Rachel E. Raphael and Dr. Mikel R. Stevens, Agronomy and Horticulture
Tospoviruses, including tomato spotted wilt virus (TSWV), infect over 550 plant species worldwide. In the commercially grown tomato (Lycopersicon 1 esculentum) TSWV decreases fruit yields and quality in up to 80-90% of the crop in South Africa2 with similar losses in Hawaii3 and other areas around the world.
Resistance to TSWV was introduced from L. peruvianum Miff, a wild tomato species containing Sw-5, a single dominant gene for resistance.4,5 This gene has economic potential because of its additional resistance to tomato chlorotic spot virus (TCSV) and ground ring spot virus (GRSV).5 Finding the genomic location of such a gene would prove valuable not only to molecular biologists, but also to plant breeders and ultimately tomato growers.
In the search for Sw-5’s exact location, plant breeders and molecular biologists have made considerable progress through wide crosses between the wild L. peruvianum and the commercial tomato to incorporate the gene from the former into the latter. After as many as 30 generations of selection, the tomato lines containing Sw-5 are considered to be all L. esculentum except for a small amount of DNA containing resistance to TSWV.4 Additionally, molecular markers linked to SW-5 have been identified. RFLP markers CT220 and bands from RAPD primer #421 are linked within 1 cM of the gene. Other markers are linked at broader distances.6 Additional markers well within 1 cM of Sw-5 are needed to saturate the regions surrounding this gene in preparation for its cloning.
Genetically directed representational difference analysis (GDRDA) was developed for cloning tightly linked molecular markers. When first performed on mice, it located markers as close as 0.2 cM from the target locus.7 GDRDA relies on the differences between two pools of DNA. In this research, separate pools of susceptible and resistant plant DNA were used. The expected result would be tightly linked genetic markers amplified from the region of the resistant genome that is not found in the susceptible genome.
Theoretically GDRDA can provide the markers needed to saturate a genetic map surrounding the SW-5 locus. Dr. Mikel Stevens had developed near-isogenic fines of resistant and susceptible tomato plants from which DNA was isolated for this purpose. The L. peruvianum DNA conferred the resistance in the tomato. This means that the gene and DNA tightly linked to it are very different from that of the susceptible plants. Furthermore, Dr. Stevens’s population had been studied for plants that have had crossover events between the genetic markers and the gene itself, allowing for selection of plants having a shorter distance of L. peruvium DNA flanking SW-5. Thus, any markers identified could be very tightly linked to Sw-5. The GDRDA protocol was obtained from the Cold Spring Harbor Laboratory with adjustments as needed.
The protocol for GDRDA is very lengthy and complex. It includes such molecular biology techniques as restriction digests, polymerase chain reactions (PCR), ligations, and hybridizations with DNA purification between each step. An incorrect temperature, pH or DNA concentration can result in a poor reaction or loss of DNA in any of the aforesaid steps. Unfortunately, because this was our first attempt with the protocol, we did not realize the assortment of ways in which DNA can be lost. To offset the losses, a sample from each step was analyzed by gel electrophoresis to pinpoint trouble spots. This analysis enabled improvement of lab technique with the restriction digests and the PCR. Time constraints prevented completion of the protocol, so no markers could be cloned for study.
If obtained, the desired results from the GDRDA protocol could accelerate further Sw-5 research by enabling gene isolation and enhancing screening of plants for disease resistance; however, results may prove elusive. Personal communications with two other plant labs that have attempted the technique show GDRDA to be an unpredictable and difficult procedure. Of the two labs, only one obtained results one time, which were published in 1994. The other lab had difficulty with 8 the hybridization step and did not obtain results. This procedure seems to have merit only as a final resort for identification of molecular markers.
References
- Wijkamp, I. and D. Peters. 1993. Phytopathology 83:986-991. 1
- van Zijl, J.J.B. et. al. 1986. Acta. Hort. 194:69-75. 2
- Cho, J.J. et. al. 1989. Plant Dis. 73:375-383. 3
- Stevens, M.R. et. al. 1992. Euphytica 59:9-17. 4
- Boiteux, L.S., and L. de B. Giordano. 1993. Euphytica 71:151-154. 5
- Stevens, M.R. et. al. 1995. Theor. Appl. Genet. 90:451-456. 6
- Lisitsyn, N.A. et. al. 1994. Nature Gen. 6:57-63. 7
- Delaney, D.E. et. al. 1994. Genome 38: 458-466.