Hair length in dogs has been known for many years to be primarily controlled by a limited number of genes, but none of the genes have been identified. One of these genes produces a recessively inherited long-haired phenotype that has been thought to explain the bulk of hair-length variation among many breeds. Sequence analysis of the FGF5 gene in short and long-haired corgis resulted in the identification of two coding region differences: a duplication in a relatively non-conserved region of the gene and a missense mutation, resulting in the substitution of Phe for Cys, in a highly conserved region. Genotyping of 218 dogs from three breeds fixed for long hair, eight breeds fixed for short hair and five breeds in which long hair is segregating provided evidence that the missense mutation is associated with the hair-length differences among these breeds.
Background: Cross-species primers have been used with moderate success to address a variety of questions concerning genome structure, evolution, and gene function. However, the factors affecting their success have never been adequately addressed, particularly with respect to producing a consistent method to achieve high throughput. Using 1,147 mammalian cross-species primer pairs (1089 not previously reported), we tested several factors to determine their influence on the probability that a given target will amplify in a given species under a single amplification condition. These factors included: number of mismatches between the two species (the index species) used to identify conserved regions to which the primers were designed, GC-content of the gene and amplified region, CpG dinucleotides in the primer region, degree of encoded protein conservation, length of the primers, and the degree of evolutionary distance between the target species and the two index species.
SummaryThe dopamine receptor 5 gene (DRD5) holds much promise as a candidate locus for contributing to neuropsychiatric disorders and other diseases influenced by the dopaminergic system, as well as having potential to affect normal behavioral variation. However, detailed analyses of this gene have been complicated by its location within a segmentally duplicated chromosomal region. Microsatellites and SNPs upstream from the coding region have been used for association studies, but we find, using bioinformatics resources, that these markers all lie within a previously unrecognized second segmental duplication (SD). In order to accurately analyze the DRD5 locus for polymorphisms in the absence of contaminating pseudogene sequences, we developed a fast and reliable method for sequence analysis and genotyping within the DRD5 coding region. We employed restriction enzyme digestion of genomic DNA to eliminate the pseudogenes prior to PCR amplification of the functional gene. This approach allowed us to determine the DRD5 haplotype structure using 31 trios and to reveal additional rare variants in 171 unrelated individuals. We clarify the inconsistencies and errors of the recorded SNPs in dbSNP and HapMap and illustrate the importance of using caution when choosing SNPs in regions of suspected duplications. The simple and relatively inexpensive method presented herein allows for convenient analysis of sequence variation in DRD5 and can be easily adapted to other duplicated genomic regions in order to obtain good quality sequence data.
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