N. G. Holmes

Melanocortin 1 receptor variation in the domestic dog

Abstract. The melanocortin 1 receptor (Mc1r) is encoded by the Extension locus in many different mammals, where a loss-of-function causes exclusive production of red/yellow pheomelanin, and a constitutively activating mutation causes exclusive production of black/brown eumelanin. In the domestic dog, breeds with a wild-type E allele, e.g., the Doberman, can produce either pigment type, whereas breeds with the e allele, e.g., the Golden Retriever, produce exclusively yellow pigment. However, a black coat color in the Newfoundland and similar breeds is thought to be caused by an unusual allele of Agouti, which encodes the physiologic ligand for the Mc1r. Here we report that the predicted dog Mc1r is 317 residues in length and 96% identical to the fox Mc1r. Comparison of the Doberman, Newfoundland, Black Labrador, Yellow Labrador, Flat-coated Retriever, Irish Setter, and Golden Retriever revealed six sequence variants, of which two, S90G and R306ter, partially correlated with a black/brown coat and red/yellow coat, respectively. R306ter was found in the Yellow Labrador, Golden Retriever, and Irish Setter; the latter two had identical haplotypes but differed from the Yellow Labrador at three positions other than R306ter. In a larger survey of 194 dogs and 19 breeds, R306ter and a red/yellow coat were completely concordant except for the Red Chow. These results indicate that the e allele is caused by a common Mc1r loss-of-function mutation that either reoccurred or was subject to gene conversion during recent evolutionary history, and suggest that the allelic and locus relationships for dog coat color genes may be more analogous to those found in other mammals than previously thought.

Chromosome-specific paints from a high-resolution flow karyotype of the dog

Using peripheral blood lymphocyte cultures and duallaser flow cytometry, we have routinely obtained high-resolution bivariate flow karyotypes of the dog in which 32 peaks are resolved. To allow the identification of the chromosome types in each peak, chromosomes were flow sorted, amplified and labelled by polymerase chain reaction with partially degenerate primers and hybridized onto metaphase spreads of a male dog. The chromosome paints from 22 of the 32 peaks each hybridized to single homologue pairs and eight peaks each hybridized to two pairs. Paints from the remaining two peaks hybridized to only one homologue each in the male metaphase spread, thus corresponding to the sex chromosomes X and Y. All of the 38 pairs of autosomes and the two sex chromosomes of the dog could be accounted for in these painting experiments. The positions of chromosomes 1–21 were assigned to the flow karyotype (only chromosomes 1–21 have as yet been officially designated). The high-resolution flow karyotype and the chromosome paints will facilitate further standardization of the dog karyotype. The ability to sort sufficient quantities of dog chromosomes for the production of chromosome-specific DNA libraries has the potential to accelerate the physical and genetic mapping of the dog genome.

Towards construction of a canine linkage map: Establishment of 16 linkage groups

1Norwegian Kennel Klub and Department of Morphology, Genetics and Aquatic Biology, Section of Genetics, P.O. Box. 8146 Dep., N-0033 Oslo, Norway ZDepartment of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Box 7023, 750 07 Uppsala, Sweden 3Department of Animal Science and Animal Health, Division of Animal Genetics, The Royal Veterinary and Agricultural University, Btilowsvej, 13, DK-1870, Fredriksberg C, Copenhagen, Denmark ~The Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK 5Centre for Preventive Medicine, Animal Health Trust, PO Box 5, Newmarket, Suffolk CB8 7DW, UK 6Medical Technology and Medicine, Michigan State University, B228 Life Science, East Lansing, Michigan 48824-1317, USA 7Institute of Animal Breeding, University of Berne, Bremgartenstrasse 109 a, 3012 Berne, Switzerland

The application of FISH techniques for physical mapping in the dog (Canis familiaris)

The abundance of CA/GT repeats in the DNA of the2 dog (Canis familiaris) has established the importance of polymorphic microsatellites in the development of a low density map of the canine genome. The assignment of linkage groups of markers to chromosomes by physical mapping requires reliable cytogenetic techniques for routine production of metaphase cells. The dog has 78 chromosomes, many of which are smaller and more contracted than those of other mammals. Although the molecular study of inherited disease in dogs has important implications for both improved welfare in dogs and the provision of animal models for human diseases, the small size and large number of chromosomes in the canine genome has discouraged the inclusion of cytogenetic analysis in the planning of relevant research protocols. In this report, Fluorescence In Situ Hybridization (FISH) techniques have been optimized for the physical mapping of probes in C. familiaris. A method to obtain a good yield of early and midmetaphases from short-term peripheral blood cultures and the optimal conditions for hybridization and detection of probes is described. Thirteen mi-crosatellite-containing cosmid probes from a canine genomic library in pWE15, a highly repetitive probe (human ribosomal DNA pHr14E3), and a human X Chromosome (Chr) paint have been mapped. Six microsatellites, two ribosomal sites, and the human paint have been assigned to specific chromosomes.

ADAMTS17 mutation associated with primary lens luxation is widespread among breeds

Primary lens luxation (PLL) is a well-recognized, painful and potentially blinding inherited ocular condition in dogs. We screened PLL-affected dogs of 30 different breeds, to identify those which carried a previously described c.1473+1 G>A mutation in ADAMTS17 that is associated with PLL in Miniature Bull terriers, Lancashire Heelers, and Jack Russell terriers. This ADAMTS17 mutation was identified in PLL-affected dogs from 14 additional breeds. PLL-affected dogs from some breeds (most notably the Shar pei and the Brittany spaniel) did not carry the G1473+1A ADAMTS17 mutation, indicating they must suffer from a genetically distinct form of the condition. We also estimated the frequency of this ADAMTS17 mutation in some of the breeds. Our findings indicate the mutation segregates in a large number of different breeds of dog, many of which are terriers or breeds with terrier co-ancestry, but some of which have more diverse origins. Our results also indicate that the mutation is present at high frequency within most of the breeds in which it segregates. In the miniature bull terrier breed estimates of mutation frequency ranged from 0.27 to 0.39, corresponding to 7.3-15.2% PLL-affected dogs in this breed. We also identified an increased risk of PLL associated with heterozygosity at ADAMTS17, suggesting that carriers carry a low risk of developing PLL.

DNA marker C04107 for copper toxicosis in a population of Bedlington terriers in the United Kingdom

The DNA microsatellite marker C04107, linked to the Bedlington terrier copper toxicosis locus, is used diagnostically in the USA to detect the disease allele. This marker has been typed in Bedlington terriers of known disease status in the United Kingdom, and it is concluded that it should be useful in eradicating the disease from the breed in the UK. The marker also identified a dog which had been diagnosed on the basis of a liver biopsy as having the disease, as being unaffected; a second liver biopsy confirmed that the dog did not have copper toxicosis.

Refined genetic and comparative physical mapping of the canine copper toxicosis locus

Abstract. Recently, the copper toxicosis (CT) locus in Bedlington terriers was assigned to canine chromosome region CFA10q26, which is homologous to human chromosome region HSA2p13-21. A comparative map between CFA10q21-26 and HSA2p13-21 was constructed by using genes already localized to HSA2p13-21. A high-resolution radiation map of CFA10q21-26 was constructed to facilitate positional cloning of the CT gene. For this map, seven Type I and eleven Type II markers were mapped. Using homozygosity mapping, the CT locus could be confined to a 42.3 cR3000 region, between the FH2523 and C10.602 markers. On the basis of a partial BAC contig, it was estimated that 1-cR3000 is equivalent to approximately 210 kb, implying that the CT candidate region is therefore estimated to be about 9 Mb.

An integrated cytogenetic, radiation-hybrid, and comparative map of dog Chromosome 5

Abstract. The development of a detailed genome map for the domestic dog (Canis familiaris, CFA) is a prerequisite for the continued use of this species as a model system for the study of inherited traits. We present an integrated cytogenetic, radiation-hybrid, and comparative map of dog Chromosome (Chr) 5 (CFA 5). The map comprises 14 gene markers, selected from loci previously mapped within the corresponding evolutionarily conserved chromosome segments (ECCS) of the human genome. Large-insert clones representing each marker were first isolated and mapped by fluorescence in situ hybridization (FISH) analysis to determine their subchromosomal localization on CFA 5. Thirteen gene markers were subsequently mapped by using a commercially available whole genome radiation hybrid (WG-RH) panel for the dog. Nine anonymous markers were also assigned to CFA 5 by both FISH and WG-RH analysis. The 22 markers formed six RH-linkage groups, spanning each of the four ECCS comprising this 99 megabase chromosome. All cytogenetic, WG-RH, and comparative mapping data were in agreement and were combined to determine both the most likely locus order within each linkage group, and also the gross relative orientation of the corresponding ECCS. This study provides a resource for the transfer of information from the human transcript map to that of the dog, and extends existing data regarding the structural relationships between CFA 5 and its evolutionary counterparts within the human genome.

Construction and integration of radiation-hybrid and cytogenetic maps of dog Chromosome X

Chromosome (chr) X is under-represented in current maps of the genome of the domestic dog (Canis familiaris). To address this problem, we have constructed a small-insert, genomic DNA library in pBluescript from flow-sorted canine Chr X DNA. Fluorescence in situ hybridization (FISH) studies confirmed that the library was highly enriched for Chr X. Clones containing microsatellites were identified and sequenced. Database searches detected significant sequence identity between four X-derived clones and genes previously characterized in other species. Thirty-seven markers derived from these clones were mapped on Chr X by FISH, and of these, 28 were mapped by using the female-derived T72 whole-genome radiation hybrid (RH) panel (Research Genetics). Four X-linked canine genes from publicly available data were also mapped. Eight RH linkage groups with LOD >4.0 were identified, and FISH data were used to locate the groups on the chromosome; four groups could be unambiguously orientated by FISH data. In each case, the FISH and RH data were mutually consistent. The data suggest strongly conserved synteny between canine and human X Chrs. The pseudoautosomal region has been further characterized, and the putative or actual locations of nine genes of clinical relevance have been suggested.

Changes in a photoreceptor polypeptide correlating with an early-onset retinal dystrophy in the cat

A preparation of rod outer segments has been used to study the polypeptides characteristic of an early-onset retinal dystrophy in cats (Rdy) by two-dimensional gel electrophoresis. Comparison of 2-D gels of rod outer segment preparations from retinas of normal and Rdy animals shows several differences. In particular, a polypeptide of Mr 51 kDa and pI 7.5 is present at increased levels in preparations from Rdy cats at 6 weeks, 9 weeks and 12.5 weeks of age but not at 3.5 weeks.