Matthew D. Robbins and Dr. Mikel R. Stevens, Agronomy and Horticulture
Fusarium crown and root rot, or “crown rot”, is a disease caused by the fungus Fusarium Oxysporum f.sp. radicis-lycopersici (FORL) and infects 36 different species of plants of at least four families.1 Crown rot was first detected in the cultivated tomato (Lycopersicon esculentum Mill.) in Japan in 1969 and since has spread to many other countries.1 Because of the loss in production in many areas of the world, control of this disease has become increasingly important. Presently, the most widely used method to control the disease in the field is through the use of pesticides. The preferable method to control crown rot is through natural plant genes that resist the disease (genetic resistance).
Genetic resistance to crown rot was introduced into the cultivated tomato from the wild species L. peruvianum (L.) Mill. serendipitously through several efforts to breed for resistance to Tobacco Mosaic Virus (TMV). The source of resistance to crown rot was found to be a single dominant gene, Frl, that was linked, or connected, to the Tm-2 gene which provides resistance to TMV.5 Both of these genes were determined to be on chromosome 9 of the tomato genome.3
Still, little was known about the specific location of Frl. A knowledge of the specific location of Frl would allow the gene to be cloned through map-based cloning. If Frl is cloned, the plant mechanism that provides resistance to FORL may be understood, and this gene could be transferred to other species that are susceptible to FORL. In this way, genetic control of FORL could potentially be accomplished in many crops, which may reduce the use of pesticides and help overcome the loss in tomato production from crown rot.
In an effort to locate Frl, Gennaro Fazio, a former BYU graduate student, found 4 RAPD (random amplified polymorphic DNA) markers between Tm-2 and Frl.2 From Gennaro’s and other studies, Frl was determined to be near the centromere and Tm-2 on chromosome 9 in a region with a genetic map distance of about 10 cM.
I was able to continue Gennaro’s work as an ORCA project in 1998 and reduce the general area in which Frl is thought to be located through the use of RFLP’s (restriction fragment length polymorphism) and AFLP=s (amplified fragment length polymorphism). Plants with Frl were screened against plants without Frl with several RFLP markers, and Frl was determined to be in a region between two RFLP markers with a genetic map distance of only 5.9 cM. While searching for Frl with RFLP’s, I began to search with AFLP’s. The goal using AFLP=s was to use very specific plants identified from Gennaro’s study that would allow us to find markers that were tightly linked to Frl. After several different AFLP attempts, a marker was found that is less than 1 cM from Frl.
Although my ORCA project of 1998 produced valuable data, it left much to be done to clone Frl through map-based cloning. The next logical steps to clone Frl would be: 1) saturate the region around the gene with molecular markers, 2) make a library of the tomato genome cut into small pieces, which is easier to work with than whole chromosomes, 3) use the molecular markers to find pieces of the library containing the gene, and 4) clone those pieces. This is a long process that would take well over one semester to complete, but I proposed to complete the first step as a 1999 ORCA project. I planned to use AFLP’s, which are more powerful than RFLP=s to locate markers less than 1 cM away from Frl.
To perform the AFLP study, specific plants were used from Gennaro Fazio’s study2 and the DNA from these plants was pooled so that any marker produced by AFLP analysis would theoretically be within 1 cM of Frl. I tested a total 62 different AFLP analyses and found that 51 of them produced a total of 146 markers. I scored the markers as: 3 = intense, 2 = moderate, and 1= weak. Of the 146 markers, 44 were 3’s, 30 were 2’s, and 72 were 1’s. Several indications confirm that all of these markers are within 1 cM from Frl. The goal of saturating the area around the gene with molecular markers was accomplished.
I attempted to convert some of these AFLP markers into single copy PCR (polymerase chain reaction) markers. PCR markers are a lot easier to work with and are much less expensive than AFLP’s. Of the 17 AFLP markers I worked with, 5 of them produce PCR products, but none of these differentiate between plants with and plants without Frl.
While working with AFLP’s, I also continued working with RFLP’s. As part of my 1998 ORCA project, Frl was determined to be between two RFLP markers with a genetic map distance of 5.9 cM. Using an updated RFLP linkage map from Pillen et al.4, and further RFLP analysis, I determined that Frl is flanked by two RFLP loci with a genetic map distance of 0.9 cM between them.
Through funding from other sources, Dr. Stevens was able to purchase a library of the tomato genome cut into small pieces, which allowed me to skip step two (listed above) in the map-based cloning process. Currently, I am screening this library (step 3) with some of the AFLP markers and I will be able to continue this project as my Master’s thesis.
I would like to express my appreciation to The Office of Research and Creative Activities (ORCA) for the opportunity to work on this project for two years. I am fortunate to have been able to not only reach my goal of becoming one step closer to cloning Frl, but I am several steps closer. I have learned a great deal from this project and I feel a sense of satisfaction at being able to work on a problem that potentially may help overcome a world-wide loss in tomato production from crown rot.
References
- Brayford, D., 1996. Mycopathologia 133:61-63.
- Fazio, G., 1997. Thesis to Brigham Young University, Department of Agronomy and Horticulture.
- Laterrot, H., Y. Couteaudler, 1989. TGC (Tomato Genetics Cooperative) reports 39:21.
- Pillen et al. In: Genome Mapping in Plants. Pp. 281-308. Paterson A, ed. Biotechnology Intelligence Unit. R.G. Landes Co., Austin Texas.
- Scott, J.W., J.D. Farley, 1983. HortScience 18:114-115.