Plant & Animal Genome VI Conference Plant & Animal Genome VI Conference
P81
Department of Plant, Soil, and Environmental Sciences, Montana State University, Bozeman, MT 59717.
Abundant amplified fragment length polymorphisms (AFLPs)for specific chromosomes are found in wheat nullitetrasomic stocks and wheat-barley addition lines. Conversion of specific AFLPs to sequence-tagged-site (STS) PCR primers would be useful for many genetic linkage applications. The goal of our experiments was to develop a system allowing efficient conversion of AFLP markers to STS-PCR markers. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines, using 17 AFLP primer combinations. On average, 36.8% of the scored AFLP fragments in wheat nullitetrasomic stocks and 10.8% in wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 600 chromosome specific AFLP markers. An efficient method was developed for the cloning of the AFLP fragments. Ten AFLP fragments for specific barley chromosomes were isolated from polyacrylamide gels, reamplified, cloned and sequenced. Amplification with STS primers, designed from these sequences, of the wheat-barley addition lines revealed that three out of ten amplified fragments specific to the predicted barley chromosome; five out of 10 amplified a similar-sized fragment on all barley chromosomes but were absent in wheat; and one out of ten amplified a similar-sized fragment in both wheat and barley. For the latter case, polymorphisms existed between wheat and barley upon digestion of PCR products with restriction enzymes. Our experiments allow an estimation of the ratio of repetitive DNA to single copy DNA among genomic fragments targeted by AFLP, and aid in the conversion of AFLPs to STS-PCR markers.