Electrophoretic karyotypes and molecular genetic analysis of Tilletia caries and T. controversa Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/8336h5520

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  • Electrophoretic karyotypes were obtained from intact cells of four wild type strains of Tilletia caries and T. controversa, and 5 inter specific hybrid progeny using clamped homogeneous electric field, pulsed field gel electrophoresis (CHEF PFGE). Each karyotype was polymorphic and unique relative to the other strains. However, the relative size range of all the chromosomes was consistent and typically ranged from approximately 850 to 4,490 kilobasepairs (Kb) for all strains, accept for two atypically small chromosomes in one strain of T. caries. The minimum estimated number of chromosomes was 19 or 20 for strains of T. controversa, 14 to 20 for T. caries, and from 19 to 22 for the hybrid progeny. The minimum estimated total genome size ranged from 28 to 39 megabasepairs (mb) for T. caries, 34 to 40 mb for T. controversa, and 36 to 42 mb for the hybrid progeny. Southern hybridization analysis performed with cloned, single copy homologous DNA fragments identified a single similar-sized chromosome in all strains. The maximum percent variability of the linkage groups defined by the single copy probes was 10% or less. The rDNA from Neurospora crassa hybridized with 2 or 3 chromosomes in the wild type stains of T. caries and T. controversa, and with 1 to 3 chromosomes in the hybrid progeny. The maximum percent variability among chromosomes that hybridized with the rDNA probe ranged from 35 to 40%. Either 1 or 2 chromosomes were identified by the single copy actin gene from Aspergillus nidulans, and the maximum percent variability ranged from 4 to 14% for these chromosomes. It was not possible to differentiate between the karyotypes of strains of either T. caries or T. controversa using the physical appearance of the karyotypes, the number and sizes of chromosomes, the genome sizes, or by Southern hybridization analysis. Analysis of the karyotypes of the hybrid progeny revealed that all were unique relative to each other and the parental stains, providing circumstantial evidence for the presence of recombinant chromosomes. That the genome size and chromosomes numbers of the hybrid progeny were similar to their monokaryotic parental strains strongly argues that the reduction division stage of meiosis had occurred. Seven teliospore samples from Oregon and Turkey were examined for their germination and autofluorescence properties. The teliospore samples were analyzed to determine if low temperature germination (0-4° C) and autofluorescing spore-wall reticulations associated with spores of T. controversa were usually linked. Two of the seven teliospore samples showed these characteristics to be unlinked. In an attempt to clone the mating type genes of T. caries and T. controversa, total genomic DNA was probed with a fragment of the b west mating type gene from Ustilago hordei. Although single 1.4 kb BamHI fragment from both pathogens was cloned by homology and sequenced, its role in mating type function remains uncertain.
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