dominant ecotypespecific PCRbased markers. Plant J. 4(2):403410. Lee Y.H., H.J. Jeon, K.H. Hong, B.D. Kim. 1995. Use of random amplified polymorphic DNA for linkage group analysis in an interspecific cross hybrid F2 generation of Cucurbita. J. Kor. Soc. Hortic. Sci. 36:323 330. Li H., H. Zhang, G. Gong, Y. Li, C. Cui. 2006. Research of molecular mark ers linked to the dwarf gene in squash. In: G.J. Holmes (ed) Cucurbitaceae 2006. Universal Press, Raleigh, North Carolina, pp 133138. Maynard, D.N. and D.L. Cantliffe. 1989. Squash silverleaf and tomato irregular ripening: New vegetable disorders in Florida. Vegetable Crops Fact Sheet. Florida Coop. Ext. Serv. VC 37. McAuslane, H.J. 1996. Influence of leaf pubescence on ovipositional preference of Bemisia argentifolii (Homoptera: Aleyrodidae) on soybea n. Environ. Entomol. 25:834841. McAuslane, H.J. 2009. Sweetpotato Whitefly B Biotype of Silverleaf Whitefly, Bemisia tabaci (Gennadius) or Bemisia argentifolii Bellows and Peering (Insect: Hemiptera: Aleyroididae) (EENY 129). University of Florida Institute of Food and Agricultural Sciences, Gainesville. July 2009. < http://edis.ifas.ufl.edu/IN286 >. McAuslane, H.J., J. Chen, R.B. Carle and J. Schmalstig. 2004. Influence of Bemisia argentifolii (Homoptera: Aley rodidae) infestation and squash silverleaf disorder on zucchini seedling growth. Plant Resistance 97(3):10961105. McAuslane, H.J., S.E. Webb and G.W. Elmstrom. 1996. Resistance in germplasm of Cucurbita pepo to silverleaf, a disorder associated with Bemis ia argentifolii (Homoptera: Aleyrodidae). Fla. Entomol. 24:11351143. McCollum, T.G., P.J. Stoeffela, C.A. Powell, D.J. Cantliffe, S. Hanif Khan. 2004. Effects of silverleaf whitefly feeding on tomato fruit ripening. Postharvest Biol. Tech. 31:183190.

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66 Moh ler, V. and G. Schwarz. 2005. Genotyping tools in plant breeding: from restriction fragment length polymorphisms to single nucleotide polymorphisms, p. 2338. In: H. Lorz and G. Wenzel (eds.). Molecular marker systems in plant breeding and crop improvement Springer, Berlin. Mossler, M.A. and O.N. Nesheim. 2003. Florida Crop/Pest Management Profile: Squash (CIR1265). University of Florida Institute of Food and Agricultural Sciences, Gainesville. July 2009. < http://edis.ifas.ufl.edu/PI046 >. Moury, B., S. Pfl ieger, A. Blattes, V. Lefebvre and A. Polloix. 2000. A CAPS marker to assist selection of tomato spotted wilt virus (TSWV) resistance in pepper. Genome 43:137142. Oumouloud A., M.S. ArnedoAndres, R. Gonzelez Torres, J.M. Alvarez JM. 2008. Development of molecular markers linked to the Fom 1 locus for resistance to Fusarium race 2 in melon. Euphytica 164:347 356. Paris, H.S. 1989. Historical records, origins, and development of the edible cultivar groups of Cucurbita pepo (Cucurbitaceae). Econ. Bot. 43(4): 423443. Paris H.S., P.J. Stoffella, C.A. Powel. 1993. Susceptibility to leaf silvering in the cultivar groups of summer squash. Euphytica 69:6972. Paris H.S. 1996. Summer squash: history, diversity, and distrubtion. HortTechnology 6(1):6 13. Paris, H.S. 2001. History of the cultivar groups of Cucurbita pepo, p. 71165. In: J. Janick (ed.). Plant breeding reviews. John Wiley & Sons, Inc. Hoboken, NJ.. Paris H.S., N. Yonash, V. Portnoy, N. Mozes Daube, G. Tzuri, N. Katzir. 2003. Assessment of genetic relati onships in Cucurbita pepo ( Cucurbitaceae) using DNA markers. Theor. Appl. Genet. 106:971978. Paris. H.S. 2005. The genes of pumpkin and squash. HortScience 40(6):16201630. Paris, H.S. 2008. Summer squash, p. 351379. In: J. Prohens and F. Nuez (eds.). Vegetables I: Asteraceae, Brassicaceae, Chenopodiaceae and Cucurbitaceae. Springer, New York, NY Paris, H.S., H. Nerson, and Y. Burger. 1987. Leaf silvering of Cucurbita. Can. J. Plant Sci. 67:593598. Paris, H.S., P.J. Stoffella and C.A. Powell. 1993. Di fferential susceptibility to leaf silvering in Cucurbita pepo. HortScience 28(6):657568. Paris, H.S., N. Yonash, V. Portnoy, N. Mozes Daube, G. Tzuri and N. Katzir. 2003. Assessment of genetic relationships in Cucurbit pepo (Cucurbitaceae) using DNA marke rs. Theor. Appl. Genet. 106:971978.

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67 Peet, Mary. 2001a. Squash, Gourd and Pumpkin: Sustainable Practices for Vegetable Production in the South Botany. NCSU, Raleigh. Oct 2008. < http://www.cals.ncsu.edu/sustainable/peet/profiles/botsquas.html >. Peet, Mar y. 2001b. Squash, Gourd and Pumpkin: Sustainable Practices for Vegetable Production in the South Production Practices. NCSU, Raleigh. http://www.cals.ncsu.edu/sustainable/peet/profiles/ppsquash.html Oct. 2008. Perring, T.M., A.D. Cooper, R.J. Rodriguez, C.A. Farrar, T.S. Bellows, Jr. 1993. Identification of a whitefly species by genomic and behavioral studies. Science. 259(5091):7477. Ruan, Y.M., J. Xu and S.S. Liu. 2006. Effects of antibiotics on fitness of the B biotype and a nonB biotype of the whitefly Bemisia tabaci Entomol. Exp. Appl. 121:159166. Schmalstig, J.G. and H.J. McAuslane. 2001. Developmental anatomy of zucchini leaves with squash silverleaf caused by Bemisia argentifolii the silverleaf whitefly. J. Am. Soc. Hortic. Sci. 126:5445 54. Schuster, D.J., J.B. Kring, and J.F. Price. 1991. Association of the sweetpotato whitefly with a silverleaf disorder of squash. HortScience. 30:316317. Schuster, D.J., T.F. Mueller, J.B. Kring and J.F. Price. 1990. Relationship of the sweetpotato whit efly to a new tomato fruit disorder in Florida. HortScience 25:16181620. Simons, J.N. P.J. Stoefella, K.D. Shuler and R.N. Raid. 1988. Silver leaf of squash in South Florida. Proc. Fl. State Hortic. 101:397399. Stephens, J.M. 2003. Squash, Zucchini Cuc urbita pepo L. (HS675). University of Florida Institute of Food and Agricultural Sciences, Gainesville. Oct 2008. < http://edis.ifas.ufl.edu/mv142 >. Seruwagi, P. J.P. Legg, M.N. Maruthi, J. Colvin, M.E.C. Rey, and J.K. Brown. 2005. Genetic diversity of Bem isia tabaci (Gennadius) (Hemiptera: Aleyrodidae) populations and presence of the B biotype and a nonB biotype that can induce silverleaf symptoms in squash in Uganda. Ann. Appl. Biol. 147:253265. Staub, J.E., M.D. Robbins and T.C. Wehner. 2008. Cucumber, p. 341 282. In: J. Prohens and F. Nuez (eds.). Vegetables I: Asteraceae, Brassicaceae, Chenopodiaceae and Cucurbitaceae. Springer: New York, NY Teetes, G.L 2009. Plant Resistance to Insects: A Fundamental Component of IPM. In: E. B. Radcliffe,W. D. Hutc hison & R. E. Cancelado (eds.) Radcliffe's IPM World Textbook. University of Minnesota, St. Paul. July 2009. < http://ipmworld.umn.edu>.

PAGE 68

68 Tezuka T., K. Waki, K. Yashiro, M. Kuzuya, T. Ishikawa, Y. Takatsu, M. Mijagi. 2009. Construction of a linkage map and identification of DNA markers linked to Fom 1, a gene conferring resistance to Fusarium oxysporum f.sp. melonis race 2 in melon. Euphytica 168:177188. USDA. 2008a. Squash: National Statistics National Agricultural Statistics Service, Washington, DC. July 2009. < http://www.nass.usda.gov:8080/QuickStats/index2.jsp > USDA. 2009. National Agricultural Statistics Vegetables 2008 Summary. U.S. Department of Agriculture, Beltsville. July 2009. < http://usda.mannlib.cornell.edu/usda/current/VegeSumm/VegeSumm 0128 2009.pdf >. VanOoijen J.W., R.E. Voorrips. 2001. JoinMap 3.0, Software for the calculation of genetic linkage maps. Pl ant Research International, Wageningen, the Netherlands. Ven, W.T.G. van de, C.S. LeVasque, T.M. Perring, and L.L. Walling. 2000. Local and systemic changes in squash gene expression in response to silverleaf whitefly feeding. Plant Cell. 12:14091423. Wang, Y.H., R.A. Dean and T. Joobeur. 2006. Genetic mapping and molecular breeding in Cucurbits, p. 213239. In: J. Janick (ed.). Plant Breeding Reviews. John Wiley & Sons, Inc. Hoboken, NJ. Webb, S.E., F. Akad, T.W. Nyoike, O.E. Liburd and J.E. Polston. 2007 Transmitted Cucurbit leaf crumple virus in Florida (EENY 477). University of Florida Institute of Food and Agricultural Sciences, Gainesville. July 2009. < http://edis.ifas.ufl.edu/IN716 >. Yokomi, R.K., K.A. Hoelmer and L.S. Osborne. 1990. Relationship between the sweetpotato whitefly and the squash silverleaf disorder. Phytopathol. 80:895900. Yokomi, R.K., L.S. Osborne and K.A. Hoelmer. 1989. Squash silverleaf and its association with the sweetpotato whitefly. Phytopathology. 79:11611162. Yu S., F. Zha ng, R. Yu, Y. Zou, J. Qi, X. Zhao, Y. Yu, D. Zhang, L. Li. 2009. Genetic mapping and localization of a major QTL for seedling resistance to downy mildew in Chinese cabbage ( Brassica rapa ssp. pekinensis ). Mol. Breeding 23:573590. Zhang, Y. and J.R. Stom mel. 2001. Development of SCAR and CAPS markers linked to the beta gene in tomato. Crop Sci. 41:16021608. Zheng, X.Y., D.W. Wolff, S. Baudracoo Arnas and M. Pitrat. 1999. Development and utility of cleaved amplified polymorphic sequences (CAPS) and restri ction fragment length polymorphisms (RFLPs) linked to the Fom 2 Fusarium wilt resistance gene in melon ( Cucumis melo L.). Theor. Appl. Genet. 99:453456.

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69 Zraidi, A. G. Stift, M. Pachner, A. Shojaeiyan, L. Gong and T. Lelley. 2007. A consensus map for Cucur bita pepo. Mol. Breeding. 20:375388. Zraidi A., T. Lelley. 2004. Genetic map for pumpkin Cucurbita pepo using random amplified polymorphic D NA markers, p 507514 In: A. Lebeda and H.S. Paris (eds.). Proceedings of Cucurbitaceae 2004, 8th Eucarpia meeting on Cucurbit Genetics and Breeding, Palacky University, Olomouc, Czech Republic

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70 BIOGRAPHICAL SKETCH Kristen Nicole Young was born in Sydney, FL. After graduating from Durant High School, she completed her bachelors degree at the University of Florida in Gainesville, FL with a major in horticultural science. For her masters degree, she concentrated on plant breeding and genetics. She received her masters degree in horticultural science in the spring of 2010.