A high-resolution 15,000(Rad) radiation hybrid panel for the domestic cat.

The current genetic and recombination maps of the cat have fewer than 3,000 markers and a resolution limit greater than 1 Mb. To complement the first-generation domestic cat maps, support higher resolution mapping studies, and aid genome assembly in specific areas as well as in the whole genome, a 15,000(Rad) radiation hybrid (RH) panel for the domestic cat was generated. Fibroblasts from the female Abyssinian cat that was used to generate the cat genomic sequence were fused to a Chinese hamster cell line (A23), producing 150 hybrid lines. The clones were initially characterized using 39 short tandem repeats (STRs) and 1,536 SNP markers. The utility of whole-genome amplification in preserving and extending RH panel DNA was also tested using 10 STR markers; no significant difference in retention was observed. The resolution of the 15,000(Rad) RH panel was established by constructing framework maps across 10 different 1-Mb regions on different feline chromosomes. In these regions, 2-point analysis was used to estimate RH distances, which compared favorably with the estimation of physical distances. The study demonstrates that the 15,000(Rad) RH panel constitutes a powerful tool for constructing high-resolution maps, having an average resolution of 40.1 kb per marker across the ten 1-Mb regions. In addition, the RH panel will complement existing genomic resources for the domestic cat, aid in the accurate re-assemblies of the forthcoming cat genomic sequence, and support cross-species genomic comparisons.

International Equine Gene Mapping Workshop Report: a comprehensive linkage map constructed with data from new markers and by merging four mapping resources.

A comprehensive male linkage map was generated by adding 359 new, informative microsatellites to the International Equine Gene Map half-sibling reference families and by combining genotype data from three independent mapping resources: a full sibling family created at the Animal Health Trust in Newmarket, United Kingdom, eight half-sibling families from Sweden and two half-sibling families from the University of California, Davis. Because the combined data were derived primarily from half-sibling families, only autosomal markers were analyzed. The map was constructed from a total of 766 markers distributed on the 31 equine chromosomes. It has a higher marker density than that of previously reported maps, with 626 markers linearly ordered and 140 other markers assigned to a chromosomal region. Fifty-nine markers (7%) failed to meet the criteria for statistical evidence of linkage and remain unassigned. The map spans 3,740 cM with an average distance of 6.3 cM between markers. Fifty-five percent of the intervals are < or = 5 cM and only 3% > or = 20 cM. The present map demonstrates the cohesiveness of the different data sets and provides a single resource for genome scan analyses and integration with the radiation hybrid map.

Evaluation of canine COL4A3 and COL4A4 as candidates for familial renal disease in the Norwegian elkhound.

The collagen type IV alpha3 and alpha4 chains (COL4A3 and COL4A4) are part of the specialized glomerular basement membrane in the kidney. In human these genes are responsible for Alport syndrome (a type of hereditary nephritis). Histopathological similarities between kidneys of Norwegian elkhound dogs affected with familial renal disease and human Alport syndrome were the basis for a candidate gene approach in Norwegian elkhounds. Three microsatellites-tightly linked to canine COL4A3 and COL4A4--were developed. The microsatellites were used to analyze linkage between COL4A3 and COL4A4 and familial renal disease in a Norwegian elkhound pedigree segregating this disease. Presence of one recombinant between familial renal disease and COL4A3/COL4A4 suggests that these genes are not likely candidates for familial renal disease in this breed.

Localizing the X-linked orange colour phenotype using feline resource families.

Many genes influencing mammalian coat colours are well conserved. While genes responsible for pelage phenotypes in one species provide strong evidence for a candidate gene in a different species, the X-linked orange phenotype of the domestic cat is unique within mammals. The orange locus (O) undergoes X-inactivation, producing females that express both wildtype black (wt) and orange (variant) phenotypes when heterozygous (tortoiseshell). The orange locus has not yet been localized on the X chromosome. Tortoiseshell male cats have been identified but have been shown to be sex chromosome trisomies (XXY). To localize the cat orange locus, 10 feline-derived X-linked microsatellites were analysed in two extended cat pedigrees consisting of 79 and 55 individuals, respectively, segregating for the orange phenotype. Linkage analyses excluded close association of orange in the vicinity of the nine informative X-linked microsatellites. One marker was not polymorphic within either family. Several markers suggested exclusion (Z < -2.0) at distances of 7.5-33 cM. Exclusion analyses suggested a possible location for orange a 14 cM region near Xcen. Recombination distances of markers in the segregating feline pedigrees were reduced as compared with the feline interspecies backcross family. Thus, the presented pedigrees may be useful as reference families for the domestic cat because more accurate recombination rates for domestic cats can be determined.

Genomic characterization of equine interleukin-4 receptor alpha-chain (IL4R).

Three overlapping fragments of the equine interleukin-4 receptor alpha chain gene (IL4R) were cloned and sequenced. The resulting 3553 bp cDNA sequence exhibited homology to human, murine and bovine IL4R. The equine IL4R exhibits many conserved features when compared to other species, including intron-exon boundary positions and amino acid sequence motifs characteristic of type I cytokine receptors. The IL4R gene was localized to horse chromosome ECA13 by synteny mapping on a somatic cell hybrid panel. Evidence for an alternative splice variant of IL4R was found in the genomic sequence and subsequently verified using RT-PCR on equine monocyte RNA. A polymorphism screen of the largest exon, homologous to exon 12 of the human IL4R gene, was performed using DNA from 60 horses of various breeds which yielded 11 coding-region single nucleotide polymorphisms (SNPs), 7 synonymous and 4 non-synonymous. Three of the four non-synonymous SNPs occur at high frequencies and are found very near a conserved tyrosine residue.

The second generation of the International Equine Gene Mapping Workshop half-sibling linkage map.

A low-density, male-based linkage map was constructed as one of the objectives of the International Equine Gene Mapping Workshop. Here we report the second generation map based on testing 503 half-sibling offspring from 13 sire families for 344 informative markers using the CRIMAP program. The multipoint linkage analysis localized 310 markers (90%) with 257 markers being linearly ordered. The map included 34 linkage groups representing all 31 autosomes and spanning 2262 cM with an average interval between loci of 10.1 cM. This map is a milestone in that it is the first map with linkage groups assigned to each of the 31 automosomes and a single linkage group to all but three chromosomes.

Linkage of the grey coat colour locus to microsatellites on horse chromosome 25.

The progressive loss of colour in the hair of grey horses is controlled by a dominantly inherited allele at the Grey locus (GG). In this study, two paternal Quarter Horse (QH) families segregating for the GG allele were genotyped with a set of 101 microsatellite markers spanning the 31 autosomes and the X chromosome. This genome scan demonstrated linkage of Grey to COR018 (RF=0.02, LOD=12.04) on horse chromosome 25 (ECA25). Further chromosome-specific analysis of seven total QH families confirmed the linkage of Grey to a group of ECA25 markers and the map order of NVHEQ43-(0.24)-UCDEQ405-(0.09)-COR080-(0.05)-GREY-(0.14)-UCDEQ464 was produced. Although G was found to be linked to TXN and COR018 in the chromosome-specific analysis, the data were not sufficiently informative to place either marker on our ECA25 map with significant LODs. Our results excluded the equine tyrosinase related protein 1 (TYRP1) and melanocyte protein 17 (Pmel17) genes as possible candidates for the grey phenotype in horses.

PCR multiplexed microsatellite panels to expedite canine genetic disease linkage analysis.

Modern dog breeds possess large numbers of genetic diseases for which there are currently few candidate genes or diagnostic tests. Linkage of a microsatellite marker to a disease phenotype is often the only available tool to aid in the development of screening tests for disease carriers. Detection of linkage to a specific disease phenotype requires screening of large numbers of markers across known affected and unaffected animals. To establish high throughput genome scanning this study placed 100 canine microsatellite markers, arranged by fragment size and fluorescent dye label, into 12 PCR multiplexed panels. The highest degree of multiplexing was 11 markers per panel while the lowest was five markers per panel; each panel was run in one gel lane on automated DNA sequencers. Selection of the markers was based upon chromosomal or linkage group locations, degree of polymorphism, PCR multiplex compatibility and ease of interpretation. The marker set has an average spacing of 22.25 centiMorgan (cM). Marker polymorphism was evaluated across 28 American Kennel Club (AKC) recognized breeds. The utility of buccal swab vs. blood samples was also validated in this study as all template DNA was derived from swabs obtained and submitted by participating dog breeders and owners. The PCR multiplexed microsatellite panels and sampling method described in this report will provide investigators with a cost effective and expedient means of pursuing linkage studies of specific canine genetic diseases.

The cream dilution gene, responsible for the palomino and buckskin coat colours, maps to horse chromosome 21.

The colour locus historically referred to as C in the horse is linked to microsatellites markers on horse chromosome 21. Preliminary results demonstrated linkage of Ccr, thought to be the cream dilution variant of the C locus, to HTG10. An analysis of horse chromosome 21 using additional families confirmed and established a group of markers linked to Ccr. This work also improved the resolution of previously reported linkage maps for this chromosome. Linkage analysis unambiguously produced the map order: SGCV16-(19.1 cM)-HTG10-(3.8 cM)-LEX60/COR73-(1.3 cM)-COR68-(4.5 cM)- Ccr-(11.9 cM)-LEX31. Comparative and synteny data suggested that the horse C locus is not tyrosinase (TYR).

Synteny and regional marker order assignment of 26 type I and microsatellite markers to the horse X- and Y-chromosomes.

The hypothesis that the conservation of sex-chromosome-linked genes among placental mammals could be extended to the horse genome was tested using the UCDavis horse-mouse somatic cell hybrid (SCH) panel. By exploiting the fluorescence in-situ hybridization (FISH) technique to localize an anchor locus, X-inactivation-specific transcript (XIST) on the horse X chromosome, together with the fragmentation and translocation of the X- and Y-chromosome fragments in a somatic cell hybrid panel, we regionally assigned 13 type I and 13 type II (microsatellite) markers to the horse X- and Y-chromosomes. The synteny groups that correspond to horse X- and Y-chromosomes were identified by synteny mapping of sex-specific loci zinc finger protein X-linked (ZFX), zinc finger protein Y-linked (ZFY) and sex-determining region Y (SRY) on the SCH panel. A non-pseudoautosomal gene in the human steroid sulfatase (STS) was identified in both X- and Y-chromosome-containing clones. The regional order of the X-linked type I markers examined in this study, from Xp- to Xq-distal, was [STS-X, the voltage-gated chloride channel 4 (CLCN4)], [ZFX, delta-aminolevulinate synthase 2 (ALAS2)], XIST, coagulation factor IX (F9) and [biglycan (BGN), equine F18, glucose-6-phosphate dehydrogenase (G6PD)] (precise marker order could not be determined for genes within the same brackets). The order of the Y-linked type I markers was STS-Y, SRY and ZFY These orders are the same arrangements as reported for the human X- and Y-chromosomes, supporting the conservation of genomic organization between the human and the horse sex chromosomes. Regional ordering of X-linked type I and microsatellite markers provides the first integration of type I and type II markers in the horse X chromosome.

Report of the International Equine Gene Mapping Workshop: male linkage map.

The goal of the First International Equine Gene Mapping Workshop, held in 1995, was the construction of a low density, male linkage map for the horse. For this purpose, the International Horse Reference Family Panel (IHRFP) was established, consisting of 12 paternal half-sib families with 448 half-sib offspring provided by 10 laboratories. Blood samples were collected and DNA extracted in each laboratory and sent to the Lexington laboratory (KY, USA) for dispatch in aliquots to 14 typing laboratories. In total, 161 markers (144 microsatellites, seven blood groups and 10 proteins) were tested for all families for which the sire was heterozygous. Genealogies and typing data were sent for analysis to the INRA laboratory (Jouy-en-Josas, France) according to a specific format and entered into a database with input verification and output processes. Linkage analysis was performed with the CRIMAP program. Significant linkage was detected for 124 loci, of which 95 were unambiguously ordered using a multipoint analysis with an average spacing of 14.2 CM. These loci were distributed among 29 linkage groups. A more comprehensive analysis including synteny group data and FISH data suggested that 26 autosomes out of 31 are covered. The complete map spans 936 CM.

A synteny map of the horse genome comprised of 240 microsatellite and RAPD markers.

To generate a domestic horse genome map we integrated synteny information for markers screened on a somatic cell hybrid (SCH) panel with published information for markers physically assigned to chromosomes. The mouse-horse SCH panel was established by fusing pSV2neo transformed primary horse fibroblasts to either RAG or LMTk mouse cells, followed by G418 antibiotic selection. For each of the 108 cell lines of the panel, we defined the presence or absence of 240 genetic markers by PCR, including 58 random amplified polymorphic DNA (RAPD) markers and 182 microsatellites. Thirty-three syntenic groups were defined, comprised of two to 26 markers with correlation coefficient (r) values ranging from 0.70 to 1.0. Based on significant correlation values with physically mapped microsatellite (type II) or gene (type I) markers, 22 syntenic groups were assigned to horse chromosomes (1, 2, 3, 4, 6, 9, 10, 11, 12, 13, 15, 18, 19, 20, 21, 22, 23, 24, 26, 30, X and Y). The other 11 syntenic groups were provisionally assigned to the remaining chromosomes based on information provided by heterologous species painting probes and work in progress with type I markers.

Two autosomal trisomies in the horse: 64,XX,-26,+t(26q26q) and 65,XX,+30.

The phenotypic effects in a yearling Arab filly of a newly described equine autosomal trisomy syndrome for chromosome 30 (65,XX,+30) consisted of small size and severe angular deviation of front legs accompanied by mild polydactyly, but no mental dullness. This case was associated with advanced maternal age. Additional banding studies of a second trisomy case confirmed the assignment to chromosome 26 (64,XX,-26,+t(26q26q)) and evidence of her fertility was presented.