Loren C. Skow

Genome Sequence, Comparative Analysis, and Population Genetics of the Domestic Horse

A Horse Is a Horse, of Course The history of horse domestication is closely tied to the history of the human society. Wade et al. (p. 865) report on the sequencing and provide a single nucleotide polymorphism map of the horse (Equus caballus) genome. Horses are a member of the order perissodactyla (odd-toed animals with hooves). The analysis reveals an evolutionarily new centromere on equine chromosome 11 that displays properties of an immature but fully functioning centromere and is devoid of centromeric satellite sequence. The findings clarify the nature of genetic diversity within and across horse breeds and suggest that the horse was domesticated from a relatively large number of females, but few males. The horse genome reveals an evolutionary new centromere and conserved chromosomal sequences relative to other mammals. We report a high-quality draft sequence of the genome of the horse (Equus caballus). The genome is relatively repetitive but has little segmental duplication. Chromosomes appear to have undergone few historical rearrangements: 53% of equine chromosomes show conserved synteny to a single human chromosome. Equine chromosome 11 is shown to have an evolutionary new centromere devoid of centromeric satellite DNA, suggesting that centromeric function may arise before satellite repeat accumulation. Linkage disequilibrium, showing the influences of early domestication of large herds of female horses, is intermediate in length between dog and human, and there is long-range haplotype sharing among breeds.

Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences

Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.

A mouse model for β-thalassemia

Abstract A mutation that produces an absolute deficiency of normal β-major globin polypeptides has been recovered from a DBA/2J male mouse. Most mice homozygous for the deficiency survived to adulthood and reproduced but were smaller at birth than their littermates and demonstrated a hypochromic, microcytic anemia with severe anisocytosis, poikilocytosis, and reticulocytosis and the presence of inclusion bodies in a high proportion of circulating erythrocytes. Mice heterozygous for the deficiency demonstrated a mild reticulocytosis but were not clinically anemic. Analysis of globin chain synthesis in vitro by 3 H-leucine incorporation revealed that β-globin synthesis was nearly normal (95%) in heterozygotes and about 75% of normal in deficiency homozygotes. Molecular characterization of the mutation by restriction analysis revealed a deletion of about 3.3 kb of DNA, including regulatory sequences and all coding blocks for β-major globin. Based on genetic and hematological criteria, mice homozygous for the mutant allele, designated Hbb th-1 , represent the first animal model of β-thalassemia (Cooleys anemia), a severe genetic disease of humans.

Construction of a 5000 rad whole-genome radiation hybrid panel in the horse and generation of a comprehensive and comparative map for ECA11

Abstract. A 5000rad 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.

A physical map of the bovine genome

BackgroundCattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project.ResultsA bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly.ConclusionFurther refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.

A 4,103 marker integrated physical and comparative map of the horse genome

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse × hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.

Syntenic conservation of HSP70 genes in cattle and humans

A phage library of bovine genomic DNA was screened for hybridization with a human HSP70 cDNA probe, and 21 positive plaques were identified and isolated. Restriction mapping and blot hybridization analysis of DNA from the recombinant plaques demonstrated that the cloned DNAs were derived from three different regions of the bovine genome. One region contains two tandemly arrayed HSP70 sequences, designated HSP70-1 and HSP70-2, separated by approximately 8 kb of DNA. Single HSP70 sequences, designated HSP70-3 and HSP70-4, were found in two other genomic regions. Locus-specific probes of unique flanking sequences from representative HSP70 clones were hybridized to restriction endonuclease-digested DNA from bovine-hamster and bovine-mouse somatic cell hybrid panels to determine the chromosomal location of the HSP70 sequences. The probe for the tandemly arrayed HSP70-1 and HSP70-2 sequences mapped to bovine chromosome 23, syntenic with glyoxalase 1, 21 steroid hydroxylase, and major histocompatibility class I loci. HSP70-3 sequences mapped to bovine chromosome 10, syntenic with nucleoside phosphorylase and murine osteosarcoma viral oncogene (v-fos), and HSP70-4 mapped to bovine syntenic group U6, syntenic with amylase 1 and phosphoglucomutase 1. On the basis of these data, we propose that bovine HSP70-1,2 are homologous to human HSPA1 and HSPA1L on chromosome 6p21.3, bovine HSP70-3 is the homolog of an unnamed human HSP70 gene on chromosome 14q22-q24, and bovine HSP70-4 is homologous to one of the human HSPA-6,-7 genes on chromosome 1.

Mapping of the mouse fibronectin gene (Fn-1) to chromosome 1: conservation of the Idh-1-Cryg-Fn-1 synteny group in mammals.

Restriction fragment length polymorphisms (RFLPs) were observed in BamHI-digested mouse DNA probed with a cDNA for human fibronectin. Analysis of the inheritance of fibronectin RFLPs in AKXD and SWXJ recombinant inbred strains of mice mapped the locus, Fn-1, to the midregion of mouse chromosome 1 about 4 cM distal from the loci encoding gamma-crystallins (Cryg). Loci homologous to genes in the centromeric third of mouse chromosome 1 are also syntenic in rats, humans, and cattle and may, therefore, mark a large conserved chromosomal segment of the mammalian genome.

Identification of copy number variants in horses

Copy number variants (CNVs) represent a substantial source of genetic variation in mammals. However, the occurrence of CNVs in horses and their subsequent impact on phenotypic variation is unknown. We performed a study to identify CNVs in 16 horses representing 15 distinct breeds (Equus caballus) and an individual gray donkey (Equus asinus) using a whole-exome tiling array and the array comparative genomic hybridization methodology. We identified 2368 CNVs ranging in size from 197 bp to 3.5 Mb. Merging identical CNVs from each animal yielded 775 CNV regions (CNVRs), involving 1707 protein- and RNA-coding genes. The number of CNVs per animal ranged from 55 to 347, with median and mean sizes of CNVs of 5.3 kb and 99.4 kb, respectively. Approximately 6% of the genes investigated were affected by a CNV. Biological process enrichment analysis indicated CNVs primarily affected genes involved in sensory perception, signal transduction, and metabolism. CNVs also were identified in genes regulating blood group antigens, coat color, fecundity, lactation, keratin formation, neuronal homeostasis, and height in other species. Collectively, these data are the first report of copy number variation in horses and suggest that CNVs are common in the horse genome and may modulate biological processes underlying different traits observed among horses and horse breeds.

Gene discovery and comparative analysis of X-degenerate genes from the domestic cat Y chromosome.

Mammalian sex chromosomes are the remnants of an ancient autosomal pair present in the ancestral mammalian karyotype. As a consequence of random decay and chromosome rearrangements over evolutionary time, Y chromosome gene repertoires differ between eutherian lineages. To investigate the gene repertoire and transcriptional analysis of the domestic cat Y chromosome, and their potential roles in spermatogenesis, we obtained full-length cDNA sequences for all known Y genes and their X chromosome gametologues and used those sequences to create a BAC-based physical map of the X-degenerate region. Our results indicate the domestic cat Y chromosome has retained most X-degenerate genes that were present on the ancestral eutherian Y chromosome. Transcriptional analysis revealed that most feline X-degenerate genes have retained housekeeping functions shared by their X chromosome partners and have not been specialized for testis-specific functions. Physical mapping data indicate that the cat SRY gene is present as multiple functional copies and that very little of the felid Y chromosome may be single copy. X-Y gene divergence time estimates obtained using Bayesian methods confirm an early origin of Stratum 1 genes prior to the origin of therian mammals. We observed no statistical difference in the ages of Stratum 2 and Stratum 3 gene pairs, suggesting that eutherian and marsupial Stratum 2 genes may have been independently retained in each lineage.