L. V. Millon

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 ≤5 cM and only 3% ≧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.

A horse whole-genome-radiation hybrid panel: chromosome 1 and 10 preliminary maps.

In recent years there has been increasing interest in mapping thehorse genome, particularly to identify disease and performance-enhancing genes. Although a number of horse mapping tools havebeen developed and have proved very useful (genetic maps, so-matic cell hybrid panels, and a BAC library), the benefits ofwhole-genome–radiation hybrid (WG-RH) mapping have not beenavailable. Its advantages over genetic linkage mapping are: (i) theresolving power is not lost in regions of the genome with a lowrecombination rate, because it does not rely on meiotic recombi-nation events; (ii) it is especially useful in animals such as thehorse with relatively long generation times and single births; and(iii) genetic and physical maps can be integrated, as both poly-morphic and non-polymorphic markers can be placed on the samemap. The WG-RH panel constructed in this study is the first suchpanel to be reported for the horse, and its preliminary character-ization demonstrates its usefulness for horse genome mapping.The panel was constructed by the fusion of horse embryonicendothelial primary lung cells (male) to the established hamsterfibroblast cell line A23 (Westerveld et al. 1971) by using themethod described in McCarthy et al. (1997). A series of fusions,involving irradiation (3000 rads) of donor cells prior to fusion withequal numbers of recipient cells, generated ∼160 hybrids in total.From these 160 hybrids, 94 were selected at random and screenedby using 20 widely distributed markers (representing 17 chromo-somes). Any hybrids that did not produce an amplification productwith these markers were screened by FISH to determine whetherthey contained horse DNA from other chromosomes or regions ofchromosomes (not represented by the 20 markers); hybrids foundto be negative by the FISH screen were replaced at random fromthe remaining unused hybrids, and the same PCR and FISH screen-ing procedure was repeated until 94 hybrids were assembled.These 94 hybrids were used for preliminary characterization as amapping panel. It is expected that the majority of the hybrids(>95%) characterized here will be represented in the TM99 panelof 94 hybrids that is being grown on a large scale by ResearchGenetics Inc. (Huntsville, Ala. 35801).The amount of horse DNA retained by the hybrids in the panelwas evaluated by examining the retention of the 20 widely dis-tributed horse markers. On average each marker was retained in27.8% of the hybrids (ranging from 10.6% to 71.3%; Table 1),implying that the panel as a whole retains the equivalent of ap-proximately 26 horse genomes. These retention frequencies com-pare well with those found for the human and mouse RH panels,which have been used successfully for creating whole-genomemaps (Gyapay et al. 1996; McCarthy et al. 1997).The mapping ability of the panel was assessed by producingRH maps for the two horse chromosomes with the most markersavailable, and comparing these with the latest genetic maps (Swin-burne et al. 2000a). In total, 39 markers on Chromosome (Chr) 1and 15 markers on Chr 10 were analyzed (Table 1). The averageretention of markers was 15.4% (ranging from 5.3% to 29.8%) onChr 1, and was higher, 25.4% (ranging from 16.0% to 44.7%) onChr 10. An increase in the retention frequency on smaller chro-mosomes was also noted in human and mouse RH panels (Gyapayet al. 1996; McCarthy et al. 1997). For each chromosome, linkagegroups with at least 4-LOD units support were identified; four suchgroups were found on Chr 1 and two were detected on Chr 10(Figs. 1 and 2). Within each of these linkage groups, frameworkmarkers were ordered with 3-LOD units support, and most of thenon-framework markers were ordered with 2-LOD units support.Some non-framework markers had lower statistical support fortheir order (shown in italics, Figs. 1 and 2). Similarly, the relativeorder of some linkage groups was suggested by linkage analysisbut had low statistical support [Chr 1 (groups B and C) and Chr 10(groups A and B)]. The relative order of the other RH linkagegroups was determined by comparison with the genetic map andFISH localizations.The genetic map can give a misleading impression of markerdensity because genetic distances may be small owing to regionshaving a low meiotic recombination rate. In such regions the mark-ers may actually be physically far apart and, therefore, at a lowerdensity than predicted by the genetic map. Since RH panels requirea high density of markers, this might account for the low statisticalsupport for the ordering of some markers and linkage groups. Alow density of available markers could also explain why two mark-ers (HLM5, ICA22) could not be placed on the RH map. HLM5 hasalready been shown to be 24 cM from its nearest neighboringmarker on the genetic map. Three other markers could not beplaced on the RH map (UM004, HTG12, and HMS15), but wereable to be placed on the genetic map in a region equivalent to RHlinkage group D; this discrepancy between the two maps should beresolved as both maps are characterized further.This study has demonstrated the ability of the horse WG-RHpanel to produce an accurate genome map as there is good agree-ment of the genetic and physical maps with the RH maps for Chrs1 and 10 (Figs. 1 and 2). Only a few differences between the mapswere observed, and experiences with human mapping suggest thatsuch differences are common and are resolved as more markers areincorporated into the maps (Walter et al. 1994). The WG-RH panelwas able to order some markers that co-segregate on the geneticlinkage map, i.e., Chr 1 markers LEX39 and ICA18, and ICA41 andICA32, and Chr 10 markers LEX8 and COR015, and NVHEQ18

Synteny and Regional Marker Order Sssignment 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, SRYand 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.

Centric fission in the karyotype of a mother-daughter pair of donkeys (Equus asinus)

A mother-daughter pair of donkeys was found to have identical karyotypes with a diploid number of 63. The difference from the species karyotype could be explained by a centric fission event in the third largest autosomal pair.

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 15,000Rad 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 15,000Rad 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 15,000Rad 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.

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

Abstract Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Ωcm−1) 3–4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

Canine COL4A3 and COL4A4: Sequencing, mapping and genomic organization

Canine α3 and α4 chains of collagen type IV genes (COL4A3 and COL4A4) are expressed in the renal glomerular basement membrane, where they provide a critical structural and functional matrix for other basement membrane components. These genes are candidates for hereditary nephritis (Alport syndrome) in several dog breeds (e.g. English Cocker Spaniel and Bull Terrier). Using RACE and PCR, the cDNA of both genes was cloned and sequenced. Both COL4A3 and COL4A4, as well as canine NPPC (Natriuretic Peptide Precursor C), were mapped to CFA25 using an RH panel. Conservation of the tight linkage of COL4A3 and COL4A4 as seen in human and mouse was verified in the dog. Intron–exon boundaries in both genes were determined by BLAST analysis of the Canis Familiaris Trace Archive. The elucidation of the cDNA sequences, genomic organization and the open reading frames of canine COL4A3 and COL4A4 provide the groundwork for screening these genes for mutations in hereditary nephritis in dogs.

Dog leukocyte antigen class II-associated genetic risk testing for immune disorders of dogs: simplified approaches using Pug dog necrotizing meningoencephalitis as a model.

A significantly increased risk for a number of autoimmune and infectious diseases in purebred and mixed-breed dogs has been associated with certain alleles or allele combinations of the dog leukocyte antigen (DLA) class II complex containing the DRB1, DQA1, and DQB1 genes. The exact level of risk depends on the specific disease, the alleles in question, and whether alleles exist in a homozygous or heterozygous state. The gold standard for identifying high-risk alleles and their zygosity has involved direct sequencing of the exon 2 regions of each of the 3 genes. However, sequencing and identification of specific alleles at each of the 3 loci are relatively expensive and sequencing techniques are not ideal for additional parentage or identity determination. However, it is often possible to get the same information from sequencing only 1 gene given the small number of possible alleles at each locus in purebred dogs, extensive homozygosity, and tendency for disease-causing alleles at each of the 3 loci to be strongly linked to each other into haplotypes. Therefore, genetic testing in purebred dogs with immune diseases can be often simplified by sequencing alleles at 1 rather than 3 loci. Further simplification of genetic tests for canine immune diseases can be achieved by the use of alternative genetic markers in the DLA class II region that are also strongly linked with the disease genotype. These markers consist of either simple tandem repeats or single nucleotide polymorphisms that are also in strong linkage with specific DLA class II genotypes and/or haplotypes. The current study uses necrotizing meningoencephalitis of Pug dogs as a paradigm to assess simple alternative genetic tests for disease risk. It was possible to attain identical necrotizing meningoencephalitis risk assessments to 3-locus DLA class II sequencing by sequencing only the DQB1 gene, using 3 DLA class II–linked simple tandem repeat markers, or with a small single nucleotide polymorphism array designed to identify breed-specific DQB1 alleles.