Impact Of Host Plant Xylem Fluid On Xylella Fastidiosa Multiplication ...

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Spring 2004 and a total of 4,500 seeds have been collected and placed into cold stratification. Should the initial subset of the family segregate in a 1:1 resistant to susceptible ratio as expected the expanded family of approximately 2,000 to 3,000 genotypes will be an excellent choice for fine resolution placement of the PdR1 resistance gene. This would be the first step toward placement of resistance markers (flanking the PdR1 locus) onto a bacterial artificial chromosome (BAC) within a genomic library in a procedure termed “chromosome landing” (Tanksley et al. 1995). Plans for construction of the library are underway. Objective 2 The original genetic linkage map was based primarily on AFLP markers with 375 placed on the map, with an additional 32 ISSR, 25 RAPD and 9 SSR markers (Doucleff et al. 2004). Our efforts expanded to more reliable SSR markers in order to construct a repeatable framework map useful for more precise placement of primary resistance genes, QTL analysis and marker-assisted selection. Among the marker classes added to the map 310 SSR markers have been tested, 155 were polymorphic in the parents and all have been added to the map; 90 EST derived SSR markers have been tested, 60 of them were polymorphic and 46 have been added to the map; 20 EST markers (provided by Doug Adams) have been tested and 16 were added to the map (Table 1). A total of 217 markers (SSR, EST-SSR and ESTP) tested on 188 genotypes have now been utilized for map construction. The 217 SSR markers included some that have been previously published and many that were developed by Vitis Microsatellite Consortium and are as yet unpublished. All markers were tested on a small set of 8 DNA samples including both parents and run on 6 % polyacrylamide gels. DNA on the gels was visualized by silver staining with a commercial kit (Promega). We have tested and used all available informative genomic microsatellite markers for the 9621 population. Meanwhile, we also initiated collaboration efforts with the research group at INRA (Montpellier, France) to obtain primer sequences of SSR markers developed at their facility. To develop ESTP (expressed sequence tagged polymorphism) markers, sequences of grape cDNA were obtained from Dr. Doug Adams (Department of Viticulture and Enology, UC Davis). Potential PCR primers were designed using the computer program PRIMER 0.5. Primers were selected to have similar properties to facilitate standard conditions for PCR reactions. Primers are 20 to 23 nucleotides long with GC contents of 50-60% and melting temperature ranging from 59-64°C. Amplification and polymorphism for each EST was tested on 2% agarose gels. If length base polymorphisms were not revealed, then a set of 10 different restriction enzymes (HindIII, EcoRI, Ava II, BstNI, DraI, Hae III, Hinf1, Msp I, EcoRV, Rsa I) were tested to find restriction site based polymorphism among parents D89090-15 and F8909-17. Objective 3 There are now a large number of EST derived SSR markers available, in addition to the genomic SSR markers from the Vitis Microsatellite Consortium. The EST derived SSR markers are more valuable if the cDNA sequence from which the EST was derived has a known function as determined by comparisons with homologs from other EST databases. We plan on selecting EST-SSR markers that show homology to genes which control disease resistance along with those that control other important morphological, physiological and agronomic traits. So far we have tested 90 EST-SSR markers from three different sources (Table 1) and 45 of informative markers were added to the entire core set of 9621 population. Our goal is to screen an additional 100-150 EST-SSR markers with putative known function and we are adding to the map as they are completed. Objective 4 In order to develop the core framework map based on SSR markers, preliminary linkage analysis for each parent was carried out with MAPMAKER 2.0. Each segregating locus was paired with a “dummy” locus, resulting in a doubled data set. Linkage groups obtained from the doubled data set were then divided into two symmetrical sets of groups and one set was chosen for further detail. The "first order" and "compare" commands were used to determine the probable order of all markers in each linkage group. The integrated linkage analysis to obtain the sex-average map was performed with JOINMAP 2.0 (LOD 5.0 and recombination frequency 0.45). Using the fixed sequence command, the order of markers was determined relative to the established order obtained from the initial MAPMAKER analysis. Map units in centimorgans (cM) were derived from the Kosambi (K) mapping function. The integrated consensus map analysis was carried out with JOINMAP 3.0. The consensus linkage map was developed with 217 markers (155 SSR markers, 45 EST-SSR, 16 ESTP markers and the Pierce’s disease resistance locus). A total of 214 markers fall in 19 linkage groups and only 3 markers were unlinked. Total map length is 1300 cM with average distance between markers of 5.9 cM. All markers were evenly distributed. The current map is depicted in Figure 1. The largest linkage group was comprised of 30 markers and smallest group consisted of 4 markers (Table 2). The locus for Pierce’s disease resistance mapped to linkage group 14 with flanking markers on each side (Figure 1). Many additional markers have been added but have not been included on the map. To saturate a narrow region around the PdR1 locus resistance locus with molecular markers, the strategy of bulk segregant analysis (BSA) (Michelmore et al. 1991) in concert with the AFLP marker system has been initiated in cooperation with our report titled “Optimizing marker-assisted selection (MAS) for resistance to <strong>Xylella</strong> fastidiosa to accelerate breeding of PD resistant grapes.” Work has begun within two segregating families from susceptible by resistant crosses. <strong>On</strong>e family, C8909-07 by F8909-08, segregates 1:1 resistant to susceptible and a good correlation between resistance and resistance marker alleles has been established. A bulk of the DNA from the 12 most susceptible and a bulk of the DNA the 12 most - 69 -
esistant genotypes are in process and will be tested for AFLP polymorphisms utilizing florescent primers and visualized on a PE 3100 sequencer. Table 1. Data on number of markers mapped for the 9621 (D8909-15 x F8909-17) mapping population. Molecular Markers Genomic SSR VMC published/unpublished 134 VVMD 10 VVS 2 INRA 9 EST derived SSR Southern Cross University, Australia 4 INRA, France 7 Genome Facility (U.C. Davis) 35 ESTP markers Doug Adams/NCBI data base 16 Grand Total 217 Table 2. Details of the 9621 genetic linkage map. Linkage groups 19 Linked markers 214 Total map length 1300 cM Average distance between markers 5.98 cM Largest group (PD linkage group) 30 markers 80cM (group14) Smallest group 4 markers 18cM (group 15) Figure 1a. Riaz & Walker2004 SSR based genetic linkage map of 9621 (8909-15 X8909-17) - 70 -
Page 1 and 2: IMPACT OF HOST PLANT XYLEM FLUID ON
Page 3 and 4: 0.18 600 Optical density 0.16 0.14
Page 5 and 6: OPTIMIZING MARKER-ASSISTED SELECTIO
Page 7 and 8: esistant and susceptible genotypes
Page 9: MAP BASED IDENTIFICATION AND POSITI
Page 13 and 14: BREEDING PIERCE’S DISEASE RESISTA
Page 15 and 16: Progeny from crosses of field resis
Page 17 and 18: a = (1=low, 4= high); b = (1=green,
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Page 23 and 24: v.8) for differences due to the pla
Page 25 and 26: SHARPSHOOTER FEEDING BEHAVIOR IN RE
Page 27 and 28: correlated with all other findings
Page 29 and 30: EFFECTS OF FEEDING SUBSTRATE ON RET
Page 31 and 32: REFERENCES Bextine, B. and T. Mille
Page 33 and 34: will also provide insights into the
Page 35 and 36: OBJECTIVES 1. Determine glassy-wing
Page 37 and 38: GWSS per 30 s sweep (seasonal avera
Page 39 and 40: ELISA for the presence of GWSS egg
Page 41 and 42: Project Leader: Thomas P. Freeman E
Page 43 and 44: Freeman, T. P., R. A. Leopold, D. R
Page 45 and 46: pathogen, when they move into viney
Page 47 and 48: A NOVEL IMMUNOLOGICAL APPROACH FOR
Page 49 and 50: 1.6 GWSS Adults That Fed on Protein
Page 51 and 52: Hagler, J.R. & S.E. Naranjo. 2004.
Page 53 and 54: RESULTS: Oviposition Survey Wild gr
Page 55 and 56: Host specificity testing: No-choice
Page 57 and 58: currently employed morphological ch
Page 59 and 60: increase our present understanding
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BIOLOGY AND MORPHOMETRIC ANALYSIS O
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Table 1. Mean a developmental durat
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EFFECTS OF USING CONSTANT AND CYCLI
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REFERENCES Gautam, R. D. 1986. Effe
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PARASITISM OF THE GLASSY-WINGED SHA
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Table 1. Parasitism by G.ashmeadi o
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Project Leader: Robert F. Luck Dept
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A more interesting analysis using t
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MYCOPATHOGENS AND THEIR EXOTOXINS I
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POPULATION DYNAMICS AND INTERACTION
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No egg masses were recorded on olea
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EXPLORATION FOR FACULTATIVE ENDOSYM
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Although Baumannia and Wolbachia we
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EFFECTS OF SUBLETHAL DOSES OF IMIDA
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Table 2. Mortality and flight perfo
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A NOVEL METHOD TO INDUCE OVIPOSITIO
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REFERENCES Brodbeck, B. V., P. C. A
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Cohorts H. coagulata neonates were
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REFERENCES Blua, M. J. and D. J. W.
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Figure 2 shows the average numbers
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REPRODUCTIVE BIOLOGY AND PHYSIOLOGY
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REFERENCES Blua, M. J., Phillips, P
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RESULTS Some plant families had no
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Table 5. Winter, spring and summer
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16S rDNA is by far the most sequenc
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DNA MICROARRAY AND MUTATIONAL ANALY
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Inhibition of biofilm is BSA concen
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CULTURE-INDEPENDENT ANALYSIS OF END
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Borneman, J., M. Chrobak, G. Della
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P (CFU P OBJECTIVE 1. Determine the
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Purcell, AH and SR Saunders. 1999a.
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Figure 1: Southern blot of tolC::ap
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Project Leader: David Gilchrist Dep
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III. Production of transgenic plant
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RESULTS Construction of cDNA Librar
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CONCLUSIONS Genetic resistance and
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Mutagenesis of Xylella The EZ::TN T
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ISOLATION OF BACTERIOPHAGES SPECIFI
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Project Leader: Michele M. Igo Sect
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outer membrane fractions using two-
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1995; Purcell and Saunders, 1999).
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DEVELOPMENT OF SSR MARKERS FOR GENO
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Strain Name Host of Origin County o
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ROLE OF ATTACHMENT OF XYLELLA FASTI
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wild-type cells was not conclusive
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DETERMINATION OF GENES CONFERRING H
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S1 S2 S3 S4 Nb123456789bb123456789b
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MULTILOCUS SEQUENCE TYPING TO IDENT
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GENOME-WIDE IDENTIFICATION OF RAPID
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Americas and since most of the plan
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EFFECTS OF CHEMICAL MILIEU ON ATTAC
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CONCLUSIONS Our overall objective i
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RESULTS Objective 1. We conducted t
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Redak, R. A., Purcell, A. H., Lopes
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In parallel, an “activating trans
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PATTERNS OF XYLELLA FASTIDIOSA INFE
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% of vessels 100 80 60 40 20 Mugwor
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DOCUMENTATION AND CHARACTERIZATION
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Magnolia002 showed more identity (9
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PLASMID ADDICTION AS A NOVEL APPROA
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GENETIC VARIABILITY OF XYLELLA FAST
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- 246 -
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probing activities). In preliminary
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the slow release valve was opened a
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12. Hopkins DL (1977) Diseases caus
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The almost complete absence of info
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QUANTIFYING LANDSCAPE-SCALE MOVEMEN
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Table 1. The mean (±SD) ELISA read
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EPIDEMIOLOGICAL ASSESSMENTS OF PIER
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multiply to relatively high (easily
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SPATIAL DATABASE CREATION AND MAINT
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Project Leader: Thomas M. Perring D
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Figure 2. Individual leaves from a
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The state variables, process functi
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DEVELOPMENT OF A FIELD SAMPLING PLA
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Figure 1. Three main dispersion pat
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- 278 -
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OBJECTIVES 1. Track the movement of
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REFERENCES 1. Beard, C.B., Cordon-R
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cases, positive phloem samples were
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EXPLOITING XYLELLA FASTIDIOSA PROTE
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Figure 2. Polymer disk accumulation
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CHARACTERIZATION OF NEONICOTINOIDS
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EVALUATION OF RESISTANCE POTENTIAL
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Project Leader: Doug Cook Dept. of
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Based on the in silico analysis, de
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CONTROL OF PIERCE’S DISEASE THROU
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Figure 1. Oleander ‘White’ afte
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RESULTS During the reporting period
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DEVELOPMENT OF AN ARTIFICIAL DIET A
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DESIGN OF CHIMERIC ANTI-MICROBIAL P
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Neutrophil elastase Cecropin B Chim
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and WTXb is very low (0.00-0.40%) a
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100 100 0.056 North America 100 0.0
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GENETIC DIFFERENTIATION AMONG GEOGR
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Table 1. Single-populations descrip
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MOLECULAR DISTINCTION BETWEEN POPUL
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These novel observations strongly s
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SEQUENCE DIVERGENCE IN TWO MITOCHON
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Table 3. Pairwise sequence distance
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DEVELOPMENT OF MOLECULAR DIAGNOSTIC
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A. B. Relative Density of SCAR Band
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THE ALIMENTARY TRACK OF GLASSY-WING
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We have dissected and identified al
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REALIZED LIFETIME PARASITISM AND TH
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REPRODUCTIVE AND DEVELOPMENTAL BIOL
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Mean adult longevity (days) 25 20 1
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the proposed exploratory work; thes
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SEARCHING FOR AND COLLECTING EGG PA
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REFERENCES Hoddle, M. S. and S. V.
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some Gram(+) bacteria, but are inac
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7. Hultmark, D., et al., Insect Imm
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Isolation of Fungal Pathogens Soil
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IDENTIFICATION OF MECHANISMS MEDIAT
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concentrations of 1.5 x 10 7 bacter
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anchor it in the outer membrane (sh
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SYMBIOTIC CONTROL OF PIERCE’S DIS
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MANAGEMENT OF PIERCE’S DISEASE OF
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pfF mutants are taken up by insects
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Simpson, A. J. G., F. C. Reinach, P
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Al-Wahaibi, A.K., Owen, and J. G. M
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patterns differed among the lines,
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Punja, Z.K. 2001. Genetic engineeri
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mind, we conducted an additional st
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REFERENCES Toscano, N., Byrne, F.,
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RESULTS AND CONCLUSIONS The program
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COMPATIBILITY OF INSECTICIDES WITH
Page 321 and 322:
More tests are in progress to addre
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Impact Of Host Plant Xylem Fluid On Xylella Fastidiosa Multiplication ...

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