To determine the source of the abnormal coat in the PWD, we tested for the presence of variant alleles in both the RPSO2 and FGF5 genes using DNA isolated from 5 PWD with IC and 29 with normal coats. Of the latter, 19 were predicted to be carriers of the critical variant as they had produced IC offspring. The remaining 10 of 29 were expected to carry breed normal genotypes at both RSPO2 and FGF5, based on the lack of IC dogs among their progeny.
We observed that all the PWDs, regardless of coat type, were homozygous for the T allele at FGF5, which is associated with long hair on the body. In addition, we found no association with the FGF5 gene or surrounding region on canine chromosome 32 (CFA32) with IC ().
Simple sequence repeat markers near FGF5. Positions given are on chromosome 32 based on the CanFam2 assembly. P values are for the single most significant allele from each marker
By comparison, none of the 5 dogs with IC carried the expected insertion in the 3′ UTR of the RSPO2 gene, which is associated with furnishings, whereas all the 29 PWD with standard coats carried the insertion. The 19 obligate carriers, as expected, each carried 1 copy of the wt allele and 1 copy of the variant ().
Figure 2 Size discrimination of amplicons containing the indel in the 3′ UTR of RSPO2 at position 11,634,766. These 3 graphs define: (A) the variant (furnishings) allele—will not produce IC, (B) carrier—can produce IC when mated to another (more ...)
We next analyzed a family of PWD that segregated IC and found that phenotype and genotype correlated perfectly (). All affected dogs being homozygous for the wt allele. This suggested that the lack of insertion in the RSPO2 gene is the sole cause of IC in PWD.
To assess the prevalence of the wt allele in the PWD population, we genotyped an additional 253 dogs selected at random from the “Georgie Project” collection of PWDs (http://www.georgieproject.com/
). As expected, the majority, 195, were homozygous for the insertion and an additional 58 were heterozygous. No additional dogs were identified that were homozygous for the wt allele. Based on the randomly chosen dogs, we calculated an allele frequency of ~12% for the wt allele at RSPO2
in the PWD population. Assuming Hardy–Weinberg (HW) equilibrium, 1.4% of PWDs should display IC. Based on information collected through the Georgie Project, approximately 0.5% of PWD report the IC phenotype. Although the number of affected dogs reported is 3 times lower than the number expected, the distribution of genotypes in the dataset is not significantly different than expected based on HW calculations (χ2 P
= 0.19). The true frequency of IC dogs is likely higher than what we report here, as owners with IC dogs are less likely to have participated in the parent study (Georgie Project) because their dogs did not meet the breed standard (http://www.georgieproject.com/
). However, if we assume that the reported numbers are representative of the breed as a whole, then the reduced appearance of affected dogs could indicate that current selection against the allele only applies to the affected individuals, not to carriers of the IC allele.
Simulations based on historical number of founders and an average effective population size suggest that the presence of the wt allele at modern frequencies would require introduction of the allele into the population within the last 40 generations (). This event most likely occurred during breed formation in the US, as the majority of simulations end in fixation of the desired allele within 10–15 generations, once immigration is halted. This does not rule out the presence of a wt allele in the original founders; although without reintroduction and with selection against the IC type, this allele would have quickly been lost.
Results of gene flow simulations modeled after the historical recreation of the PWD breed and subsequent importation into the United States