Douglas F. Antczak

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.

Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse

The rich fossil record of equids has made them a model for evolutionary processes. Here we present a 1.12-times coverage draft genome from a horse bone recovered from permafrost dated to approximately 560–780 thousand years before present (kyr bp). Our data represent the oldest full genome sequence determined so far by almost an order of magnitude. For comparison, we sequenced the genome of a Late Pleistocene horse (43 kyr bp), and modern genomes of five domestic horse breeds (Equus ferus caballus), a Przewalski’s horse (E. f. przewalskii) and a donkey (E. asinus). Our analyses suggest that the Equus lineage giving rise to all contemporary horses, zebras and donkeys originated 4.0–4.5 million years before present (Myr bp), twice the conventionally accepted time to the most recent common ancestor of the genus Equus. We also find that horse population size fluctuated multiple times over the past 2 Myr, particularly during periods of severe climatic changes. We estimate that the Przewalski’s and domestic horse populations diverged 38–72 kyr bp, and find no evidence of recent admixture between the domestic horse breeds and the Przewalski’s horse investigated. This supports the contention that Przewalski’s horses represent the last surviving wild horse population. We find similar levels of genetic variation among Przewalski’s and domestic populations, indicating that the former are genetically viable and worthy of conservation efforts. We also find evidence for continuous selection on the immune system and olfaction throughout horse evolution. Finally, we identify 29 genomic regions among horse breeds that deviate from neutrality and show low levels of genetic variation compared to the Przewalski’s horse. Such regions could correspond to loci selected early during domestication.

DNA of bovine papillomavirus type 1 and 2 in equine sarcoids: PCR detection and direct sequencing

SummaryNucleotide sequences of bovine papillomavirus (BPV) DNA amplified by the polymerase chain reaction (PCR) from samples of equine sarcoid skin tumours were determined. All naturally occurring sarcoids (n=58 tumours from 32 horses and 2 donkeys) contained BPV-DNA. All but 3 of the genome fragments belonged to the BPV type 1 strain (BPV-1); the remaining were BPV type 2. Similar results were obtained with cutaneous bovine papillomas used as controls (n=20). One of the horses, carrying 2 sarcoids, was particularly interesting; one tumour contained BPV-1 DNA whilst the other sarcoid yielded BPV-2 DNA, suggesting that horses are not immune to super-infection. BPV-DNA was even amplified from the sarcoid samples which had yielded negative results in previous investigations when DNA isolated from the lesions was used in Southern blot hybridization with BPV probes. In addition, there was no detectable BPV-DNA in any equine or bovine tissue examined other than sarcoids or cutaneous bovine papillomas. Biopsies of normal skin surrounding lesions yielded exclusively negative results. The described nucleotide differences represent a natural genomic variation of this BPV type between geographically distant locations. The identical variations recovered from cattle and horses in Switzerland, a finding of great epidemiological interest, strongly suggest that a uniform variant of BPV-1 is one of the etiologic agents of equine sarcoid and bovine papilloma in a given region.

Recurrent airway obstruction (RAO) in horses is characterized by IFN-γ and IL-8 production in bronchoalveolar lavage cells

In horses prone to developing recurrent airway obstruction (RAO), we tested the hypotheses that the cytokine profile in the bronchoalveolar lavage (BAL) cells of affected horses would reflect a polarized Th-2 response; that cytokine and chemokine alterations would occur within 24 h of allergen exposure; and that allergen exposure would induce alterations in the expression of the transcription factor t-bet (t-box-expressed in T-cells). The expression levels of interleukin-4 (IL-4), IL-13, Interferon-gamma (IFN-gamma), t-bet, IL-8 and granulocyte-macrophage colony stimulating factor (GM-CSF) were measured in BAL cells obtained from control and RAO-susceptible horses during an asymptomatic phase and at 24 h and 5 weeks post-stabling and hay exposure. At each sampling time, BAL neutrophil percentages in the RAO-group exceeded controls. In the RAO-group, only IL-13 expression was decreased 2-fold during the asymptomatic phase. No differences in cytokine or chemokine expression were detected during the acute exposure phase. During the chronic phase, IFN-gamma and IL-8 expression levels were 2.5- and 3-fold greater, respectively, in the RAO-group. No other differences in gene expression were detected. We conclude that the cytokine profile of the airway cells does not reflect a polarized Th-2 response; that increases in IFN-gamma result from a t-bet independent pathway and that chemokines from epithelial or interstitial cells may contribute to early neutrophil influx.

Report of the Second Equine Leucocyte Antigen Workshop, Squaw Valley, California, July 1995

The final assignment of antibody clusters for leucocyte antigens and immunoglobulins, as described in detail in Sections 3 and 4, is summarized in Table 4. Together with other mAbs developed outside of ELAW II (Table 9) this pool of reagents represent a powerful array of tools for the study of equine immunity. The Second Equine Leucocyte Antigen Workshop made considerable advances in pursuing the objectives of establishing the specificities of mAbs and achieving consensus on the nomenclature for equine leucocyte and immunoglobulin molecules. Of equal importance, several productive collaborations were fostered among the participating laboratories and observers. Overall, enormous advances have been made in the past decade since mAbs specific for equine leucocyte antigens and immunoglobulins were first reported. There remains enormous scope and need for further studies of equine leucocyte antigens and immunoglobulins, both for the purposes of comparative immunology and for the good of the horse. In the future novel techniques will be required to develop reagents for specific target antigens such as the orthologues of the CD25 or CD45 isoforms. In studies of equine immunoglobulins the functional role of the IgG isotypes must be better established, reagents for IgE must be developed, and cloning of the immunoglobulin heavy chain genes will be essential if the complexities of the IgG sub-isotypes are to be elucidated. The tasks still facing the currently small group of equine immunologists throughout the world remain formidable, and will only be tackled successfully in a spirit of collaboration.

The Complete Map of the Ig Heavy Chain Constant Gene Region Reveals Evidence for Seven IgG Isotypes and for IgD in the Horse

This report contains the first map of the complete Ig H chain constant (IGHC) gene region of the horse (Equus caballus), represented by 34 overlapping clones from a new bacterial artificial chromosome library. The different bacterial artificial chromosome inserts containing IGHC genes were identified and arranged by hybridization using overgo probes specific for individual equine IGHC genes. The analysis of these IGHC clones identified two previously undetected IGHC genes of the horse. The newly found IGHG7 gene, which has a high homology to the equine IGHG4 gene, is located between the IGHG3 and IGHG4 genes. The high degree of conservation shared between the nucleotide sequences of the IGHG7 and IGHG4 genes is unusual for the IGHG genes of the horse and suggests that these two genes duplicated most recently during evolution of the equine IGHG genes. Second, we present the genomic nucleotide sequence of the equine IGHD gene, which is located downstream of the IGHM gene. Both the IGHG7 and IGHD genes were found to be expressed at the mRNA level. The order of the 11 IGHC genes in the IGH-locus of the horse was determined to be 5′-M-D-G1-G2-G3-G7-G4-G6-G5-E-A-3′, confirming previous studies using λ phage clones, with the exception that the IGHG5 gene was found to be the most downstream-located IGHG gene. Fluorescence in situ hybridization was used to localize the IGHC region to Equus caballus (ECA) 24qter, the horse chromosome corresponding to human chromosome 14, where the human IGH locus is found.

Whole-genome SNP association in the horse: identification of a deletion in myosin Va responsible for Lavender Foal Syndrome.

Lavender Foal Syndrome (LFS) is a lethal inherited disease of horses with a suspected autosomal recessive mode of inheritance. LFS has been primarily diagnosed in a subgroup of the Arabian breed, the Egyptian Arabian horse. The condition is characterized by multiple neurological abnormalities and a dilute coat color. Candidate genes based on comparative phenotypes in mice and humans include the ras-associated protein RAB27a (RAB27A) and myosin Va (MYO5A). Here we report mapping of the locus responsible for LFS using a small set of 36 horses segregating for LFS. These horses were genotyped using a newly available single nucleotide polymorphism (SNP) chip containing 56,402 discriminatory elements. The whole genome scan identified an associated region containing these two functional candidate genes. Exon sequencing of the MYO5A gene from an affected foal revealed a single base deletion in exon 30 that changes the reading frame and introduces a premature stop codon. A PCR–based Restriction Fragment Length Polymorphism (PCR–RFLP) assay was designed and used to investigate the frequency of the mutant gene. All affected horses tested were homozygous for this mutation. Heterozygous carriers were detected in high frequency in families segregating for this trait, and the frequency of carriers in unrelated Egyptian Arabians was 10.3%. The mapping and discovery of the LFS mutation represents the first successful use of whole-genome SNP scanning in the horse for any trait. The RFLP assay can be used to assist breeders in avoiding carrier-to-carrier matings and thus in preventing the birth of affected foals.

An ordered BAC contig map of the equine major histocompatibility complex

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.

Paternally expressed genes predominate in the placenta.

The discovery of genomic imprinting through studies of manipulated mouse embryos indicated that the paternal genome has a major influence on placental development. However, previous research has not demonstrated paternal bias in imprinted genes. We applied RNA sequencing to trophoblast tissue from reciprocal hybrids of horse and donkey, where genotypic differences allowed parent-of-origin identification of most expressed genes. Using this approach, we identified a core group of 15 ancient imprinted genes, of which 10 were paternally expressed. An additional 78 candidate imprinted genes identified by RNA sequencing also showed paternal bias. Pyrosequencing was used to confirm the imprinting status of six of the genes, including the insulin receptor (INSR), which may play a role in growth regulation with its reciprocally imprinted ligand, histone acetyltransferase-1 (HAT1), a gene involved in chromatin modification, and lymphocyte antigen 6 complex, locus G6C, a newly identified imprinted gene in the major histocompatibility complex. The 78 candidate imprinted genes displayed parent-of-origin expression bias in placenta but not fetus, and most showed less than 100% silencing of the imprinted allele. Some displayed variability in imprinting status among individuals. This variability results in a unique epigenetic signature for each placenta that contributes to variation in the intrauterine environment and thus presents the opportunity for natural selection to operate on parent-of-origin differential regulation. Taken together, these features highlight the plasticity of imprinting in mammals and the central importance of the placenta as a target tissue for genomic imprinting.

Speciation with gene flow in equids despite extensive chromosomal plasticity

Significance Thirty years after the first DNA fragment from the extinct quagga zebra was sequenced, we set another milestone in equine genomics by sequencing its entire genome, along with the genomes of the surviving equine species. This extensive dataset allows us to decipher the genetic makeup underlying lineage-specific adaptations and reveal the complex history of equine speciation. We find that Equus first diverged in the New World, spread across the Old World 2.1–3.4 Mya, and finally experienced major demographic expansions and collapses coinciding with past climate changes. Strikingly, we find multiple instances of hybridization throughout the equine tree, despite extremely divergent chromosomal structures. This contrasts with theories promoting chromosomal incompatibilities as drivers for the origin of equine species. Horses, asses, and zebras belong to a single genus, Equus, which emerged 4.0–4.5 Mya. Although the equine fossil record represents a textbook example of evolution, the succession of events that gave rise to the diversity of species existing today remains unclear. Here we present six genomes from each living species of asses and zebras. This completes the set of genomes available for all extant species in the genus, which was hitherto represented only by the horse and the domestic donkey. In addition, we used a museum specimen to characterize the genome of the quagga zebra, which was driven to extinction in the early 1900s. We scan the genomes for lineage-specific adaptations and identify 48 genes that have evolved under positive selection and are involved in olfaction, immune response, development, locomotion, and behavior. Our extensive genome dataset reveals a highly dynamic demographic history with synchronous expansions and collapses on different continents during the last 400 ky after major climatic events. We show that the earliest speciation occurred with gene flow in Northern America, and that the ancestor of present-day asses and zebras dispersed into the Old World 2.1–3.4 Mya. Strikingly, we also find evidence for gene flow involving three contemporary equine species despite chromosomal numbers varying from 16 pairs to 31 pairs. These findings challenge the claim that the accumulation of chromosomal rearrangements drive complete reproductive isolation, and promote equids as a fundamental model for understanding the interplay between chromosomal structure, gene flow, and, ultimately, speciation.