Selection of RAPD markers for investigation of genetic population structure in fusiform rust fungus infecting loblolly pine
research to determine patterns of genetic differentiation among and within field populations of Cronartium quercuum f. sp. fusiforme using RAPD markers is currently underway in the molecular genetics laboratory at the Southern Institute of Forest Genetics. Fungal tissue was collected as a drop of spermatia or scrapings of a discrete hymenium from a single gall on each tree sampled at a location. Collections were made on twenty or more loblolly pines at 25 geographic locations, widely dispersed throughout the natural range of this host species. DNAs are presently being extracted from these tissue samples. The extracted DNAs are being amplified using the polymerase chain reaction (PCR) and 10-mer oligonucleotide primers to produce RAPD products that have potential for use as genetic markers. From bulked samples, twenty-one such RAPD markers have been identified that show consistent, clear band separations and polymorphisms that closely correspond to those produced by genetic markers previously shown to segregate as Mendelian factors in a C. q. fusiforme population derived from a smgle urediniospore culture. It is likely, however, that some of our extractions contain contaminant DNA, from host trees and also possibly from insects and other fungi. This extrinsic DNA might be amplified by PCR in addition to the targeted DNA. In this paper, we describe techniques that are being employed to provide reasonable assurance that the RAPD markers we use for analysis of allele and haplotype frequencies are C. q. fusiforme allelic segregants.
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