UBC Theses and Dissertations
Identification and characterization of RAPD markers linked to the V1 avirulence gene of Ustilago hordei Lin, Danny C. C.
The basidiomycetous fungus Ustilago hordei is the causal agent of covered smut of barley and oats. The resistance or susceptibility of the barley host to this phytopathogen is determined by the resistance genes in the plant and the corresponding avirulence genes in the pathogen. A map-based cloning strategy was adopted for the isolation of the avirulence genes, VI and V6, in U. hordei. Randomly amplified polymorphic DNA (RAPD) analysis of bulked segregant pools for W, vi, V6 and v6 was performed to obtain molecular markers linked to these alleles. Polymerase chain reaction (PCR) amplifications with 890 available RAPD primers identified 2 markers cosegregating closely with the vi allele and one marker tightly linked to the VI allele, but no markers linked to either V6 or v6 were obtained. One marker, designated 743-1.0, mapped 5.5 cM from vi while two allelic markers,- 359-1.55 and 359-2.0, were 3.7 cM from the VI and vi alleles. Hybridization studies of the identified markers determined that all three RAPD products were part of a repetitive DNA element which was present on all chromosomes of the U. hordei genome. Nucleotide sequence information from the markers was obtained and used to design sequence characterized amplified region (SCAR) primers. These primers were useful for the further characterization of the markers and for the screening of genomic libraries using PCR. A cosmid library of parental strain Uh 4857-4 (VI, V2, V6, MAT-1) was constructed for the purpose of a chromosome walk toward the VI allele. A screen of 2496 clones of the cosmid library by a combined approach of hybridization and direct PCR with the SCAR primers did not result in the isolation of cosmids carrying marker 359-1.55. Successful PCR amplification of a 1.55 kb SCAR product from pooled cosmid clones, however, suggests that this cosmid clone is present within the library. The results of the work described here have subsequently led to the isolation of two overlapping cosmids carrying marker 359- 2.0 from another cosmid library. These cosmid clones will be invaluable tools for the isolation of the VI avirulence gene.
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