Physical mapping of chromosomes in plant-pathogenic fungi by fluorescence in situ hybridization (FISH)

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Techniques of fluorescence in situ hybridization (FISH) including fiber-FISH were developed with two plant-pathogenic fungi, Nectria haemataococca and Cochliobolus heterostrophus, and physical mapping of their chromosomes was attempted with the established techniques. The summarized results obtained in this study are as follows.1. The method of preparing DNA fibers for fiber-FISH was established, in which protoplasts-containing agarose blocks for pulsed-field gel electrophoresis (PFGE) or PFGE-separated chromosomal DNA bands were used as the source of DNA fibers.2. Making contig map with cosmid clones for 1.6-Mb CD-chromosome of N. haematococca was shown to feasible by using multi-color fiber-FISH.3. Physical distance of RFLP linkage markers that locate near the breaking points of reciprocal translocation was determined with fiber-FISH in C. heterostrophus, and compared with the distance obtained in a genetic map. The results showed that crossing-over was not repressed near breaking points in meiosis and that the inserted alien fragment is unlikely to be large as expected previously.4. Physical mapping by FISH using interphase nuclei as targets was attempted with cosmid and plasmid probes in N. haematococca. Although the probe hybridization was successfully detected on the nuclei, mapping resolution was much lower than that of fiber-FISH. FISH on the interphase nuclei was thought to be useful to analyze the behavior and territory of chromosomes in the nucleus.5. Two-color FISH on the prometaphase or metaphase mitotic chromosomes of C. heterostrophus revealed that PKS1 and DEK1 which are involved in T-toxin production reside separately on the different chromosomes. It was also shown that chromosomes 6 and 12 are reciprocally translocated.