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Molecular and Field Analysis of Nematode Resistance in Peanut (Arachis hypogaea L.)

Permanent Link: http://ufdc.ufl.edu/UFE0021492/00001

Material Information

Title: Molecular and Field Analysis of Nematode Resistance in Peanut (Arachis hypogaea L.)
Physical Description: 1 online resource (122 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: arachis, hybridization, meloidogyne, molecular, peanut, r, subtractive, suppression
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Root-knot nematodes (Meloidogyne arenaria) are the most important nematode pathogens of cultivated peanut (Arachis hypogaea L.). Following interspecific hybridization using wild peanut species and backcrossing with the cultivar Florunner, the root-knot nematode resistant cultivars COAN and NemaTAM were released by Texas A & M University. The first objective of this study was to identify differentially expressed genes in the root-knot nematode resistant cultivar NemaTAM and the root-knot nematode susceptible cultivar Florunner following nematode inoculation. Using suppression subtractive hybridization (SSH), a cDNA library was constructed enriched with differentially expressed sequences from root-knot nematode challenged root tissues of NemaTAM and Florunner. This work resulted in the identification of 140 NemaTAM-specific clones and 123 Florunner-specific clones. Gene ontology studies revealed that a large proportion of the NemaTAM-specific sequences encoded pathogen defense proteins (PR proteins) and detoxification /oxidative stress proteins, while the Florunner-specific sequences were associated with genes encoding non-pathogenesis related proteins. This work provides the foundation for further identification and study of genes involved in the molecular defense mechanism of root-knot nematode resistance in peanut. The second objective was to incorporate the root-knot nematode resistance found in COAN into Florida breeding lines that were high in oleic acid using molecular markers. Crosses involving COAN and susceptible genotypes were made and subsequent generations were screened with both a sequence characterized amplified region (SCAR) marker (Z3/265) and a restriction fragment length polymorphism (RFLP) marker (R2430E). Forty-five of the 50 lines tested (90%) were positive for both molecular markers, and among these positive lines, 88% displayed field resistance to Meloidogyne arenaria indicating successful implementation of marker-assisted selection. The final objective was to clone and characterize peanut resistance genes (R-genes) from NemaTAM that may contribute to this cultivar?s resistance to the root-knot nematode. Degenerate PCR primers were used to amplify the conserved nucleotide binding site (NBS) encoding regions of R-genes from NemaTAM. Analysis of 145 genomic clones revealed that 26% of the sequences had homology to genes belonging to the section Arachis, another 18% to genes found in other legumes, and 41% to genes found in other plants. Of the remaining clones, 15% had sequences related to genes found in microorganisms. Interestingly, one NemaTAM clone shared conserved motifs found in the well-characterized tomato root-knot nematode resistance gene, Mi. This cloned peanut R-gene was further characterized.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Gallo, Maria.
Local: Co-adviser: Gorbet, Daniel W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021492:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021492/00001

Material Information

Title: Molecular and Field Analysis of Nematode Resistance in Peanut (Arachis hypogaea L.)
Physical Description: 1 online resource (122 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: arachis, hybridization, meloidogyne, molecular, peanut, r, subtractive, suppression
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Root-knot nematodes (Meloidogyne arenaria) are the most important nematode pathogens of cultivated peanut (Arachis hypogaea L.). Following interspecific hybridization using wild peanut species and backcrossing with the cultivar Florunner, the root-knot nematode resistant cultivars COAN and NemaTAM were released by Texas A & M University. The first objective of this study was to identify differentially expressed genes in the root-knot nematode resistant cultivar NemaTAM and the root-knot nematode susceptible cultivar Florunner following nematode inoculation. Using suppression subtractive hybridization (SSH), a cDNA library was constructed enriched with differentially expressed sequences from root-knot nematode challenged root tissues of NemaTAM and Florunner. This work resulted in the identification of 140 NemaTAM-specific clones and 123 Florunner-specific clones. Gene ontology studies revealed that a large proportion of the NemaTAM-specific sequences encoded pathogen defense proteins (PR proteins) and detoxification /oxidative stress proteins, while the Florunner-specific sequences were associated with genes encoding non-pathogenesis related proteins. This work provides the foundation for further identification and study of genes involved in the molecular defense mechanism of root-knot nematode resistance in peanut. The second objective was to incorporate the root-knot nematode resistance found in COAN into Florida breeding lines that were high in oleic acid using molecular markers. Crosses involving COAN and susceptible genotypes were made and subsequent generations were screened with both a sequence characterized amplified region (SCAR) marker (Z3/265) and a restriction fragment length polymorphism (RFLP) marker (R2430E). Forty-five of the 50 lines tested (90%) were positive for both molecular markers, and among these positive lines, 88% displayed field resistance to Meloidogyne arenaria indicating successful implementation of marker-assisted selection. The final objective was to clone and characterize peanut resistance genes (R-genes) from NemaTAM that may contribute to this cultivar?s resistance to the root-knot nematode. Degenerate PCR primers were used to amplify the conserved nucleotide binding site (NBS) encoding regions of R-genes from NemaTAM. Analysis of 145 genomic clones revealed that 26% of the sequences had homology to genes belonging to the section Arachis, another 18% to genes found in other legumes, and 41% to genes found in other plants. Of the remaining clones, 15% had sequences related to genes found in microorganisms. Interestingly, one NemaTAM clone shared conserved motifs found in the well-characterized tomato root-knot nematode resistance gene, Mi. This cloned peanut R-gene was further characterized.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Gallo, Maria.
Local: Co-adviser: Gorbet, Daniel W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0021492:00001


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1 MOLECULAR AND FIELD ANALYSIS OF NEMATODE RESISTANCE IN PEANUT (Arachis hypogaea L .) By SIVANANDA VARMA TIRUMALARAJU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 Sivananda Varma Tirumalaraju

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3 To my professors Dr. M.V.C. Gowda, Dr. S.L. Dwivedi, Dr. Morag Ferguson, Dr. Eric McGaw, Dr. John Kirby and Dr. Maria Gallo.

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4 ACKNOWLEDGMENTS At the very outset, I express the lions share of my heartfe lt gratitude and profound indebtedness to Dr. Maria Gallo, esteemed chai r of my advisory committee, for her steadfast support, persistent encouragement and scientific guidance during my association with her. My sincere and special thanks to Dr. Fredy Altpeter Dr. Daniel W. Gorbet, Dr. Donald W. Dickson, and Dr. Jeffrey A. Rollins who are members of my advisory committee for their creative suggestions. I would also like to thank Dr. Je rry M. Bennett, Chair and Professor of the Agronomy Department for his kind cooperation during my study. I express a deep sense of gratitude and sin cere thanks to Mr. Jeff Seib, Ms. Kimberly Lottinville, Dr. Maria de Lourdes Mendes, Dr Marcelo Carvalho and Dr. Masahiko Murakami for their kind help and cooperation. I also extend my thanks to my lab members, Dr. Chengalrayan Kudithipudi, Dr. Il-Ho Kang, Dr. Mukesh Jain, Shanon May, Sunil Joshi, Fanchao Yi, Bhuvan Pathak, Dr. Victoria James, Diana Dr ogan, Dr. Pratibha Srivastava, Friderike Wilde and Sahar Khan for selfless help and friendship. I sincerely thank Mr. George Person, Mr Harry Wood, Mr. Serrafin Aguirre and Mr. Justin McKinney for their help durin g my research. I am deeply grat eful to my respected parents, family and friends for their love constant support and patience.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION AND RATIONALE.................................................................................12 2 REVIEW OF LITERATURE.................................................................................................15 Molecular Markers and Linkage Maps in Peanut...................................................................15 Nematode Resistance Introgressi on Using Molecular Markers.............................................18 Management of Meloidogyne arenaria Race 1 in Peanut......................................................19 Plant R Genes, Classes and Cons erved Functional Domains.................................................24 3 CHARACTERIZATION BY SUPPRESSI ON SUBTRACTIVE HYBRIDIZATION OF TRANSCRIPTS THAT ARE DIFFERENTIALLY EXPRESSED IN ROOTS OF ROOT-KNOT NEMATODE RESISTANT AND SUSCEPTIBLE CULTIVARS OF PEANUT ( Arachis hypogaea L.)...........................................................................................30 Introduction................................................................................................................... ..........30 Materials and Methods.......................................................................................................... .32 Plant Materials and Root-knot Nematode Inoculation....................................................32 RNA Extraction...............................................................................................................32 Suppression Subtractive H ybridization (SSH) and Differentially Expressed Gene Cloning........................................................................................................................ .33 cDNA Sequence Analysis and Homology Search...........................................................35 Northern Blot Analysis....................................................................................................35 Reverse Transcritase-Polymerase Chain Reaction..........................................................36 Results........................................................................................................................ .............37 Differential Screening for cDNA Specif ic to NemaTAM and Florunner Following Inoculation with Meloidogyne arenaria race 1............................................................37 Sequence Analysis and Functional Annotation...............................................................37 Validation of Differential Gene Expression Results.......................................................39 Discussion..................................................................................................................... ..........40

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6 4 MOLECULAR MARKER-BASED SELECTI ON FOR COMBINING ROOT-KNOT NEMATODE RESISTANCE WITH HIGH OLEIC ACID CONTENT IN FLORIDA BREEDING LINES................................................................................................................61 Introduction................................................................................................................... ..........61 Materials and Methods.......................................................................................................... .65 Plant Materials................................................................................................................ .65 Experiment 1............................................................................................................65 Experiment 2............................................................................................................65 Root-knot Nematode Resistance Test.............................................................................66 DNA Extraction from Seeds and Leaves.........................................................................67 Sequence Characterized Amplified Region Analysis......................................................67 Oleic Acid Analysis.........................................................................................................68 Restriction Fragment Lengt h Polymorphism Analysis...................................................68 Results........................................................................................................................ .............69 Discussion..................................................................................................................... ..........71 5 CLONING AND CHARACTERIZATION OF R -GENES IN PEANUT ( ARACHIS HYPOGAEA L.)......................................................................................................................81 Introduction................................................................................................................... ..........81 Materials and Methods.......................................................................................................... .83 Plant Materials................................................................................................................ .83 Genomic DNA Extraction...............................................................................................84 RNA Extraction...............................................................................................................84 Reverse Transcriptase Polymerase Chain Reaction......................................................85 Polymerase Chain Reaction.............................................................................................85 Cloning and Transformation............................................................................................86 Sequencing and Annotation.............................................................................................86 Results........................................................................................................................ .............87 Sequence Analysis and Identif ication of Genomic Clone NRP3D-4 ..............................88 Expression of the NRP3D-4 Gene in Peanut Infected by M. arenaria ...........................89 Discussion..................................................................................................................... ..........90 APPENDIX A EST CLONES FROM THE ROOT-KNOT NEMATODE RESISTANT CULTIVAR NEMATAM WITH AN E-VALUE BELOW 1E-04...........................................................103 B EST CLONES FROM THE ROOT-KNOT NEMATODE SUSCEPTIBLE CULTIVAR FLORUNNER WITH AN EVALUE BELOW 1E-04........................................................106 LIST OF REFERENCES.............................................................................................................108 BIOGRAPHICAL SKETCH.......................................................................................................122

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7 LIST OF TABLES Table page 3-1 Reverse transcriptase-polymerse ch ain reaction primer combinations..............................46 3-2 Identification of fifty-seven EST clone s from the root-knot nematode resistant cultivar NemaTAM............................................................................................................47 3-3 Identification of seventy-five EST clone s from the root-knot nematode susceptible cultivar Florunner............................................................................................................. ..51 4-1 Sample number representation, parents i nvolved and crosses made for seed from F1 plants......................................................................................................................... .........74 4-2 Gall and egg mass index scale (1-4) used to evaluate peanut ge notypes for resistance to Meloidogyne arenaria ...................................................................................................75 4-3 Field reaction of peanut breeding lines to Meloidogyne arenaria evaluated at the Plant Science Unit University of Florida, FL., 2001......................................................75 4-4 Crosses tested using th e SCAR marker for root-k not nematode resistance.......................76 4-5 Comparison of oleic acid content, domi nant SCAR marker Z3/265 and co-dominant RFLP marker R2430E data with r oot-knot nematode field reaction.................................77 5-1 Primer sequences used to amplify RGAs in NemaTAM...................................................97 5-2 Identification of genomic clones from the root-knot nematode resistant cultivar NemaTAM........................................................................................................................ .98 A-1 Expressed sequence tag clones from the root-knot nematode resistant cultivar NemaTAM with a cut off E-value below 1E-04..............................................................104 B-1 Expressed sequence tag clones from the root-knot nematode susceptible cultivar Florunner with a cut off E-value below 1E-04................................................................107

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8 LIST OF FIGURES Figure page 2-1 Breeding scheme used to develop the root -knot nematode resist ant peanut cultivars into COAN and NemaTAM...............................................................................................29 3-1 Double-stranded cDNA synthe sis and Rsa I digestion......................................................56 3-2 Agarose gel electrophoresis of pr imary and secondary PCR products..............................57 3-3 Differential screening results.............................................................................................58 3-4 Comparison of major fu nctional categories of Nema TAM and Florunner expressed sequences...................................................................................................................... .....59 3-5 Subcategories within the response to st ress plant defense f unctional category for NemaTAM and Florunner..................................................................................................59 3-6 Gene expression analysis of differe ntial clones (SSH-NEPL5-A01, SSH-NE-PL5C05, SSH-NE-PL5-E03, and SSH-NE-PL5F07) obtained from the NemaTAM subtracted library............................................................................................................. ..60 3-7 Gene expression analysis of differe ntial clones (SSH-FL-PL5-B06, SSH-FL-PL5B10, SSH-FL-PL5-E06, and SSH-FL-PL5-F 01) obtained from the Florunner subtracted library............................................................................................................. ..60 4-1 The restriction fragment length polymor phism locus R2430E linked to resistance to Meloidogyne arenaria in peanut breeding lines................................................................79 4-2 Sequence characterized amplified region marker analysis................................................79 4-3 Restriction fragment length polymorphism marker analysis.............................................80 5-1 Polymerase chain reaction products obt ained after optimization of degenerate primers P1B-fwd and P3D-rev...........................................................................................92 5-2 Sequence categorization based on homology searches in GenBank.................................92 5-3 Major functional categories of NemaTAM........................................................................93 5-4 Restriction map of peanut ( Arachis hypogaea L.) genomic clone NRP3D-4....................93 5-5 Nucleotide and deduced amino aci d sequences of the genomic clone NRP3D-4 ..............94 5-6 Hydropathy profile of the pr otein deduced from NRP3D-4..............................................95 5-7 Graphical overview of peanut NRP3D-4 representing location of the conserved NBARC domain..................................................................................................................... .95

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9 5-8 Comparisons of the deduced amino acid sequence of the NemaTAM protein NRP3D-4 with sequences from A. cardenasii (AY157789), A. stenosperma (AY157947), and Lycopersicon Mi-1.1 root-knot nema tode resistance protein (AAC67237)..................................................................................................................... .96 5-9 Reverse transcriptase-polymerase chain reaction analysis of the expression of NRP3D-4 in NemaTAM and Florunner............................................................................97

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10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MOLECULAR AND FIELD ANALYSIS OF NEMATODE RESISTANCE IN PEANUT ( ARACHIS HYPOGAEA L.) By Sivananda Varma Tirumalaraju May 2008 Chair: Maria Gallo Major: Agronomy Root-knot nematodes ( Meloidogyne arenaria ) are the most important nematode pathogens of cultivated peanut ( Arachis hypogaea L.). Following interspecifi c hybridization using wild peanut species and backcrossing with the cultiv ar Florunner, the rootknot nematode resistant cultivars COAN and NemaTAM were released by Texas A&M University. The first objective of this study was to identify differentially expresse d genes in the root-kno t nematode resistant cultivar NemaTAM and the root-knot nematode susceptible cultivar Florunner following nematode inoculation. Using suppression subtra ctive hybridization (SSH), a cDNA library was constructed enriched with differentially ex pressed sequences from root-knot nematode challenged root tissues of NemaTAM and Florunner. This work resulted in the identification of 140 NemaTAM-specific clones and 123 Florunne r-specific clones. Ge ne ontology studies revealed that a large proportion of the NemaTA M-specific sequences encoded pathogen defense proteins (PR proteins) and det oxification /oxidative stress protei ns, while the Florunner-specific sequences were associated with genes encoding non-pathogenesis related proteins. This work provides the foundation for further identification and study of genes involved in the molecular defense mechanism of root-knot nematode resistance in peanut.

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11 The second objective was to in corporate the root-knot nema tode resistance found in COAN into Florida breeding lines that were high in oleic acid using molecular markers. Crosses involving COAN and susceptible genotypes were made and subsequent generations were screened with both a sequence characterized amp lified region (SCAR) marker (Z3/265) and a restriction fragment length polymorphism (RFLP) marker (R2430E). Forty-five of the 50 lines tested (90%) were positive for both molecula r markers, and among these positive lines, 88% displayed field resistance to Meloidogyne arenaria indicating successful implementation of marker-assisted selection. The final objective was to clone and characterize peanut resistance genes ( R -genes) from NemaTAM that may contribute to this cultiv ars resistance to the root-knot nematode. Degenerate PCR primers were used to amplif y the conserved nucleotide binding site (NBS) encoding regions of R -genes from NemaTAM. Analysis of 145 genomic clones revealed that 26% of the sequences had homology to genes belonging to the section Arachis another 18% to genes found in other legumes, and 41% to genes found in other plants. Of the remaining clones, 15% had sequences related to genes found in microorganisms. Interestingly, one NemaTAM clone shared conserved motifs found in the we ll-characterized tomato root-knot nematode resistance gene, Mi This cloned peanut R -gene was further characterized.

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12 CHAPTER 1 INTRODUCTION AND RATIONALE Cultivated peanut ( Arachis hypogaea L.) is an internationa lly prominent crop for both human consumption and oil seed production. Pea nut ranks fifth in the world among oil seed crops (Marcio et. al., 2004). World-wide it is grown on 26.4 million hect ares with a production of 36 million tons (FAO, 2004). Peanut is curren tly being cultivated in over 110 countries primarily between 40o N and 40o S for edible oil, high seed prot ein, vitamins, and confectionary use. In the United States, it is grown in on approximately 0.6 million hectares with total production of approximately 2 million metric tons annually (http://www.fas.usda.gov/psd/comple te_tables/OIL-table11-186.htm ). Additionally, per person consumption of peanut products is about six pounds annually (Beyer et al., 2001). It is estimated that the peanut crop contributes approximately $1 billion to the U.S. economy each year (Sasser and Freckman, 1987). On a global scale, for all crops, losses attributed to plant-parasitic nematodes are estimated to be $78 billion annually (Sasse r and Freckman, 1987). Specifical ly for peanut, economic losses due to root-knot nematodes ( Meloidogyne species) have been significant in many production regions (Florida, Georgia, Alabama, Texas and North Carolina) with es timated annual monetary losses of $102 million. Also, a 50% decrease in peanut yield poten tial has been observed due to M. arenaria alone (Dickson and De Waele, 2005). Root -knot nematode infection of peanut plants results in heavy galling of pegs, pods and roots. This in turn re duces peanut quality and yield (Rodriguez-Kaba na et al., 1986). Cultivated peanut is susceptible to various pathogens, including root-knot nematodes, due to its genetic uniformity. Conversely, wild Arachis species are highly polymorphic and represent a tremendous reservoir of resistance genes. Ther efore, several efforts have been made to

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13 introgress valuable genes from wild species into cultivated peanut. One such effort led to the development of two root-knot nematode resist ant cultivars, COAN (Simpson and Starr, 2001) and NemaTAM (Simpson et al., 2003). Because all of the wild spec ies involved in the development of COAN and NemaTAM were found to be susceptible to root-knot nematodes, except for Arachis cardenasii it was hypothesized that root-knot nematode resistance was due to introgression of re sistance gene(s) from A. cardenasii (Garcia et al., 1996). To date, however, there has been a lack of information regarding a comparative response, at the molecular level, of root-knot nematode resistant and susceptible peanut cultivars to root-knot nematode challenge. Consequently, the first objective of the pr esent study was to inve stigate the molecular mechanisms that may be functioning in the r oot-knot nematode resistant cultivar NemaTAM, and the root-knot nematode susceptible cultivar Florunner. Additionally, peanut cultivars that combine the desirable high oleic fa tty acid trait (Norden et al., 1987) with resistan ce to root-knot nematodes are unavail able in the southeastern U. S. Thus, the second objective of this research was to incorporate high oleic fatty acid seed oil and root-knot nematode resistance into the Florida peanut germplasm. Initially, peanut seeds that were classified according to their oleic acid content (Moore and Knauft, 1989) were subsequently screened for root -knot nematode resistance by empl oying marker-assisted selection using a previously identified SC AR marker, Z3/265 (Garcia et al ., 1996), and an RFLP marker, R2430E (Church et al., 2000). RFLP analysis is more labor inte nsive and costly than SCAR analysis. SCAR marker analysis is simple, rapid and cost effec tive for screening a large number of breeding populations. However, the particular SCAR marker (Z3/265) us ed in this analysis, which is derived from A. cardenasii is 10 14 cM from the root-knot nematode resistance gene(s) introgressed into cultivated peanut (Gar cia et al, 1996). Therefor e, more tightly-linked

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14 flanking markers would be desi rable to provide plant breede rs with more precision when selecting for root-knot nematode resistant plants within segregating popula tions. Hence, the third objective of this research was to identify resi stance genes in NemaTAM that may be tightlylinked to the root-knot ne matode resistance trait.

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15 CHAPTER 2 REVIEW OF LITERATURE Molecular Markers and Li nkage Maps in Peanut Marker-assisted selection (MAS) is a techno logy that can improve the efficiency of conventional plant breeding. Molecu lar markers are particularly a dvantageous for traits where traditional field selection is difficult, expensiv e, or lacks precision (Crouch, 2001). The most essential requirements for developing MAS br eeding programs include: (1) polymorphisms in germplasm with useful characteri stics, (2) the identification of flanking markers closely linked to the gene/quantitative trait loci (3) a simple polymerase chai n reaction (PCR)-based marker technology for cost effective screening of larg e breeding populations, a nd (4) proper screening techniques for phenotyping of mapping populations (Dwivedi et al., 2003). Molecular markers offer several advantages over morphologica l markers such as phenotypic neutrality, independence from environmental conditions, indepe ndence of detection related to plant age, and segregation at a 1:1 ratio between marker se gregation and the genetic constitution of the individual (Dwivedi et al., 2003). One of the main advantages of using molecu lar markers is the time gained to introgress resistance genes into cultivars (Tanksley et al., 1989; Melchinger, 1990). The use of DNA markers allows selection of the resistant offs pring with the lowest amounts of the donor genome in every generation (Tanksley et al., 1989). Molecular markers are esp ecially useful in root-knot nematode resistance breeding because they: (1) minimize the need for screening large numbers of individuals once the marker-t rait relationship is establishe d, (2) eliminate the need for destructive sampling of plants for root-knot nema tode evaluations, (3) ease the identification and transfer of recessive genes, (4) reduce linka ge drag by allowing selection of segregating

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16 populations with minimum donor DNA, and (5) f acilitate map-based cloning of root-knot nematode resistance genes. Only recently have molecular techniques been employed in peanut to interpret its genome organization. Although great variation for mor phological and physiological traits has been observed in cultivated peanut, little polymorphism has been de tected at the molecular level relative to other crops (Kochert et al., 1991). However, extensive variation has been observed in wild Arachis species (Kochert et al., 1991; Halwar d et al., 1991, 1993; Pa ik-Ro et al., 1992; Stalker et al., 1994; He and Prakash, 1997; Hopkins et al., 199 9; Subramanian et al., 2000). Both RFLPand PCR-based markers have been used to assess polymorphic variation in cultivated peanut and wild Arachis species. Halward et al. ( 1991) employed RAPDs and RFLPs to study genetic variation among wild Arachis species and unadapted ge rmplasm resources of cultivated peanut from South America, Africa, and China. They observed abundant polymorphism among wild Arachis species, but very little within the cultivated peanut germplasm. Using RAPDs, Lanham et al. ( 1992) detected 49 polymorphic loci between cultivated peanut (TMV 2) and a synthetic amphiploid (B x C)2 developed from a cross between Arachis batizocoi and Arachis diogoi Of these 49 polymorphic loci, only two were polymorphic in the cultivated peanut germplasm Paik-Ro et al. (1992) studied RFLP variation in six peanut species within the section Arachis ( A monticola, A. batizocoi, A. cardenasii, A. glandulifera, A. duranensis and A. hypogaea ) Using 23 random genomic and seed cDNA probes, they reported that most of the genomic probes and more than half of the seed cDNA probes showed polymorphism among the accessions of the tetraploid species. However, no polymorphism could be detected within or between A. hypogaea, A. monticola, and interspecific derivatives. Halward et al. (1993), using primers of ar bitrary sequence, studied polymo rphic variation in two peanut

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17 cultivars, 25 unadapted A. hypogaea germplasm, the wild allotetraploid progenitors of cultivated peanut A. monticola A. glabrata (another tetraploid species) from section A. rhizomatosae and 29 diploid wild species of Arachis They observed abundant polymor phism among wild relatives, but did not find any polymorphism in the cultivated peanut germplasm. Bhagwat et al. (1997) used 12 primers a nd studied random amplified polymorphic DNA (RAPD) profiles between peanut cultivar Spanish Improved a nd its mutants obtained following X-ray irradiation. They detected 1182 fragments of which 65 were polymorphic. He and Prakash (1997), using and amplified fragment length polymorphism (AFLP) and DNA amplification fingerprinting (DAF), studied DNA polymorphism in six diverse accessions of cultivated peanut from three botanical varieties ( hypogaea fastigiata and aequatoriana ). The analysis detected 111 polymorphic AFLP markers with an averag e of 6.7 polymorphic bands per primer, while DAF analysis detected 63 polymorphic marker s with an average of 3.7 DAF polymorphic bands per primer. Hopkins et al. ( 1999) identified six polymorphic simple sequence repeat (SSR) primers and detected 10 putative SSR loci in cul tivated peanut. Similarl y, Dwivedi et al. (2001) studied RAPD profiles of 26 cultivated p eanut accessions and detected 41% genetic dissimilarity. Other studies also revealed the presence of some DNA polymorphism in cultivated peanut using SSR (Hopkins et al., 1999) and RAPD analysis (Subramanian et al., 2000). Halward et al. (1993) were the first to re port on a RFLP-based genetic linkage map of peanut. They used both random genomic DNA and cDNA clones from the peanut cultivar GK 7 (subsp. hypogaea var. hypogaea ). In total, 100 genomic and 300 cDNA clones were evaluated on F2 populations derived from the interspecific cross between A. stenosperma and A. cardenasii Only 15 genomic and 190 cDNA clones revealed polymorphism among the mapping parents, and of these, 132 markers were further used to study segregation. This work resulted in 117

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18 markers that spanned across 11 linkage groups w ith a total map distan ce of approximately 1063 cM. Subsequently, Burow et al. (2001) reported the development of a RFLP-based tetraploid genetic linkage map derived from a cross betw een Florunner and a synthetic amphidiploid {[( A. batizocoi K9484 x ( A. cardenasii GKP 10017 x A. diogoi GKP 10602)]4X}. This map consists of 370 RFLP loci mapped over 23 linkage groups with a total map distance of 2210 cM. Nematode Resistance Introgressi on Using Molecular Markers In soybean, PCR-based markers cl osely linked to alleles at the Gro1 and H1 loci, conferring resistance to soybean cyst nematode have been developed for use in resistance breeding (Niewhner et al., 1995). For another ma jor pathogen of soybean, the southern rootknot nematode ( Meloidogyne incognita ), two major QTLs conferring resistance have been mapped using RFLP markers (Concibidio, et al., 1997). In barley, two nematode ( Heterodera avenae ) resistance genes Ha1 and Ha2 have been mapped to chromosome 5 using molecular markers (Barr et al., 1998). In peanut, Garcia et al. (1995) closely m onitored introgression of A. cardenasii chromosome segments into 46 lines derived from a cross between A. hypogaea and A. cardenasii using 73 RFLP probes and 70 RAPD primers. They reported that A. cardenasii introgressed segments represent approximately 360 cM in the diploid peanut ge nome by using 34 RFLP probes and 45 RAPD primers that detected A. cardenasii segments in one or more introgression lines. Subsequently, two SCAR marker s linked to root-knot nematode ( Meloidogyne arenaria race 1) resistance, Z3/265 (Garcia et al., 1996) and RKN440 (Burow et al., 1996), have been developed. Choi et al. (1999) reported that RFLP probes R2430E, S11137E, and R2545E were linked with resistance to root-knot nematodes in BC5F2 populations of the cross Florunner (rootknot nematode susceptible cultivar) x TxAG 7 (a parent of the root-knot nematode resistant cultivar COAN). Recently, Chu et al. (2007) report ed the identification of a root-knot nematode

PAGE 19

19 resistant dominant marker, 197/909, in crosses in volving COAN and various susceptible parents. These studies demonstrated the ut ility of molecular markers to ta g and subsequently enhance the introgression of desira ble traits from wild Arachis into cultivated peanut. Management of Meloidogyne arenaria Race 1 in Peanut Cultivated peanut is infected by severa l species of root-knot nematodes, but Meloidogyne arenaria (Neal) Chitwood (peanut root-knot nematode ) is the most common species infecting peanut (Dickson, 1998). In the United States it is the most important nematode pathogen of peanut in Alabama, South Carolina, Georgia, Texas and Florida (Sturgeon, 1986). Based on the ability to infect and re produce on peanut, two races: M. arenaria race 1 and race 2, were proposed (Sasser, 1982). Of the two races, only ra ce 1 has the ability to infect and reproduce on peanut. The female of the root-knot nematode is capable of producing up to 2,500 eggs at the infection site. The embryo inside the egg develops into a first-stage juvenile (J1). The J1 molts inside the egg shell to a second stage-juvenile (J2). The J2 hatc hes, becomes mobile and is the infective stage. The J2 body is slender, tapering to bluntly rounded anterior and sharply pointed posterior end. After three additional molts, an ad ult is produced. The male body is variable in length and tapers to bluntly rounded ends (C liff and Hirschmann, 1985). Microscopic studies of several populations of males of M arenaria revealed a low, rounded, posterior sloping head cap, which was nearly as wide as the head region (Eisenback, 1980). The mail tail is short without bursa. Males have slender articu lated spicules that protrude from the cloaca, and their gubernaculum is small, slender and crescent-shaped. The female of M. arenaria is pear-shaped with a pearly white body and prominent neck, but without a tail pr otuberance (Dickson, 1998).

PAGE 20

20 Peanut plants that are heavily infected by root-knot nematode show severe stunting, chlorosis and wilting symptoms. Abnormal swellings (galls), appear on the roots, pegs and pods (Dickson, 1998). Root-knot nematodes in peanut are most commonly managed by crop rotation and the application of nematicides (Abde l-Momen et al., 1998). Because th e cash value for peanut is relatively low, crop rotation may be the most profitable way of managing M. arenaria race 1 (Dickson and De Waele, 2005). Tropical fo rage grasses, such as bahiagrass ( Paspalum notatum Flugge), are reported to be one of the best crops to manage popul ation densities of M. arenaria in Florida (Norden et al., 1977; Dickson and He wlett, 1989). Kokalis-B urelle et al. (2002) reported that peanut followed by two years of switch grass suppressed M. arenaria population densities in peanut growing fiel ds. Also, field corn was reported to be marginally effective in suppressing M. arenaria population densities (Johnson et al., 1999; Dickson, 1998). Cotton is another crop that can be rotated with peanut since M. arenaria does not infect cotton (Sasser and Carter, 1982, Dickson and De Waele, 2005). Ho wever, one should not rely solely on crop rotation because some root-knot nematode populat ions survive the winter without a host, and numerous weeds support nematode reproduction to some extent (Dickson and De Waele, 2005). In severely infested fields, and when othe r cultural methods of ma nagement do not work, then chemical control by nematicides is the most effective and reliable method of managing rootknot nematodes in peanut (Dickson and De Waele, 2005). Farmers in the southeastern U. S. have widely relied on nematicides to control M. arenaria populations (Dunn, 1988; RodriguezKabana and Ivey, 1986; Dickson an d Hewlett, 1988). In general, two types of nematicides are available, fumigants and nonfumigants. Until their use was suspended in the 1970s and 1980s, the fumigants 1, 2-dibromo-3-chloropropane (DBCP) and ethylene dibromide (EDB) were

PAGE 21

21 available. They were suspended because of human toxicology and environmental concerns (Dickson and Hewlett, 1988; Dick son and De Waele, 2005). Current ly, 1, 3-dichloropropene (1, 3-D) is the only fumigant available on the market for use on peanut. A preplant broadcast application of 1, 3-D at a relativ ely high rate (84 liters/ha) on M. arenaria infested fields consistently increased peanut yields compared to nontreated controls (Dickson and Hewlett, 1988). Several nonfumigant nematicides such as aldica rb, and oxamyl are currently registered for nematode control in peanut (Dickson a nd De Waele, 2005). Among these nonfumigant nematicides, aldicarb is the most effective when applied preplant or postplant (Dickson, 1998). A postplant application of aldicarb (10 kg a.i./ha) increased peanut yields significantly over the nontreated control (Dickson and Hewlett, 1989). Although nematicides are one of the best management tactics for controlling M. arenaria their continued use in the future has become uncertain because of their economics and e nvironmental hazards (Chen et al., 1996). The nonfumigants are less reliable when M. arenaria population densities are very high (Dickson and Hewlett, 1988). Biological control of M. arenaria is another alternative tactic that is receiving attention (Chen and Dickson, 1998). Worldwide, Pasteuria spp., have the ability to infect and suppress a wide variety of nematodes (Sayre and Starr, 1988). By field observati ons (Bird and Brisbane, 1988; Dickson et al., 1991; Oostendorp et al .,1991) and by using microplot and greenhouse experiments (Channer and Gowen,1988; Chen a nd Dickson, 1998; Davies et al.,1988), it was demonstrated that P. penetrans has the ability to control plant-parasitic nematodes. Pasteuria penetrans is one of the most effective bi ological agents that parasitize M. arenaria in peanut fields in Florida and Georgia (Minton and Sayre, 1989; Dickson et al., 1994). Pasteuria.

PAGE 22

22 penetrans is an endospore forming oblig ate bacterial parasite on Meloidogyne spp. (Chen et al., 1996; Dickson et al., 1994). Although J2 deve lop into females, when parasitized by P. penetrans their fecundity is greatly redu ced (Bird, 1986). Dickson et al. ( 1991) observed increased peanut yields over a period of 5 years in fields n ear Williston, FL that were infested with M. arenaria and P. penetrans Chen et al. (1996), using micropl ot experiments, showed that P. penetrans applied at the rate of 10,000 and 100,000 e ndospores/g in soil infested with M. arenaria race 1 had increased peanut pod yields of 94% and 57% respectively. These results clearly demonstrate the potential of P. penetrans to suppress M. arenaria race 1 in peanut. Further evaluation of P. penetrans revealed that initial endospor e infection of J2 during planting is the main suppressive mechanism of M. arenaria race 1 in peanut (Chen et al., 1997). Additionally, Cetintas and Dickson (2004) studied the long term effects of continuous growth of peanut (4 years) in bahiagrass, rhizomal peanut, and weed fallow plots on persistence of P. penetrans They found weed fallow plots supported the greatest e ndospore densities followed by bahiagrass and rhizomal peanut. They further studied th e vertical distributio n of endospores of P. penetrans and found that rain and irrigation wate r has the potential to move e ndospores deeper into the soil. However, they found the majority of endospores in the top 0 30 cm of soil (Cetintas and Dickson, 2005). The control of M. arenaria by P. penetrans in peanut fields did not appear to be effective in terms of yield until the third growing season (Dickson, 1998; Weibelzahl-Fulton, 1998). Due to economic considerations, simplicity, and ease of use for farmers, nematode resistant cultivars are often the best method of managing M. arenaria (Taylor, 2003). Baltensperger et al. (1986) first reported resistance to M. arenaria in Arachis glabrata Benth., however this species is not cross compatible with cultivated pea nut. In a later study, Nelson et al.

PAGE 23

23 (1989) reported M. arenaria resistance in 21 Arachis species and two inte rspecific hybrids. Although these resistance sources suppress M. arenaria population densities by only 40 60% (Noe et al., 1992), their yield pote ntials are equal to that of the susceptible standards in the absence of M. arenaria pressure and are superior in the presence of M. arenaria pressure (Holbrook et al., 1995). The Texas A&M University peanut breeding program has given considerable attention toward searching, collecting, introduc ing, preserving and evaluating wild Arachis germplasm. Initially, efforts were mainly directed toward ut ilization of the wild species for improvement of the cultigens (Simpson, 1991). All attempts to in trogress root-knot nematode resistance from A. cardenasii and A. chacoensis into Tamnut 74 (a Spanish market type) failed due to a lack of fertility. So, further efforts were focuse d on the development of resistance to M. arenaria through a diploid route (Simpson, 1991). An A. cardenasii x A. chacoensis diploid hybrid was crossed to A. batizocoi The resulting diploid three way hybrid, upon colchicine treatment, produced a tetraploid progeny expressing elevated hybrid vigor, pollen stability (92%), and high fertility. In another attempt, F1 obtained from a cross betw een two diploid (AA) wild Arachis species, A. cardenasii and A. diogoi, were crossed with another diploid (BB) wild species A. batizocoi The diploid (AB) F2, thus obtained, when doubled by colchicine treatment, resulted in the tetraploid interspecific hybrid, TxAG-6, whic h was then crossed with Florunner to produce another interspecific hybrid desi gnated as TxAG-7. This hybrid, when back crossed five times with Florunner, led to the deve lopment of COAN, the first rootknot nematode resistant peanut cultivar (Simpson and Starr, 2001). Two furthe r backcrosses of COAN with Florunner led to NemaTAM (Simpson et al., 2003). Because all of the wild species involved in these crosses were susceptible to root-knot nematodes, except for A. cardenasii, it was hypothesized that resistance

PAGE 24

24 in COAN and NemaTAM was due to introgression of segment(s) of DNA from A. cardenasii (Garcia et al., 1996; Fig. 2-1). In nema tode infested fields both of the M. arenaria race 1 resistant cultivars, COAN and NemaTAM, may have greater yield potential than susceptible cultivars (Dickson and De Waele, 2005). However, in fields not infested with root-knot nematodes, the yield potential of COAN is lower than the highest yielding suscep tible cultivars (Starr et al., 2002). Stalker et al. (1994) also focused th eir efforts to develop resistance to M. arenaria using A. cardenasii as the source of resistance through a hexa ploid route. Several of the breeding lines that were developed had complex segregati on patterns, indicating that resistance to M. arenaria in A. cardenasii is probably conditioned by multiple, dominant, major genes (Starr and Simpson, 1991). Similarly, it was reporte d that resistance to M. arenaria derived from A. cardenasii is controlled by more than two genes with major effects (Garcia et al., 1996; Choi et al., 1999). These observations indicate that more than two genes with major effects confer resistance in A. cardenasii However, resistance in the F2 generation from TxAG6 x A. hypogaea and a derived BC3 population segregated in a 3:1 ratio, indicating a single do minant gene (Church et al., 2000). Bendezu and Starr (2003) also reported that resistance to M. arenaria in COAN is controlled by a single dominant gene. Therefor e, it appears likely that only one gene was introgressed into COAN and subsequently in to NemaTAM due to conventional breeding and selection. NemaTAM and Florunner, being nearly is ogenic, should differ mainly with respect to root-knot nematode resistance. Plant R Genes, Classes and Conserved Functional Domains During plant-pathogen interactions, resist ance and susceptible reactions are often controlled by the compatibility inter action of plant resistance genes ( R genes) and their corresponding pathogen-encoded avirulence genes ( Avr genes). Matched specificity between R

PAGE 25

25 genes and Avr genes trigger signal transduction pathways leading to further activation of host defense mechanisms (Lamb, 1996). Each plant can have many R genes and similarly a pathogen can have many Avr genes. One of the most important char acteristics of the matched interaction between an R gene and an Avr gene is the activation of a hype rsensitive reaction, during which plant cells surrounding the pathoge n infection site undergo progr ammed cell death (Jones and Dangl, 1996). Other features of a defense res ponse include production of salicylic acid, Ca2+ and other ion fluxes, generation of activated oxygen sp ecies such as super oxide (Dixon et al., 1994), and synthesis of antimicrobial metabolites a nd harmful enzymes such as chitinases and glucanases (Dixon and Lam b, 1990; Dixon et al., 1994). Several R genes that confer resistance to a wide spectrum of pathogens, including viruses, bacteria and fungi have been cloned from differen t plant species. The prot ein products of most of these R genes share common sequence motifs that are involved in signal transduction and protein-protein interactions (Michelmore 1996; Hammond-Kosack and Jones 1997; Martin et al. 2003). Based on their deduced amino acid sequences and the combination of R protein structural domains, R genes have been classified into fi ve major classes (Soriano et al., 2005). The first, and the most frequent class, is comprised of genes that code for proteins containing the n ucleotide b inding s ite-l eucine-r ich r epeat (NBS-LRR) domain (Bent et al., 1994; Whitham et al., 1994). The NBS region consists of conserved motifs su ch as the phosphatebinding P-loop (Kinase mo tif) with the consensus sequence Gx GxxGR(T/S), where x stands for any residue, a hydrophobic domain (sequence GLPLxL) and two additional kinase domains, kinase 2 and kinase 3a (Soriano et al., 2005). The kinase 2 domain is proposed to coordinate the metal ion binding required for phospho-transfer reacti ons. Similarly, the arginine in the kinase 3a domain interacts with the purine base of ATP in other proteins (Traut, 1994). Because of the

PAGE 26

26 structural similarity of the NB S region with cell death genes in Caenorhabditis elegans and humans, it is hypothesized to be involved in hypersensitive (HR) cell death upon recognition of Avr genes in plants (Biezen et al., 2002). The LRR region consists of conserved motifs with leucine, and in some cases, proline and asparagine (Soria no et al., 2005). This portion is hypothesized to be involved in protein-protein interactions (Kobe and Deisenhofer, 1994), and to have a pathogen-specific recogniti on function (Takken and Joosten, 2000). Proteins belonging to the NBS-LRR class ca n be further divided into sub-classes depending on their N-termini. These are proteins having a leucine-zipper (LZ) or other coiledcoil sequence (CC) at their N-termini, and those having N-termini referred to as TIR because of their similarity to the Drosophila Toll and the mammalian Interleukin 1 receptor (Bent, 1996; Hammond-Kosack and Jones, 1997; Hulbert et al., 2001). Several R genes belonging to the NBS-LRR class have been cloned and characterized in plants. Among these are the tobacco N gene conferring resistance to tobacco mosaic virus (TMV) (Whitham et al. 1994), Arabidopsis RPM1 and RPS2 conferring resistance to the strains of the bacterial blight pathogen Pseudomonas syringae and RPP5 conferring resistance to the fungus Peronospora parasitica (Bent et al., 1994; Mindri nos et al., 1994), the I2C-1 locus in tomato conferring resistance to the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici race 2 (Ori et al., 1997), the L6 and M genes conferring resistance to flax rust caused by the fungus Melampsora lini (Lawrence et al. 1995; Anderson et al. 1997), and the Xa1 gene conferring resistance to the ri ce bacterial blight pathogen Xanthomonas oryzae (Oshimura et al. 1998). The second class of R genes codes for proteins contai ning extra-cytoplasmic LRR domains and a C-terminal transmembrane domain (Dixon et al. 1996). This class includes the gene

PAGE 27

27 families, Cf-2 Cf-4, Cf-5 and Cf-9 of tomato conferring race-specifi c resistance to the leaf mold pathogen Cladosporium fulvum (Hammond-Kosack et al., 1996). These genes confer resistance by specifically recognizing the avr ge ne products secreted by the fungus C. fulvum (Van den Ackerveken et al., 1992; Jones et al., 1994; Dixon et al., 1996). The third class of R genes encodes proteins with a seri ne/ threonine protei n kinase domain with no LRR domain (Martin et al., 1993). Th is class includes the first race-specific R gene, the tomato Pto gene that governs resistance to the bacterial speck pathogen Pseudomonas syringae pv. Tomato (Martin et al., 1993). Pto upon recognizing the avrPto, codes for a 321-amino acid protein containing a serine/threonine -specific protein kinase domain. Pto is capable of phosphorylating additional proteins which then leads to cellular defense responses against Pseudomonas syringae pv. Tomato (Bogdanove, 2002). The fourth class of R genes code for products that c ontain both the extra cellular LRR domain and the cytoplasmic protein kinase (Son g et al., 1995). This class includes the rice Xa21 gene that governs resistance to over 30 stra ins of the bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae (Song et al., 1995). This gene product possesses both the LRR feature of the Cf-9 protein and a Pto-like seri ne/threonine kinase domain. Theref ore, it was proposed that the Xa21 LRR domain must be involved in rec ognition of extra cellular pathogen avr -gene products, and the kinase must be involved in phosphorylati ng additional proteins th at lead to cellular defense responses against Xanthomonas oryzae pv. oryzae (Hulbert et al., 2001). The fifth, and final, class includes R genes that do not fit into any of the above classes. The best example is the first cloned R gene, the maize Hm1 that encodes a toxin reductase (Johal and Briggs, 1992). This gene confers resistance to Cochliobolus carbonum by encoding a NADPHdependent reductase that inactiv ates a potent plant toxin produ ced by the plant upon infection. In

PAGE 28

28 this case, the defense response does not involve specific recognition of an avr-gene product by the Hm1 gene product (Johal and Briggs, 1992). Several R genes against nematodes have been cloned and characterized. Other than the first nematode R gene identified, Hs1pro-1 all the other nematode R genes share structural homology with other plant disease resistance genes (Baker et al., 1997; Cai et al., 1997; Ellis and Jones, 1998; Williamson, 1998). Hs1pro-1 only contains a le ucine-rich domain but not a true LRR. Additionally, it has an N-terminal signal sequenc e that might facilitate cytoplasmic membrane localization (Cai et al., 1997). Most of the other nematode R genes code for members of a family characterized by NBS-LRRs (Lagudah et al., 1997; Milligan et al., 1998). For example, the potato cyst nematode resistance gene, Gpa2 encodes a product with NBS and LRR domains (Van der Vossen et al., 2000). Similarly, the well characterized tomato nematode resistance Mi protein belongs to NBS-LRR family and has an additional N-terminal region that encodes a leucine zipper domain, which might be involved in protein dimerization (Atkinson et al., 2003). Another tomato nematode resistance gene, Hero codes for a 148 kDa protei n that also belongs to the NBS-LRR family with an additional N-te rminal leucine zipper domain (Atkinson et al., 2003).

PAGE 29

29 Figure 2-1. Breeding scheme used to develop the root-knot nematode resi stant peanut cultivars into COAN and NemaTAM (Burow et al., 1996). A. cardenasii ( AA ) x A. dio g oi ( AA ) F1 diploid hybrid (AA) x A. batizocoi (BB) F2 diploid hybrid (AB) colchicine doubled TxAG-6 x Florunner TxAG-7 x Florunner COAN x Florunner (AABB) (AABB) (AABB) (AABB) (AABB) (AABB) NemaTAM (AABB)

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30 CHAPTER 3 CHARACTERIZATION BY SUPPRESSI ON SUBTRACTIVE HYBRIDIZATION OF TRANSCRIPTS THAT ARE DIFFERENTIALLY EXPRESSED IN ROOTS OF ROOT-KNOT NEMATODE RESISTANT AND SUSC EPTIBLE CULTIVARS OF PEANUT ( ARACHIS HYPOGAEA L.) Introduction Plant-parasitic nematodes are a serious threat to numerous crops around the world and are responsible for average annual crop losses of as much as $78 billion (Sasser and Freckman, 1987). Monetary losses due to nema todes in cultivated peanut ( Arachis hypogaea L.) alone are estimated at $102 million (Sasser, 1990). In the United States, economic losses due to root-knot nematodes ( Meloidogyne species) can be significant in many peanut-producing states (e.g. Alabama, Florida, Georgia, North Carolina, and Texas). Peanut is parasitized by three major Meloidogyne species, viz. M. javanica M. hapla and M. arenaria and each is capable of severely s uppressing yields. Among these root-knot nematodes, M. hapla is the least damaging, whereas M. javanica and M. arenaria are more destructive (Dickson and DeWaele, 2005). For example, when M. arenaria is present at damaging levels, that is as low as a single juvenile/100 cm3 of soil in the case of Florida (McSorley et al.,1992), an approximate 50% decr ease in peanut yield potential can be observed (Dickson and DeWaele, 2005). In peanut, two host races of M. arenaria have been defined based on their ability (race 1) or inability (race 2) to reproduce on the peanut cultivar Florunner (Sasser, 1954). Polyploidization has reproductivel y isolated cultivated tetrap loid peanut from its diploid wild relatives that contain a rese rvoir of resistance genes. The e ffect of this genetic bottle-neck has been a low level of genetic variability in cultivated peanut and th is has resulted in high vulnerability to root-knot nematodes in general, and M. arenaria specifically. Although chemical control of root-knot nematodes using nematicides is most effective (Dickson and Hewlett, 1988),

PAGE 31

31 it has limitations due to governmental regulations and environmental concer ns (Chu et al., 2007). Consequently, deployment of resistant cultivar s has become a more attractive and desirable management tactic (Zijlstra et al., 2000). An effo rt to incorporate rootknot nematode resistance from the wild species, A. cardenasii into cultivated peanut led to the development of the interspecific hybrids, TxAG-6 and TxAG-7 (S impson et al., 1993; Starr et al., 1995). A backcross breeding program was then used to inco rporate the nematode resi stance found in these hybrids into peanut breeding populations, which ultim ately lead to the release of COAN, the first peanut cultivar with resistance to M. arenaria (Simpson and Starr, 2001). Further back-crossing of COAN with Florunner led to th e release of the near isogenic cultivar, NemaTAM (Simpson et al., 2003). It was first reported that the resistance to M. arenaria derived from A. cardenasii is controlled by two genes with majo r effects (Garcia et al., 1996; C hoi et al., 1999). Garcia et al. (1996) reported that two domi nant genes, designated as Mae and Mag, may restrict egg number and galling, respectively, in a segregating F2 population derived from the cross of 4x ( Arachis hypogaea X Arachis cardenasii )-GA 6 and PI 261942. However, Bendezu and Starr (2003) reported that resistance to M. arenaria race 1 in COAN is controlled by a single dominant gene. They also observed that most of the second-st age juveniles (J2) found in the roots of COAN were restricted to the cortical tissue with only one out of 90 J2 observed being associated with vascular tissues. However, greater than 70% of th e J2 were associated with vascular tissues in Florunner (Bendezu and Starr, 2003). They also reported that root-knot nematode resistance in COAN might be due to constituti ve factors in the roots. Howeve r, there have been no gene characterization studies involving root-knot nematode infection in peanut. Consequently, the objective of the present study was to investig ate the molecular mechanisms that may be

PAGE 32

32 functioning in the root-knot nemat ode resistance and susceptible res ponses in peanut as exhibited by NemaTAM and Florunner, respectively. Suppr ession subtractive hybridization (SSH) was used to identify differentially expressed ES Ts in roots of both NemaTAM and Florunner following exposure to root-knot ne matodes. Results obtained lead to a hypothesis for a root-knot nematode resistance mechanism that may be operating in NemaTAM. Materials and Methods Plant Materials and Root-knot Nematode Inoculation Individual plants of NemaTAM and Florunne r were grown in clay pots containing pasteurized soil (Arredondo and Sparr type; sand 95%, silt 3%, clay 2% and organic matter 1.5%; pH 6.5) maintained in a greenhouse at 26 32 oC, where they were watered daily and fertilized once a week with N-P-K (2 4-8-16; Miracle-Gro, Marysville, OH). Ten days after planting, each pot was inoculated wi th a suspension of 5000 eggs of M. arenaria race 1 that had been cultured on tomato ( Solanum esculentum Mill. cv. Rutgers). Eggs were pipetted into three holes (0.6-cm diameter x 4-cm depth) made close to the roots, but distribute d at an equal distance from the base of the plant. Root-knot nematode inoculum was prepared using the NaOCl method as described by Hussey and Barker (1973). Whole roots from five plants per cultivar were collected for RNA extraction at five time points: 0, 12, 24, 48, and 72 h after inoculation. RNA Extraction Total RNA was isolated from the root samples described above using the GuanidineHCl method of Logemann et al. (1987) with slight modification. Roots (3 g) were ground in liquid nitrogen followed by the addition of 15 ml of 6 M Guanidine buffer and 15 ml of phenol/chloroform/isoamyl alcohol (PCI; 25:24: 1). The mixture was shaken for 20 min and centrifuged for 20 min (20, 000 x g) at 4 oC to separate the phases. The supernatant was gently removed and 1.5 vol of absolute ethanol and 0.3 vol of 1M acetic acid (pre-cooled at 4 oC) were

PAGE 33

33 added, and the RNA was precipitated overnight at oC. The pellet obtained after a 20 min centrifugation (20, 000 x g) was resuspended in 5 ml of diethylpyrocar bonate (DEPC)-treated water and the RNA was reprecipit ated by incubation for 1 h at oC after the addition of 300 l of 4 M lithium chloride and 5 ml of cold absolute ethanol. After washing the pellet with 10 ml of cold 70% ethanol, the RNA was air dried at room temperature, resuspended in 1 ml of sterile distilled water, and stored at oC until use. Suppression Subtractive Hybridization (SSH) a nd Differentially Expressed Gene Cloning For each cultivar, equal amounts of root RNA were pooled from the five sampled plants separately for each time point and SSH libraries were constructed by Evrogen (Moscow, Russia) in the following manner. Amplified ds cDNA was prepared from NemaTAM and Florunner RNA using the SMART approach (Zhu et al ., 2001). SMART Oligo II oligonucleotide and cDNA synthesis (CDS) primer were used for firs t-strand cDNA synthesis. In both cases, firststrand cDNA synthesis involved 0.3 g RNA in a total reaction volume of 10 l. One microlitre of diluted (5x) first-strand cDNA was then used for PCR amplification with SMART PCR primer (Fig. 3-1). Nineteen P CR cycles (each cycle included 95 oC for 7 s; 65 oC for 20 s; 72 oC for 3 min) were performed. SMART-amplified cDNA samples of the tester (NemaTAM) and driver (Florunner) cDNA populations were di gested with the re striction endonuclease Rsa I (Gibco) to obtain short, bl unt-ended fragments (Fig. 3-1). Subtractive hybridization was performed using the SSH method in both direc tions (NemaTAM vs. Florunner and Florunner vs. NemaTAM) as described in Diatchenko et al 1996 and 1999. Briefly, for each direction, two tester populations were created by ligation of different suppression adapters (Adapters 1 and 2R). These tester populations were mi xed with 30X driver excess (driver cDNA had no adapters) in two separate tubes, denatured and allowed to renature. After the firs t hybridization, these two samples were mixed and hybridized together. Subtracted cDNA was then amplified by primary

PAGE 34

34 and secondary PCR. The primary PCR cons isted of 26 cycles with primer 1 (5' CTAATACGACTCACTAT AGGGC-3') for subt racted NemaTAM cDNA and Florunner cDNA, respectively (Fig. 3-2). Th e secondary (nested) PCR consis ted of 11 cycles with nested primers 1 (5'TCGAGCGGCCGCCC GGGCA GGT') and 2R (5'AGCGTGGTCGCGG CCGAGGT') for subtracted NemaTAM and Flor unner cDNA samples, respectively (Fig. 3-2). The Mirror Orientation Selection (MOS) procedure was performed for both SSH subtracted libraries as descri bed in Rebrikov et al. (2000). Br iefly, 22 PCR cycles with MOS PCR primer (5'GGTCGCG GCCG AGGT') were performed for subtracted NemaTAM cDNA samples and 23 PCR cycles for subtracted Florun ner cDNA samples. In order to obtain genes that were expressed in NemaTAM, and at lower levels or not at all in Florunner, the reverse subtraction was also carried ou t, using Florunner cDNAs as the tester. NemaTAM as tester and Florunner as driver and vice versa were used during forward and reve rse subtractions. Two subtracted cDNA samples enriched with diffe rentially expressed se quences (NemaTAM specific and Florunner -specific) obtained by MOS PCR were used for library construction (Fig. 3-2). The PCR products after purification were cloned into the vector pAL17 (Evrogen) to produce the subtractive c DNA library, and subsequently transformed into Escherichia coli In each case, approximately 40 ng of purified cDNA was cloned into pAL17 and used for E. coli transformation. For both libraries the ratio of wh ite colonies (recombinant plasmids) to blue colonies (non-recombinant plasmids) was 70:30. Nine ty-five percent of white colonies contained plasmids with insert. Four hundred and eighty (five 96-well plates) ra ndomly picked white colonies, each from both the tester (NemaTAM-specific) library and the driver (Florunner-specific) library, were used for differential screening which was perf ormed by Evrogen (Moscow, Russia) as follows.

PAGE 35

35 All plates were grown in 100 l of LB-Amp ( 75 g/ml) medium for six hours at 37 C. Medium aliquots of 1 l each were used for PCR amp lification with pAL17 forward and pAL17 reverse primers. After the plates were supplied with 20% glycerol and stored at 0 C, 2.0 l of each PCR-amplified insert (about 100 ng DNA) was a rrayed in a 96-well fo rmat onto duplicated nylon membranes (Hybond-N+ nylon membranes, Am ersham International, Little Chalfont, Bucks, UK). After air drying, membranes were de natured in 0.6 M NaOH for 3 min, neutralized in 0.5 M TrisHCl (pH 7.5) for 3 min, and rinsed in distilled water for 1 min. Samples were UV cross linked to membranes for 3 min at 70,000 micro-joules/cm2 (SPECTROLINKER, XL-1000 UV Cross linker, Spectronics Corporati on, Westbury, NY) and then stored at 4 oC for later use. Differential screening of potenti al clones that were highly sp ecific to NemaTAM and Florunner was done by probing nylon membranes dotted by pin replication (Bio-Rad Hercules, CA) of ordered clones of each 96-we ll micro-titration plate with 32P-labeled (labeled by random-primed labeling using the Prime-a-Gene Labeling System ; Promega, Madison, WI) subtracted cDNA of NemaTAM and Florunner. Based upon NemaTAMand Florunner-spe cific signals, clones were selected and sequenced. cDNA Sequence Analysis and Homology Search The ESTs were sequenced using the M13 forward primer at the Interdisciplinary Center for Biotechnology Research (University of Florida, Gainesville, FL ). Sequences were compared with sequences in the NCBI da tabase using BLAST algorithms (http://www.ncbi.nlm.nih.gov/ ). Northern Blot Analysis Northern blot analysis was conducted by Ev rogen (Moscow, Russia) as follows. Equal amounts of RNA (10 g) from root samples collect ed at the five different time points (0, 12, 24, 48, and 72 h) and extracted as described above were separated on a 1.0% agarose/formaldehyde gel at 60 V for 2 h in 1X MOPS buffer followed by transfer to positively charged nylon

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36 membranes (Hybond-NX, Amersham Biosciences, Pi scataway, NJ) by upward capillary transfer with 20X SSC, and then UV cross linke d for 3 min at 70,000 micro-joules/cm2 (Spectrolinker, XL-1000 UV Cross linker, Spectronics Corpor ation, Westbury, NY). Sp ecific probes labeled with a -32P-dCTP were generated by purified PCR amp lification from a plasmid containing the gene fragments of interest (NemaTAM: SSH-NE-PL5-A01, SSH-NE-PL5-C05, SSH-NE-PL5E03, SSH-NE-PL5-F07; Florunner: SSH-FLPL5-B06, SSH-FL-PL5-B10, SSH-FL-PL5-E06, SSH-FL-PL5-F01) of the relevant differen tially expressed clones. After a 30-min prehybridization step performed in ExpressH yp solution (Clontech, Palo Alto, CA) and hybridization with the probe in the same solution for 1 h at 65 oC, the membrane was washed twice in 2X SSC and 0.1% SDS at room temperature for 15 min a nd then washed in 0.1X SSC and 0.1% SDS at 65 oC for 20 min. RT-PCR Reverse transcription of total RNA was carried out using iScript reverse transcriptase (BioRad, Hercules, CA) with oligo-dT as the primer according to the manufacturers instructions. First-strand cDNA from 25 ng total RNA was us ed in each PCR reaction with the primer combinations listed in Table 3-1. PCR was carried out in a MJ-100 thermocycler (MJ Research, Watertown, MA) with th e following program: 94 oC for 1min, followed by 25 or 30 cycles at 94 oC for 15 sec, 60 oC for 20 sec, 72 oC for 15 sec and a final extension at 72 oC for 1 min. PCR products were loaded onto a 1% agarose gel and run in 1X TBE c ontaining ethidium bromide at 75 V for 3 hr.

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37 Results Differential Screening for cDNA Specific to NemaTAM and Florunner Following Inoculation with Meloidogyne arenaria race 1 In total, 960 clones from the NemaTAMand Florunnerspecific libraries were used for differential screening. Of these, 140 clones from the forward library showed a higher level of expression in the root-knot nematode resistant cultivar NemaTAM, while another 123 clones from the reverse library were more highly expr essed in the root-knot nematode susceptible cultivar Florunner. A representative exam ple highlighting the difference between the hybridization patterns with the forward and reverse probes is shown in Fig. 3-3. In order to reduce false positives to the lowest level, a seri es of differential screenings and identification were performed. Clones that showed different hy bridization intensities between the two probes and which displayed at least a threefold higher transc ription level between the cultivars were sequenced. Sequence Analysis and Functional Annotation A total of 263 sequences with an averag e insert size of 550 bp were obtained. These sequences were used in homology searches (B lastn and Blastx) of various databases (nonredundant sequences in NCBI, EST sequences in NCBI, and the potat o and rice EST database at TIGR) with a cut off E-value of 1E-04 (Tables 3-2 and 3-3). A total of 132 sequences were identified using these criteria, 57 from the NemaTAM-specific library and 75 from Florunnerspecific library. As shown in Table 3-2, ~ 30% of the NemaTAM-specific clones matched sequences found in Arabidopsis thaliana and another 21% could be found in other legumes such as Medicago, Vigna, Pisum, Phaseolus and Glycine. Only one of the NemaTAM-specific sequences belonged to Arachis hypogaea (~ 2%). Approximately 32% of the clones have sequences with significant homology to genes found in other plants such as Zea diploperennis

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38 Bowringia mildbraedii, Sorghum bicolor, Sol anum cardiophyllum, Zingiber officianalis, Nicotiana tabacum, Prunus persica, Cucurbita pepo and Trifolium pratense The remaining clones, 15% have sequences related to genes found in other organisms such as Porcupine (rodent), Drosophila pseudoobscura (fruit fly) Burkholderia ambifaria (bacterium), and Cholorobium phaebacteroides (bacterium). As illustrated in Table 3-3, ~ 23% of the Fl orunner-specific sequences were homologous to Arabidopsis thaliana and another 40% belonged to other legumes such as Medicago truncatula, Vigna radiata, Pisum sativa, Phaseolus vulgaris, and Glycine max. Only 3% of the Florunnerspecific sequences belonged to Arachis hypogaea Twenty-seven percent of the other clones have sequences with significant homology to genes found in Sophora alopecuroides Pterocarpus rotundifolius, Cucurbita pepo, Oryza sativa, Nicotiana tabacum, Solanum cardiophyllum, Prunus persica, Beta procumbe ns, Vitis aestivalis, Gymnodenia conopsea, and Malus domestica The remaining clones, 3% have seque nces related to genes found in other organisms such as Strongylocentrotus purpuratus (sea urchin). Putative functions of ~ 55% (49 out of 90) of the NemaTAM-specific sequences and 78% (65 out of 83) of the Florunner-specific sequenc es were assigned (Tables 3-2 and 3-3). The NemaTAMand Florunner-specific sequences we re classified into six primary functional categories according to the putat ive function of their homologous genes in the databases, and the distribution of these genes is illustrated in Fig. 3-4. For NemaTAM, the largest set of genes (38%) was assigned to the response to stress/p lant defense category, followed by genes with unknown function (45%), metabolism (9%), signal transduction and cellular communication (4%), transcriptional activity (2%), and proteolysis (2%). For Flor unner, the largest set of genes (30%) was also assigned to the response to stress/plant defense category, followed by genes

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39 involved in signal transducti on and cellular communication ( 24%), unknown function (22%), transcriptional activity (11%), hydrolase activity (7%), and metabolism (6%). The sequences of both NemaTAM and Florunner that showed no si gnificant homology to sequences deposited in public databases are listed in APPENDIX A and B, respectively. Looking more closely at the major functional category, response to stress/pla nt defense, it can be broken down into the distribution illustrated in Fig. 3-5. For NemaTA M, pathogenesis related (PR) transcripts occupied the largest percentage of the five categories (36%). Th is is followed by genes involved in oxidative stress (23%); unive rsal stress (23%), lectins (9 %), and senescence associated function (9%). For Florunner, lec tins occupied the largest percen tage (52%), followed by rootknot nematode response activity (24%), oxidative stre ss (20%), and senescence associated function (4%). Validation of Differential Gene Expression Results Northern blot analysis was pe rformed to validate the differential screening results. Four clones from each subtracted library were used for analysis, and the results are shown in Figs. 3-6 and 3-7. Clones selected from the NemaTAMsubtracted library (SSH-NE-PL5-A01, SSH-NEPL5-C05, SSH-NE-PL5-E03, SSH-NE-PL5-F07; Fig. 3-6) and the Florunner subtracted library (SSH-FL-PL5-B06, SSH-FL-PL5-B10, SSH-FL-PL5E06, SSH-FL-PL5-F01; Fig. 3-7) showed appropriate levels of differe ntial expression. NemaTAM-speci fic clones when blasted in GenBank showed homology to genes encoding proteins of unknown function. Among Florunnerspecific clones: SSH-FL-PL5-B06 showed homology to a catalase encoding gene, SSH-FL-PL5B10 to a gene encoding an unknown protein a nd the remaining two clones, SSH-FL-PL5-E06, SSH-FL-PL5-F01, showed homology to genes en coding the Hs1Pro1 protein and a syringolideinduced protein, respectively.

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40 Further validation of randomly selected di fferential clones was done by RT-PCR. Total RNA from NemaTAM and Florunner was used. As a control probe, the highly conserved ribosomal subunit 18S, which is expressed in equa l amounts in both cultivars, was used (Figs. 36 and 3-7). Once again, these clones exhibited cultivar-specific expression as originally determined by SSH. Discussion The transcription profiles of infected roots of the root-knot nemat ode resistant peanut cultivar NemaTAM and the suscep tible cultivar Florunner were compared by SSH analysis. The number of transcripts coding for proteins involv ed in plant defense (inc luding PR proteins) was comparatively higher in NemaTAM than in Florunne r. These transcripts include the pathogenesis related mRNAs encoding a major timothy grass po llen allergen, pathogene sis-related protein 1, S-adenosyl-L-methionine: trans-caffeoyl-CoenzymeA 3-O-methyltransferase, disease resistance protein (LRR), arachidic acid-induced DEA1 pr otein, and an ATPRB1/P utative pathogenesisrelated protein. In addition, substantially mo re clones containing cDNAs encoding enzymes responsible for mitigating oxidative stress such as oxidoreductase/zinc ion, NADH: ubiquinone oxidoreductase, copper chaperone homol og CCH/Detoxifies heavy metals, NADPH peroxidase/oxidase, and patatinlike protein were more highly e xpressed in NemaTAM than in Florunner. The expression of PR proteins in the roots of NemaTAM, and possibly other parts of the plant, is likely responsible fo r restricting the entry, establishm ent and development of secondstage juveniles of M. arenaria race 1 (Bendezu and Starr, 2003) They may also be responsible for the failure of the root-knot nematode to comple te its life cycle in the giant cells. In plants, formation of PR proteins plays an important role in both compatible and incompatible interactions and the defense agai nst pathogens. This response to pathogen infection is usually

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41 systemic because the PR proteins not only accumu late at sites of pathogen localization, but also in non-inoculated parts of the plant (Zinoveva et al., 2004). Additionally, Zinoveva et al. (2001) observed that PR-related enzyme activity increases upon nematode in fection, and that it is higher in nematode resistant plants compared to their susceptible counterparts under the same conditions. Similarly, an earlier study by Qui et al. (1997) on tw o soybean cultivars which differ in their susceptibility to M. incognita infection revealed that higher PR protein activity conferred resistance to root-knot nematodes. These results indicate that PR proteins are involved in defense against plant-parasitic nematode s and probably play a major role in NemaTAMs resistance to the root-knot nematode. It has also been well documented that an oxi dative burst plays a central role in plant defense against pathogens, and that reactive oxygen speci es cause damage to the pathogen (Apel and Hirt, 2004). Therefore, it is not surprisi ng that NemaTAM highly ex presses a number of clones encoding peroxidases, oxidoreductases, and heavy metal detoxifiers (Table 3-2). Moreover, Sabine et al. (1997) using pat hogenesis-related experi ments suggested the involvement of oxidoreductases in defense against pathogens. NemaTAM also expressed a number of Univer sal Stress Proteins (USP); however USP expression was absent in Florunne r. In prokaryotes, USP genes are known to be stimulated and required for stress resistance, survival during growth limitation, and oxidative stress resistance (Nystrom and Neidhart, 1994; Nachin et al., 2005). Taken together, differential expr ession of PR and oxidative st ress responsive proteins in NemaTAM suggests that these proteins play a prominent role in defense against M. arenaria race 1. Because a higher level of e xpression of PR proteins requires increased levels of salicylic acid (SA), which is usually followed by universal and oxidative stress responses, it is proposed

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42 that SA receives a signal from R-genes and tr iggers universal stress and oxidative stress responsive genes further triggering downstream expression of pathogenesis-related genes. Therefore, the SA pathway might be ope rating in NemaTAM for resistance against M. arenaria This hypothesis needs to be tested by studying the levels of specific PR gene expression both in uninoculated and root-knot nematode inoculated NemaTAM and Florunner. In addition to the genes described above, seve ral other cDNAs were identified that are potentially related to a root-knot nematode defense response. For example, two clones SSHPL1E05 and SSHPL1D11 (Table 3-2), whic h encode a CCHC-type cytokinin binding protein CBP57 and a transmethyl re gulation protein, respectively ma y interact with other defense genes triggering downstream gene expression. Three putative signaling proteins (SSHPL1D01, SSHPL1B11 and SSHPL1B06 (Table 3-2) may also be involved in cellular communication or signal transduction in plant defense. Among the genes that were represented more of ten in Florunner than in NemaTAM, it is most striking that there are no PR transcripts encoding plant de fense proteins. However, non-PR transcripts, such as those for lectins and Hs1pr o1, were highly expressed in Florunner. There are several reports of lectins havi ng biological activity against nema todes. For example, MarbanMendoza et al. (1987) reported th at concanavalin A suppresses M. incognita when used as a soil amendment in tomato. Burrows et al. (1998) reported that snowdrop lectin (GNA) when expressed in potato decreased the number of females of Globodera pallida Lectins are believed to have potential as external anti-nematode e ffectors by causing behavioral disruption (Atkinson et al., 2003). Although the predic ted protein to be encoded by Hs1pro-1 has a leucine-rich region and a putative transmembrane domain, it does not belong to the NBS-LRR class of plant R genes nor does it fit any other existing R protein class (Thurau et al., 2003). Therefore, it has been

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43 proposed that Hs1pro-1 represents an entirely new class of R genes (Jung et al., 1998). Thurau et al. (2003) also reported that the promoter of Hs1pro-1 upon activation triggered nematoderesponsive gene expression in sugar beet ( Beta vulgaris L.) and Arabidopsis thaliana That study clearly demonstrated that upon nematode infection, Hs1pro-1 was locally induced; and its transcriptional activity reached its maximum one day after nematode infection. Based on these results, it appears that enhanced expression of Hs1pro-1 in feeding structures might be crucial for nematode resistance. Taking th ese results into consideration Florunner may exhibit resistance to other plant-parasitic nematodes such as Globodera pallida but remain susceptible to M. arenaria due to the absence of specific R genes. Florunner expressed a number of genes i nvolved in signal transduction and cellular communication, such as the calcium-binding EF-h and and syringolide-induced (SIH) proteins. Analysis by Day et al. (2002) i ndicated that EF-hand-containing proteins are probably the key transducers mediating Ca2+ action. Hagihara et al. (2004) reported that Ca2+ is required for hypersensitive cell death (HCD) and the induction of SIH genes. These results suggest that the induction of SIH genes could be activated throug h the syringolide specific signaling pathway and the SIH gene products may play an important role in the processes of HCD. Based on this study, it is proposed that upon the perception of stress from primary and secondary signaling molecules following root-knot nematode infection in Fl orunner there is an increase in calcium concentration activating EF-ha nd-containing proteins that are probably the key transducers mediating Ca2+ action. This event is followed by syri ngolide-specific signaling pathway and the SIH gene products may play an importa nt role in the processes of HCD. Among additional Florunner-specific tr anscripts that amplified were Phi and XET genes reported to be regulated by ABA (Sano and Nagata 2002; Wu et al., 1994). It appears that ABA

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44 induced gene expression of the Phi-1 gene alleviate fluctuations in intracellular pH (Sano and Nagata, 2002). These results suggest that Flor unner may counteract the non-synchronous cell division caused by root-knot nema tode attack by expression of Phi-1 It can be further proposed from the above studies that stress due to r oot-knot nematode attack might result in ABAmediated acidification of the cytoplasm. Cytoplasmic acidification further triggers Phi-1 and Phi-2 genes which are reported to induce semi-s ynchronous cell division resulting in giant cells/feeding sites for M. arenaria (Sano and Nagata, 2002). Furthe r functional characterization of the Phi-1 gene should unravel the role of phosphate in inducing cell divi sion in plant cells. Xyloglucan endotransglycosyl ase (XET) has been proposed to cause cell wall-loosening by cleaving xyloglucan molecules and there by promoting micro fi bril separation (Smith and Fry, 1989; Nishitani and Tominaga, 1991; Fry et al., 1992). On the other hand, Sharp et al. (1994) reported that exogenous application of ABA restored the activity of XET which in turn resulted in recovery of the root elongati on rate. Based on these previous re ports and expression analysis of XET, It is proposed that XET gene expres sion may be regulated vi a ABA signaling by one of the transcription factors of M. arenaria to loosen the cell walls for efficient feeding of cell contents. In summary, 263 differentially expressed genes were detected in two near-isogenic peanut cultivars, NemaTAM and Florunner, which diffe r only with respect to their resistance to M. arenaria race 1. The major conclusion of this study is that, in addition to the expression of a number of well-characterized PR proteins, high-level expression of genes encoding USPs may be major factors in rendering NemaTAM resistan t to infection by the root-knot nematode M. arenaria race 1. These transcripts were absent in the root-knot nematode susceptible cultivar

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45 Florunner. However, the expression of lectins, Hs1pro-1 and SIH responsive genes in Florunner indicates a basal level of plant defense against M. arenaria Elucidating the molecular basis of rootknot nematode resistance found in NemaTAM should contribute to the understand ing of nematode-plant interac tions and assist in breeding future nematode-resistant peanut cultivars. Furt hermore, this information provides candidate genes that can be further analyzed for th eir utility against root-knot nematodes.

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46 Table 3-1. RT-PCR primer combinations. Clone Forward Primer Reverse Primer SSH1-NE2-PL53-A014 5-TACTCTGGCATCGCAAAATG-3 5-AGGAGGTTCTGCTCCAATGA-3 SSH-NE-PL5-C05 5GCCGAGGTACACATCAGTGG -3 5GGCCGAGGTACATAAGGAGA -3 SSH-NE-PL5-E03 5GTATGGCAAAGCCTTCCTGA -3 5CGAAATAGGGGAACACATGG -3 SSH-NE-PL5-F07 5CGCGGGGATATACAATCTCA -3 5CACAATTGGACGTGGATACG -3 SSH-FL5-PL5-B06 5ACCAACTTGCAACACAACCA -3 5TTGCAGAGAGGTTTGTGGAAT -3 SSH-FL-PL5-B10 5GGCAATTCTTTCCGTTTTCA -3 5GTCATCAGGGCCGTTAGAGA -3 SSH-FL-PL5-E06 5CCAGATTGTGGAGTCCTGGT-3 5CACCTCCACTAGCTCCTTCG -3 SSH-FL-PL5-F01 5-TGCAAATGGTGCTTCTCTTG -3 5-AACCAAGCCTTCAGCAACAC-3 1SSH = Suppression Subtra ctive Hybridization; 2NE = NemaTAM; 3PL=Plate 5; 4AO1= Clone A01 in plate 5; 5FL=Florunner.

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47Table 3-2. Identification of fifty-seve n EST clones from the root-knot nema tode resistant cultivar NemaTAM. Clone ID Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Response to Stress Pathogenesis related (PR) proteins SSHPL1H08 Ne469375 INIOB 3E-04 Phleum pretense Timothy grass pollen allergen SSHPL1H03 Ne469380 ABA34077 8E-06 Zea diploperennis Pathogenesis-related protein 1 SSHPL1G07 Ne469388 AAA62426 4E-05 Arabidopsis thaliana S-adenosyl-L-methionine:trans-caffeoylCoenzymeA3-O-methyltransferase SSHPL1E07 Ne469412 XP_476483 1E-15 Oryza sativa Putative Pathogenesis-related protein SSHPL1D09 Ne469422 CAA65419 1E-17 Arabidopsis thaliana Pathogenesis-related protein SSHPL1C07 Ne469436 ABD28507 4E-11 Medicago truncatula Disease Resistance Protein(LRR) SSHPL1B10 Ne469445 CAI51313 4E-22 Capsicum chinense Arachidic acid-induced DEA1 protein SSHPL2A07 Ne472920 XP_476500 1E-15 Oryza sativa Pathogenesis-related protein 1 SSHPL2A04 Ne472923 NP_179064 8E-08 Arabidopsis thaliana ATPRB1/Putative pathogenesis-related protein SSHPL2A01 Ne472926 CAI51313 2E-14 Capsicum chinense Arachidic acid-induced DEA1 protein SSHPL2B10 Ne472984 AA033592 5E-08 Arachis hypogaea Putative cold stress responsive protein SSHPL2C01 Ne472981 AAW38998 8E-15 Arabidopsis thaliana Pathogenesis related protein Lectins SSHPL1H11 Ne469372 1QMOH 4E-07 Dolichos lab lab Lectin SSHD07 Ne469424 P42088 7E-35 Bowringia mildbraedii Lectin SSHPL2A03 Ne472924 P42088 1E-40 Bowringia mildbraedii Lectin Senescence associated function SSHPL2B03 Ne472991 AAR13310 4E-35 Phaseolus vulgaris B12D-Like protein SSHPL2B01 Ne472993 AAR13310 3E-35 Phaseolus vulgaris B12D-Like protein SSHPL2D01 Ne472969 AAR13310 8E-35 Phaseolus vulgaris B12D-Like protein

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48 Table 3-2. (Continued) Clone ID Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Oxidative stress SSHPL1A07 Ne469384 NP_197201 4E-30 Arabidopsis thaliana Oxidoreductase/Zinc ion SSHPL1F10 Ne469397 EAL33043 2E-06 Drosophila pseudoobscura NADH:Ubiquinone oxidoreductase SSHPL1F09 Ne469398 1OXWC 7E-15 Solanum cardiophyllum Patatin-like Protein1 SSHPL1E01 Ne469418 CAA89262 8E-87 Arabidopsis thaliana NADH Oxidoreductase SSHPL1C11 Ne469432 AAF15286 3E-35 Glycine max Copper Chaperone Homolog CCH/Detoxifies heavy metals SSHPL1C02 Ne469441 NP910315 1E-05 Oryza sativa NADPH peroxidase/Oxidase SSHPL1B12 Ne469443 NP_199172 2E-14 Sorghum bicolor Patatin-like Protein SSHPL2C03 Ne472979 AAF15286 7E-38 Glycine max Copper Chaperone Homolog CCH/Detoxifies heavy metals Universal stress SSHPL1A02 Ne469369 NP_566108 1E-31 Arabidopsis thaliana Universal stress protein (Usp) SSHPL1G08 Ne469387 ABA34077 8E-06 Cholorobium phaebacteroides Universal stress protein (Usp) SSHPL1C12 Ne469431 ABE85050 8E-29 Medicago truncatula Universal stress protein (Usp) SSHPL1C08 Ne469435 ABE85050 3E-29 Medicago truncatula Universal stress protein (Usp) SSHPL1C04 Ne469439 ABE85050 2E-28 Medicago truncatula Universal stress protein (Usp) SSHPL1C03 Ne469440 ABE85050 4E-29 Medicago truncatula Universal stress protein (Usp) SSHPL1BO7 Ne469448 NP_566108 2E-29 Arabidopsis thaliana Universal stress protein (Usp) SSHPL1BO7 Ne469448 NP_566108 1E-29 Arabidopsis thaliana Universal stress protein (Usp)

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49 Table 3-2. (Continued) Clone ID Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Transcriptional Activity SSHPL1E05 Ne469414 ABE83455 7E-07 Medicago truncatula Zinc finger, CCHC-type SSHPL1D11 Ne469420 BBA03710 3E-35 Nicotiana sylvestris Cytokinin binding proteinCBP57 Signal Transduction and Cellular Communication SSHPL1D01 Ne469430 NP_178306 1E-68 Arabidosis thaliana Transporter protein/Putative endomembrane protein SSHPL1B11 Ne469444 XP_470213 5E-46 Oryza sativa Tonoplast intrinsic protein SSHPL1B06 Ne469449 BAD22802 8E-59 Prunus persica Nitrate transporter Metabolism SSHPL1B02 Ne469357 NP_200261 3E-78 Arabidopsis thaliana Flavodoxin-like Quinone reductase 1 SSHPL1H07 Ne469376 4MDHB 2E-11 Sus scrofa heart (procupine) Cytoplasmic Malate dehydrogenase SSHPL1G11 Ne469384 ZP_006888621E-58 Burkholderia ambifaria 5-methyl tetrahydropteroyltriglutamate homocysteine SSHPL1F08 Ne469399 ZP_010439801E-20 Idiomarina baltica Carbamoylphosphate synthase small subunit SSHPL1E04 Ne469415 NP_199172 5E-16 Arabidopsis thaliana nutrient reservoir/patatin like phospholipase SSHPL1E03 Ne469416 BAD38550 7E-49 Oryza sativa nutrient reservoir/putative patatin like homolog SSHPL1B04 Ne469451 AT46998 3E-20 Glycine max Triosephosphate isomerase SSHPL2C02 Ne472980 AAY86360 1E-38 Acacia species Cinnamoyl-CoA reductase

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50 Table 3-2. (Continued) Clone ID Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Proteolysis SSHPL2B12 Ne472982 NP_565330 2E-57 Arabidopsis thaliana Peptidase/Subtilase/proteolysis SSHPL2D06 Ne472964 BAE71300 3E-23 Trifolium pretense Putative snRNP 70K protein Unknown Function SSHPL1H06 Ne469377 1OXWC 2E-07 Solanum cardiophyllum Hypothetical protein SSHPL1B01 Ne469358 NP_189632 3E-28 Arabidopsis thaliana Unknown protein SSHPL1A04 Ne469367 NP_198805 4E-45 Arabidopsis thaliana Unknown protein SSHPL1A03 Ne469368 NP_568343 8E-06 Arabidopsis thaliana Unknown protein SSHPL1G06 Ne469389 EAL31846 2E-49 Drosophila pseudoobscura GA14712-PA gene product SSHPL1D05 Ne469426 NP_910315 1E-05 Oryza sativa Unknown protein SSHPL2C04 Ne472978 NP_568343 1E-05 Arabidopsis thaliana Unknown protein SSHPL2D08 Ne472962 NP_910315 6E-06 Oryza sativa Unknown protein

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51Table 3-3. Identification of seventy-five EST clones from the root-knot nema tode susceptible cultivar Florunner. Clone ID. Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Response to Stress Lectins SSHPL3B02 FL468983 AAY68291 2E-06 Sophora alopecuroides Lectin SSHPL3A05 FL468992 AAY68291 2E-29 Sophora alopecuroides Lectin SSHPL3H07 FL469002 AAY68291 1E-04 Sophora alopecuroides Lectin SSHPL3H03 FL469006 ABE89758 2E-18 Medicago truncatula Concanavalin A-like lectin / glucanase SSHPL3G05 FL469016 AAT57665 3E-45 Pterocarpus rotundifolius Lectin SSHPL3D12 FL469045 AAT57665 1E-45 Pterocarpus rotundifolius Lectin SSHPL3D11 FL469046 AAT57665 2E-42 Pterocarpus rotundifolius Lectin SSHPL3C12 FL469057 AAT57665 5E-43 Pterocarpus rotundifolius Lectin SSHPL3C10 FL469059 AAT57665 4E-44 Pterocarpus rotundifolius Lectin SSHPL3C05 FL469064 AAT57665 2E-45 Pterocarpus rotundifolius Lectin SSHPL3C02 FL469067 AAT57665 3E-47 Pterocarpus rotundifolius Lectin SSHPL2F07 FL472939 AAT57665 5E-46 Pterocarpus rotundifolius Lectin SSHPL2E12 FL472946 AAT57665 1E-47 Pterocarpus rotundifolius Lectin Nematode responsive gene activity SSHPL3E02 FL469043 AAG44839 6E-25 Glycine max Putative Hs1pro1-like receptor SSHPL3D07 FL469050 AAG44839 3E-23 Glycine max Putative Hs1pro1-like receptor SSHPL3D05 FL469052 AAG44839 1E-26 Glycine max Putative Hs1pro1-like receptor SSHPL3C09 FL469060 AAF67003 4E-07 Pisum sativa Putative Hs1pro1-like homolog SSHPL2E08 FL472950 AAG44839 8E-27 Glycine max Putative Hs1pro1-like receptor SSHPL3A10 FL468987 AAZ39087 3E-25 Beta procumbens Nematode resistance protein Senescence associated function SSHPL3F12 FL469021 BAD42919 2E-52 Arabidopsis thaliana Senescence-associated protein

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52 Table 3-3 (Continued) Clone ID. Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Oxidative stress SSHPL3D04 FL469053 P48350 4E-51 Cucurbita pepo Catalase isozyme1 SSHPL3A12 FL468985 AAZ29552 5E-76 Oryza sativa Catalase-C SSHPL3H11 FL468998 ABE83062 8E-11 Medicago truncatula Heavy metal detoxi fication protein SSHPL2F04 FL472942 AAC06243 3E-08 Nicotiana tabacum Osmotic stress-induced protein SSHPL2E11 FL472947 CAD42909 9E-69 Prunus persica Catalase Transcriptional Activity SSHPL3A02 FL468995 ABE86987 6E-13 Medicago truncatula DNA-binding WRKY SSHPL3H09 FL468900 ABE86850 5E-12 Medicago truncatula Basic helix loop helix (bHLH) factor SSHPL3H06 FL469003 ABE_93963 9E-47 Medicago truncatula Zinc finger,C2H2-type SSHPL3H05 FL469004 ABE86271 2E-33 Medicago truncatula Homeodomain related SSHPL3F04 FL469029 ABD66513 1E-30 Gymnodenia conopsea Eukaryotic translati on initiation factor SSHPL3E05 FL469040 ABE85589 2E-05 Medicago truncatula Zinc finger protein SSHPL3D03 FL469054 ABE93963 3E-50 Medicago truncatula Zinc finger,C2H2-type SSHPL3C11 FL469058 AAR92477 1E-18 Vitis aestivalis Putative WRKY transcription factor 30 SSHPL3B12 FL469069 AAM15234 8E-06 Arabidopsis thaliana Putative bHLH transcription factor

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53 Table 3-3 (Continued) Clone ID. Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Signal Transduction and Cellular Communication Calcium-binding EF-Hand SSHPL3B01 FL468985 ABE78935 3E-46 Medicago truncatula Calcium-binding EF-hand SSHPL3A11 FL468986 ABE78935 1E-45 Medicago truncatula Calcium-binding EF-hand SSHPL3G08 FL469013 ABE78935 6E-39 Medicago truncatula Calcium-binding EF-hand SSHPL3G07 FL469014 NP_173355 8E-26 Arabidopsis thaliana DNA binding protein SSHPL3F09 FL469024 ABE78935 1E-29 Medicago truncatula Calcium-binding EF-hand SSHPL3E03 FL469042 ABE78935 1E-32 Medicago truncatula Calcium-binding EF-hand SSHPL3C06 FL469063 ABE78935 3E-30 Medicago truncatula Calcium-binding EF-hand SSHPL2F03 FL472943 ABE78935 2E-32 Medicago truncatula Calcium-binding EF-hand SSHPL2D10 FL472960 ABE78935 5E-37 Medicago truncatula Calcium-binding EF-hand Phi-1 protein (Phosphate induced activity) SSHPL3A07 FL468990 CAJ13706 1E-46 Capsicum chinense Phi-1 protein SSHPL3A01 FL468996 CAJ13706 2E-16 Capsicum chinense Phi-1 protein SSHPL3H01 FL469008 CAJ13706 7E-115 Capsicum chinense Phi-1 protein SSHPL3G09 FL469012 CAJ13706 3E-30 Capsicum chinense Phi-1 protein SSHPL2F08 FL472938 BAA33810 2E-28 Nicotina tabacum Phi-1 protein Auxin related activity SSHPL3G05 FL469018 O24543 5E-43 Vigna radiate Auxin induced protein 22E SSHPL3D06 FL469051 AAZ20292 2E-11 Arachis hypogaea Auxin repressed protein SSHPL3C08 FL469061 AAZ20292 2E-25 Arachis hypogaea Auxin repressed protein Syringolide-induced proteins SSHPL3G02 FL469019 BAB86896 2E-55 Glycine max Syringolide-induced protein 13-1-1 SSHPL3F10 FL469023 BAB86896 4E-38 Glycine max Syringolide-induced protein 13-1-1 SSHPL2E10 FL472948 BAB86896 6E-59 Glycine max Syringolide-induced protein 13-1-1

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54 Table 3-3 (Continued) Clone ID. Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Nodulin SSHPL3F07 FL469026 AAC32828 9E-05 Glycine max Nodulin Hydrolase Activity SSHPL3F05 FL469028 NP_172939 8E-43 Arabidopsis thaliana Hydrolase SSHPL3DO9 FL469049 NP_173266 1E-53 Arabidopsis thaliana Hydrolase SSHPLED01 FL469056 NP_173266 3E-52 Arabidopsis thaliana Hydrolase SSHPL3C01 FL469068 NP_173266 1E-42 Arabidopsis thaliana Hydrolase SSHPL2F02 FL472944 NP_173266 1E-51 Arabidopsis thaliana Hydrolase SSHPL2D11 FL472960 NP_173266 5E-50 Arabidopsis thaliana Hydrolase Unknown Function SSHPL3H10 FL468999 XP_792559 1E-09 Strongylocentrotus purpuratus Hypothetical protein SSHPL3F01 FL469032 AAU44522 4E-06 Arabidopsis thaliana Hypothetical protein AT4G27810 SSHPL3E12 FL469033 ABE92227 3E-46 Medicago truncatula Conserved hypothetical protein SSHPL3B03 FL469078 ABE92227 4E-34 Medicago truncatula Conserved hypothetical protein SSHPL3G06 FL469015 NP_564307 2E-63 Arabidopsis thaliana Unknown protein SSHPL3G06 FL469044 NP_566063 3E-44 Arabidopsis thaliana Unknown protein SSHPL3B08 FL469073 NP_196762 1E-39 Arabidopsis thaliana Unknown protein SSHPL2F09 FL472937 NP_179949 4E-16 Arabidopsis thaliana Unknown protein SSHPL2E05 FL472953 NP_182061 3E-17 Arabidopsis thaliana Unknown protein SSHPL2E04 FL472954 NP_195718 1E-05 Arabidopsis thaliana Unknown protein

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55 Table 3-3 (Continued) Clone ID. Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function Others SSHPL3D02 FL469055 AAN07898 1E-100 Malus domestica Xyloglucan endotransglycosylase (XET) SSHPL2F10 FL472936 ABC68415 2E-39 Glycine max Cytochrome P450 Monoxygenase SSHPL2D09 FL472961 ABE82347 4E-21 Medicago truncatula Las1-like protein/ Cell morphogenesis and cell surface growth SSHPL2E06 FL472952 NP_181933 1E-10 Arabidopsis thaliana Riboflavin synthase

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56 Figure 3-1. Double-stranded cDNA synthesis and Rsa I digestion. Lanes: M = 1 Kb DNA size marker; 1 = SMART-amplified NemaTAM cDNA (tester); 2 = SMART-amplified Florunner cDNA (driver); 3 = Rsa I digest ed NemaTAM cDNA; 4 = Rsa I digested Florunner cDNA. 0.25 Kb 0.75 Kb 0.5 Kb M 1 2 3 4

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57 Figure 3-2. Agarose gel electrophoresis of prim ary and secondary PCR products. Lanes: M = 1 Kb DNA size markers; 1 = Primary P CR of NemaTAM subtracted cDNA; 2 = Primary PCR of Florunner subtracted cDNA; 3 = Secondary PCR of NemaTAM subtracted cDNA; 4 = Secondary PCR of Florunner subtracted cDNA; 5 = Mirror Orientation Selection PCR of NemaTAM subtracted cDNA; 6 = Mirror Orientation Selection PCR of Florunner subtracted cDNA; 7 = Unsubtracted NemaTAM cDNA; 8 = Unsubtracted Florunner cDNA. 0.25 Kb 0.5 Kb 0.75 Kb M 1 2 3 4 5 6 7 8 M

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58 Figure 3-3. Differential screening results. Ninety-six PCR-amplified inserts each of NemaTAM and Florunner (about 100 ng DNA) were arrayed in a 96-well format onto duplicated nylon membranes and hybridized with eith er P-32-labelled subtracted cDNAs of NemaTAM (NE probe) or Florunner (FL probe). 1 2 3 4 5 6 7 8 9 101112 A B C D E F G H A B C D E F G H F L p r o b e 1 2 3 4 5 6 7 8 9 10 11 12 N E p r o b e

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59 0 5 10 15 20 25 30 35 40 45 50Response to stress Transcriptional activity Signal transduction and cellular communication Metabolism Proteolysis Hydrolase activity Unknown functionMajor functional categoriesNumber of sequences NemaTAM Florunner Figure 3-4. Comparison of major functional ca tegories of NemaTAM and Florunner expressed sequences. Note: The unknown function cate gory is comprised of clones which showed significant homology to hypotheti cal and unknown proteins, and the clones which gave no significa nt hits in GenBank. Figure 3-5. Subcategories within the response to stress plant defense functional category for NemaTAM and Florunner. Senesence associated function{9%} Pathogenesis related function(PR-proteins){36%} Universal stress{23%} Lectins{9%} Oxiative stress{23%} Lectins{52%} Nematode responsive activity{24%} Senesence associated function{4%} Oxiative stress{20%} 36% 9% 9% 23% 23% NemaTAM 52% 24 % 4% 20% Florunner

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60 Figure 3-6. Gene expression anal ysis of differential clones (SSH-NE-PL5-A01, SSH-NE-PL5C05, SSH-NE-PL5-E03, and SSH-NE-PL5F07) obtained from the NemaTAM subtracted library. A = Northern blot an alysis of differential clones; B = RT-PCR analysis of differential clones; Lane 1, 3, 5, 7 = NemaTAM; Lane 2, 4, 6, 8 = Florunner. Figure 3-7. Gene expression an alysis of differential clone s (SSH-FL-PL5-B06, SSH-FL-PL5B10, SSH-FL-PL5-E06, and SSH-FL-PL5-F 01) obtained from the Florunner subtracted library. A = Northern blot an alysis of differential clones; B = RT-PCR analysis of differential clones; Lane 1, 3, 5, 7 = NemaTAM; Lane 2, 4, 6, 8 = Florunner. A01 C05 E03 F07A B 18S B A 18S 1 2 3 4 5 6 7 8 B06 B10 E06 F01 1 2 3 4 5 6 7 8

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61 CHAPTER 4 MOLECULAR MARKER-BASED SELECTI ON FOR COMBINING ROOT-KNOT NEMATODE RESISTANCE WITH HIGH OLEIC ACID CONTENT IN FLORIDA BREEDING LINES Introduction Cultivated peanut ( Arachis hypogaea L.), is grown in over 110 countries between 40o N and 40o S for both human consumption and as an oil se ed crop. It occupies approximately 31% of the total cropped area for oil seeds and accounts for 36% of to tal oil seed production in the world, ranking fifth among oil seed crops (Marcio et al., 2004). In the United States, peanuts are mostly grown in the south where they have sign ificant economic impact. Consumption of peanut in the United States is about 6 pounds annually per person (B eyer et al., 2001). Consumers in the United States have become increasingly health conscious with respect to their food intake (Isleib et al ., 2004). Consequently, the peanut industry has a greater concern regarding the composition of peanut seed, so as to enhance the marketability of various peanut products. Peanut seed composition is largely a function of lipid chemistry. Peanut contain approximately 50% oil (Cobb and Johnson, 1973). Pa lmitic acid (16:0), oleic acid (18:1), and linoleic acid (18:2) are the major fatty acids f ound in peanut seeds and th ey may comprise > 90% of the total fatty acids (Ahmed and Young, 1982; Norden et al., 1987). Peanuts contain mostly unsaturated fatty acids which are associated with improved cardiovascular h ealth (Paterson et al., 2004). For instance, it has been reported that di ets with high monounsaturated fatty acids (i.e. 18:1) and low saturated fatty aci ds reduce plasma cholesterol le vels (Moore and Knauft, 1989), serum cholesterol levels (OByr ne et al., 1997) and low-density lipoproteins (LDL) in humans (Grundy, 1986; Kris-Etherton et al ., 1999). Peanut oil with reduced linoleic acid and high oleic acid content has several advantages, such as enhanced nutritional quality and improved oil stability (St Angelo and Ory, 1973; Grundy, 1986) enhanced shelf life (Moore and Knauft,

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62 1989) decreased risk of rancidity of the roasted peanut s (OKeefe et al., 1993; Braddock et al., 1995), significant human health advantages ( cardiovascular) and enhanced marketability (Andersen and Gorbet, 2002). Norden et al. (1987) first reported a peanut line, UF435, with 80% oleic and 2% linoleic acid. High oleic peanut varieties, SunOleic 95R (Gorbet and Knauft, 1997) and SunOleic 97R (Gorbet, 2000), were obtained from a cross between the high oleic breeding line UF435 and a component line of Sunrunner (Gorbet and Knau ft, 1997). Moore and Knauft (1989) reported that the high oleic acid trait is controlled by two recessive genes ol1 and ol2, and if both genes are present, the oleic acid content increases to grea ter than 70% and linoleic acid content to below 5%. Although high oleic varietie s have improved lipid chemistry and are popular among some peanut processors, they are highly su sceptible to root-knot nematodes. In addition to genetic determinants and abioti c factors, peanut seed quality and production are affected by numerous biotic st resses such as insects, fungi, vi ruses, bacteria and nematodes. Peanut root-knot nematodes are major parasite s in all peanut-growing regions of the world. Economic losses due to root-knot nematodes can be substantial in many peanut-producing areas in the United States (e.g. Flor ida, Georgia, Alabama, Texas a nd North Carolina). The two major Meloidogyne species, M. arenaria (peanut root-knot nematode) and M. hapla (Northern rootknot nematode), parasitize peanut and are respon sible for causing most of the severe yield decreases. The reduction in peanut yield and quality is the result of heavy galling of roots, pegs, and pods following root-knot nematode infection of the root system (Rodriguez-Kabana et al., 1986). Although nematicides like Telone (1, 3 Dichloropropene) can control root-knot nematodes, they may be harmful to other bene ficial organisms in the soil and risky to the applicator. Moreover, nematicides increase prod uction costs and are hazardous to farmers.

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63 Therefore, the development and deployment of root-knot nematode-resistant peanut cultivars in combination with crop rotation is the most desi rable alternative to re duce root-knot nematode damage. However, screening for resistance to root-knot nematode infection within A. hypogaea has not identified good resistant sources (Holbrook and Noe, 1992). Fortunately, high levels of resistance were observed in seve ral wild, diploid species (Nels on et al., 1989; Stalker et al., 1987). Two interspecific hybrids, TxAG-6 and TxAG7, which are highly resistant to root-knot nematodes, as well as major foliar diseases, lik e leaf spot and rust, we re developed by Simpson et al. (1993). TxAG-6 was backcrossed with the cu ltivar Florunner to develop two new cultivars COAN (Simpson and Starr, 2001) and NemaTAM (Simpson et al., 2003). Although they are resistant to M. arenaria race1, these cultivars have reduced yi eld potential and are low in their oleic/ linoleic fatty acid ratio (NemaTAM is 1.3, Florunner is 1 .6; Simpson et al., 2003). With these factors under consideration, breeding e fforts were focused on combining root-knot nematode resistance with the high oleic acid trait (80% 18:1) into agronomi cally superior peanut varieties. This effort should result in the develo pment of peanut cultivars with better qualities for consumers as well as improved efficiency for producers. Markers such as Random Amplified Polymorp hic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP) have been used to monitor in trogression of wild Arachis chromosome segments into cultivated pea nut (Garcia et al., 1995). Several RAPD (RKN 229, RKN 410, and RKN 440) and RFLP (R2430E, R 2545E, and S1137E) markers have been reported to be linked to root-knot nematode resistance loci in peanut (Burow et al., 1996; Choi et al., 1999). Some of these markers, RKN 229, RK N 410 (Burow et al., 1996) and R2430E (Choi et al., 1999) are linked to the root-knot nematode resistance locus in the breeding lines and

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64 cultivars derived from TxAG-6. These reports in dicate that root-knot ne matode resistance in these genotypes is inherited as a single dominant gene that was derived from A. cardenasii (Dickson and DeWaele, 2005). The above markers were used to select indi viduals for root-knot nematode resistance from segregating populations in the development of COAN and NemaTAM (Garcia et al., 1996; Church et al ., 2000). However, these studies did not examine the use of both RAPD and RFLP markers in the same breeding material to de termine which would be more efficient. Peanut cultivars that are high oleic and resistant to root-kno t nematode are currently not available. Consequently, an objective of the Univ ersity of Florida pea nut breeding program was to develop peanut breeding lines that have root-knot nematode ( M. arenaria ) resistance along with high oleic acid content. The interspecific hybrids, TxAG-6 and TxAG-7, which were used in the development of COAN, were used as parent s for root-knot nematode resistance in Florida breeding material. Populations were initially selected for resistan ce to leaf spot, tomato spotted wilt virus, and agronomic traits, but not for root-knot nematode re sistance. As mentioned earlier, there were reports identifying molecular markers li nked to a root-knot nematode resistance locus, but very limited data were available to compar e the efficiency of marker-assisted selection procedures to other selection techniques. In th e present study, the utili ty of marker-assisted selection using a previously id entified SCAR marker, Z3/265 (Garcia et al., 1996) and the RFLP marker R2430E (Church et al., 2000) is reporte d for combining root-knot nematode resistance with high oleic acid content in peanut.

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65 Materials and Methods Plant Materials Experiment 1 Six University of Florida breeding lines (F1334; F79x4-6; F94x30-8-2-1-b3; F94x30-8-31-b2; F94x30-8-2-2-b3; F94x30-52-3-3-b3), and the Texas A&M University breeding line, TP301-1-8, were planted in field plots in 2001 at the Plant Science Unit, Teaching and Research Center, University of Florida, Gainesville, FL. The field plots were two 5.0 m long rows with 90 cm inter rows with four replications. The fi eld was infested with root-knot nematodes, M. arenaria race 1 based on established protocols for reproduction (Starr et al, 1995). During the development of the F94x30 lines, the interspecifi c hybrid TxAG-6 was used as a parent. Peanut cultivars Florunner and COAN were included in this test as root-knot ne matode susceptible and resistant controls, respectivel y. Ten plants were labeled in each plot. Young leaves were collected from those plants, frozen in liquid N2, and stored at oC until use. Root-knot nematode reproduction was measured on the same 10 plants previously labeled in each replication and used for molecular ma rker analysis, as described below. Experiment 2 Crosses were made in 2002 by the University of Florida breeding program involving the root-knot nematode resistant pa rent COAN and root-knot nema tode susceptible parents HULL (Gorbet, 2007a), ANorden (Gorbet, 2007b), F 89XOL14-1-4-1-1-1-2 and 92x0L19-8-1-1 (Table 4-1). A total of 740 seed from F1 plants of these crosses were assayed for the presence of the SCAR marker, Z3/265, as described belo w. All seed from these crosses (F2) that had the marker were subsequently planted in 2003 (F2 generation) at the Plant Science Unit, Teaching and Research Center, University of Florid a, Gainesville, FL. Leaves from 50 F2 plants were selected based on their desirable agronomic traits and subject ed to RFLP analysis and seed were analyzed

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66 for oleic acid, as described below, before their advancement to the F3 generation. Seeds from F2 plants and their parents were pl anted in a root-knot nematode-inf ested field in 2004, at the Plant Science Unit, Teaching and Research Center, Univers ity of Florida, Gainesvi lle, FL as described for experiment one above in a Randomized Complete Block Design (RCBD) with four replications. Each replication consisted of 13 ro ws that were 3.6 m long with 1 m between rows and 30 cm between plants in rows, givi ng a total number of approximately 28 F3 plants per plot. Each row consisted of four lines separated by 1 m. Plot size was 11 m x 12 m. Root-knot Nematode Resistance Test One hundred days after planting, plants were harvested and evaluated for root-knot nematode resistance. The resistance to M. arenaria race 1 was measured based on the number of egg masses and galls present on peanut roots and pegs. Plants were harvested and the soil was washed from the roots with tap water. Roots were then placed into 3000 ml beakers containing approximately 900 ml of 0.05% Phloxine B / L solution for 3 5 min (Daykin and Hussey, 1985). Root gall and egg mass ratings were assigned based on the scale described in Table 4-2. Briefly, each plant was rated according to the nu mber of egg masses and galls found on roots, pegs and pods. A plant given a rating of 1 (no ga lls or egg masses on roots, pegs and pods) was considered highly resistant, a plant rated as 2 (1-10 egg masses and/or galls on roots and less than 10 egg masses and/or galls on pegs and pods) was considered resistant, a rating of 3 (11-100 egg masses and/or galls on roots and between 10 -50 egg masses and/or galls on pegs and pods) indicated that the plants were susceptible and a plant rated as 4 (> 100 eg g masses and/or galls on roots and > 50 egg masses and/or galls on pegs and pods) was considered highly susceptible. Root-knot nematode count data were subjected to analysis of variance and the treatment means were compared by Tukeys studentized range test (SAS Institute, 1985). Statistical difference was assumed at P = 0.05.

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67 DNA Extraction from Seeds and Leaves Total genomic DNA was extracted from seed acc ording to Chenault et al. (2007). Briefly, a small section (0.02 g) of seed was removed ta king care not to damage the embryo for planting purposes, and placed in a 1.5 ml eppendorf containing 400 l of extraction buffer (100 mM Tris pH 8.0; 50 mM EDTA pH 8.0; 500 mM NaCl, and 10 mM mercapto ethanol added fresh) at 60 oC. The seed samples were ground into a milkywhite solution using pestle and mortar, and transferred to a fresh tube containing 200 l of 20% SDS for 10 min. This was followed by the addition of prechilled 5 M potassium acetate (5 ml), two phenol/chloroform/isoamyl (25:24:1) alcohol extractions, and a fina l precipitation with ice cold isopropanol. DNA was pelleted by centrifugation at 23000 g for 20 min at 4 oC. After air drying, and rinsing in 70% ethanol, the pellet was resuspended in buffer ( 10 mM Tris (pH 8.0), 1 mM EDTA). Genomic DNA extraction from leaves was pe rformed according to the Dellaporta method (1983) with the following modifi cations. Briefly, DNA was extrac ted from 1.2 g of leaf tissue with 15 ml extraction buffer (100 mM Tris-H CI, pH 8.0, 50 mM EDTA, and 500 mM NaCI) at 60 oC followed by prechilled 5 M potassium acetate (5 ml) treatment, two chloroform/isoamyl alcohol extractions and a fina l precipitation with ice cold isopropanol. DNA was pelleted by centrifugation at 20,000 x g for 20 min at 4 oC and rinsed twice with 70% ethanol. After air drying, the DNA was re-suspended in water followed by RNAase (20 pg/ml) treatment and centrifugation at 20,000 x g for 15 min at 4 oC. The DNA pellet was re-suspended in TE buffer and stored at oC. SCAR Analysis Forward primer, SCZ3-FO1 5-CAGCACCG CAGCATAAAAAC-3 and reverse primer, SCZ3-RO2 5-CAGCACCGCACACATTCTGG-3 were used to amplify a SCAR marker with an expected size of 310 bp (Garcia et al., 1996 ). PCR reactions were performed with a MJ

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68 Research thermocycler (Waltham, MA) under the following conditions: 94 oC for 5 min, 30 cycles at 94 oC for 1 min, 60 oC for 1 min, 72 oC for 2 min, and a final extension at 72 oC for 3 min. The root-knot nematode susceptible cultivar, Florunner, was used as a negative control and the root-knot nematode resistant cultivar, Ne maTAM was used as a positive control for all experiments. Oleic Acid Analysis Seeds from fifty F2 plants that were positive for the SCAR marker and agronomically superior were then analyzed by George D. Person (laboratory technician, Un iversity of Florida) using fourier transform infrar ed (FTIR) spectrometer (Nexus 870 FTIR, Thermo Nicolet, Madison, WI) for oleic acid content. Seeds were then characterized as hi gh oleic (> 75% oleic acid content), medium oleic (60 75% oleic acid), and low oleic (< 60% oleic acid). These seeds were then planted at the University of Florida, Pine Acres Research Station, Citra, FL. Leaf tissue was collected from 50 of the resulting pl ants. The tissue was quick frozen in liquid nitrogen and stored at oC until use for RFLP analysis. RFLP Analysis Peanut genomic DNA (20 g) was digested overnight with EcoRI according to the manufacturers instruct ions (New England Biolabs, Beverl y, MA). Digested DNA was subjected to electrophoresis for 16 h using 0.8% agaros e gels and transferred onto Hybond N+ membranes (Amersham, Arlington Heigts, IL) by capillary blotting (Southern, 1975) and UV cross linked for 3 min at 70,000 micro-joules/cm2. The RFLP probe, R2430E, was kindly provided by Dr. Gregory T. Church (Texas A&M Univers ity). Probe DNA (50 ng) was labeled with -P32 dCTP by random primer extension (Feinberg and Vogelte in, 1983). Pre-hybridiza tion and hybridization were performed at 65 oC with 7% SDS and denatured salmon sperm DNA (Church and Gilbert, 1984). Samples were washed three times for 20 min each at 65 oC with 0.5X SSC and 0.1% SDS.

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69 Hybridized blots were autora diographed using X-ray film (K odak XAR-5) and two intensifying screens at 70 oC for three to 10 d. Results The root-knot nematode resistant and suscep tible alleles detected using R2430E were distinct and easy to score for lines F1334, F79x4-6, F94x30-8-2-1-b3, F94x30-8-3-1-b2, F94x308-2-2-b3 and F94x30-5-2-3-3-b3. All lines with a susceptible RFLP genotype (Fig. 4-1) had a susceptible phenotype based on root -knot nematode reproduction as described in Table 4-2. Field results showed that the gall and egg mass index for the root-knot nematode resistant cultivar COAN was 1, whereas the mean gall and egg ma ss index on roots and pod s of the susceptible cultivar Florunner was 3.2 (Table 4-3). Reproduction of M. arenaria on each of the lines as measured by the number of galls and egg masses on roots ranged from 3.0 to 3.9 with greater than 11% egg masses on pods and pegs whic h was more than the reproduction found on Florunner ( P < 0.001) (Table 4-3). No resistance to M. arenaria was observed in any of the six Florida breeding lines. Consequently, to incorporate root-knot nemat ode resistance into the Florida germplasm along with the high oleic trait, several crosses in volving the root-knot nemat ode resistant cultivar COAN and susceptible high oleic cultivars HU LL (Gorbet, 2007a), ANorden (Gorbet, 2007b), UF 98326 and lines F89xOL14-1-4-1-1-1-2, and 92x0L 19-8-1-1 (Table 4-3) were made and the seed from F1 plants was screened for root-knot nema tode resistance with the SCAR marker Z3/265 (Garcia et al., 1996). This marker was ch osen over the RFLP mark er due to its ease for screening. A total of 740 seed from F1 plants were screened for Z3/265 and 193 were positive for the presence of the marker (Table 44; Fig. 4-2). All the seeds from F1 plants positive for the presence of Z3/265 were subsequently advanced to the F2 generation. Leaves from 50 plants

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70 (Table 4-5) that were agronomi cally sound and disease free were co llected and subjected to oleic acid and RFLP analysis with the main objectiv es of determining a relationship between the presence of the SCAR marker a nd RFLP marker as a means to test for reliability and zygosity, and for further advancing to the F3 generation. Thirty of the 50 plan ts were high oleic (> 75%), 9 were medium oleic (65 70%) and 11 (< 60%) were low oleic (Table 4-5). There was no relationship between root-knot nematode resist ance and the high oleic aci d trait (Table 4-5). Using R2430E, individuals plants were scored as homozygous for resistance (RR) if only the band associated with resistance was presen t; heterozygous for resistance (RS) if both the band for resistance and susceptibility were presen t, and susceptible (SS) if the band associated with susceptibility was present (F ig. 4-3). Five (10%) out of 50 F2 selections were homozygous for the susceptible marker and six (12%) were homozygous for the resistance marker, with the remainder (74%) being heterozygous (Table 4-5) Three selections homozygous for the S-allele were susceptible and the remaining heterozygous and R-allele homoz ygotes were resistant (Table 4-5). Finally, these 50 F2 selections were planted in a fiel d highly infested with root-knot nematodes ( M. arenaria race 1). Resistance to M. arenaria race 1 was measured based on number of galls present on peanut roots and pegs (Table 4-2). Fo rty-four selections (88%) were found to be resistant and three selections (18%) were susceptible (Table 4-5). For the resistant control NemaTAM, the mean peg and pod gall inde x was 1 and root gall index was 1, whereas for the susceptible control Florunner, the mean peg and pod gall index was 1 and root gall index was 3 (Table 4-5). Reproduction of M. arenaria on each of the selections as measured by the peg and pod gall index and root gall index ranged from 0.0 to 1 and 1 to 3 respectively (Table 4-5).

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71 Reproduction of M. arenaria on each of the selections as measured by the number of galls on roots, pegs and pods ranged from 0 to greater than 100. Discussion Results of RFLP analysis of six Florida breed ing lines indicated that none of this Florida breeding material inherited the locus for root-knot nematode re sistance from TxAG-6. A possible explanation for this result is th at the root-knot nematode resistan ce gene was lost in segregating generations subsequent to the F2 because no selection for root -knot nematode resistance was used to advance these materials until the scr eening employed in this study. Also TxAG-6 was heterozygous for the resistance and the gene may not have been transferred in the cross. This result prompted the use of marker-assisted sele ction for screening the earlier generations for root-knot nematode resistance with the main obj ective of introgression of root-knot nematode resistance into lines carryi ng the high oleic acid trait. The SCAR marker Z3/265, and the RFLP R 2430E analyses of filial generations (F1 and F2) of crosses involving the rootknot nematode resistant cultiv ar COAN and the susceptible cultivars HULL (Gorbet, 2007a), and ANorden (G orbet, 2007b) and lines F89xOL14-1-4-1-1-12 and 92x0L19-8-1-1, indicated co-s egregation of these markers. Based on this, it can be hypothesized that both the markers might be linke d to the same root-knot nematode resistance gene. Resistance in some selections (Table 4-5), despite the absence of the R2430E marker was possible because R2430E is 4.2 cM away from the re sistance locus, and th is indicates a possible recombination of 4% between marker and root -knot nematode resistan ce (Choi et al., 1999; Church et al., 2000). RFLP data were somewhat mo re reliable than SCAR marker data as there was strong relationship (90% of F2 selections that were RFLP marker positive were root-knot nematode M. arenaria resistant) between the presence of the RFLP marker and root-knot

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72 nematode resistance as measured in the field (T able 4-5). Moreover, the RFLP marker allowed for the selection of homozygous individuals for root-knot nematode resi stance (Church et al., 2000). However, using the RFLP marker resulted in a higher frequency of lost data due to several factors such as insufficient DNA availa bility, incomplete digest ion of DNA, and poor hybridization (Church et al., 2000). In addition, RFLP analysis is more cumbersome and costly than SCAR analysis. SCAR marker analysis is s imple, rapid and cost effective when it comes to screening large numbers of breedi ng lines. The primary drawback of SCAR markers is that they are dominant and do not permit the scoring of heterozygous individuals. However, the SCAR marker Z3/265 derived form A. cardenasii is linked at 10 + 2.5 cM and 14 + 2.9 cM from the root-knot nematode resistance genes Mag and Mae, respectively (Garcia et al., 1996), which indicates a possible recombina tion frequency of 10 to 14%. This high rate of recombination might result in false positives (Chu et al., 2007). Recently, Chu et al. (2007) developed a dominant marker 197/909, which is about 5.8 cM from the root-knot nematode resistance gene. This still leaves room for a recombination fre quency of 5.8%. Therefore, a more tightly linked SCAR marker would be desirable. Also matu re peanut leaf has high amounts of phenolic compounds and polysaccharides; th ese compounds interfere with Taq polymerase (Sharma et al., 2000; Chenault et al., 2007) affecting the reprod ucibility of Z3/265 marker. So in order to overcome this problem peanut seed DNA was us ed in our study to perform SCAR marker analysis. According to the root gall index of the F3 generation (Table 4-5) there is a discreet reaction with respect to root-kno t nematode resistance, but the pe g and pod gall index is not as uniform. This can be explained by the recent repo rt that the resistance gene in COAN has two

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73 major effects when compared to susceptible cult ivars, penetration and further development after penetration (Bendezu and Starr, 2003). Marker-assisted selection helps in identif ying those individuals in which desirable recombination events have occurred during backcr ossing of the nematode resistance gene into elite peanut cultivars. In addi tion, marker-assisted selection has the potential to significantly reduce the cost associated with field planting, maintenance and fiel d trails. Furthermore, in this study marker assisted selection allowed for pean ut genotypes to be screened without relying on inoculation tests in advanced ge nerations, which in turn reduces the time for developing rootknot nematode resistan t peanut cultivars.

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74 Table 4-1. Sample number representation, parent s involved and crosses made for seed from F1 plants. Sample # Cross # Parents 1 01 X 14 -1 Hull X COAN 2 01 X 14 -1 Hull X COAN 3 01 X 14 -1 Hull X COAN 4 01 X 14 -1 Hull X COAN 5 01 X 14 -1 Hull X COAN 6 01 X 22 -1 COAN X ANorden 7 01 X 22 -1 COAN X ANorden 8 01 X 22 -1 COAN X ANorden 9 01 X 22 -1 COAN X ANorden 10 01 X 22 -1 COAN X ANorden 11 01 X 22 -2 COAN X ANorden 12 01 X 22 -2 COAN X ANorden 13 01 X 22 -2 COAN X ANorden 14 01 X 22 -2 COAN X ANorden 15 01 X 22 -2 COAN X ANorden 16 01 X 22 -3 COAN X ANorden 17 01 X 22 -3 COAN X ANorden 18 01 X 22 -3 COAN X ANorden 19 01 X 22 -3 COAN X ANorden 20 01 X 22 -3 COAN X ANorden 21 01 X 23 -1 COAN X UF 98326 22 01 X 23 -1 COAN X UF 98326 23 01 X 23 -1 COAN X UF 98326 24 01 X 23 -1 COAN X UF 98326 25 01 X 23 -1 COAN X UF 98326 26 01 X 24 -1 COAN X F89xOL14-1-4-1-1-1-2 27 01 X 24 -1 COAN X F89xOL14-1-4-1-1-1-2 28 01 X 24 -1 COAN X F89xOL14-1-4-1-1-1-2 29 01 X 24 -1 COAN X F89xOL14-1-4-1-1-1-2 30 01 X 24 -1 COAN X F89xOL14-1-4-1-1-1-2 31 01 X 24 -2 COAN X F89xOL14-1-4-1-1-1-2 32 01 X 24 -2 COAN X F89xOL14-1-4-1-1-1-2 33 01 X 24 -2 COAN X F89xOL14-1-4-1-1-1-2 34 01 X 24 -2 COAN X F89xOL14-1-4-1-1-1-2 35 01 X 24 -2 COAN X F89xOL14-1-4-1-1-1-2 36 01 X 26 -1 COAN X 92x0L19-8-1-1 37 01 X 26 -1 COAN X 92x0L19-8-1-1 38 01 X 26 -1 COAN X 92x0L19-8-1-1 39 01 X 26 -1 COAN X 92x0L19-8-1-1 40 01 X 26 -1 COAN X 92x0L19-8-1-1

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75 Table 4-1 (Continued) Sample # Cross # Parents 41 01 x 26 -2 COAN X 92x0L19-8-1-1 42 01 x 26 -2 COAN X 92x0L19-8-1-1 43 01 x 26 -2 COAN X 92x0L19-8-1-1 44 01 x 26 -2 COAN X 92x0L19-8-1-1 45 01 x 26 -2 COAN X 92x0L19-8-1-1 46 01 x 26 -3 COAN X 92x0L19-8-1-1 47 01 x 26 -3 COAN X 92x0L19-8-1-1 48 01 x 26 -3 COAN X 92x0L19-8-1-1 49 01 x 26 -3 COAN X 92x0L19-8-1-1 50 01 x 26 -3 COAN X 92x0L19-8-1-1 51 Florunner Susceptible Control 52 NemaTAM Resistant Control Table 4-2. Gall and egg mass index scale (1-4) used to evaluate peanut genotypes for resistance to Meloidogyne arenaria (Dr. D.W. Dickson, Pers. Comm.). Grade No of galls or egg masses on roots No of galls or egg masses on pegs and pods Reaction 1 0 0 Highly Resistant 2 1 10 <10 Resistant 3 11100 10 50 Susceptible 4 > 100 > 50 Highly Susceptible Table 4-3. Field reaction of peanut breeding lines to Meloidogyne arenaria evaluated at the Plant Science Unit University of Florida, FL., 2001. Genotypesa Gall and egg mass index (Average/STDV)b Egg mass on pods and pegs (%) Classificationc F1334 3.90/0.23 a 11-50 S F94x30-8-2-2-b3 3.90/0.31 a 11-50 S F94x30-5-2-3-3-b3 3.80/0.41 a 11-50 S F94x30-8-3-1-b2 3.70/0.57 a > 50 HS F94x4-6 3.60/0.50 a 11-50 S F94x30-8-2-1-b3 3.60/0.50 a > 50 HS Florunner 3.20/0.36 b > 50 HS TP301-1-8 1.00/0.00 c 0 R COAN 1.00/0.00 c 0 R aT301-1-8 = resistant breeding line; COAN = resistant genot ype; Florunner = susceptible genotype. cHS = highly susceptible, R = resistant, S =susceptible. bMeans within a column followed by the same lett er are not statistically different at P = 0.05 based on Tukey's studentized range test.

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76 Table 4-4. Crosses tested using the SCAR ma rker for root-knot nematode resistance. Cross TotalNo. of seed tested No. of seed positive for the SCAR marker % Positive HullXCOAN 137 35 26 COANXANorden 223 19 8.5 COANXUF98326 78 39 50 COANXF89/OL14-1-4-1-1-1 184 34 18 COANX920L19-8-1-1 112 66 59 Total 740 193 26

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77Table 4-5. Comparison of oleic acid content, dominant SCAR marker Z3/265 and co-domin ant RFLP marker R2430E data with rootknot nematode field reaction. Sample # Seed oleic acid contenta Z3/265 Seed marker datab R2430E leaf Southern datac Peg and pod gall index Root gall index Root-knot nematodefield Reaction 1 H + 1 3 Susceptible 2 H + + 1 1 Resistant 3 H + + 1 1 Resistant 4 H + + 1 1 Resistant 5 H + 1 2 Resistant 6 H + + 1 1 Resistant 7 H + + 1 1 Resistant 8 H + + 1 1 Resistant 9 H + + 1 1 Resistant 10 H + + 0 0 0 11 H + + 1 2 Resistant 12 H + + 1 1 Resistant 13 H + + 1 1 Resistant 14 H + + 1 2 Resistant 15 M + + 1 1 Resistant 16 M + + 1 1 Resistant 17 L + 1 3 Susceptible 18 L + + 1 1 Resistant 19 L + + 1 1 Resistant 20 L + + 1 1 Resistant 21 H + 0 1 1 Resistant 22 H + + 1 1 Resistant 23 H + + 1 1 Resistant 24 H + + 1 1 Resistant 25 H + + 1 2 Resistant 26 H + + 1 1 Resistant 27 L + + 1 2 Resistant

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78Table 4-5 (Continued) Sample # Seed oleic acid contenta Z3/265 Seed marker datab R2430E leaf Southern datac Peg and pod gall index Root gall index Root-knot nematodefield Reaction 28 L + + 1 1 Resistant 29 L + + 1 1 Resistant 30 L + + 1 2 Resistant 31 H + + 1 1 Resistant 32 H + + 1 1 Resistant 33 M + + 0 0 0 34 M + + 1 1 Resistant 35 M + + 1 1 Resistant 36 M + + 1 1 Resistant 37 M + + 1 1 Resistant 38 L + + 1 2 Resistant 39 L + + 1 1 Resistant 40 L + + 0 0 0 41 H + + 1 1 Resistant 42 H + + 1 1 Resistant 43 H + + 1 1 Resistant 44 H + + 1 1 Resistant 45 M + + 1 1 Resistant 46 H + 0 1 1 Resistant 47 H + + 1 1 Resistant 48 H + 1 3 Susceptible 49 H + + 1 1 Resistant 50 M + 1 1 Resistant 51 L 1 3 Susceptible 52 M + + 1 1 Resistant aH = High oleic, M = Medium oleic, L = Low oleic bMarker present (+), Marker absent (-) cHeterozygous positive (+ ), Homozygous positive (+), Homozygous negative (-), No results (0); 0= No data

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79 Figure 4-1. RFLP locus R2430E linked to resistance to Meloidogyne arenaria in peanut breeding lines. R = resistant and S = susceptible alle les. Lane 1 is the root-knot nematode resistant cultivar COAN; lane 2 is the suscep tible cultivar Florunner; Lanes 3 to 9 are the breeding lines F1334; F94x4-6; F 9430-8-2-1-b3; F94x30-8-3-1-b2; F94x30-8-22-b3; F94x30-5-2-3-3-b3, F 94x30-5-2-3-3-b3, respectively; lane 10 is the root-knot nematode resistant breeding line T301-1-8. Figure 4-2. SCAR marker analys is. M = 1kb Ladder. Amplicons of Z3/265 marker: Lane 1 = COAN, Lane 2 = Florunner, Lanes 3-8 = F2 seed tested. 250 bp 500 bp 750 bp 1000 bp 8 M 1 2 3 4 5 6 7 8 RS SS SSSS SR 1 2345678910

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80 Figure 4-3. RFLP marker anal ysis. RR = homozygous resist ant (++); SS = homozygous susceptible (--); RS = he terozygous resistant (+-). RR RS RS RS SS RR SS RR + + + + +-++ -+ +

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81 CHAPTER 5 CLONING AND CHARACTERIZATION OF R -GENES IN PEANUT ( ARACHIS HYPOGAEA L.) Introduction Peanut ( Arachis hypogaea L.) production is affected by ma ny biotic constraints such as, insects, microbial pathogens and root-knot nema todes. In the approximately 35% of crop losses that occurs annually, 12% is due to diseases, 7% is due to insects, and 11% is due to nematodes. Root-knot nematodes cause significant yield loss es in many peanut-producing areas (e.g. Florida, Georgia, Alabama, Texas and North Carolina). In the United States, estimated annual monetary losses due to nematodes in peanut is $102 million (Sasser, 1990). As much as a 30% decrease in peanut yield potential has been observed due to M. arenaria alone (Nelson et. al., 1990). Root-knot nematode infection of the root syst em of peanut plants results in heavy galling of roots, pegs, and pods. This in turn reduces peanut quality a nd yield (RodriguezKabana et al., 1986). Root-knot nematodes have a broad host ra nge so controlling them by crop rotation is difficult. Although nematicides are effective, their continued usage is uncertain due to environmental hazards and economic concerns (C hen et al., 1996). Moreover, the application of some nematicides has been abandoned in some ar eas because of their a ssociated toxicity to farmers (Jung et al., 1998). Consequently, the be st method of controlling the root-knot nematode, M. arenaria is by deployment of root-knot nema tode resistant peanut cultivars. Several resistance genes ( R genes) against nematodes have been cloned and mapped. Other than the first nematode R gene identified, Hs1pro-1 all the other nematode R genes share structural homology with other plant disease resistance genes (B aker et al., 1997; Cai et al., 1997; Ellis and Jones, 1998; Williams on, 1998). Most of these nematode R genes code for members of a family characterized by a nucleotid e-binding site (NBS) a nd leucine-rich repeats (LRRs) (Lagudah et al., 1997; Milligan et al., 1 998). For example, the potato cyst nematode

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82 resistance gene, Gpa2 encodes a product with NBS and LRR domains (Van der Vossen et al., 2000) and shows high homology with the virus resistance gene Rx (Davis et al., 2000) Several studies have also shown that this resistance against potato vi rus X operates on a gene-for-gene basis in which the R gene encodes a putative receptor th at recognizes the vi ral coat protein (Bendahmane et al., 1999). Functio nal and genomic studies of the R genes above revealed that the NBS-LRR, LRR, and LRR-Kinase superfam ilies are highly conserved and ubiquitous in plants (Young, 2000). Breeding for root-knot nematode resistance in peanut based on inoculation and phenotypic selection is a challenging proce ss due to irregular di stribution of root-kno t nematode populations in the field and several other field constraints. Currently, knowledge of peanut resistance genes ( R ) is limited and their use for peanut genetic improvement lags behind most crop plants. A better understanding of the molecular and ge netic basis of nematode resistance ( R ) genes would enhance the effective use of R genes as molecular markers in peanut breeding for nematode resistance. However, effective use of these R genes as molecular markers first requires isolation and characterization of these genes. To date large scale ge nome wide analysis of disease re sistance gene-like sequences in Arachis species has been conducted by Bertioli et al (2003) and Yuksel et al. (2005). Seventyeight resistance gene homologue s (RGHs) were generated with degenerate primers targeting NBS region from A. hypogaea var. Tatu and four wild relatives ( A. duranensis, A. cardenasii, A. stenosperma and A. simpsonii ) (Bertioli et al., 2003). Yuksel et al. (2005) designed primers targeting consensus sequences of various classes of resistance genes and isolated 234 resistance gene analogs (RGAs). Their phyl ogenetic analysis of these RGAs indicated their evolutionary proximity to RGAs from ot her legume species, including Glycine max Lotus japonicus

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83 Medicago truncatula and Phaseolus vulgaris In the current study, the objective was to clone R genes from the root-knot nematode resistant cul tivar, NemaTAM, which may confer resistance to the root-knot nematode, M. arenaria race 1, using degenerate primers for conserved NBS-LRR domains reported to be associated with resistance genes (D onald et al, 2002). Materials and Methods Plant Materials Individual plants of the root -knot nematode resistant cult ivar NemaTAM were grown in clay pots containing pasteurized soil (Arredondo and Sparr type; sa nd 95%, silt 3%, clay 2% and organic matter 1.5%; pH 6.5) for leaf genomic DNA extraction a nd PCR amplification of RGAs. Thirty days after planting, genomic DNA was extracted from young leaves of NemaTAM. Similarly, for RT-PCR and gene expression studi es, total RNA was extracted from roots of individual plants of NemaTAM and the root-kno t nematode susceptible cultivar Florunner grown in clay pots containing pasteurized soil (Arredon do and Sparr type; sand 95%, silt 3%, clay 2% and organic matter 1.5%; pH 6.5). For both experi ments, all of the pots were kept in a greenhouse at 27-32 oC. They were watered daily and fert ilized (Miracle-Gro, Marysville, OH) once a week with N-P-K (24-8-16). For RT-P CR / gene expression studies, 10 days after planting, each pot was inoculated wi th a suspension of 5000 eggs of M. arenaria race 1 which had been cultured on tomato ( Solanum esculentum Mill. cv. Rutgers). The eggs were pipetted into three holes (0.6-cm diameter x 4-cm depth) made close to the roots, but distributed at equal distance from the base of the plants. Root-knot nematode inoculum was prepared using the NaOCl method as described in Hussey and Barker (1973). Total RNA was isolated from roots of NemaTAM and Florunner at five time points: 0, 12, 24, 48, and 72 h after inoculation/infection with M. arenaria race 1.

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84 Genomic DNA Extraction For genomic DNA extraction, leaves stored at oC were ground and treated according to the Dellaporta method (1983) with the follo wing modifications. Brie fly, DNA was extracted from 1.2 g of leaf tissue with 15 ml extraction buffer (100 mM Tris-HCI, pH 8.0, 50 mM EDTA, and 500 mM NaCI) at 60 oC followed by prechilled 5 M potassium acetate (5 ml) treatment and two chloroform/isoamyl alcohol extractions and final precipitation with ice cold isopropanol. DNA was pelleted by centrifugati on at 20,000 x g for 20 min at 4 oC and rinsed twice with 70% ethanol. After air drying, the DNA was re-suspe nded in water followed by RNAase (20 pg/ml) treatment and centrifugation at 20,000 x g for 15min at 4 oC. The DNA pellet was re-suspended in TE buffer and stored at oC. Each sample yielded 30-50 g of DNA. The DNA extracted was utilized for amplifying RGAs as described below. RNA Extraction Total RNA for gene expression studies was isolated from root samples using the GuanidineHCl method as described by Logemann et al. (1987) with slight modifications. Roots (3g) were ground in liquid nitrogen, then 15 ml of 6M Guanidine buffer and 15 ml of phenol/chloroform/isoamyl alcohol (PCI; 25:24:1) were added, and the mixture was shaken for 20 min and centrifuged for 20 min at the rate of 20, 000 x g to separa te the phases. The supernatant was gently removed and 1.5 vol of ab solute ethanol and 0.3 vol of 1M acetic acid (pre-cooled at 4 oC) were added and the RNA was precipitated overnight at 20 oC. The pellet obtained after centrifugati on at the rate of 20, 000 x g was resu spended in 5 ml of DEPC-treated water and precipitated by incubation for 1 h at 20 oC after the addition of 300 l of 4M LiCl and 5 ml of cold 100% ethanol. After washing th e pellet with 10 ml of cold 70% ethanol, the RNA was air dried, resuspended in 1 ml of distilled sterile water and stored at 80 oC.

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85 RT-PCR Reverse transcription of total RNA was carried out using iScript reverse transcriptase (BioRad, Hercules, CA) with oligo-dT as the primer according to the manufacturers instructions. First-strand cDNA from 25 ng total RNA was used in each PCR reaction with the following primer combinations: forward F5-atctgaaagcatggg tttgc-3 and reverse R5-gctgctctcccatttgattc3 for NRP3D-4 ; and for 18S control forward F 5aacggctaccaccacatccaaggaaggc-3 and reverse R 5gcgcgtgcggcccagaacatctaag-3. PCR wa s carried out in a MJ-100 thermocycler (MJ Research, Watertown, MA) with the following program: 94 oC for 1min, followed by 25 or 30 cycles at 94 oC for 15 sec, 59 oC for 20 sec, 72 oC for 15 sec and a final extension at 72 oC for 1 min. PCR products were loaded onto a 1% agarose gel and run in 1X TBE containing ethidium bromide at 75 V for 3 hr. Polymerase Chain Reaction Amplification of RGAs was performed in 20 l reactions containing 15-20 ng/ l of NemaTAM DNA, 10 pmole/ l of degenerate primers P1B-fw d, P3D-rev, P3A-rev, P1A-fwd and P3D-rev and RNBS-D-rev (Table 5-1; Bertio li et al., 2003), 2 mM of dNTPs, 10 mM MgCl2 and 10 x buffer and Taq polymerase (1u/ l) (Invitrogen, Carlsbad, California). Annealing temperatures for each reaction were optimized using a Gradient Cycler (MJ Research PTC-200, Watertown, MA). The annealing te mperature that yielded a suffi cient amount of PCR product at the desired range was selected. The following pr imer combinations were used for PCR: LM638 with P3D-rev, P1B-fwd with P3D-rev, P1A-fw d with P3D-rev, P1B-fw d with P3A-rev, LM638 with P3A-rev, and LM638 with RNBS-D-rev (Ber tioli et al., 2003). PCR was carried out in a MJ-100 thermocycler (MJ Research, Watertown, MA ) with the following program: 5 min at 94 oC, followed by 30 cycles of 1 min 94 oC, 1 min at 50 oC, 2 min at 72 oC and final extension of 5

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86 min at 72 oC. PCR products were run on a 1% agarose gels containi ng ethidium bromide (EtBr0.5 l of 10 mg/ml) in 1x TBE at 75 V for 3 hr for visualization. Cloning and Transformation PCR products resolved by electrophoresis on a 1% agarose gel in 1x TBE buffer were excised. Fragments thus obtai ned were purified using QIA quick gel extraction columns according to the manufacturers instructions (Qiagen, Valencia, California). Purified PCR fragments were ligated into pGEM using th e pGEM-T Easy cloning kit and propagated in Escherichia coli DH5cells according to the manufacturers instructions (Promega, Madison, Wisconsin). Transformed cells were subsequently grown with constant shaking at 300 rpm in 1 ml of SOC medium (2% tryptone, 0.5% yeast extract, 8.5 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose) without antibiotics (Sambrook and Russell, 2001). Recombinant plasmids were selected by plating bacteria ont o LB medium containing ampicillin (100 g/ml) and X-GAL (50 mg/ml). White colonies containing the recombinant plasmids were selected and grown at 37 oC overnight in LB liquid medium contai ning ampicillin (100 g/ml) for plasmid DNA extraction which was done using the QIApre p Spin Mini Prep kit (Qiagen). Randomly selected white clones were se quenced as described below. Sequencing and Annotation Randomly selected white clones were sequen ced using the M13 forward primer by the Interdisciplinary Center for Biot echnology Research (ICBR; Universi ty of Florida, Gainesville). Sequences obtained were exposed to a VecScreen algorithm (http://www.ncbi.nlm.nih.gov/ ) to remove vector contamination. Using ClustalW program overlapping sequences were discarded. Genomic sequences obtained were identified by homology searches (Blastn and Blastx) in different databases (non-redundant sequences in National Center for Biotechnology Information

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87 (NCBI), EST sequences in NCBI, and the potato and rice EST database at The Institute for Genomic Research (TIGR)). Results A combination of degenerate primers (Bertioli et al., 2003) was used for DNA amplification of RGA sequences from the root-knot nematode resistant cultivar, NemaTAM. The resulting PCR products range in size from 500 bp to 800 bp. A representa tive gel is shown in Fig. 5-1. Genomic sequences obtained upon cloning and se quencing of the purified PCR products were identified by homology searches (Blastn and Blastx) in differe nt databases and results are summarized in Table 5-2 and Fig. 5-2. Homologies with the NBS-LRR of R genes or with the RGL sequences that had been cloned from other plant species were found. In total, 234 genomic clones were sequenced, and 145 s howed significant hits to known sequences in the NCBI database. Homology searches revealed that 26 % of the sequences bel onged to the section Arachis and another 18% belonged to other legumes such as Medicago truncatula, Vigna mungo and Cicer arietinum. Fourty one percent of the cl ones have sequences with significant homology to genes found in Manihot esculanta, Zingi ber officinalis, Populus tomentosa, Ipomea batatus, Coffea arabacia, Oryza sativa, Poinciru s trifoli, Prunus avium, Triticum aestivum, Solanum demissum, Cicer arietinum, and Arabidopsis thaliana Of the remaining clones, 15% have sequences related to genes found in microorganisms such as Erwinia amylovora and Campylobacter jejuni (Fig. 5-2). Interestingly, one Nema TAM genomic clone shared conserved motifs found in the well-characterized toma to root-knot nematode resistance gene, Mi (Milligan et al., 1998). Putative functions of 88% of the NemaTAM sequences were assigned. The remaining 12% of the sequences in this study did not show significant homology to those in public databases.

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88 Sequences were classified into four different major groups accord ing to the putative function of their homologous genes in the databases. The dist ribution of the genes is illustrated in Fig.5-3. The largest set of genes (70%) was assigned to th e disease resistance/response to stress /plant defense group. Within this group, genes that be long to the NBS-LRR class (53%) occupied the largest percentage, followed by TIR (8%), CC-NBS (5%), and TIR-NBS (4%). Catalases (9%) and Reverse Transcriptase (9%) formed the next largest groups. The remaining 12% of sequences were grouped as hypothetical proteins. Sequence analysis and identi fication of genomic clone NRP3D-4 A clone of approximately 771 bp amplified w ith P1B-fwd and P3D-rev and designated as NRP3D-4 showed high homology to previously identified resistance genes from wild Arachis species (Bertioli et al., 2003). Figur e 5-4 shows the restriction map of NRP3D-4 The genomic clone lacks unique restriction sites for Bam HI, Xho I, Xho II, Eco RI, Sma I and Xba I. The unique restriction sites present are Sac I, Sac II, Pst I, Hind III, Sau3A1, Dra I, Dra III, and Not I. The partial nucleotide sequence of NRP3D-4 and its deduced amino acid sequence are shown in Fig. 5-5. The position of the first poten tial ATG initiation codon in the sequence is found at position 33. The 3 untranslated region contains a se quence that is identical to the consensus polyadenylation site (AATAAA) located at position 651 bp. The first ATG starts an open reading frame at nucleotide 33, and enc odes a polypeptide of 193 amino acids with a calculated molecular mass of 20.9 kDa and a pI of 8.27. The hydropathy profile of the protein, deducted by the algorithm of Kyte and Doolittle (1982), indicates th at it is largely hydrophilic in nature (Fig. 5-6). Moreover, according to the k-NN algorithm (Horton and Nakai, 1997) used in the PSORT program, the protein was predicted, with a probability of ~50%, to be an intracellular and cytoplasmic in localization. Additional analysis based on cons ensus sequences for conserved functional domains (PROSITE databases) showed that the NRP3D-4 protein contains an NB-

PAGE 89

89 ARC domain that begins at amino acid 20 (Kin ase Ia) and ends at amino acid 200 (HD motif) (Fig. 5-7). This region consists of conser ved motifs such as the phosphate-binding P-loop (Kinase Ia motif), with the cons ensus sequence GxGxxGR(T/S) wher e x stands for any residue, a hydrophobic HD motif with consensu s sequence GLPLxL and two a dditional kinase domains, kinase 2 with consensus sequence LLVLDDVW/D and kinase 3a with consensus sequence GSRIII/ATTRD (Soriano et al., 2005). Mutation in Kianse Ia domain causes reduced ATP binding indicating possible role of this motif in ATP binding (Tameling et al., 2002). The kinase 2 domain is proposed to coordinate the metal i on binding required for phospho-transfer reactions. Similarly, the arginine in the kinase 3a domain interacts with purine base of ATP in other proteins (Traut, 1994). Comparisons of the nucleotide sequence and the predicted amino acid sequence against other known sequences were conducted with BL AST and the latest versions of GenBank, EMBL, GenPep and SwissProt databases. Initia l analysis of the NRP3D4-deduced amino acid sequence showed significant identi ty (99% and 98%) with the de duced amino acid sequences of two clones (AY157789 and AY157947) which were isolated from Arachis cardenasii and Arachis stenosperma encoding two putative resistance proteins (Bertioli et al.,2003) (Fig.5-8). It was also found that the NB-ARC domain of NRP3D-4 has hi gh similarity to the NB-ARC domains of these two proteins (F ig. 5-8). There was also 52% id entity at the amino acid level with the root-knot nematode resi stance protein Mi-1.1 protein of Lycopersicon esculentum (AAC67237). Expression of the NRP3D-4 gene in peanut infected by M. arenaria The transcript level of NRP3D-4 in roots of root-knot nemat ode resistant and susceptible cultivars was determined after infection with M. arenaria race 1 by semi-quantitative RT-PCR analysis. Total RNA was isolated from roots of the root-knot nematode-resistant cultivar

PAGE 90

90 NemaTAM and the susceptible cultivar Florunn er at five time points: 0, 12, 24, 48, and 72 h after inoculation/infection with M. arenaria race 1. Based on the RT-PCR products, NRP3D-4 was expressed at 0, 12 and 24 hours after i noculation in NemaTAM. NRP3D-4 was not expressed in Florunner roots at a ny time point tested (Fig.5-9). Discussion Knowledge of the cultivated peanut genom e is limited and only in recent years have molecular techniques been used to interpret its genome organi zation. Consequently, the best strategy to identify candidate R genes/ NBS en coding sequences from peanut is to use the information obtained from model plants and public data bases. In the present study, degenerate primers designed by Bertioli et al (2003) that bind to regions en coding NBS motifs were used to isolate R genes from NemaTAM which is known to be resistant to root-knot nematodes due to introgression of sequences from A. cardenasii (Bendezu and Starr, 2003). Among the defense related genes amplified, a majority belonged to the TIR type (12%) and none belonged to the non-TIR type. This finding is in agreement with the previous reports by Bertioli et al., (2003) and Yuksel et al., (2005) that legumes in general contain fewe r non-TIR type genes than TIR type genes. The R gene sequences isolated from NemaTAM can be explored further as potential markers to trace resistance genes in peanut br eeding programs through marker-assisted selection. For instance, Seah et al. (2001) used R gene derived markers to trace integrated rust resistance in wheat. Similarly, Rgene markers have been us ed to assess genetic diversity and for QTL analysis (Feuillet et al ., 1997; Chen et al., 1998). Homology searches further revealed that 38 (26%) of the genomi c clones belonged to Arachis species, out of which, 18 (13%) clones showed high homology to A. cardenasii. The remaining 20 clones belonged to Arachis stenosperma and Arachis hypogaea Among the clones

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91 that showed homology to A. cardenasii one clone NRP3D-4 showed significant homology to a well-characterized tomato rootknot nematode resistance gene, Mi and two cDNA clones (AY157789 and AY157947) that code for putative R proteins from A. cardenassi and A. stenosperma (Fig.5-8). Hydropathicity plot analysis indicates that the NRP3D-4 protein is hydrophilic and possibly located in the cytoplasm due to the absence of signal sequences (Fig.55). Similarly, Williamson (1998) reported that Mi and related NBS-LRR pr oteins lack signal sequences and are thus likely to be cytoplasmically located. In NemaTAM roots, NRP3D-4 is expressed prior to nemat ode infection and continues to be expressed at 12 and 24 h post infestation. Howe ver, at 48 and 72 h after infection there is no detectable expression of NRP3D-4 In Florunner roots, NRP3D4 transcripts were not detected any time point. This indicates that NRP3D-4 may not be present in Florunner or it may be present but not expressed. Taken together, the above results suggest that NRP3D -4 is a major sequence introgressed from A. cardenassi into NemaTAM may be res ponsible for resistance to M. arenaria atleast at the early stages of interaction. However, a dditional studies are necessary to determine NRP3D-4 expression at the protein level fo llowing root-knot nematode inf ection and its specific cellular localization within NemaTAM roots. Also, a direct relationship needs to be established between expression of NRP3D-4 and root-knot nematode resistance either through future forward or reverse genetic analyses.

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92 Figure 5-1. PCR products obtained after optimiza tion of degenerate primers P1B-fwd and P3Drev. Lane 1 = 1 KB DNA Ladder; Lane 2 = Amplified product at annealing temperature 50 oC; Lane 3 = Amplified product at annealing temperature 50.3 oC; Lane 4 = Amplified product at annealing temperature 51.4 oC; Lane 5 13 = No amplified products between annealing temperatures 53.2 oC to 70.5 oC. Figure 5-2. Sequence categorization base d on homology searches in GenBank. 500bp 750bp 1 2 3 4 5 6 7 8 9 10 11 12 13 A rachi s 26% Other legumes 18% Other plants 41% Microorganisms 15% A rachis Other leguminous plants Other plants Microorganisms

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93 Figure 5-3. Major Functional Categories of Ne maTAM. NBS = Nucleotide Biding Site; LRR = Leucine Rich Repeats; TIR = Toll and Interleukin-1 Receptor; CC = Coiled-Coil Figure 5-4. Restriction map of peanut ( Arachis hypogaea L.) genomic clone NRP3D-4. 53% 8% 5% 4% 9% 9% 12% NBS-LRR type {53%} TIR type {8%} CC-NBS type {5%} TIR-NBS {4%} Catalase {9%} Reverse Transcriptase {9%} Hypothetical proteins {12%}

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94 ttgggcccgacgtcgcatgctcccggccgccatggccgcgggattggg atg ggggggggg M G G G gggaaaacgactcttgcctctgttgtctgtaaaagaatcagaagagaatttgaagattat G K T T L A S V V C K R I R R E F E D Y tgcattctccgagttggagatgtttcaaaggaaggagatctagttaacttacaaaatcaa C I L R V G D V S K E G D L V N L Q N Q cttctttcacatctgaaactaaggagcagagtaattgaaactttagtccaaggaagggac L L S H L K L R S R V I E T L V Q G R D aacatcagaaaccttctgtacaaaaaaaggattcttattgtcctagatgatgtgagaact N I R N L L Y K K R I L I V L D D V R T atagaacagctagagaatttggtaggaaacaaggaatggtttggtccgggaagcagaata I E Q L E N L V G N K E W F G P G S R I gttgttacaactcgggacaagaacctgcttagttcacatggtgcgtttaaaatctatgag V V T T R D K N L L S S H G A F K I Y E atggaagtcttgaacactgatgaatcccttcagctctttcatcaagaagctttcaaagga M E V L N T D E S L Q L F H Q E A F K G gagctaccaaaagaagagtacttggagttgtcgaaaaggtttgtgagctatactggaggc E L P K E E Y L E L S K R F V S Y T G G ctccccttgacccccaacctaatcactagtgcggccgcctgcaggtcgaccatatgggag L P L T P N L I T S A A A C R S T I W E agctcccaacgcgttggatgcatagct tga gtattctatagtgtcacc taaataa cttgg S S Q R V G C I A cgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacaca acatacgagccggaagca taa agtgtaaagcctggggtgcctaatgagtg Figure 5-5. Nucleotide and deduced ami no acid sequences of the genomic clone NRP3D-4 .From top to bottom, the (atg) in itiation codon, (IRREFE) mitocho ndrial targeting sequence, (SKL) signal for peroxisomal protein, the first stop codon (tga), and the putative polyadenylation signal (taaataa) are in bold. Two additional stop codons are underlined. The deduced amino acid sequen ce is below the nucleotide sequence.

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95 Figure 5-6. Hydropathy profile of th e protein deduced from NRP3D-4. Figure 5-7. Graphical overview of peanut NRP3D-4 representing location of the conserved NBARC domain. This domain N-terminal motif cons ists of 181 residues (20 through 200; E value 1e-20). Numbers indicate amino acid residue.

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96 K inase 1a NRP3D-4 --------------LGPTSHAP GRHGRGIGG G G GKTTLA SVVCKRIRREFEDYCILRVGD 46 A.cardenasii ------------------------------------LA SVVCKRIRREFEDYCILRVGD 27 A.stenosperma ------------------------------------LA SVVCKRIRREFEDYCILRVGD 27 Lycopersicon TVGFEEETNLILRKLTSGSADL DVISITGMP G S GKTTLA YKVYN--DKSVSSRFDLRAWC 58 ****** : :.... **. Kinase 2 NRP3D-4 VSKEGDLVNLQNQLLSHLKLRSRVIETLVQGRDNIRN LLYKKRI LIVLDDV R TIEQLENL 106 A.cardenasii VSKEGDLVNLQNQLLSHLKLRSRVIETLVQGRDNIRN LLYKKRI LIVLDDV R TIEQLENL 87 A.stenosperma VSKEGDLVNLQNQLLSHLKLRSRVIETLVQGRDNIRN LLYKKRI LIVLDDV R TIEQLENL 87 Lycopersicon TVQGCDEKKLLNTIFSQVSDSDSKLSENIDVADKLRK QLFGKRY LIVLDDV W DTTTWDEL 118 : :* ::*::. :. :: *::*: *: ** ******* ::* Kinase 3a NRP3D-4 VGNKEWFGP GSRIVVTTRDK NLL SSHGAFKIYE-EVLNTDESLQLFHQEAFKGELPKEE 164 A.cardenasii VGNKEWFGP GSRIVVTTRDK NLL SSHGVFKIYEMEVLNTDESLQLFHQEAFKGELPKEE 146 A.stenosperma VGNKEWFGP GSRIVVTTRDK NLL SSHGVFKIYEMEVLNTDESLQLFHQEAFKGELPKEE 146 Lycopersicon TRPFPESKK GSRIILTTREKEVAL HGKLNTDPLDLRLLRPDESWELLEKRAFGNESCPDE 178 ****::***:* .: : .:*..*** :*:.:.** .* :* HD motif NRP3D-4 YLELSKRFVSY TGGLPLTP NLITSAAACRSTIWESSQRVG----------CIAVFYSVTI 214 A.cardenasii YLELSKRFVSY TGGLPL ------------------------------------------163 A.stenosperma YLELSKRFVSY TGGLPL ------------------------------------------163 Lycopersicon LLDVGKEIAEN CKGLPLVA DLIAGVIAGREKKRSVWLEVQSSLSSFILNSEVEVMKVIEL 238 *::.*.:.. **** NRP3D-4 AWRNHGHSCFLCEIVIRSQFHTTYEPEASVKPGVPNE--251 A.cardenasii ---------------------------------------A.stenosperma ---------------------------------------Lycopersicon SYDHLPHHLKPCLLYFASFPKDTSLTIYELNVYFGAEGFV 278 Figure 5-8. Comparisons of the deduced amino acid sequence of the NemaTAM pr otein NRP3D-4 with sequences from A. cardenasii (AY157789), A. stenosperma (AY157947), and Lycopersicon Mi-1.1 root-knot nematode resistance protein (AAC67237).

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97 Figure 5-9. RT-PCR analysis of the expression of NRP3D-4 in NemaTAM and Florunner. A) Expression pattern of NRP3D-4 in r oots of NemaTAM 0, 12, 24, 48, 72 h after infestation with M. arenaria B) Expression pattern of NR P3D-4 in roots of Florunner 0, 12, 24, 48, 72 h after infestation with M. arenaria Table 5-1. Primer sequences used to amplif y RGAs in NemaTAM (Bertioli et al., 2003). Primer Motif Motif sequence Primer sequencea P1a-fwd P-loop GM[PG]G[IVS] GKTT GGIATGCCIGGIIIIGGIAARACIAC P1b-fwd P-loop GM[PG]G[IVS] GKTT GGIATGGGIGGIIIIGGIAARACIAC P3a-rev GLPL GLPL[TAV][LAV ][KND]AIITYIRIIYIAGIGGYAAICC P3d-rev GLPL GLPL[TAV][LAV ][KND]AIITYIRIIRYYAAIGGIAGICC LM638 P-loop GGVGKTT GGIGGIGTIGGIAAIACIAC RNBS-D-rev RNBS-D CFLYCALFP GGRAAIARISHRCARTAIVIRAARC aI, inosine. Codes for degenerate positions are: R, A/G; Y, C/ T; S, G/C; H, A/C; V, A/C 0 12 24 48 72 NRP3D-4 18S 0 12 24 48 72 NRP3D-4 18S A B

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98Table. 5-2. Identification of genomic clones from th e root-knot nematode resistant cultivar NemaTAM. Clone # Accession # of genomic clone Accession # of matching sequence Best Evalue (BLASTX) Source of matching sequences Matching sequence from database Resistant Proteins NRGH3B12 Ne36037 AAN85395.17E-75 Arachis cardenasii Resistance Proteins (NB-ARC domain) NRGH3H11 Ne36038 AAN85041 6E-36 Arachis cardenasii Resistance Proteins (NB-ARC domain) NRGH3H10 Ne363039 AAN85377 3E-09 Arachis cardenasii Resistance Proteins (NB-ARC domain) NRGH3H09 Ne363040 AAN85401 6E-17 Arachis cardenasii Resistance Proteins (NB-ARC domain) NRGH3H03 Ne363045 AAN5401 2E-33 Arachis cardenasii Resistance Protein NRGH3H02 Ne363046 AAO20377 3E-55 Arachis hypogaea Resistance Protein ( NBS LRR type) NRGH3H01 Ne363047 AAX81299 3E-52 Arachis hypogaea Resistance Protein pLTR ( NBS LRR type) NRGH3G11 Ne363049 AAX71066 7E-81 Arachis hypogaea Resistance Protein PRG (NBS-LRR) NRGH3G10 Ne363050 AAO38219 3E-36 Manihot esculanta Resistance Protein ( NBS LRR type)(RCa8) NRGH3G09 Ne363051 AAO20357 2E-76 Arachis stenosperma Resistance Protein (TIR-NBS) NRGH3G08 Ne363052 AAO20357 3E-72 Arachis stenosperma Resistance Protein (TIR-NBS) NRGH3G05 Ne363055 AAO20357 4E-81 Arachis stenosperma Resistance Protein NRGH3G01 Ne363058 AAX71086 7E-73 Arachis hypogaea Resistance Protein NRGH3F10 Ne363061 AAO20357 6E-78 Arachis stenosperma Resistance Protein NRGH3F06 Ne363063 AAO20357 3E-81 Arachis stenosperma Resistance Protein PLTR NRGH3F03 Ne363066 AAX81299 6E-78 Arachis hypogaea Resistance Protein PLTR NRGH3F01 Ne363068 AAO20357 5E-46 Arachis stenosperma Resistance Protein (NBS-LRR) NRGH3E12 Ne363069 AAO20357 5E-74 Arachis stenosperma Resistance Protein (TIR-NBS) NRGH3E10 Ne363071 AAN85380 6E-65 Arachis cardenasii Resistance Protein NRGH3E08 Ne363073 AAV70155 3E-60 Arachis cardenasii Resistance Protein like Protein NRGH3E07 Ne363074 AAO38219 3E-40 Manihot esculanta Resistance Protein RCG8 (NBS-LRR) NRGH3E06 Ne363075 CAN63338 7E-28 Glycine max NBS-LRR type disease resistance protein NRGH3E02 Ne363079 AAO38219 5E-35 Manihot esculanta Disease Resistance Protein RCA8 NRGH3D12 Ne363081 AA85395 4E-85 Arachis cardenasii Resistance Protein NRGH3D07 Ne363085 ABE86887 6E-42 Medicago truncatula Disease Resistance Protein NRGH3D06 Ne363086 AAO38219 8E-35 Manihot esculanta Resistance Protein RCA8 NRGH3C11 Ne363092 AAO38219 2E-55 Manihot esculanta Resistance Protein RCA8

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99Table 5-2(Continued) Clone # Accession # of genomic clone Accession # of matching sequence Best Evalue (BLASTX) Source of matching sequences Matching sequence from database NRGH3C10 Ne363093 AAY81228 7E-32 Arachis hypogaea Resistant Putative PLTR NRGH3C09 Ne363094 AAN85395 3E-89 Arachis cardenasii Resistance Protein NRGH3C08 Ne363095 AAN85395 5E-64 Arachis cardenasii Resistance Protein NRGH3C07 Ne363096 AAO38219 8E-40 Manihot esculanta Resistance Protein RCA8 NRGH3C06 Ne363097 AAO38220 2E-40 Manihot esculanta Resistance Protein RCA8 NRGH3C03 Ne363100 AAN85395 8E-80 Arachis cardenasii Resistant Protein NRGH3C02 Ne363101 AAV70115 1E-52 Arachis cardenasii Resistant Protein like Protein NRGH3C01 Ne363102 AAN85395 6E-76 Arachis cardenasii Resistant Protein like Protein NRGH3H12 Ne363103 AAN85401 8E-35 Arachis cardenasii Resistant Protein like Protein NRGH3B08 Ne363107 ABE84400 8E-48 Medicago truncatula Disease Resistance Protein NRGH3B07 Ne363108 AAO38219 3E-40 Arachis esculanta Resistant Protein NRGH3B06 Ne363109 AAN85380 2E-69 Arachis cardenasii Resistant Protein NRGH3B01 Ne363112 AAX71046 1E-29 Arachis hypogaea Resistant Protein NRGH3A12 Ne363113 AAN85395 4E-49 Arachis cardenasii Resistant Protein NRGH3A11 Ne363114 AAV70115 9E-68 Arachis cardenasii Resistant Protein like Protein NRGH3A08 Ne363116 AAO38219 4E-39 Manihot esculanta Resistant Protein RCA8 NRGH3A06 Ne363118 AAO20349 9E-26 Arachis stenosperma Resistant Protein NRGH3A04 Ne363120 AAO38219 2E-14 Manihot esculanta Resistant Protein RCA8 NRGH3A02 Ne363122 AAN85380 5E-55 Arachis cardenasii Resistant Protein NRGH2A06 Ne262150 QO2940 1E-08 Burkholderia ceparia Beta lactamase precursor NRGH2A09 Ne262153 ABD33309 1E-08 Populus tomentosa NBS-LRR type disease resistance protein NRGH2B04 Ne262160 ABO21765 7E-14 Vigna mungo Disease resistance protein (NBS-LRC) NRGH2B07 Ne262163 ABN42888 7E-32 Phaseolus vulgaris Resistance Protein NRGH2C01 Ne262169 AAO23077 1E-33 Glycine max Resistance Protein (NB-ARC) ( LRR Protein) NRGH2C03 Ne262171 CAC86491 5E-11 Cicer arietinum Resistance Protein RGA-B NRGH2C07 Ne262175 ABD28507 4E-25 Medicago truncatula LRR type resistance protein

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100Table 5-2(Continued) Clone # Accession # of genomic clone Accession # of matching sequence Best Evalue (BLASTX) Source of matching sequences Matching sequence from database NRGH2C09 Ne262177 ABN42879 1E-41 Phaseolus vulgaris Resistance Protein (NB-ARC) NRGH2C10 Ne262178 ABC59491 8E-34 Populus tomentosa NBS-LRR type disease resistance protein NRGH2C11 Ne262179 AA023077 2E-28 Glycine max Resistance Protein (NB-ARC) NRGH2D01 Ne262181 AAZ07904 5E-15 Ipomea batatus NBS-LRR type resistance protein NRGH2D02 Ne262182 ABC59503 7E-37 Populus tomentosa NBS-LRR type resistance protein NRGH2D06 Ne262186 ABA54554 3E-04 Triticum aestivum NBS-LRR type resistance protein NRGH2D07 Ne262187 ABC59503 6E-39 Populus tomentosa NBS-LRR type resistance protein NRGH2D09 Ne262189 ABB00419 6E-17 Capsicum annum NB-ARC resistance protein NRGH2E02 Ne262194 AAT39962 3E-16 Solanum demissum late blight resistance protein NRGH2E03 Ne262195 ABC59502 2E-38 Populus tomentosa NBS-LRR disease resistance protein NRGH2E02 Ne262196 ABC59503 3E-11 Populus tomentosa NBS-LRR disease resistance protein NRGH2E05 Ne262197 AAZ07904 2E-26 Ipomea batatus NBS-LRR disease resistance protein NRGH2E07 Ne262199 AAO95247 7E-19 Solanum tuberosum Resistance Protein (NB-ARC) NRGH2E08 Ne262200 CAC82607 2E-07 Coffea arabacia NBS-LRR type resistance protein NRGH2E09 Ne262201 ABN42889 4E-46 Phaseolus vulgaris NBS-LRR type resistance protein NRGH2E12 Ne262204 ABC59503 6E-39 Populus tomentosa NBS-LRR type disease resistance protein NRGH2F01 Ne262205 AAT39962 1E-06 Solanum demissum late blight resistance protein NRGH2F02 Ne262206 AAZ07904 6E-16 Ipomea batatus NBS-LRR type resistance protein NRGH2F03 Ne262207 IT48898 4E-05 Arabidopsis thaliana disease resistance protein RPP8 (NB-ARC) NRGH2F06 Ne262210 ABN42889 1E-36 Phaseolus vulgaris NBS-LRR disease resistance protein NRGH2F08 Ne262212 ABC59503 4E-13 Populus tomentosa NBS-LRR disease resistance protein NRGH2F09 Ne262213 CAC82598 3E-21 Coffea arabacia NB-ARC resistance protein NRGH2F10 Ne262214 ABC59503 1E-30 Populus tomentosa NBS-LRR type resistance protein NRGH2F11 Ne262215 ABC59503 7E-32 Populus tomentosa NBS-LRR type resistance protein NRGH2F12 Ne262216 AAZ07904 5E-18 Ipomea batatus NBS-LRR type resistance protein NRGH2G01 Ne262217 AAT39962 2E-15 Solanum demissum late blight resistance protein NRGH2G02 Ne262218 AAT39962 7E-16 Solanum demissum late blight resistance protein NRGH2G05 Ne262221 AAZ07910 5E-07 Ipomea batatus NBS-LRR type disease resistance protein NRGH2G06 Ne262222 AAZ07910 2E-18 Ipomea batatus NBS-LRR type disease resistance protein

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101Table 5-2(Continued) Clone # Accession # of genomic clone Accession # of matching sequence Best Evalue (BLASTX) Source of matching sequences Matching sequence from database NRGH2G10 Ne262226 AAT69649 1E-27 Oryza sativa NBS-LRR type resistance protein NRGH2G11 Ne262227 AAU89649 2E-15 Poncirus trifoli NB-ARC type resistance protein NRGH2G12 Ne262228 ABN42889 1E-47 Phaseolus vulgaris NB-ARC type resistance protein NRGH2H01 Ne26229 AAZ99766 5E-13 Triticum aestivum NBSLRR type RGA ( resistant protein) NRGH2H09 Ne262237 AAU89649 8E-15 Poncirus trifoli NB-ARC type resistance protein NRGH2H11 Ne262239 ABC59503 7E-29 Populus tomentosa NBS-LRR type resistance protein NRGH2H12 Ne262240 AAZ07904 4E-19 Ipomea batatus NBS-LRR type resistance protein NRGH1B02 Ne250949 ABH06479 4E-11 Prunus avium NBS-LRR type resistance protein NRGH1B04 Ne250951 AB148864 1E-80 Arachis hypogaea NBS-LRR type resistance protein NRGH1C07 Ne250965 ABN42879 2E-06 Phaseolus vulgaris NB-ARC type resistance protein NRGH1C10 Ne250968 ABN42879 7E-05 Phaseolus vulgaris NB-ARC type resistance protein NRGH1E07 Ne250980 ABI18151 4E-57 Arachis hypogaea NBS-LRR type resist ant protein GnRGC5 TIR type Resistance Protein NRGH2A12 Ne262156 ABD28703 2E-29 Medicago truncatula TIR type resistance protein NRGH2B08 Ne262164 ABD28703 1E-30 Medicago truncatula TIR type resistance protein NRGH2C05 Ne262173 ABD28703 2E-28 Medicago truncatula TIR type resistance protein NRGH2C06 Ne262174 ABD28703 2E-36 Medicago truncatula TIR type resistance protein NRGH1B06 Ne250953 ABD28703 1E-24 Medicago truncatula TIR type resistance protein NRGH1B07 Ne250954 ABD28703 3E-37 Medicago truncatula TIR type resistance protein NRGH1C04 Ne250962 ABD28703 4E-13 Medicago truncatula TIR type resistance protein NRGH1C08 Ne250966 ABD28703 8E-41 Medicago truncatula TIR type resistance protein NRGH1E10 NE250983 ABD28703 3E-32 Medicago truncatula TIR type resistance protein Beta Lactamase Precursor NRGH2A06 Ne262150 QO2940 1E-08 Burkholderia ceparia Beta lactamase precursor Catalase NRGH3H06 Ne363043 Q59296 3E-09 Campylobactor jejuni Catalase NRGH3H05 Ne363044 Q59296 2E-09 Campylobactor jejuni Catalase NRGH3G07 Ne363053 XP_731877 2E-09 Campylobactor jejuni Catalase

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102Table 5-2(Continued) Clone # Accession # of genomic clone Accession # of matching sequence Best Evalue (BLASTX) Source of matching sequences Matching sequence from database NRGH3G04 Ne363056 ABK60177 2E-09 Campylobactor jejuni Catalase NRGH3G02 Ne363057 ABK60177 2E-09 Campylobactor jejuni Catalase NRGH3C12 Ne363091 Q59296 2E-09 Campylobactor jejuni Catalase NRGH3B11 Ne363104 Q59296 2E-09 Campylobactor jejuni Catalase NRGH3B09 Ne363106 Q59296 2E-09 Campylobactor jejuni Catalase Hypothetical Protein NRGH2B12 Ne262168 AAA72620 1E-08 Synthetic Pr otein Diptheria T oxin Protein DT-201 NRGH2B03 Ne262159 AAA72620 1E-08 Synthetic Construct Produces Diphtheria toxin NRGH3G07 Ne363053 XP_731877 3E-12 Cicer arietinum Reverse Transcriptase NRGH3G04 Ne363056 ABK60177 1E-16 Zingiber officinalis Putative Reverse Transcriptase NRGH3F07 Ne363062 ABK60177 1E-16 Zingiber officinalis Putative Reverse Transcriptase NRGH3E11 Ne363070 ABK60177 1E-16 Zingiber officinalis Putative Reverse Transcriptase NRGH3E09 Ne363072 ABK60177 8E-17 Zingiber officinalis Putative Reverse Transcriptase NRGH3E05 Ne363076 ABK60177 1E-14 Zingiber officinalis Putative Reverse Transcriptase NRGH3D10 Ne363083 ABK60177 1E-16 Zingiber officinalis Putative Reverse Transcriptase NRGH3D05 Ne363087 ABK60177 1E-16 Zingiber officinalis Putative Reverse Transcriptase NRGH3C12 Ne363091 CAD59768 8E-08 Cicer arietinum Putative Reverse Transcriptase NRGH3C05 Ne363098 ABK60177 1E-16 Zingiber officinalis Putative Reverse Transcriptase NRGH3B03 Ne363110 ABK60177 1E-16 Zingiber officinalis Putative Reverse Transcriptase NRGH2A03 Ne262147 CAL37000 3E-09 Platanus acerifolia Putative Reverse Transcriptase NRGH2B05 Ne262161 CAL37000 3E-09 Platanus acerifolia Putative Reverse Transcriptase NRGH1E08 Ne250981 CAL37000 3E-09 Platanus acerifolia Reverse transcriptase Ribonuclease H NRGH2A08 Ne262152 NP758764 2E-12 Erwinia amylovora Ribonuclease H NRGH2A09 Ne262153 ABD33309 1E-08 Medicago truncatula Ribonuclease H NRGH1B05 Ne250952 ABI33309 8E-04 Medicago truncatula Ribonuclease H

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103 APPENDIX A EST CLONES FROM THE ROOT-KNOT NEMATODE RESISTANT CULTIVAR NEMATAM WITH A CUT O FF E-VALUE BELOW 1E-04

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104Table A-1. EST clones from the root-knot nematode resistant cultivar NemaTA M with a cut off E-value below 1E-04 Clone ID Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function SSHPL1F07 Ne469400 XP_754657 2.7 Aspergillus fumigatus RNA recognition motif SSHPL1A08 Ne469363 BAC14473 0.97 Oceanobacillus iheyensis Late competence protein SSHPL1H01 Ne469382 EAL30259 0.73 Drosophila pseudoobscura Unknown protein SSHPL2A12 Ne472915 ABE93082 1.3 Medicago truncatula Unknown protein SSHPL2C12 Ne472970 BAA00448 1.5 Mus musculus Unknown protein SSHPL2A10 Ne472917 BAD13492 5 Chlamydomonas reinhardtii Unknown protein SSHPLA06 Ne469365 AAT71913 2.2 Rock bream iridovirus Unknown protein SSHPLA09 Ne469362 XP_754657 0.25 Aspergillus fumigatus RNA recognition motif SSHPL1H09 Ne469374 ABA86508 8.1 Drosophila melanogaster Ribonuclease inhibitor SSHPL1A11 Ne469360 AAH54715 0.57 Danio rerio Hypothetical protein SSHPL1H12 Ne469371 XP_505388 0.74 Yarrowia lipolytica Hypothetical protein SSHPL1G03 Ne469392 EAM73150 3.6 Kineococcus radiotolerans Hypothetical protein SSHPL1E06 Ne469413 XP_505388 0.14 Yarrowia lipolytica Hypothetical protein SSHPL1E09 Ne469410 ABE88542 42.4 Medicago truncatula Hypothetical protein SSHPL2A06 NE472921 XP_534180 0.77 Canis familiaris Hypothetical protein SSHPL2A05 Ne472922 XP_640433 8.5 Dictyostelium discoideum Hypothetical protein SSHPL1H04 Ne469379 EAM73150 1.3 Kineococcus radiotolerans Hypothetical protein SSHPL1E08 Ne469411 AAW44411 0.32 Cryptococcus neoformans Hypothetical protein SSHPL1E12 Ne469407 XP_505388 0.32 Yarrowia lipolytica Hypothetical protein SSHPLE11 Ne469408 XP_754657 1.6 Aspergillus fumigatus RNA recognition motif SSHPL1D06 Ne469425 XP_700570 1.2 Danio rerio Recombination signal recognition protein SSHPL1D03 Ne469428 ZP_00787826 2.7 Streptococcus agalactiae Restriction enzyme SSHPL1D12 Ne469419 NP_573462 2 Mus musculus Ion transport protein SSHL1C09 Ne469434 XP_744235 2 Plasmodium chabaudi Hypothetical protein

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105Table A-1(Continued) Clone ID Accession # of EST clone Accession # of matching sequence E-value Sourceof matching sequence Function SSHPL1C06 Ne469437 CAJ06381 1.2 Leishmania major Hypothetical protein SSHPL1B03 Ne469452 XP_711078 0.59 Cadida albicans Hypothetical protein SSHPL2D05 Ne472965 ZP_01122517 0.042 Robiginitalea biformata Hypothetical protein SSHPL2B11 Ne472983 AAH95705 9.3 Danio rerio Hypothetical protein SSHPL2C09 Ne472973 XP_637648 3.2 Dictyostelium discoideum Homeodomain (Hox) protein SSHPL2B03 Ne472990 ABB81899 5.4 Sclerophasma paresisensis NADH dehydrogenase SSHPL2C08 Ne472974 ZP_01050141 5.5 Cellulophaga species DNA polymerase SSHPL2C07 Ne472975 XP_0010660855.5 Rattus norvegicus G-protein receptor SSHPL2C06 Ne472976 AAV3683838 5.5 Drosophila melanogaster Unknown protein

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106 APPENDIX B EST CLONES FROM THE ROOT-KNOT NEMATODE SUSCEPTIBLE CULTIVAR FLORUNNER WITH A CUT O FF E-VALUE BELOW 1E-04

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107Table B-1. EST clones from the root-knot nematode susceptible cultivar Florunne r with a cut off E-value below 1E-04 Clone ID Accession # of EST clone Accession # of matching sequence E-value Source of matching sequence Function SSHPL3A06 FL468991 ABE83065 0.039 Medicago truncatula DNA-binding protein SSHPL2E02 FL472956 EAS32068 6.5 Coccidioides immitis Hypothetical protein SSHPL3E09 FL469036 AAC32828 0.025 Glycine max Ammonium transporter SSHPL3E11 FL469034 BAD94515 1 Oncorhynchus keta Peroxisome receptor SSHPL3E06 FL469039 NP_001035725 5.1 Danio rerio Hypothetical protein SSHPL3B09 FL469072 BAC42411 0.025 Arabidopsis thaliana Unknown protein SSHPL2F11 FL472935 EAS32068 3.8 Coccidioides immitis Hypothetical protein SSHPL2E01 FL472957 AAB39638 0.001 Glycine max Zinc finger protein

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122 BIOGRAPHICAL SKETCH Sivananda Varma Tirumalaraju was born in Rajahmundry, India. He graduated from Acharya N.G. Ranga Agricultural University with B. Sc. (Ag) degree ma joring in agricultural sciences in1999. In 2002, he completed his M. Sc. (Ag) in Genetics and Plant Breeding at University of Agricultural Sciences, India. The title of his Mast ers dissertation was Identification of PCR-Based DNA Markers Linked with Resi stance to Rust in Groundnut ( Arachis hypogaea L.). During his Masters degree, he joined International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) as a research scholar and scientific officer until 2002. In the fall of 2002, he got enrolled in the Cell and Molecular Biology program at University of Arkansas and later on continue d his doctoral studies from August 2003 at the University of Florida. He is the second son of Jagannadha Raju Tirumalaraju and Indira Tirumalaraju.