Distribution, abundance and analysis of polymorphic microsatellite DNA in Aspergillus flavipes and Pythium ultimum
| dc.contributor.author | Fernandez, Marcel Alexander | en_US |
| dc.date.accessioned | 2007-05-15T15:16:46Z | |
| dc.date.available | 2007-05-15T15:16:46Z | |
| dc.date.issued | 1997-05-01T00:00:00Z | en_US |
| dc.degree.discipline | Microbiology | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.abstract | The objective of this research project was to develop tools for strain identification in the fungi Pythium ultimum and Aspergillus flavipes. The principal drive was to identify genetic loci containing simple sequence arrays (microsatellites) which could be amplified from numerous isolates to reveal polymorphisms. A genomic library of P. ultimum (BR471) was screened with d(GT)$\sb9$ and d(CT)$\sb9$ probes. A d(GT/CA) motif was found to occur at least once every 86 kb and a d(CT/GA) motif every 137 kb on average. A clone hybridizing to the d(GT)$\sb9$ probe was sequenced to reveal a 200 bp region with five d(GT/CA)$\sb{\rm n}$ motifs interspersed with unique and repetitive sequences. Primers complementary to flanking sequences were employed to amplify the regions from all P. ultimum var. ultimum isolates tested (25) and from one isolate P. ultimum var. sporangiiferum. Many length polymorphisms were detected in the amplification products of all isolates tested. Three of these isolates were characterized by sequen ing the polymorphic region. Variance in the number of d(GT/CA) dinucleotides as well as a deletion extending into the sequence flanking the d(GT/CA) array was observed. For strain differentiation in Aspergillus flavipes, amplification of genomic DNA with single random primers (RAPD) produced unique sets of electrophoretic profiles for each of the nine isolates of the species. Thirteen primers were used. Reproducible profiles for each isolate showed extreme polymorphism with very few coincident bands between pairs of isolates and none shared by all nine isolates. The genome of A. navipes was investigated using hybridization of a genomic library with four synthetic oligonucleotide probes, d(GT)$\sb9,$ d(CT)$\sb9,$ d(AT)$\sb9,$ and d(GC)$\sb9.$ Results from genomic dot blots showed the existence of abundant d(GT/CA) and d(CnGA) simple sequence motifs, but no apparent d(AT/TA) or d(GC/CG). Hybridization of the probes to Southern blots of restriction profiles of genomic DNA revealed that the simple sequence motifs are widely dispersed in the genome, that their distribution is highly polymorphic, and that some of them may be members of repetitive DNA families. DNA fragments from library clones which hybridized to the simple sequence probes d(GT)$\sb9$ and d(CT)$\sb9$ were cloned and sequenced. Primers flanking the simple sequence repeats were synthesized and used for amplification of two d(GT/CA) and two d(CT/GA) motifs. d(GT/CA) and d(CT/GA) loci are polymorphic as a consequence of site-specific length variation located within the dinucleotide repeat. Flanking regions of these motifs are highly conserved. However, minor differences in sequence homology were identified in the regions flanking the simple sequence motifs. One microsatellite locus contained a d(GT/CA)$\sb{40}$ repeat. This repeat showed the largest degree of length variation in comparison to the other loci investigated. In another simple sequence locus, two d(CT/GA) motifs were found flanking a d(GT/CA) motif. Sequence comparison of this locus to that in other isolates of A. flavipes showed that polymorphism of a simple sequence motif can include its flanking regions. (Abstract shortened by UMI.) | en_US |
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| dc.format.extent | 184 bytes | |
| dc.format.mimetype | application/pdf | |
| dc.format.mimetype | text/plain | |
| dc.identifier.uri | http://hdl.handle.net/1993/782 | |
| dc.language.iso | eng | en_US |
| dc.rights | open access | en_US |
| dc.title | Distribution, abundance and analysis of polymorphic microsatellite DNA in Aspergillus flavipes and Pythium ultimum | en_US |
| dc.type | doctoral thesis | en_US |