DEVELOPMENT OF RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS FOR GENETIC-MAPPING IN PACIFIC YEW (TAXUS-BREVIFOLIA)

A genetic linkage map was constructed for Pacific yew (Taxus brevifoli a Nutt.) based on random amplified polymorphic DNA (RAPD) markers. A s eries of optimization experiments were conducted to develop a highly r epeatable protocol for Pacific yew. In these experiments, a high MgCl2 concentration (5.5 mM) together with a low primer concentration (0.2 mu m) in the polymerase chain reaction (PCR) mixture yielded the best amplification products. PCR amplification products were further improv ed by treating the template DNAs with RNase. Experiments showed that b ovine serum albumine had the same effect as RNase on PCR amplification . The segregating mapping population consisted of 39 haploid megagamet ophytes from a single mother tree. DNA extracted from a subset of 6 me gagametophytes was screened with 345 ten-base oligonucleotide primers of arbitrary sequence. Of the screened primers, 28% revealed at least one polymorphic locus. Eighty-six of these primers revealed at least o ne polymorphic locus and were used with the entire set of megagametoph yte DNAs. One-hundred-two loci were scored and segregated in the expec ted 1.1 ratio (1.19 locus per primer). Linkage analysis was conducted using MAPMAKER. Forty-one of 102 markers were distributed into 17 link age groups and covered 305.8 centimorgans. The remaining 61 unlinked m arkers should be assigned to linkage groups as more markers are added to the map.