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Y chromosome polymorphisms such as microsatellites or single nucleotide polymorphisms represent a paternal counterpart to mitochondrial DNA (mtDNA) for evolutionary and phylogeographic studies. The use of Y chromosome haplotyping in natural populations of species other than humans is still hindered by the lack of sequence information necessary for polymorphism screening. Here we used representational difference analysis (RDA) followed by a screen of a bacterial artificial chromosome (BAC) library for repetitive sequences to obtain polymorphic Y-chromosomal markers. The procedure was performed for the domestic horse (Equus caballus) and we report the first six Y-chromosomal microsatellite markers for this species. Three markers were also useful for haplotyping taxa of the zebra/ass lineage. Y-chromosomal microsatellite markers show a single haplotype in the domestic horse, whereas notable variation has been observed in the other members of the genus Equus.

A first-generation radiation hybrid (RH) map of the equine (Equus caballus) genome was assembled using 92 horse x hamster hybrid cell lines and 730 equine markers. The map is the first comprehensive framework map of the horse that (1) incorporates type I as well as type II markers, (2) integrates synteny, cytogenetic, and meiotic maps into a consensus map, and (3) provides the most detailed genome-wide information to date on the organization and comparative status of the equine genome. The 730 loci (258 type I and 472 type II) included in the final map are clustered in 101 RH groups distributed over all equine autosomes and the X chromosome. The overall marker retention frequency in the panel is approximately 21%, and the possibility of adding any new marker to the map is approximately 90%. On average, the mapped markers are distributed every 19 cR (4 Mb) of the equine genome--a significant improvement in resolution over previous maps. With 69 new FISH assignments, a total of 253 cytogenetically mapped loci physically anchor the RH map to various chromosomal segments. Synteny assignments of 39 gene loci complemented the RH mapping of 27 genes. The results added 12 new loci to the horse gene map. Lastly, comparison of the assembly of 447 equine genes (256 linearly ordered RH-mapped and additional 191 FISH-mapped) with the location of draft sequences of their human and mouse orthologs provides the most extensive horse-human and horse-mouse comparative map to date. We expect that the foundation established through this map will significantly facilitate rapid targeted expansion of the horse gene map and consequently, mapping and positional cloning of genes governing traits significant to the equine industry.

Consensus DNA sequences from human, mouse and/or rat were used to design oligonucleotide primers for equine homologues of exons 16, 17 and 20-23 of potassium chloride co-transporter (SLC12A4) and exons 10, 11 and 3, 4, respectively, for two amino acid transporters (SLC7A10 and SLC7A9). DNA sequences of the PCR products showed high sequence identity to these regions. Equine BAC clones were obtained for SLC12A4 and SLC7A10 and mapped to equine chromosomes ECA3p13 and ECA10p15, respectively, by fluorescence in situ hybridization (FISH). Several single nucleotide polymorphisms (SNP) were found. Substitutions of A/G were found within exon 17 of SLC12A4, within intron 11 of SLC7A10 and within intron 3 of SLC7A9. The SNP associated with SLC7A10 and SLC7A9 were sufficiently polymorphic to investigate associations with erythrocyte fragility among a group of 20 thoroughbred horses. A non-parametric rank-sum test showed a weak association between erythrocyte fragility and the SNP associated with SLC7A10 (P < 0.05).

The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 137 kb of horse genomic DNA from equine chromosome 20q22 containing the closely linked equine TPX1 and AEG2 genes are described. The equine TPX1 gene consists of ten exons spanning 18 kb while the AEG2 gene consists of eight exons that are spread over 24 kb. The expression of these two genes was investigated in several tissues by reverse transcription polymerase chain reaction analysis and Western blotting. Comparative genome analysis between horse, human, and mouse indicates that all three CRISP genes are clustered on one chromosomal location, which shows conserved synteny between these species.

The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clones. For the first time, genes were mapped to ECA13p, ECA29, and probably ECA30. A total of 284 genes are now FISH mapped on the horse chromosomes. Comparison with the human map defines 113 conserved segments that include new homologous segments not identified by Zoo-FISH on ECA7 and ECA13p.

Acidic epididymal glycoprotein 1 (AEG1), also called cysteine-rich secretory protein 1 (CRISP1), is a member of the CRISP protein family which is characterized by 16 conserved cysteine residues at the C-terminus. The CRISP proteins are expressed in the male genital tract and are thought to be involved in sperm-egg fusion. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 90 kb of horse genomic DNA from equine chromosome 20q22 containing the complete equine AEG1 gene are described. The equine AEG1 gene consists of eight exons spanning 31 kb. Analysis of equine AEG1 transcripts did not reveal any evidence for alternative splicing, however three different transcription start sites are used. The first transcription start site is located 20 nt downstream of a TATA box motif. Reverse transcription polymerase chain reaction analysis demonstrated that AEG1 is expressed in different parts of the epididymis, whereas it is hardly detectable in the testis. The naturally occurring diversity of the equine AEG1 gene in different horse breeds was investigated and several polymorphisms are reported, including one that affects the amino acid sequence. Finally, sequence comparisons revealed that the intronless equine PGK2 gene for the testis-specific phosphoglycerate kinase is located approximately 39 kb downstream of AEG1.

A 5000(rad) whole-genome radiation hybrid (RH) panel was created for the horse. The usefulness of the panel for generating physically ordered maps of individual equine chromosomes was tested by typing 24 markers on horse Chromosome 11 (ECA11). The overall retention of markers on this chromosome was 43.6%. Almost complete retention of two of the typed markers--- CA062 and AHT44---clearly indicated the location of thymidine kinase gene on the short arm of ECA11. Seven of the typed markers were FISH mapped to align the RH and cytogenetic maps. With the RH-MAPPER approach, a physically ordered map comprising four linkage groups and incorporating all the markers was obtained. The study provides the first comprehensive map for a horse chromosome that integrates all available mapping data and adds new information that spans the entire length of the equine chromosome. The map clearly underlines the resolving power and utility of the panel and emphasizes the need to have uniformly distributed cytogenetic markers for appropriate alignment of RH map with the chromosome. A comparative status of the ECA11 map in relation to the corresponding human/mouse chromosome is presented.