Mark W. Neff[1,2], Mike Strathmann[1], Janet Ziegle[3], and Jasper Rine[1].
[1] Department of Molecular & Cell Biology, Division of Genetics, University of California, Berkeley, CA 94720. [2] Human Genome Center, Lawrence Berkeley National Laboratory, Berkeley, CA 94720. [3] Applied Biosystems (ABI), Foster City, CA 94404. email: neff@mendel.berkeley.edu
The morphological and behavioral traits that distinguish breeds of domestic dog are genetically defined, having been fixed by artificial selection. A linkage map of the dog genome will make breed-specific traits and canine genetic diseases amenable to mapping approaches. We and others are assembling a linkage map for the domestic dog using microsatellites, molecular markers which are highly polymorphic and relatively straightforward to genotype [1,2]. We currently genotype these markers with an ABI fluorescence-based DNA sequencing instrument. Multiplex marker sets for dog have been established based upon differences in product size and primer dye color, allowing for high throughput analysis and unambiguous allele assignment [3].
We have also investigated whether alternative types of genetic markers might supersede microsatellites, with the aim of reducing the time and cost of constructing and using linkage maps. Specifically, an alternative marker should obviate the need for DNA sequencing and primer synthesis during marker construction, and should permit a single mass genotyping of the collection of markers for each individual. RFLPs have the potential to meet these requirements. Bi-allelic RFLPs are sufficiently informative for an F[2] intercross when the parents are homozygous for different alleles. To create a library of RFLPs that are informative for a Border Collie X Newfoundland intercross, we have begun a series genomic subtractions using DNA from the parents [4]. We have also begun developing novel methods of RFLP genotyping which do not require DNA sequence information or marker-specific primers. If successful, these advances will reduce the resources necessary to construct and use linkage maps. A low cost method of genotyping will permit genetic mapping in any species that possesses interesting natural variation.
M.W.N. was supported by a DOE Human Genome Distinguished Postdoctoral Fellowship administered by ORISE, and M.S. was supported by an Alexander Hollander Postdoctoral Fellowship.
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[2] E. A. Ostrander, F. A. Mapa, M. Yee, and J. Rine (1995) One hundred and one new simple sequence repeat-based markers for the canine genome. Mammalian Genome 6:192-5.
[3] J. S. Ziegle et al. (1992) Application of automated DNA sizing technology for genotyping microsatellite loci. Genomics 14: 1026-31.
[4] M. Rosenberg, M. Przybylska and D. Straus (1994) "RFLP subtraction": a method for making libraries of polymorphic markers. Proc. Natl. Acad. Sci. USA 91: 6113-7.