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Matthew D. Robbins, Mikel R. Stevens, Gennaro Fazio and Gennaro Fazio

Fusarium crown and root rot (crown rot) develops on tomato from the fungus Fusarium oxysporum f.sp. radicis-lycopersici (FORL). Genetic resistance to crown rot was previously introduced into the cultivated tomato from the wild species Lycopersicon peruvianum and found to be a single dominant gene, Frl, on the long arm near the centromere of chromosome 9 of the tomato genome. In an effort to identify molecular markers tightly linked to the gene, Ohio 89-1 Fla 7226, Fla 7464, `Mocis', and `Mopèrou', lines homozygous for Frl (resistant), were screened with restriction fragment length polymorphism (RFLP) markers in comparison to Fla 7482B and `Monalbo', lines homozygous for Frl + (susceptible). Frl was determined to be between the RFLP markers CT208 and CD8. These two markers are separated by a genetic map distance of 0.9 cM according to Pillen et al. (1996). In addition, we screened a pool of eight resistant plants against a pool of nine susceptibles from a BC1 population segregating for Frl for amplified fragment length polymorphism (AFLP) markers. Fazio et al. (1998) previously determined that crossover events occurred in these 17 plants between Frl and a rapid amplified polymorphic DNA (RAPD) marker, UBC194. Our research has indicated that UBC194 is also between CT208 and CD8 on the centromeric side of Frl. Of the 62 AFLP primer combinations tested, 34 showed more than 63 strong polymorphisms in linkage to resistant phenotypes.

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Gennaro Fazio, Mikel R. Stevens and John W. Scott

Fusarium crown and root rot of tomato is caused by Fusarium oxysporum f.sp. radicis-lycopersici (FORL). A single dominant gene (Frl) derived from L. peruvianum L. (Mill.) was previously identified as a useful source of resistance to FORL. The objective of this research was to identify molecular markers linked to Frl and RAPD markers linked to a new source of resistance to FORL being developed from L. pennellii (Corr.) D'Arcy accession LA1277. The DNAs of resistant (Frl) and susceptible breeding lines were screened for polymorphisms using 1200 RAPD primers. Of these, only 104 yielded polymorphisms between the resistant and susceptible lines. These polymorphisms were then tested on four additional tomato lines homozygous for Frl and an additional pair of near-isogenic lines developed by Dr. Laterrot. Only 13 primers still produced consistent polymorphisms between all resistant and susceptible lines. Four of these polymorphisms (RAPD 116, 194, 405, 655) were determined to be linked to Frl in an F5 segregating population using an inoculation procedure devised to clearly differentiate susceptible and resistant plants. The linkage between ah and Frl reported by Laterrot [Laterrot and Moretti Tomato Genet. Coop. Rep. 45:29 (1995)] places Frl on the long arm of chromosome 9 of the tomato genome. The parent lines were also tested with a sequence tagged site (STS) of TG101, which is tightly linked to Tm2a [Young et al., Genetics 120:579-585 (1988)] and yielded polymorphic codominant bands. This STS was also tested on the F5 segregating population and it cosegregated with the resistance and with the RAPD markers. Breeding of the second source of resistance is still in progress. The DNAs of 30 resistant BC1F5 plants derived from LA1277 were bulked and compared to the recurrent susceptible parent DNA using 800 RAPD primers. Of the 800 RAPD primers, 72 yielded consistent polymorphisms. None of the 72 primers were found to produce polymorphisms similar to those identified from the analysis of Frl, thus suggesting the possibility different genetic control being involved with FORL resistance from LA1277.

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Mikel R. Stevens, John W. Scott, John J. Cho, Bradley D. Geary and Frederic D. Memmott

Tomato spotted wilt virus (TSWV), a tospovirus, is a thrips-vectored disease infecting more than 1000 species of both monocots and dicots, including many species of agriculture importance. TSWV is the limiting factor for tomato (Lycopersicum esculentum Mill.) production in several areas of the world. For a number of years, the Sw-5 gene (derived from L. peruvianum Mill.) has provided acceptable control of this disease. Recently, Sw-5 derived resistance has been overcome by virulent pathogen isolate(s) in tomato production areas such as Spain and Italy. In earlier studies, we identified a potential new source of resistance to TSWV derived from L. chilense Dun. accession LA 1938. In a set of recent field studies, it was demonstrated that this putative new source of resistance was highly resistant to TSWV in Hawaii, Florida/Georgia, and South Africa. Furthermore, greenhouse screening trials have clearly demonstrated that the L. chilense source of TSWV resistance is resistant to isolates that overcome tomatoes homozygous for Sw-5. In these same greenhouse and field studies, there is uniform evidence that this resistance is dominant. Subsequent greenhouse studies suggest that this resistance is controlled by a single gene. Studies have been initiated to verify the inheritance of the gene(s) and to develop linked molecular markers. Furthermore, studies are under way in Australia to test this resistance on non-TSWV tospoviruses. If the data demonstrate that this is a single dominant gene we suggest this gene be designated Sw-7.

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Mikel R. Stevens, Shawn A. Chrisensen, Ammon B. Marshall, JoLynn J. Stevens, Peter Wenzl, Eric Hunter, Jason Carling and Andrzej Killian

Recently, a technology known as DArT (diversity array technology) has been developed to increase throughput in marker assisted selection (MAS). DArT utilizes microarray technology as a method to potentially compare thousands of molecular markers in one test to a single DNA sample. We used DArT on two sets of interspecific tomato [Solanum lycopersicum (Fla 7613) × S. pennellii (LA 716 or LA 2963)] segregating populations (BC, F2, and F1). We compared over 300 segregating plants to 3840 random tomato genomic fragments. After the 3840 markers were prepared, it took about 2 weeks of laboratory time to perform the experiments. With experience, this time can be reduced. We identified a total of 654 polymorphic markers usable for developing a DArT tomato genetic map. Depending on the particular cross, 13 to 17 linkage groups were identified (LOD 3) per population. Most recently, the amplified polymorphic DNA (AFLP) technique has been used for rapid genetic mapping of large numbers of anonymous genomic fragments. Besides the additional effort and reagents using AFLPs compared to DArT, a desired AFLP polymorphic band is often difficult to clone and process into a PCR based marker, whereas in DArT all markers are already cloned and immediately available for such experiments. A drawback to DArT is that it requires specialized software and equipment and is technically demanding. However, once the equipment and software are secured, techniques are optimized, and segregating populations developed, marker throughput is increased by orders of magnitude. Although challenging, the application of DArT can dramatically increase MAS throughput, thus facilitating quantitative trait and saturated mapping research.

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Matthew D. Robbins, Mohammed A.T. Masud, Dilip R. Panthee, Randolph G. Gardner, David M. Francis and Mikel R. Stevens

Tomato spotted wilt virus (TSWV) and Phytophthora infestans (late blight) in tomato (Solanum lycopersicum) have a worldwide distribution and are known to cause substantial disease damage. Sw-5 (derived from S. peruvianum) and Ph-3 (derived from S. pimpinellifolium) are, respectively, TSWV and late blight resistance genes. These two genes are linked (within 5 cM on several maps) in repulsion phase near the telomere of the long arm on chromosome 9. The tomato lines NC592 (Ph-3) and NC946 (Sw-5) were crossed to develop an F2 population and subsequent inbred generations. Marker-assisted selection (MAS) using three polymerase chain reaction-based codominant markers (TG328, TG591, and SCAR421) was used in F2 progeny with the goal of selecting for homozygous coupling-phase recombinant lines. From 1152 F2 plants, 11 were identified with potential recombination events between Ph-3 and Sw-5; of those, three were male sterile (ms-10). F3 progeny were generated from the remaining eight F2 recombinants, and resistance to both pathogens, or Ph-3 and Sw-5 in coupling phase, was confirmed in three of those. Recombination was suppressed fivefold in our F2 population to 1.11 cM between genes when compared with published maps of the same region. However, MAS was an efficient tool for selecting the desirable recombination events for these two pathogen resistance genes.