Progress 08/15/07 to 08/14/11
Outputs
OUTPUTS: The objective of this research was to fine map the TYLCV resistance locus Ty-3 in LA2779-derived advanced breeding lines. Of 720 recombinant inbred lines (RILs) that were genotyped, 30 informative RILs containing varied lengths of a Solanum chilense introgressed segment in the Ty-3 target region were tested for resistance to Tomato yellow leaf curl virus (TYLCV). Results suggested that Ty-3 maps to a seven cM interval between markers T0774 (18 cM) and P6-25 (~25 cM). Two resistant RILs, each containing a shortened introgression including this ~7 cM interval, were used to develop a large segregating population to more finely map Ty-3. In spring 2009, over 10,500 plants were screened with molecular markers to identify approximately 100 recombination events between markers T0774 and P6-25. Multiple cuttings were taken from each recombinant, rooted, inoculated with sweet potato whiteflies viruliferous for TYLCV for two weeks, transplanted to the field in a replicated experiment, and evaluated for disease severity. In addition, recombinants were genotyped with over 20 newly developed molecular markers corresponding to BAC and FOS end sequences within the ~7 cM target interval. Results indicated that Ty-3 resides between markers T0774 (18 cM) and cLEG-31-P16 (20 cM). Homozygous RILs were developed for each recombinant where crossing-over occurred within this ~2 cM region. Evaluation of these lines for resistance in spring 2010 clearly delimited the location of Ty-3 to a single BAC clone (HBa161K22) approximately 80 Kb in size. Two candidate genes within this BAC were identified, and efforts to clone each of these genes will soon be underway. Two resistant RILs, each having the portion of the introgression below Ty-3 removed, were crossed to a resistant RIL with the upper portion of the introgression removed. The F1 plants from each of these crosses were self-pollinated, and 1030 F2 progeny were screened for recombination at the Ty-3 locus. Three plants with desired recombination were identified and selected from these crosses. In each of these plants, the shortened upper portion of the introgression from one parent was combined with the shortened lower portion of the introgression from the other parent, yielding a chromosome with a Ty-3+ introgression that is less than 72 Kb in size. PARTICIPANTS: Sam Hutton was the postdoctoral scientist in charge of most of the work on this project. He is now an Assistant Professor of Horticultural Science at the University of Florida. Assisting him were technical staff members Jose Diaz, Dolly Cummings, and Kim Hutchinson. Other members of our lab team; Cathy Provenzano, Rosa Ayala, Tim Davis, and Rudy Jones help with some of the transplanting and crossing work. Israeli colleagues on the BARD grant mentioned above are Ilan Levin and Moshe Lapidot of the Volcani Institute. We have already exchanged germplasm and made crosses for that grant. TARGET AUDIENCES: Tomato breeders working on begomovirus resistance are the primary target audience. The germplasm developed will allow them to develop much improved resistant varieties that until now have been hampered by linkage drag. Also it will now be easier to recombine Ty-3 with other genes in the region that are now linked in repulsion. The precise mapping of Ty-3 will also be of interest to tomato geneticists working with other resistances in this area of the genome where there are numerous resistances. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Ty-3 fine mapping progress was presented in a recently funded BARD proposal where Ty-3, Ty-4, and Ty-5 will be combined in all combinations to determine optimal levels of resistance to TYLCV and tomato mottle virus (ToMoV). The cloning of Ty-3 is also an objective of this proposal. Additionally, two of the newly-developed molecular markers correspond to either end of the clone HBa161K22. Moreover, a third newly-developed marker lies within this BAC, and linkage between resistance and this marker has not yet been broken. Three breeding lines were developed which contain Ty-3 within a <72 Kb introgression and field testing revealed that these lines had no linkage drag. That is, the foliage was not extra sensitive to bacterial spot (Xanthomonas perforans)and early blight (Alternaria sp.). The vines were normal and not spindly and fruit set was normal. These breeding lines are being used widely in our backcrossing program and soon will be released for other tomato breeders. This should greatly improve the resistant varieties that will be developed in the future as they will be horticulturally similar to susceptible varieties.
Publications
- Hutton, Samuel and J.W. Scott. 2011. Tomato breeding program: present status and future directions. Proc. Florida Tomato Institute. PRO 527:19-20.
- Hutton, S.F and J.W. Scott. 2011. A collection of polymorphic markers useful for fine-mapping the Ty-3 locus from Solanum chilense accession LA2779. Rept. Tomato Genet Coop 61:12-14.
- Hutton, S.F., J.W. Scott, and D.J. Schuster. 2010. Fine mapping of a begomovirus resistance gene in tomato. Proc. Plant and Animal Genome Conference, San Diego, CA, USA p. 204 (Abstr.)
- Hutton, Sam F. and Jay W. Scott. 2011. Present status of begomovirus resistance breeding efforts at University of Florida. Proc. 43rd Tomato Breeders Roundtable http://tgc.ifas.ufl.edu/2011TBRTAbstracts.pdf p.6
- Scott, John W., S.F. Hutton, and M.R. Stevens. 2011. Mapping of new loci controlling viral diseases in tomato. Proc. Eucarpia Tomato 2011, Malaga, Spain p.15
- Hutton, S.F., J.W. Scott, and D.J. Schuster. 2011. High resolution mapping of the begomovirus resistance locus Ty-3 in tomato. Proc. Plant and Animal Genome Conference, San Diego, CA, USA p. 220 (Abstr.)
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Progress 08/15/09 to 08/14/10
Outputs
OUTPUTS: Previous results indicated that Ty-3 resides between markers T0774 (18 cM) and cLEG-31-P16 (20 cM). Homozygous RILs were developed for each recombinant where crossing-over occurred within this ~2 cM region. Evaluation of these lines for resistance in spring 2010 clearly delimited the location of Ty-3 to a single BAC clone (HBa161K22) approximately 80 Kb in size. Two candidate genes within this BAC were identified, and efforts to clone each of these genes will soon be underway. Two resistant RILs, each having the portion of the introgression below Ty-3 removed, were crossed to a resistant RIL with the upper portion of the introgression removed. The F1 plants from each of these crosses were self-pollinated, and F2 progeny were screened for recombination at the Ty-3 locus. Three plants with desired recombination were identified and selected from these crosses. In each of these plants, the shortened upper portion of the introgression from one parent was combined with the shortened lower portion of the introgression from the other parent, yielding a chromosome with a Ty-3+ introgression that is less than 80 Kb in size. This introgression is presently being incorporated into advanced breeding lines for final testing and evaluation. PARTICIPANTS: Training: Sam Hutton is the postdoctoral scientist in charge of most of the work on this project. Assisting him are technical staff members Jose Diaz, Dolly Cummings, and Kim Hutchinson. Other members of our lab team; Cathy Provenzano, Rosa Ayala, and Rudy Jones help with some of the transplanting and crossing work. Collaborations: Our Israeli colleagues on the BARD grant are Ilan Levin and Moshe Lapidot of the Volcani Institute. We have already exchanged germplasm and begun making crosses in anticipation of funding for that grant. TARGET AUDIENCES: Tomato breeders are the main target audience. Once we release seed of homozygous lines with introgressions less than 80Kb, these lines will be of great value in developing TYLCV resistant varieties using the Ty-3 gene that is free of linkage drag. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The progress we have made with the Ty-3 fine mapping was presented in a BARD proposal recently re-submitted, where Ty-3, Ty-4, and Ty-5 will be combined in all combinations to determine optimal levels of resistance to TYLCV and tomato mottle virus (ToMoV). The cloning of Ty-3 is also an objective of this proposal. Additionally, two of the newly-developed molecular markers correspond to either end of the clone HBa161K22. Moreover, a third newly-developed marker lies within this BAC, and linkage between resistance and this marker has not yet been broken. As mentioned, material is being developed which contains Ty-3 within a <80 Kb introgression, which will potentially have no associated linkage drag. This work is thus providing both information and materials which will substantially improve the efficiency with which begomovirus resistance is incorporated into superior germplasm.
Publications
- Hutton, S.F., J.W. Scott, and D.J. Schuster. 2010. Fine mapping of a begomovirus resistance gene in tomato. Proc. Plant and Animal Genome Conference, San Diego, CA, USA p. 204 (Abstr.)
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: Thirty recombinant inbred lines (RILs), containing varied lengths of a Solanum chilense introgressed segment in the Ty-3 target region, were previously tested for resistance to Tomato yellow leaf curl virus (TYLCV). Results suggested that Ty-3 maps to the interval between markers T0774 (18 cM) and P6-25 (~25 cM). Two resistant RILs, each contaning this ~7 cM interval within a shortened introgression, were used to develop a large segregating population to more finely map Ty-3. In spring 2009, approximately 10,500 plants were screened with molecular markers to identify more than 100 recombination events between markers T0774 and P6-25. Multiple cuttings were taken from each recombinant, rooted, inoculated with sweet potato whiteflies viruliferous for TYLCV for two weeks, transplanted to the field in a replicated experiment, and evaluated for disease severity. In addition, recombinants were genotyped with nearly 20 newly developed molecular markers corresponding BAC and FOS end sequences within the ~7 cM target interval. Results indicate that Ty-3 resides between markers T0774 (18 cM) and cLEG-31-P16 (20 cM). RILs are currently being developed for each recombinant and will be tested in spring 2010; evaluation of lines homozygous for the shortened segments is expected to further delimit the location of Ty-3 to within a single cM. PARTICIPANTS: Jeremy Edwards, Assistant Professor of Horticulture at the University of Florida, wrote a program to design primers for our new markers based on their sequences. This was most useful to delimit the location of Ty-3. TARGET AUDIENCES: Our primary target is/will be tomato breeders and molecular geneticists working with tomato breeding programs. Once better resistant cultivars are developed based on this work tomato growers will gain from this work. PROJECT MODIFICATIONS: Because of reduced recombination in regions where chilense and tomato cromosomes match we have had to screen very large populations to attain a modest number of recombinants. In the next year we are crossing 2 heterozygous recombinant lines which overlap (homozygous for chilense segments)in the Ty-3 region but which have chilense introgressions coming from different directions. We expect an increased recombination in the chilense overlap region and this should provide many plants with only small introgressions around Ty-3 to allow us to hone in on it's location and perhaps even clone the gene.
Impacts
The main impact of this project will be to have tomato lines that contain Ty-3 in a very small introgression that will be free of linkage drag. In the last year we have reduced the location of the Ty-3 gene 3-fold but as yet we don't have a line with as small of an introgression as we want. Next year we should be at that point and then will have a "breeder friendly" line that can be used to incorporate this resistance gene without detrimental traits linked to it.
Publications
- Hutton, Samuel, Jay W. Scott, Yuanfu Ji, and David J. Schuster. 2009 Fine mapping Ty-3, a major begomovirus resistance gene in tomato. HortScience 44:1007 [Abstr.]
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Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: Forty homozygous recombinant inbred lines (RILs) containing varied lengths of Solanum chilense introgressed segments in the Ty-3 target region were tested for resistance to Tomato yellow leaf curl virus (TYLCV). Thirty of these lines were produced from F2 recombinants, while the remaining ten were identified from advanced breeding lines previously genotyped with molecular markers. In fall 2007 these RILs were inoculated at seedling stage for two weeks with sweet potato whiteflies viruliferous for TYLCV and were then transplanted to the field. A randomized complete block design with 12-plant plots and 3 blocks was used, and disease severity was evaluated three times at three week intervals. An average of the three ratings was used for analysis, and means were separated by Duncan's multiple range test. The experiment was repeated in spring 2008. Results suggest that Ty-3 maps to the interval between markers T0774 (18 cM) and PG-25 (~25 cM). The introgression segments in fifteen of twenty-seven resistant RILs spanned this entire region, while recombination events between these two markers resulted in part of this region being present in eleven of the other resistant lines. It was noted that the remaining resistant line, whose introgression did not span any part of this region, also had a lower level of resistance than the others. All thirteen susceptible lines were deficient for this interval. This indicates that there was no detectable resistance at the Ty-1 locus in this germplasm derived from LA2779. According to the map positions provided by SGN for T0774 and the BAC from which PG-25 was designed, the interval between these two markers is approximately 7 cM. However, only six of 717 F2 plants initially screened showed recombination within this interval. Thus, the realized distance between these two markers was estimated to be 0.84 cM in our material. To more finely map the Ty3 gene, two resistant RILs were selected from the spring 2008 trial and crossed to the TYLCV-susceptible breeding line, Fla. 7776. The first RIL contains a ~20 cM introgression spanning markers C2_At2g39690 (5.3 cM) to PG-25, while the ~14 cM introgression in the second RIL spans markers T0774 to T0834 (32 cM). F1 plants from each of these crosses will be self-pollinated in fall 08, and F2 seed will be harvested. In spring 2009, approximately six thousand F2 plants from these crosses will be screened with the molecular markers T0774 and PG-25. Given the realized distance between the two markers of 0.84 cM, we estimate that approximately 50 recombinants will be identified. These F2 recombinants will be evaluated in spring 2009, and their derived F4 RILs will be tested in fall 2009. PARTICIPANTS: J.W. Scott Professor of Horticulture is the tomato breeder who introgressed the resistance into tomato and ovesaw the molecular marker work over 10 years that got this project started. Yuanfu Ji is the Post-Doctoral scientist who conducted most of the work; developing and screening molecular markers on the germplasm, rating plants for disease severity, analyzing data, and developing recombinant populations. D.J. Schuster, Professor of Entomology maintained the viruliferous whitefly colony used for disease screening, assisted with the inoculation procedur and he provided 1/2 the field space used to grow the plants. Jose Diaz and Dolly Cummings worked half time in the lab assisting with molecular marker screening procedures. TARGET AUDIENCES: Tomato breeders from the public sector, seed companies, and processing tomato companies around the world are the main target audience. Scientists working in cytogenetics or molecular analysis would be interested in our data on reduced recombination in the introgressed chromosome segments. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Advanced breeding lines highly resistant to begomoviruses released from our research project have been utilized in tomato breeding programs worldwide. In addition, molecular markers developed from this project have also been widely applied in tomato breeding programs for marker-assisted selection. Commercial hybrids utilizing our released breeding lines and marker information (such as Carmencita in Spain and Llanero in Guatemala), have been released by seed companies.
Publications
- Ji Y, Schuster DJ, Scott JW (2007a) Ty-3, a begomovirus resistance locus near the Tomato yellow leaf curl virus resistance locus Ty-1 on chromosome 6 of tomato. Mol Breed 20:271-284
- Ji Y, Scott JW, Hanson P, Graham E, Maxwell DP (2007b) Sources of resistance, inheritance, and location of genetic loci conferring resistance to members of the tomato-infecting begomoviruses. In: Czosnek H (ed) Tomato Yellow Leaf Curl Virus Disease: Management, Molecular Biology, Breeding for Resistance. Kluwer, Dordrecht, pp 343-362
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