We are developing a genetic map of the dog based partly upon markers contained within known genes. In order to facilitate the development of these markers, we have used polymerase chain reaction (PCR) primers designed to conserved regions of genes that have been sequenced in at least two species. We have refined the method for designing primers to maximize the number that produce successful amplifications across as many mammalian species as possible. We report the development of primer sets for 11 loci in detail: CFTR, COL10A1, CSFIR, CYP1A1, DCN1, FES, GHR, GLB1, PKLR, PVALB, and RB1. We also report an additional 75 primer sets in the appendices. The PCR products were sequenced to show that the primers amplify the expected canine genes. These primer sets thus define a class of gene-specific sequence-tagged sites (STSs). There are a number of uses for these STSs, including the rapid development of various linkage tools and the rapid testing of genomic and cDNA libraries for the presence of their corresponding genes. Six of the eleven gene targets reported in detail have been proposed to serve as "anchored reference loci" for the development of mammalian genetic maps [O'Brien, S. J., et al., Nat. Genet. 3:103, 1993]. The primer sets should cover a significant portion of the canine genome for the development of a linkage map. In order to determine how useful these primer sets would be for the other genome projects, we tested the 11 primer sets on the DNA from species representing five mammalian orders. Eighty-four percent of the gene-species combinations amplified successfully. We have named these primer sets "universal mammalian sequence-tagged sites" because they should be useful for many mammalian genome projects.

Nucleotide diversity (pi), the average number of base differences per site for two homologous sequences randomly selected from a population, is an important parameter used to understand the structure and history of populations. It is also important for determining the feasibility of developing a genetic map for a species from single nucleotide polymorphisms (SNPs). Nucleotide diversity has never been estimated for dogs. Segments of twelve canine genes from ten diverse dog breeds were examined for nucleotide variation by using a pool-and-sequence method. We identified three SNPs in the coding regions (2501 bp) and 11 SNPs in the introns (2953 bp). Each of these putative SNPs was tested by restriction enzyme analysis, and all were verified. Six additional SNPs were identified in a single SINE contained in one gene. Using these data, canine sequence diversity across breeds was estimated to be 0.001 and 0.0004 in intronic and coding regions, respectively, with SNPs spaced every 400 bp on average. Discovery of useful SNPs in 7 of the 12 genes suggests that construction of a canine SNP-based map can be accomplished with current technology. Thirteen polymorphic SNPs were also found in 5847 bp in the cat, horse, ox, and pig, by using four of the same genes from which canine nucleotide diversity was estimated. These results suggest that these species may have similar amounts of nucleotide diversity.

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Identification of single nucleotide polymorphisms (SNPs) by DNA sequence comparison across breeds is a strategy for developing genetic markers that are useful for many breeds. However, the heterozygosity of SNPs identified in this way might be severely reduced within breeds by inbreeding or genetic drift in the small effective population size of a breed (population subdivision). The effect of inbreeding and population subdivision on heterozygosity of SNPs in dog breeds has never been investigated in a systematic way. We determined the genotypes of dogs from three divergent breeds for SNPs in four canine genes (ACTC, LMNA, SCGB, and TYMS) identified by across-breed DNA sequence comparison, and compared the genotype frequencies to those expected under Hardy-Weinberg equilibrium (HWE). Although population subdivision significantly skewed allele frequencies across breeds for two of the SNPs, the deviations of observed heterozygosities compared with those expected within breeds were minimal. These results indicate that across-breed DNA sequence comparison is a reasonable strategy for identifying SNPs that are useful within many canine breeds.

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