9728376 McDonald The object of this SGER research is to develop a new technique, anonymous rare-cutter restriction fragments (ARRFs) for assaying genetic variation. The ARRF technique would consist of digesting genomic DNA with two rare-cutter restriction enzymes, isolating the few small fragments from the many large fragments, and resolving the small fragments on a gel. Because there is no PCR amplification step, the intensity of a band will be proportional to the number of copies of the fragment. Therefore, single-copy nuclear fragments should be distinguishable from multiple-copy and organellar fragments, and heterozygotes should be distinguishable from homozygotes. DNA from parasites or contamination should also be easily be identified. Each pair of rare-cutter restriction enzymes has the potential to yield dozens of independent genetic markers, all of which could be assayed simultaneously. The only development steps required to apply the technique to a new species will be finding a suitable technique for preparing genomic DNA and determining which pair of enzymes yield suitable numbers of fragments. The AARF technique would be useful in a wide variety of studies in population biology where genetic markers are needed, including studies of gene flow and population structure, hybridization, mating behavior, selfing rates, and QTL mapping. These attributes of the AARF technique are major advantages over other techniques currently used for assaying variation in nuclear DNA. The ARRF technique will be developed gradually, starting with bacterial DNA (genome size about 4M base pairs), then yeast (11Mbp), then Drosophila (100 Mbp), then larger Metazoans (1000+Mbp). As the genome size gets larger, each single-copy fragments becomes a smaller fraction of the total DNA, increasing the technical challenges. Calculations indicate that the amount of DNA required for detecting ARRF bands from an organism with a genome size in the billions of base pairs is fairly large, near the upper limit of p racticality, so there is no guarantee the ARRF technique will be feasible on all organisms. If the technique works, however, it will be a simple, rich, and robust source of genetic markers that will be useful to a broad range of population biologists, as well, potentially, in studies of animal and plant disease and crop improvement.
