Progress 10/01/99 to 09/30/04
Outputs
We have used a positional cloning approach to construct a high definition linkage map and a companion physical map that identified a large cluster of TIR-NBS-LRR sequences associated with the I locus. Diverse bean cultivars bearing the dominant allele (II) share the same haplotype as detected by DNA gel blot hybridizations with a Pv-TIR probe. In contrast, cultivars with a recessive allele display diverse haplotypes that comprise half as many TIR-hybridizing fragments as the resistant cultivars. This disparity in TIR copy number creates hemizygosity and it is likely responsible for suppressing recombination within this locus. The co-existence of the two alleles in Mexican land races of black beans conforms to the `trench warfare' model of evolution for host-pathogen interactions. RNA analysis with a Pv-TIR probe showed an accumulation of an array of TIR transcripts in both susceptible and resistant beans in response to inoculations with necrotic or non necrotic strains
of the virus. These results indicated that bean plants in general can sense the virus, but only plants with the dominant allele have the paralog(s) capable of mounting an effective resistance response.
Impacts
The I locus of the common bean controls resistance to 10 different potyviruses with host that include soybean, cowpea, watermelon, zucchini, adn passionfruit. Further characterization of the multigene family will likely identify reistance genes that may be useful in all these crop species.
Publications
- No publications reported this period
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Progress 10/01/02 to 10/01/03
Outputs
We have constructed a BAC clone-based contig comprising the I locus of the common bean. This locus comprises a multigene family that belongs to the TIR-NBS-LRR class of disease resistance genes, and extends over approximately 500 Kb. Two lines of evidence indicate that resistance to BCMV is encoded by at least one copy of the TIR-NBS-LRR family. Through genetic analysis, we have determined that the resistance phenotype co-segregates perfectly with the multigene family, and that while no recombination has been detected within the multigene family, few recombinants have been detected between this megalocus and flanking single copy sequences. In addition, we have also found that transcripts from this multigene family are expressed at a higher level in the virus-unchallenged resistant line than in the susceptible line, and that the transcript level increases dramatically after inoculating with the virus.
Impacts
The I locus of the common bean controls resistance to 10 different potyviruses with host that include soybean, cowpea, watermelon, zucchini, adn passionfruit. Further characterization of the multigene family will likely identify reistance genes that may be useful in all these crop species.
Publications
- No publications reported this period
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Progress 10/01/01 to 10/01/02
Outputs
Previous genetic analyses of resistance to race 3 derived from the wild species Lycopersion pennellii have been inconsistent and overall confusing. New race 3 mutants were generated to improve our understanding of the interactions between tomato and race 3. The two known avirulence genes (avrXv3 and avrXv4) in a common race 3 accession were deleted, then each of the avr genes was independently reintroduced into the double knockout mutant. These three mutant strains and the wild type were used to screen a BC1 progeny [L. esculentum x (L. esculentum x L. pennellii)]. Results obtained in this survey were surprising. Four different phenotypes were detected: susceptible to all four strains, b) hypersensitive reaction to double knockout + avrXv4, c) HR to double knockout + avrXv3, and d) HR to double knockout. The last two phenotypes were unexpected because neither the parents, nor the F1 displayed a hypersensitive reaction to the double knockout or to avrXv3. The factors
controlling HR-mediated reaction to avrXv4 and the cryptic resistances to avrXv3 and to an unidentified avirulence gene appear to segregate independently. Advanced generations have been produced to further characterize these traits.
Impacts
Characterization of these resistances, in particular, the cryptic resistances may lead to the development of tomato cultivars with increased resistance to bacterial spot.
Publications
- No publications reported this period
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Progress 10/01/00 to 10/01/01
Outputs
Resistance to bacterial spot in tomatoes. The wild species Lycopersicon esculentum shows resistance to race 3 strains of Xanthomonas campestris pv vesicatoria. It has been demonstrated that the avirulence gene avrRxv4 is the elicitor of the HR in the wild species. Both a backcross and an F2 progeny were generated between the race3 susceptible breeding line Hawaii7998 and the LA716 accession of L. pennellii. It is known that most race 3 strain carry other avirulence strains that induce a mild response in Hawaii7998. To eliminate all interferences, the avrRxv4 gene was cloned and trans-conjugated into a mutant race 1 strain that is virulent in both Hawaii 7998 and L. pennellii. The trans-conjugant elicits an HR in L. pennellii, but not in Hawaii 7998. Using the mutant race 1 strain as the control and the transconjugant, 100 backcross plants and 200 F2 progeny were screened. The F2 progeny yielded a 3 Resistant : 1 susceptible ratio, indicating the presence of a single
dominant gene for resistance, but unexpectedly the backcross displayed a significant deviation from a Mendelian ratio (69 resistant: 31 susceptible chisq=4.84, p<0.05). A collection of segmental introgressions of L. pennellii in to L. esculentum are being tested to identify the chromosome location of the resistance gene that interacts with avrXv4. DNA from all backcross and 100 F2 plants have been extracted are are being prepared for molecular marker analysis.
Impacts
The addition of new genes for resistance to bacterial spot will retard the development of new virulent strains of bacterial spot in tomatoes.
Publications
- No publications reported this period
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Progress 10/01/99 to 09/30/00
Outputs
Two Phaseolus vulgaris genes involved in resistance to soybean mosaic virus (SMV) have been subjected to genetic analysis. Great Northern 1140 is resistant to SMV whereas the breeding line B21 has the I gene and develops systemic necrosis upon inoculation with SMV. The F1 between these lines develops local lesion after inoculation, but doesn't develop systemic necrosis. Segregation analysis of phenotypic responses to SMV inoculations in a recombinant inbred family (102 lines) obtained between these genotypes shows a ratio of: 4 healthy:2 systemic mosaic:1 healthy with local lesions:1 local lesions with systemic necrosis. These ratios suggest the presence of 3 loci: Smv resistance to SMV (reported by Molly Kyle), I, and Ssn (suppressor of systemic necrosis). This model was tested by segregation analysis of F2 progenies obtained between RI lines. Progenies between healthy and mosaic lines resulted in segregation ratios of 3 healthy: 1 mosaic; and between local lesions
and local lesion with systemic necrosis, the ratios were of 3 local lesions: 1 systemic necrosis. Smv and Ssn have been targeted for mapping with molecular markers. Recently, Xv4, a gene for resistance to race 3 of Xanthomonas campestris pv. vesicatoria carrying avrXv4 was mapped to chromosome 5 of tomato. However, the closest molecular markers were approximately 10 cM away on each side of this locus. We have used two segmental introgression lines, each carrying a L. pennellii chromosome segment containing the Xv4 gene, to screen for AFLP associated with that chromosome region. Five of the six markers have been characterized as single copy sequences and are awaiting segregation and linkage analysis with respect to Xv4. Race 2 of X. c. pv. vesicatoria has been increasingly detected in the Caribbean Region and in Central America. We have identified resistance to race 2 associated with a hypersensitive reaction in the nightshade Solanum lycopersicoides. This species is a close relative
of the cultivated tomato and a potential source of resistance to race 2. We have screened a set of chromosome addition lines (2n+1) of the night shade into L. esculentum and detected resistance to race 2 in at least two chromosomes. Neither of these chromosome addition lines has a response similar to that observed in the intergeneric hybrid.
Impacts
The I gene of the common bean controls resistance to bean common mosaic virus and a group of related potyviruses that include among others watermelon mosaic virus, soybean mosaic virus and zucchini yellow mosaic virus. Idetnification and isolation of genes involved in resistance to this virus complex can help divise strategies to develop resistant cultivars in not only beans, but a number of other crops such as cucurbits which are important to Florida agriculture. Bacterial spot of tomatoes is a constant threat to the Florida tomato industry. Development of moelcular markers tightly associated with resistance genes can increase the efficiency with which new bacterial spot-resistant cultivars can be developed.
Publications
- No publications reported this period
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Progress 10/01/98 to 09/30/99
Outputs
We have used BAC clones from a Phaseolus vulgaris BAC library to construct a contig of about 300 Kb near the I gene. The library was screened with a DNA marker tightly linked to the I gene, and then the leading termini of these clones were isolated to screen the library again, and to extend the contig. A cloning strategy for the isolation of BACs termini was developed. It is based on the presence of a unique NotI site about 300 bp on each site of the EcoRI cloning site of the BAC vector. BAC clones were digested with a combination of NotI and a second restriction enzyme. Fragments from the double digestions were resolved by agarose gel electrophoresis and blotted on nylon membranes. Membranes were hybridized with a PCR-amplified probe corresponding to one of the NotI/EcoRI segments of the vector. This probe hybridized to a fragment containing both the BAC vector NotI/EcoRI fragment and the insert terminal EcoRI/2nd enzyme fragment. BAC DNA was digested with the enzyme
combination that yielded the smallest fragment, and was ligated to an equally doubly digested pBlueScript plasmid. Transformants with the appropriate insert size were screened further by Southern analysis. Candidate clones were digested with EcoRI and a EcoRI/NotI combination. Positive clones were identified by a characteristic pattern. EcoRI digestions yield at least two fragments, bean DNA and a few bases of BlueScript up to the its EcoRI site (> 1 fragment if bean DNA has internal EcoRI sites), and the BlueScript vector plus the BAC NotI/EcoRI fragment. The double digestions yield the BAC NotI/EcoRI fragment adjacent to the cloning site, the EcoRI fragment of bean insert DNA (as above), and the rest of the BlueScript vector. Hybridization with the PCR product of the NotI/EcoRI fragment of the BAC vector should yield two bands: the 300 bp NotI/EcoRI segment, and the BlueScript vector because the probe is part of the lacZ gene. The leading termini of the contig are identified via
Southern hybridization of each of the BAC terminus against an EcoRI digest of the overlapping clones. A termini that hybridizes exclusively to the BAC where it originated is identified as one of the leading termini of the contig and is used to screen the BAC library for additional overlapping BAC clones. The current contig spans approximately 350 Kb. We have determined with an F2 population of 1665 F2 plants that the map distance between the I gene and one of the markers in the contig is 1.12 +/- 0.4 cM. Using a sub-set of informative recombinants from this population, we estimate that the leading terminus of the contig closest to the I gene is approximately 200-250 Kb away. We have discovered that the E. coli host DH10(alpha) used for the construction of the library carries the transposable element Tn10. Unfortunately, we have detected insertions of IS10 into one of the BAC clones. It appears that continuous sub-culture of these clones results in transposition into the eukaryotic
DNA. We are currently assessing the magnitude of this problem.
Impacts
The I gene of common beans confers resistance against the bean common mosaic virus and eight other distinct potyviruses. Isolation and characterization of this resistance may help us understand some aspects of the mechanism of resistance against potyviruses. This group alone represents 35 percent of the plant pathogenic viruses.
Publications
- Plyler, T.R, and Vallejos, C. E. 2000. A method for cloning restriction fragments containing the termini of BAC inserts. BioTechniques (accepted with revisions). Astua-Monge, G., Jones, V., Mackenzie, S.A. and Vallejos, C. E. Detection of transposition of Tn10 in BAC clones. (In preparation).
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