Development of a Molecular Marker for Tagging a Resistance Gene to Geminiviruses in Tomatoes

Abstract
        Each year geminiviruses cause millions of dollars in damage to tomato crops in Central America. Attempts to create a resistant plant through breeding programs have been unsuccessful. This study hopes to develop a molecular marker for the gene that controls resistance to geminiviruses. To accomplish this goal we will use a PCR tagging technique that is based on RFLP probes. The marker would enable breeders to quickly determine a tomato’s susceptibility to geminiviruses and greatly aid in the creation of a resistant hybrid.

Introduction
        Tomatoes in Central America are plagued by a series of geminiviruses that are transmitted by the whitefly, Bemisia tabaci (Jones, 2003). The effect of the disease is near total loss of crops and annual damages ranging in the millions of dollars (Morales and Anderson, 2001; Nakhla et al., 2004). In some areas of Nicaragua and Guatemala losses have been so extensive that the crop is no longer grown. Suitable resistant cultivars are currently unavailable, and methods of control are mostly restricted to insecticides that must be applied every third day (Nakhla et al., 2004). Unfortunately, these drastic measures have been ineffective in controlling the virus. The insecticides are only partially effective even as a preventative measure, yet the farmers in the area continue to spray infected crops. This costs the farmers money that they will not recoup due to the loss of the infected crop (Maxwell, D., pers. com.).
         Lycopersicon hirsutum and Lycopersicon chilense are wild species of tomato that have shown resistance to Tomato yellow leaf curl virus, which is a monopartite geminivirus (Vidavsky and Czosnek, 1998). However, the shape and size of the plant’s fruits make them unsuitable for commercial use. Breeding programs have been underway for some time with the goal of creating a resistant hybrid plant that produces a healthy fruit (Chen et al., 2003; Mejia et al., 2004; Narasegowda et al., 2003; Scott et al., 1995). After crossing parent plants, the F1 and F2 generations must be tested in order to determine if the gene for resistance has been inherited. Current methods of doing this testing involve growing the plants to maturity in a field that has been shown to produce 100% infection of susceptible plants. Each cycle takes five months and there can be an incorrect diagnosis of plant resistance due to escapes. Thus far, breeding programs have been ineffective in producing a successful resistant hybrid for Central America.
        Therefore, in order to more quickly produce a resistant hybrid, a molecular marker for the resistance gene is needed. The molecular marker could be used to track the resistance gene through successive generations with Polymerase Chain Reaction (PCR), which is a method of amplifying DNA. An accurate molecular indicator would eliminate the need to grow the plants to maturity and eliminate false positives.
        This study hopes to develop a molecular marker for resistance. To accomplish this goal we will be using the tomato breeding lines, Gh13, Gc9, and Gc173, that are resistant to the bipartite geminiviruses in Guatemala (Mejía et al., 2004; Nakhla et al., 2004). As a control, we will be using the breeding line Heinz 1706. Heinz 1706 is the tomato cultivar being sequenced in an international sequencing project (Budiman et al., 2000; Ozminkowski, 2004), and it is most likely susceptible to geminiviruses (Maxwell, D., pers. com.). Heinz 1706 is currently being tested to confirm its susceptibility in Israel and Guatemala (Maxwell, D., pers. com.). Gh13 is the F7 generation and is a homogeneous breeding line with resistance derived from L. hirsutum. Gc173 and Gc9 are at least F8 breeding lines with resistance genes introgressed from L. chilense by J. W. Scott (Scott et al., 1995).
Restriction fragment length polymorphism (RFLP)-based probes have been used to help develop a map of the tomato genome (Solanaceae Genomics Network, 2004). The results of this work have shown that there are hotspots in which genes that control resistance to disease are likely to be found (Pan et al., 1999). For this research, hotspots are defined as a place on the genome where two or more resistance genes are located in close proximity. Thus far, thirteen hotspots have been found in the tomato genome. Some hotspots have as many as five resistance genes located in close proximity. Most hotspots have two to three resistance genes (Pan et al., 1999). Previously, the Maxwell lab had tested hotspots on chromosomes 6 and 11. This study concluded that resistance to geminiviruses is not located at the hotspots on those chromosomes (Mejia et al., 2004). For this study, two new hotspots will be tested. The hotspots will be chosen based on their concentration of resistance genes.
        Specifically, two hotspots on the genome of Gh13, Gc9, and Gc173 will be tested to determine if there is a DNA introgression of L. hirsutum or L. chilense, respectively. We will test these hotspots using a PCR-based tagging method that identifies resistance genes (Czosnek et al., 2004; Nesbitt and Tanksley, 2002). The sequence of the tested hotspot will be compared against a control, susceptible tomato, Heinz 1706. Differences in the sequences as small as 3-4% would be indicative of an introgression from a wild species. If the introgression of L. hirsutum is found in Gh13 or if the introgression of L. chilense is found in Gc173 or Gc9, and these results can be verified in other lines of tomato, then we will use that hotspot as a marker for the geminivirus resistance gene.

Materials and Methods

DNA Extraction
        We will be using plant lines Gh13, Gc173 and Heinz 1706. The geminivirus resistant lines, Gh13, Gc9, and Gc173, will be supplied by Dr. L. Mejía, Universidad de San Carlos, Guatemala City. The susceptible line Heinz 1706 will be supplied by Dr. R. Ozminikowski, Heinz Seed Co., Stockton, CA. DNA will be extracted from the fresh leaves of plants grown in a plant growth chamber at the University of Wisconsin-Madison. Thirty mg of tissue will be frozen in liquid nitrogen in a microfuge tube, then ground with a sterilized Kontes™ micropestle (Kontes Glass, Vineland, NJ). The DNA will be extracted with the PUREGENE® DNA Purification Kit (Gentra Systems, Inc., Minneapolis, MN) following the manufacturer’s instructions. DNA concentrations will be adjusted to 10ng/µl and the extracts will be frozen at -20oC.

Primer Development
        Primers will be developed for two hotspots, one on chromosome 7 and one on chromosome 1. The hotspot on chromosome 7 is located between RFLP probes TG128 and TG662 on the long arm of the chromosome (Pan et al., 1999). Other RFLP probes that fall in this region are CT84 and TG572 (Pan et al., 1999). The hotspot on chromosome 1 is located on the long arm of the chromosome between RFLP probes TG125 and CT2. Other probes that fall in this region are TG24, CD15, and TG301 (Pan et al., 1999). The partial sequences of the RFLP probes are located on the Cornell website (Solanaceae Genomics Network, 2004). Appropriate DNA data bases (National Center for Biotechnology Information, 2004; Schoof et al., 2003) will be accessed to determine if these sequences are associated with known plant genes. Where possible, primers will be designed to anneal to the exon regions and amplify at least one intron. Maxwell’s lab group has completed this primer development process many times before, and the proposed experiment should pose no special problems (Czosnek, et al., 2004).

PCR Reactions
        PCR fragments from each set of primers, for each of the four genotypes, will be obtained using methods developed in the Maxwell lab (Czosnek et al., 2004). PCR parameters will be for 50-µl reactions containing: 5-µl 2.5mM deoxynucleotide triphosphates (dNTPs), 5-µl 10X buffer, 5-µl 25 mM MgCl2, 0.2-µl Taq DNA polymerase, 5-µl each forward and reverse sense primer at 10µM, 5-7 µl of DNA extract, and H20. PCR cycle parameters for fragment amplification will be as follows: denaturation at 94°C for 3 min, then 35 cycles at 94°C for 30 sec each, annealing at 50 or 53°C for 1 min, and extension at 72°C for 1 min. These cycles will be followed by a reaction at 72°C for 10 min, and then the reaction will be held at 4°C. PCR reactions will be performed in the MJ DNA Engine PT200 Thermocycler™ (MJ Research Inc., Waltham, MA).

PCR Fragment Analysis
        The PCR-amplified DNA will be run on an electrophoresis gel of 1.5% Seakem LE™ agarose (BioWhittaker Molecular Applications Rockland, ME) in 0.5X TBE buffer, stained with ethidium bromide, and visualized with a Kodak Gel Logic 200 Imaging System. This will allow us to determine the quality of the amplified DNA. If the primer pair produces multiple bands, we will redesign the primer and do PCR again. If the primer pair has produced only one band, this PCR fragment will be directly sequenced.

Sequencing and Comparison
        After successful amplification of the tomato genomic DNA, PCR fragments will be directly sequenced using Big Dye Sequencing Kit™ (Biotechnology Center, Madison, WI). Analysis of the sample sequences will be accomplished by comparison with the DNAMAN software (Lynnon Corp., Quebec, Canada). In comparing the DNA sequences of Gh13, Gc9, and Gc173 with Heinz 1706, we will be looking for an introgression of L. hirsutum or L. chilense DNA. Sequence differences as small as 3-4%, such as SNPs or indels, between Gh13, Gc9 or Gc173 and Heinz 1706 would be evidence of an introgression and could be used as a molecular marker for resistance.

Expected Results
        We expect to find that there are differences between Gh13, Gc9, or Gc173 and Heinz 1706 in at least one of the hotspots. This would indicate that an introgression of wild tomato species DNA was present in the Gh13, Gc9, or Gc173 genome. In order to determine the validity of this introgression, all known resistant lines would be tested with the PCR primers we had developed and compared to the susceptible cultivar Heinz 1706. If the introgression of L. hirsutum or L. chilense was present in all of the resistant plants then this sequenced hotspot could be used as a molecular marker for resistance.
        It is possible that no difference will be found between Gh13, Gc9, or Gc173 and Heinz 1706 at the hotspots we will be testing. This type of result would indicate that the two hotspots we had measured were not the location of the disease resistance gene. Future studies on other parts of the tomato chromosome would then be necessary in order to locate the gene responsible for resistance. There are only thirteen known hotspots for resistance to disease on the tomato genome. Therefore, this type of result would still prove valuable as it would narrow down the possible locations for the disease resistance gene.
        In addition, it is possible that the resistance gene is not located at a known hotspot for disease resistances genes. If no evidence is found for the location of a geminivirus resistance gene at a known hotspot, then a method that detects genetic variability over the whole genome, such as amplified fragment length polymorphism (AFLP)-based tagging (Parella et al., 2004), would be used to develop new starting points for the experiment. However, this would still be an interesting result because most resistance genes are located at hotspots.
        With either a positive or negative outcome, the results of this experiment will bring us closer to developing a molecular marker for resistance gene to geminiviruses. In the end this research will result in saving millions of dollars and countless tomato crops throughout Central America.

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