Towards isolation of the tomato root-knot nematode resistance gene MI via positional cloning

Root-knot nematodes of the genus Meloidogyne are severe pathogens of plants and worldwide they cause damage to many economically important crops like potato, rice, cotton, and tomato. So the control of nematodes and the protection of plants against nematode damage are matters of major concern. Some plants carry resistance genes which prevent damage by nematodes. If these resistance genes would become available for introducing into other plant species, this could be of considerable economic importance. From a fundamental point of view, nematode resistance genes are also very interesting since they control the interaction between plants and parasites in a very specific manner. The Mi -gene of tomato is a single dominant gene, located on chromosome 6, that confers resistance against several species of root-knot nematodes and may serve as a model for the study of plant-nematode interactions. Such studies, to date, are severely hampered by a lack of knowledge of the proteins and the functions of the proteins encoded by the resistance genes. If the product of the gene is not known, the isolation of such a gene is very difficult. But nowadays several approaches have been developed to isolate genes that are only characterized by phenotype and genetic position. In chapter 1 an overview is presented of the different approaches, their relevance to the isolation of the nematode resistance gene Mi is evaluated.The most relevant procedure for isolating the Mi -gene seemed to be positional cloning. This approach involves the identification of tightly linked molecular markers followed by a chromosomal walk which starts at these markers. The walk results in cloning of all sequences between two markers at either side of the gene. The gene itself can subsequently be identified from these sequences using the information encoded in the sequence. In order to proceed with the positonal cloning approach several technical requirements have to be met which were, at the onset of the present work, not available. The development and application of these new techniques is described in this thesis.First of all, an efficient method for isolating and handling of megabase-sized plant DNA had to be developed, which is described in chapter 2. Furthermore, the technical means to electrophoretically separate large DNA fragments, by so called pulsed field gel electrophoresis, were becoming commercially available at the beginning of this work. In chapter 3, the application of these techniques to physically characterize large genomic regions and the construction of long range restriction maps for two Mi -flanking markers, GP79 and Aps-1 , are described. These maps provided the first physical characterization of parts of the Mi -region that was sofar only genetically characterized. The work described in this thesis has focused on Aps-1 and GP79 since those were the earliest available tightly linked markers. By now many other markers, not available at the onset of the work, are known.The yeast artificial chromosome (YAC) cloning system allows for the cloning of fragments many hundred kb's in size. Such very large DNA fragments are an essential ingredient for chromosomal walking. The characteristics of YAC cloning are reviewed in chapter 4. For YAC cloning of plant DNA several technical adaptations were necessary, These were worked out in detail in the construction and characterization of a tomato Cla I YAC library, as described in chapter 5, and of a partial Eco RI YAC library, as described in chapter 6. Finally, in chapter 7, the lessons learned from the excercises described in the chapters 2, 3, 5 and 6 are evaluated, along with a discussion of how to proceed further now the techniques required for positional cloning the Mi -gene are available.