Characterization and Use of a Multiplex PCR-based System: Random amplified Polymorphic DNA

Sammanfattning: Random amplified polymorhic DNA (RAPD) is a PCR-based marker system that makes use of universal sets of short oligonucleotides that amplify random DNA fragments under low-stringency conditions. The RAPD marker system has found widespread use and possesses a number of very positive features, being a multiplex and universal system. The present studies characterize and evaluate the properties of the RAPD marker system in a number of different applications. RAPD markers were compared with RFLP markers in their ability to determine genetic relationships between lines of oilseed rape and were found to have the same resolving power as RFLP markers have. A high density genetic linkage map was constructed in sugar beet using both RAPD and RFLP markers. Both types of markers show a high degree of clustering over the genetic map, compatiple with a strong distal localization of recombination. In regions of high marker density, dominant RAPD markers present in either linkage phase and codominant RFLP markers subclustered relative to each other. The subclustering could be attributed both to the mapping procedure and to genuine differences in the way RAPD and RFLP markers are recruited. DNA mixtures were used to investigate how reliably RAPD markers function as genetic markers. Competition occurs in the amplification of all RAPD fragments and is a major source of genotyping errors in RAPD analysis. Mixtures of multiple primers were used to analyse the properties of the RAPD system. Competition makes mixtures of more than to primers inefficient. Two-primer mixtures produces xn(n-1)/2 different products, of which at least xn2/4 are unique, and can generate the extremly large numbers of markers needed in bulked segregant analysis (BSA) and chromosome landing. BSA were used to isolate markers linked to a beet cystnematode resistence locus in sugar beet and fertility restorer gene for the Ogura cytoplasmic male sterility in oliseed rape. Graphical genotype information was shown to be useful in the selection of individuals used in BSA experiments and a set of dominant RAPD markers were used succesfully in a marker-assisted selection experiment