Development of the cytogenetic techniques to analyze chromosome rearrangements in plant-pathogenic filamentous fungi

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1.Background and objectivesOne of the major causes for genetic variation of plat-pathogenic filamentous fungi is the change of complement chromosomes of the genomes. Such chromosomal changes are thought to be constituted of aneuploidy and structural rearrangements of chromosomes such as translocation and inversion. Presently techniques that enable detailed cytogenetic analyzes are not available for filamentous fungi. The purpose of this study is to develope novel cytogenetic techniques to elucidate chromosomal changes, especially the rearrangements, in plant fungal pathogens. We set up three concrete goals, that is, (1) application of fluorescence in situ hybridization (FISH) to identify specific chromosomes, (2) detection of chromosomal rearrangements by FISH and pulsed field gel electrophoresis (PFGE), (3) search for the kinds and frequencies of chromosomal rearrangements in fungal populations.2.MaterialsThree plant pathogens, Fusarium solani f.sp.pisi, Botrytis cinerea and Alternari … More a alternata, were used.3.Results and conclusions(1)FISH and PFGE techniquesExperimental protocols for FISH and PFGE were successfully developed especially for F.solani. Using our PFGE prosocol the chromosomes in size range between ca.6-Mb and ca.500-kb could be clearly separated in a sigle run, which enabled us to analyze karyotypic polymorphisms of F.solani. Regarding FISH four types of probes, that is, plasmid cosmid, total nuclear DNA,PCR-amplified DNA of specific chromosomes were labeled with either biotin or DIG.With these probes specific chromosomes became detectable among the somatic metaphase complement chromosomes of the genome.(2)Identification and characterization of specific chromosomesNucleolar-organizing chromosomes was identified by FISH and PFGE using rDNA as a probe in F.solani, A.alternata and B.cinerea. The number of nucelolar-organizing chromosomes was only one in F.solani and B.cinerea, while two were detected in A.alternata. The morphological change of rDNA in the course of nuclear division was evident for F.solani.B chromosome in F.solani was detected by three types of FISH (ordinary FISH with B chromosome-specific cosmid clone, genomic in situ hybridization, and chromosome painting). This was the first success of cytological detection and visualization of B chromosome in fungi.Simultaneous identification of two chromosomes was accomplished by two-color FISH using probes of plasmid-cosmid clones or chromosome painting coupled with hybridization detection by FITC and rhodamine.In this study we have not succeeded in showing the chromosomal rearrangement yet mainly because of the shortage of time. Less