A major quantitative trait locus (QTL) controlling resistance to Pierce's disease (PD) of... more A major quantitative trait locus (QTL) controlling resistance to Pierce's disease (PD) of grape, caused by the bacterium Xylella fastidiosa (Xf), was identified on a Vitis linkage map and denoted as 'Pierce's disease resistance 1' (PdR1). Placement of the locus was accomplished by evaluating a family of full-sib progeny from a cross of two PD-resistant interspecific hybrids with resistance inherited from Vitis arizonica. Resistance was measured under greenhouse conditions by direct quantification of Xf numbers in stem tissues as well as by evaluation of disease symptoms based on leaf scorch and a cane maturation index (CMI). A large QTL (LOD 17.2) accounting for 72% of the phenotypic variance in bacterial numbers was localized to linkage group 14 of the male parent F8909-17. The approximate 95% confidence interval around the QTL peak extended 5.7 cM when using composite interval mapping. The other disease evaluation methods (leaf scorch and CMI, respectively) placed the resistance QTL to the same region on linkage group 14, although at wider 95% confidence intervals (6.0 and 7.5 cM), lower peak LOD scores (11.9 and 7.7) and accounting for less phenotypic variance (59 and 42%). This is the first report of an Xf resistance QTL mapped in any crop species. The relevance of the markers located in the region spanning the QTL will be discussed, addressing their usefulness for the development of PD-resistant grape cultivars.
Quality protein maize (QPM) describes a range of maize cultivars with twice the content of limiti... more Quality protein maize (QPM) describes a range of maize cultivars with twice the content of limiting amino acids lysine and tryptophan compared with conventional maize, and has been developed to help reduce human malnutrition in areas where protein deficiency is prevalent and where maize is the major protein source in the diet, as in various parts of Sub-Saharan Africa (SSA). The International Maize and Wheat Improvement Center (CIMMYT), in collaboration with the International Institute of Tropical Agriculture (IITA) and the National Agricultural Research Systems (NARS) in 17 countries of SSA, has developed a broad range of QPM cultivars responding to the needs of different countries and agroecological zones. Commercial QPM seed is currently available in all collaborating countries and, based on average 2003-2005 seed production, approximately 200,000 hectares of land are being planted to QPM cultivars.
Efforts at identifying molecular markers linked to Xylella fastidiosa (Xf) resistance are continu... more Efforts at identifying molecular markers linked to Xylella fastidiosa (Xf) resistance are continuing. Our primary focus is on resistance derived from b43-17, a Vitis arizonica/candicans type collected near Monterrey, Nuevo Leon, Mexico. The '9621' V. rupestris x V. arizonica hybrid mapping family (PD resistant D8909-15 x PD resistant F8909-17) was used to localize PdR1, a primary PD resistance locus within the linkage map of the male parent F8909-17 (progeny of b43-17) and identify candidate linked resistance markers. In more recent research, a comparative mapping strategy between the '9621' linkage map and other SSR maps within Vitis was used to identify 9 SSR markers within 10 cM of the resistance locus. Resistance from the female parent D8909-15 has not yet been localized to a genetic map. The strategy of bulk segregant analysis (BSA) in concert with the AFLP marker system has been initiated to saturate the region around the resistance locus and is expected to yie...
Efforts at identifying genetic markers tightly linked to Xylella fastidiosa (Xf) resistance are c... more Efforts at identifying genetic markers tightly linked to Xylella fastidiosa (Xf) resistance are continuing. These efforts are primarily focused on the 9621 mapping population (D8909-15 x F8909-17) and are in cooperation with fine-scale mapping efforts discussed in our report "Map based identification and positional cloning of Xylella fastidiosa resistance genes from known sources of Pierce's disease resistance in grape." This project is adding 200 SSR markers to the 9621 map and has positioned Xf resistance from the male parent F8909-17 on the lower arm of linkage group 14, where Xf resistance is flanked by multiple markers. Resistance from the female parent D8909-15 maps as a quantitative trait locus. The addition of additional SSR marker is expected to reduce the distance between SSR markers and Xf resistance to about 2 cM and lead to the development and utilization of very specific sequence characterized amplified region (SCAR) primers. These markers will be applied...
Pierce's disease (PD), caused by the bacterium Xylella fastidiosa, is an important disease of gra... more Pierce's disease (PD), caused by the bacterium Xylella fastidiosa, is an important disease of grapevines in California, across the southern United States, and into South America. In regions where disease pressure is high, the cultivation of Vitis vinifera cultivars is difficult or impossible. This study reports on the introgression of PD resistance into elite wine, table, and raisin grape genetic backgrounds and on the reliability of PCR-based marker-assisted selection (MAS) to accelerate the breeding of PD-resistant grapes. This work documents the introgression of PD resistance from a homozygous resistant form of V. arizonica, b43-17. A total of 4,321 seedlings from 83 different crosses of resistant selections and high-quality susceptible V. vinifera cultivars (F1, first and second generation modified backcrosses (mBC1, mBC2) were screened with two to three flanking microsatellite markers (VVIP26 and ctg1026876, VMC2a5) in early spring 2006 and 2007. The alleles linked to resistance were unique in size and were not shared by susceptible V. vinifera selections. Based on the presence of unique resistant alleles, 1,683 seedlings from wine, table, and raisin grape background were selected. The distinctiveness of these resistant alleles allows the use of MAS to optimize the breeding of PD-resistant grape cultivars.
Symptoms of Pierce's disease (PD) were assessed under greenhouse conditions on field-resistan... more Symptoms of Pierce's disease (PD) were assessed under greenhouse conditions on field-resistant and field-susceptible grapevines in order to characterize the PD resistance phenotype in the genus Vitis. A cane maturation index (CMI) was developed to quantify the green-islands symptom, which was measured at 12 weeks post-bacterial inoculation, along with leaf scorch and percentage of xylem vessels blocked by occlusions. Canes of resistant genotypes matured normally and had a significantly lower CMI score of 0.9 (on a 0-to-6 scale) compared with 5.1 for the susceptible genotypes. The CMI scoring method had a high correlation (R(2) = 0.91) with previously characterized field performance, whereas leaf scorch had only a moderate correlation (R(2) = 0.51) with field performance. Average scorched area on leaves of the susceptible and resistant genotypes was 80 and 48%, respectively, demonstrating that leaf scorch can be extensive in resistant genotypes under the presented screening condi...
all collaborating countries and, based on average 2003-2005 seed production, approximately 200,00... more all collaborating countries and, based on average 2003-2005 seed production, approximately 200,000 hectares of land are being planted to QPM cultivars.
A framework genetic map based on genomic DNA-derived SSR, EST-derived SSR, EST-STS and EST-RFLP m... more A framework genetic map based on genomic DNA-derived SSR, EST-derived SSR, EST-STS and EST-RFLP markers was developed using 181 genotypes generated from D8909-15 (female) £ F8909-17 (male), the '9621' population. Both parents are half siblings with a common female parent, Vitis rupestris 'A. de Serres', and diVerent male parents (forms of V. arizonica). A total of 542 markers were tested, and 237 of them were polymorphic for the female and male parents. The female map was developed with 159 mapped markers covering 865.0 cM with an average marker distance of 5.
The inheritance of resistance to Xylella fastidiosa (Xf), the bacterium which causes Pierce&#... more The inheritance of resistance to Xylella fastidiosa (Xf), the bacterium which causes Pierce's disease (PD) in grapevines, was evaluated within a factorial mating design consisting of 16 full-sib families with resistance derived from Vitis arizonica interspecific hybrids. Measurements of disease progression under greenhouse conditions were based on quantitative assessment of Xf populations in stem tissues and on three phenotypic scores: leaf scorch, a cane maturation index (CMI) and an index that incorporated shoot stunting into the cane maturation index (CMSSI). Measurement of bacterial populations yielded the highest broad-sense heritability for resistance on a genotype mean basis (0.97), indicating that this measure of resistance was the least effected by environmental variation. Narrow-sense heritability of PD resistance was moderately high and measured 0.52, 0.60, 0.63 and 0.37 for Xf populations, CMI scores, CMSSI scores and leaf scorch values, respectively. Complex segregation analysis using the computer program Statistical Analysis for Genetic Epidemiology (SAGE: ) strongly indicated the existence of a major gene for PD resistance, which accounted for 91% of the total genetic variance. Conversion of the quantitative data into qualitative resistance levels and evaluation via a chi-square analysis showed that 15 of the 16 families segregated in accordance with a single gene hypothesis with a dominant allele controlling PD resistance. These data indicate that the trait should be relatively easy to pass on from parents to progeny in a breeding program for the development of PD-resistant grape cultivars, particularly when selection is based on cane maturation scores or stem Xf populations.
Development of a seed DNA-based genotyping system for marker-assisted selection (MAS) has provide... more Development of a seed DNA-based genotyping system for marker-assisted selection (MAS) has provided a novel opportunity for understanding aberrant reproductive phenomena such as hetero-fertilization (HF) by observing the mismatch of endosperm and leaf genotypes in monocot species. In contrast to conventional approaches using specific morphological markers, this approach can be used for any population derived from diverse parental genotypes. A large-scale experiment was implemented using seven F(2) populations and four three-way cross populations, each with 534 to 1024 individuals. The frequency of HF within these populations ranged from 0.14% to 3.12%, with an average of 1.46%. The highest frequency of HF in both types of population was contributed by the pollen gametes. Using three-way crosses allowed, for the first time, detection of the HF contributed by maternal gametes, albeit at very low frequency (0.14%-0.65%). Four HF events identified from each of two F(2) populations were tested and confirmed using 1032 single nucleotide polymorphic markers. This analysis indicated that only 50% of polymorphic markers can detect a known HF event, and thus the real HF frequency can be inferred by doubling the estimate obtained from using only one polymorphic marker. As expected, 99% of the HF events can be detected by using seven independent markers in combination. Although seed DNA-based analysis may wrongly predict plant genotypes due to the mismatch of endosperm and leaf DNA caused by HF, the relatively low HF frequencies revealed with diverse germplasm in this study indicates that the effect on the accuracy of MAS is limited. In addition, comparative endosperm and leaf DNA analysis of specific genetic stocks could be useful for revealing the relationships among various aberrant fertilization phenomena including haploidy and apomixis.
ABSTRACT The pattern of Xylella fastidiosa infection in resistant and susceptible grapevines repr... more ABSTRACT The pattern of Xylella fastidiosa infection in resistant and susceptible grapevines representing a diverse selection of Vitis spp. was characterized through measurements of X. fastidiosa bacterial movement and accumulation in artificially inoculated greenhouse-grown grapevines. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was optimized for quantification of X. fastidiosa populations and tested on known amounts of X. fastidiosa added to grape tissue extracts. Predicted versus known X. fastidiosa concentrations proved to be highly correlated (R(2) = 0.99). Populations of X. fastidiosa in stem internode, stem node, petiole, and leaf blade samples from the genotypes in this study were measured at 12 weeks postinoculation using the optimized ELISA procedure. Samples from each plant part were taken at eight positions along the inoculated shoots. Systemic infection was detected in both susceptible and resistant genotypes. Resistant genotypes were characterized by significant differences in X. fastidiosa populations between stem internodes and leaves (1.0 x 10(6) and 1.1 x 10(7) cells/g of sample, respectively). In contrast, the susceptible genotypes were characterized by high mean X. fastidiosa populations in both stems and leaves (5.6 x 10(7) and 4.8 x 10(7) cells/g, respectively) the latter of which were not significantly different from the resistant genotypes. A high correlation (R(2) = 0.97) between stem X. fastidiosa numbers to previously characterized field Pierce's disease (PD) performance indicates that the quantitative ELISA measurements of X. fastidiosa in greenhouse-grown grapevines should be a useful tool for predicting PD resistance under field conditions.
Leaf collection from the field, labeling and tracking back to the source plants after genotyping ... more Leaf collection from the field, labeling and tracking back to the source plants after genotyping are rate limiting steps in leaf DNA-based genotyping. In this study, an optimized genotyping method using endosperm DNA sampled from single maize seeds was developed, which can be used to replace leaf DNA-based genotyping for both genetic studies and breeding applications. A similar approach is likely to be suitable for all plants with relatively large seeds. Part of the endosperm was excised from imbibed maize seeds and DNA extracted in 96-tube plates using individuals from eight F 2 populations and seven inbreds. The quality of the resultant DNA was functionally comparable to DNA extracted from leaf tissue. Extraction from 30 mg of endosperm yields 3-10 lg DNA, which is sufficient for analysis of 200-400 agarose-gel PCR-based markers, with the potential for several million chip-based SNP marker analyses. By comparing endosperm DNA and leaf DNA for individuals from an F 2 population, genotyping errors caused by pericarp contamination and hetero-fertilization were found to average 3.8 and 0.6%, respectively. Endosperm sampling did not affect germination rates under controlled conditions, although under normal field conditions the germination rate, seedling establishment, and growth vigor were significantly lower than that of non-sampled controls for some genotypes. However, careful field management can compensate for these effects. Seed DNA-based genotyping lowered costs by 24.6% compared to leaf DNA-based genotyping due to reduced field plantings and labor costs. A substantial advantage of this approach is that it can be used to select desirable genotypes before planting. As such it provides an opportunity for dramatic improvements in the efficiency and selective gain of breeding systems based on optimum combinations of markerassisted selection and phenotypic selection within and between generations.
A genetic linkage map of grape was constructed, utilizing 116 progeny derived from a cross of two... more A genetic linkage map of grape was constructed, utilizing 116 progeny derived from a cross of two Vitis rupestris × V. arizonica interspecific hybrids, using the pseudo-testcross strategy. A total of 475 DNA markers -410 amplified fragment length polymorphism, 24 intersimple sequence repeat, 32 random amplified polymorphic DNA, and nine simple sequence repeat markers-were used to construct the parental maps. Markers segregating 1:1 were used to construct parental framework maps with confidence levels >90% with the Plant Genome Research Initiative mapping program. In the maternal (D8909-15) map, 105 framework markers and 55 accessory markers were ordered in 17 linkage groups (756 cM). The paternal (F8909-17) map had 111 framework markers and 33 accessory markers ordered in 19 linkage groups (1,082 cM). One hundred eighty-one markers segregating 3:1 were used to connect the two parental maps' parents. This moderately dense map will be useful for the initial mapping of genes and/or QTL for resistance to the dagger nematode, Xiphinema index, and Xylella fastidiosa, the bacterial causal agent of Pierce's disease.
A major quantitative trait locus (QTL) controlling resistance to Pierce's disease (PD) of... more A major quantitative trait locus (QTL) controlling resistance to Pierce's disease (PD) of grape, caused by the bacterium Xylella fastidiosa (Xf), was identified on a Vitis linkage map and denoted as 'Pierce's disease resistance 1' (PdR1). Placement of the locus was accomplished by evaluating a family of full-sib progeny from a cross of two PD-resistant interspecific hybrids with resistance inherited from Vitis arizonica. Resistance was measured under greenhouse conditions by direct quantification of Xf numbers in stem tissues as well as by evaluation of disease symptoms based on leaf scorch and a cane maturation index (CMI). A large QTL (LOD 17.2) accounting for 72% of the phenotypic variance in bacterial numbers was localized to linkage group 14 of the male parent F8909-17. The approximate 95% confidence interval around the QTL peak extended 5.7 cM when using composite interval mapping. The other disease evaluation methods (leaf scorch and CMI, respectively) placed the resistance QTL to the same region on linkage group 14, although at wider 95% confidence intervals (6.0 and 7.5 cM), lower peak LOD scores (11.9 and 7.7) and accounting for less phenotypic variance (59 and 42%). This is the first report of an Xf resistance QTL mapped in any crop species. The relevance of the markers located in the region spanning the QTL will be discussed, addressing their usefulness for the development of PD-resistant grape cultivars.
Quality protein maize (QPM) describes a range of maize cultivars with twice the content of limiti... more Quality protein maize (QPM) describes a range of maize cultivars with twice the content of limiting amino acids lysine and tryptophan compared with conventional maize, and has been developed to help reduce human malnutrition in areas where protein deficiency is prevalent and where maize is the major protein source in the diet, as in various parts of Sub-Saharan Africa (SSA). The International Maize and Wheat Improvement Center (CIMMYT), in collaboration with the International Institute of Tropical Agriculture (IITA) and the National Agricultural Research Systems (NARS) in 17 countries of SSA, has developed a broad range of QPM cultivars responding to the needs of different countries and agroecological zones. Commercial QPM seed is currently available in all collaborating countries and, based on average 2003-2005 seed production, approximately 200,000 hectares of land are being planted to QPM cultivars.
Efforts at identifying molecular markers linked to Xylella fastidiosa (Xf) resistance are continu... more Efforts at identifying molecular markers linked to Xylella fastidiosa (Xf) resistance are continuing. Our primary focus is on resistance derived from b43-17, a Vitis arizonica/candicans type collected near Monterrey, Nuevo Leon, Mexico. The '9621' V. rupestris x V. arizonica hybrid mapping family (PD resistant D8909-15 x PD resistant F8909-17) was used to localize PdR1, a primary PD resistance locus within the linkage map of the male parent F8909-17 (progeny of b43-17) and identify candidate linked resistance markers. In more recent research, a comparative mapping strategy between the '9621' linkage map and other SSR maps within Vitis was used to identify 9 SSR markers within 10 cM of the resistance locus. Resistance from the female parent D8909-15 has not yet been localized to a genetic map. The strategy of bulk segregant analysis (BSA) in concert with the AFLP marker system has been initiated to saturate the region around the resistance locus and is expected to yie...
Efforts at identifying genetic markers tightly linked to Xylella fastidiosa (Xf) resistance are c... more Efforts at identifying genetic markers tightly linked to Xylella fastidiosa (Xf) resistance are continuing. These efforts are primarily focused on the 9621 mapping population (D8909-15 x F8909-17) and are in cooperation with fine-scale mapping efforts discussed in our report "Map based identification and positional cloning of Xylella fastidiosa resistance genes from known sources of Pierce's disease resistance in grape." This project is adding 200 SSR markers to the 9621 map and has positioned Xf resistance from the male parent F8909-17 on the lower arm of linkage group 14, where Xf resistance is flanked by multiple markers. Resistance from the female parent D8909-15 maps as a quantitative trait locus. The addition of additional SSR marker is expected to reduce the distance between SSR markers and Xf resistance to about 2 cM and lead to the development and utilization of very specific sequence characterized amplified region (SCAR) primers. These markers will be applied...
Pierce's disease (PD), caused by the bacterium Xylella fastidiosa, is an important disease of gra... more Pierce's disease (PD), caused by the bacterium Xylella fastidiosa, is an important disease of grapevines in California, across the southern United States, and into South America. In regions where disease pressure is high, the cultivation of Vitis vinifera cultivars is difficult or impossible. This study reports on the introgression of PD resistance into elite wine, table, and raisin grape genetic backgrounds and on the reliability of PCR-based marker-assisted selection (MAS) to accelerate the breeding of PD-resistant grapes. This work documents the introgression of PD resistance from a homozygous resistant form of V. arizonica, b43-17. A total of 4,321 seedlings from 83 different crosses of resistant selections and high-quality susceptible V. vinifera cultivars (F1, first and second generation modified backcrosses (mBC1, mBC2) were screened with two to three flanking microsatellite markers (VVIP26 and ctg1026876, VMC2a5) in early spring 2006 and 2007. The alleles linked to resistance were unique in size and were not shared by susceptible V. vinifera selections. Based on the presence of unique resistant alleles, 1,683 seedlings from wine, table, and raisin grape background were selected. The distinctiveness of these resistant alleles allows the use of MAS to optimize the breeding of PD-resistant grape cultivars.
Symptoms of Pierce's disease (PD) were assessed under greenhouse conditions on field-resistan... more Symptoms of Pierce's disease (PD) were assessed under greenhouse conditions on field-resistant and field-susceptible grapevines in order to characterize the PD resistance phenotype in the genus Vitis. A cane maturation index (CMI) was developed to quantify the green-islands symptom, which was measured at 12 weeks post-bacterial inoculation, along with leaf scorch and percentage of xylem vessels blocked by occlusions. Canes of resistant genotypes matured normally and had a significantly lower CMI score of 0.9 (on a 0-to-6 scale) compared with 5.1 for the susceptible genotypes. The CMI scoring method had a high correlation (R(2) = 0.91) with previously characterized field performance, whereas leaf scorch had only a moderate correlation (R(2) = 0.51) with field performance. Average scorched area on leaves of the susceptible and resistant genotypes was 80 and 48%, respectively, demonstrating that leaf scorch can be extensive in resistant genotypes under the presented screening condi...
all collaborating countries and, based on average 2003-2005 seed production, approximately 200,00... more all collaborating countries and, based on average 2003-2005 seed production, approximately 200,000 hectares of land are being planted to QPM cultivars.
A framework genetic map based on genomic DNA-derived SSR, EST-derived SSR, EST-STS and EST-RFLP m... more A framework genetic map based on genomic DNA-derived SSR, EST-derived SSR, EST-STS and EST-RFLP markers was developed using 181 genotypes generated from D8909-15 (female) £ F8909-17 (male), the '9621' population. Both parents are half siblings with a common female parent, Vitis rupestris 'A. de Serres', and diVerent male parents (forms of V. arizonica). A total of 542 markers were tested, and 237 of them were polymorphic for the female and male parents. The female map was developed with 159 mapped markers covering 865.0 cM with an average marker distance of 5.
The inheritance of resistance to Xylella fastidiosa (Xf), the bacterium which causes Pierce&#... more The inheritance of resistance to Xylella fastidiosa (Xf), the bacterium which causes Pierce's disease (PD) in grapevines, was evaluated within a factorial mating design consisting of 16 full-sib families with resistance derived from Vitis arizonica interspecific hybrids. Measurements of disease progression under greenhouse conditions were based on quantitative assessment of Xf populations in stem tissues and on three phenotypic scores: leaf scorch, a cane maturation index (CMI) and an index that incorporated shoot stunting into the cane maturation index (CMSSI). Measurement of bacterial populations yielded the highest broad-sense heritability for resistance on a genotype mean basis (0.97), indicating that this measure of resistance was the least effected by environmental variation. Narrow-sense heritability of PD resistance was moderately high and measured 0.52, 0.60, 0.63 and 0.37 for Xf populations, CMI scores, CMSSI scores and leaf scorch values, respectively. Complex segregation analysis using the computer program Statistical Analysis for Genetic Epidemiology (SAGE: ) strongly indicated the existence of a major gene for PD resistance, which accounted for 91% of the total genetic variance. Conversion of the quantitative data into qualitative resistance levels and evaluation via a chi-square analysis showed that 15 of the 16 families segregated in accordance with a single gene hypothesis with a dominant allele controlling PD resistance. These data indicate that the trait should be relatively easy to pass on from parents to progeny in a breeding program for the development of PD-resistant grape cultivars, particularly when selection is based on cane maturation scores or stem Xf populations.
Development of a seed DNA-based genotyping system for marker-assisted selection (MAS) has provide... more Development of a seed DNA-based genotyping system for marker-assisted selection (MAS) has provided a novel opportunity for understanding aberrant reproductive phenomena such as hetero-fertilization (HF) by observing the mismatch of endosperm and leaf genotypes in monocot species. In contrast to conventional approaches using specific morphological markers, this approach can be used for any population derived from diverse parental genotypes. A large-scale experiment was implemented using seven F(2) populations and four three-way cross populations, each with 534 to 1024 individuals. The frequency of HF within these populations ranged from 0.14% to 3.12%, with an average of 1.46%. The highest frequency of HF in both types of population was contributed by the pollen gametes. Using three-way crosses allowed, for the first time, detection of the HF contributed by maternal gametes, albeit at very low frequency (0.14%-0.65%). Four HF events identified from each of two F(2) populations were tested and confirmed using 1032 single nucleotide polymorphic markers. This analysis indicated that only 50% of polymorphic markers can detect a known HF event, and thus the real HF frequency can be inferred by doubling the estimate obtained from using only one polymorphic marker. As expected, 99% of the HF events can be detected by using seven independent markers in combination. Although seed DNA-based analysis may wrongly predict plant genotypes due to the mismatch of endosperm and leaf DNA caused by HF, the relatively low HF frequencies revealed with diverse germplasm in this study indicates that the effect on the accuracy of MAS is limited. In addition, comparative endosperm and leaf DNA analysis of specific genetic stocks could be useful for revealing the relationships among various aberrant fertilization phenomena including haploidy and apomixis.
ABSTRACT The pattern of Xylella fastidiosa infection in resistant and susceptible grapevines repr... more ABSTRACT The pattern of Xylella fastidiosa infection in resistant and susceptible grapevines representing a diverse selection of Vitis spp. was characterized through measurements of X. fastidiosa bacterial movement and accumulation in artificially inoculated greenhouse-grown grapevines. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was optimized for quantification of X. fastidiosa populations and tested on known amounts of X. fastidiosa added to grape tissue extracts. Predicted versus known X. fastidiosa concentrations proved to be highly correlated (R(2) = 0.99). Populations of X. fastidiosa in stem internode, stem node, petiole, and leaf blade samples from the genotypes in this study were measured at 12 weeks postinoculation using the optimized ELISA procedure. Samples from each plant part were taken at eight positions along the inoculated shoots. Systemic infection was detected in both susceptible and resistant genotypes. Resistant genotypes were characterized by significant differences in X. fastidiosa populations between stem internodes and leaves (1.0 x 10(6) and 1.1 x 10(7) cells/g of sample, respectively). In contrast, the susceptible genotypes were characterized by high mean X. fastidiosa populations in both stems and leaves (5.6 x 10(7) and 4.8 x 10(7) cells/g, respectively) the latter of which were not significantly different from the resistant genotypes. A high correlation (R(2) = 0.97) between stem X. fastidiosa numbers to previously characterized field Pierce's disease (PD) performance indicates that the quantitative ELISA measurements of X. fastidiosa in greenhouse-grown grapevines should be a useful tool for predicting PD resistance under field conditions.
Leaf collection from the field, labeling and tracking back to the source plants after genotyping ... more Leaf collection from the field, labeling and tracking back to the source plants after genotyping are rate limiting steps in leaf DNA-based genotyping. In this study, an optimized genotyping method using endosperm DNA sampled from single maize seeds was developed, which can be used to replace leaf DNA-based genotyping for both genetic studies and breeding applications. A similar approach is likely to be suitable for all plants with relatively large seeds. Part of the endosperm was excised from imbibed maize seeds and DNA extracted in 96-tube plates using individuals from eight F 2 populations and seven inbreds. The quality of the resultant DNA was functionally comparable to DNA extracted from leaf tissue. Extraction from 30 mg of endosperm yields 3-10 lg DNA, which is sufficient for analysis of 200-400 agarose-gel PCR-based markers, with the potential for several million chip-based SNP marker analyses. By comparing endosperm DNA and leaf DNA for individuals from an F 2 population, genotyping errors caused by pericarp contamination and hetero-fertilization were found to average 3.8 and 0.6%, respectively. Endosperm sampling did not affect germination rates under controlled conditions, although under normal field conditions the germination rate, seedling establishment, and growth vigor were significantly lower than that of non-sampled controls for some genotypes. However, careful field management can compensate for these effects. Seed DNA-based genotyping lowered costs by 24.6% compared to leaf DNA-based genotyping due to reduced field plantings and labor costs. A substantial advantage of this approach is that it can be used to select desirable genotypes before planting. As such it provides an opportunity for dramatic improvements in the efficiency and selective gain of breeding systems based on optimum combinations of markerassisted selection and phenotypic selection within and between generations.
A genetic linkage map of grape was constructed, utilizing 116 progeny derived from a cross of two... more A genetic linkage map of grape was constructed, utilizing 116 progeny derived from a cross of two Vitis rupestris × V. arizonica interspecific hybrids, using the pseudo-testcross strategy. A total of 475 DNA markers -410 amplified fragment length polymorphism, 24 intersimple sequence repeat, 32 random amplified polymorphic DNA, and nine simple sequence repeat markers-were used to construct the parental maps. Markers segregating 1:1 were used to construct parental framework maps with confidence levels >90% with the Plant Genome Research Initiative mapping program. In the maternal (D8909-15) map, 105 framework markers and 55 accessory markers were ordered in 17 linkage groups (756 cM). The paternal (F8909-17) map had 111 framework markers and 33 accessory markers ordered in 19 linkage groups (1,082 cM). One hundred eighty-one markers segregating 3:1 were used to connect the two parental maps' parents. This moderately dense map will be useful for the initial mapping of genes and/or QTL for resistance to the dagger nematode, Xiphinema index, and Xylella fastidiosa, the bacterial causal agent of Pierce's disease.
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Papers by Alan Krivanek