Freyja Imsland

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.

Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left–right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.

Copy Number Variation in Intron 1 of SOX5 Causes the Pea-comb Phenotype in Chickens

Pea-comb is a dominant mutation in chickens that drastically reduces the size of the comb and wattles. It is an adaptive trait in cold climates as it reduces heat loss and makes the chicken less susceptible to frost lesions. Here we report that Pea-comb is caused by a massive amplification of a duplicated sequence located near evolutionary conserved non-coding sequences in intron 1 of the gene encoding the SOX5 transcription factor. This must be the causative mutation since all other polymorphisms associated with the Pea-comb allele were excluded by genetic analysis. SOX5 controls cell fate and differentiation and is essential for skeletal development, chondrocyte differentiation, and extracellular matrix production. Immunostaining in early embryos demonstrated that Pea-comb is associated with ectopic expression of SOX5 in mesenchymal cells located just beneath the surface ectoderm where the comb and wattles will subsequently develop. The results imply that the duplication expansion interferes with the regulation of SOX5 expression during the differentiation of cells crucial for the development of comb and wattles. The study provides novel insight into the nature of mutations that contribute to phenotypic evolution and is the first description of a spontaneous and fully viable mutation in this developmentally important gene.

The Rose-comb Mutation in Chickens Constitutes a Structural Rearrangement Causing Both Altered Comb Morphology and Defective Sperm Motility

Rose-comb, a classical monogenic trait of chickens, is characterized by a drastically altered comb morphology compared to the single-combed wild-type. Here we show that Rose-comb is caused by a 7.4 Mb inversion on chromosome 7 and that a second Rose-comb allele arose by unequal crossing over between a Rose-comb and wild-type chromosome. The comb phenotype is caused by the relocalization of the MNR2 homeodomain protein gene leading to transient ectopic expression of MNR2 during comb development. We also provide a molecular explanation for the first example of epistatic interaction reported by Bateson and Punnett 104 years ago, namely that walnut-comb is caused by the combined effects of the Rose-comb and Pea-comb alleles. Transient ectopic expression of MNR2 and SOX5 (causing the Pea-comb phenotype) occurs in the same population of mesenchymal cells and with at least partially overlapping expression in individual cells in the comb primordium. Rose-comb has pleiotropic effects, as homozygosity in males has been associated with poor sperm motility. We postulate that this is caused by the disruption of the CCDC108 gene located at one of the inversion breakpoints. CCDC108 is a poorly characterized protein, but it contains a MSP (major sperm protein) domain and is expressed in testis. The study illustrates several characteristic features of the genetic diversity present in domestic animals, including the evolution of alleles by two or more consecutive mutations and the fact that structural changes have contributed to fast phenotypic evolution.

Worldwide frequency distribution of the ‘Gait keeper’ mutation in the DMRT3 gene

For centuries, domestic horses have represented an important means of transport and served as working and companion animals. Although their role in transportation is less important today, many horse breeds are still subject to intense selection based on their pattern of locomotion. A striking example of such a selected trait is the ability of a horse to perform additional gaits other than the common walk, trot and gallop. Those could be four-beat ambling gaits, which are particularly smooth and comfortable for the rider, or pace, used mainly in racing. Gaited horse breeds occur around the globe, suggesting that gaitedness is an old trait, selected for in many breeds. A recent study discovered that a nonsense mutation in DMRT3 has a major impact on gaitedness in horses and is present at a high frequency in gaited breeds and in horses bred for harness racing. Here, we report a study of the worldwide distribution of this mutation. We genotyped 4396 horses representing 141 horse breeds for the DMRT3 stop mutation. More than half (2749) of these horses also were genotyped for a SNP situated 32 kb upstream of the DMRT3 nonsense mutation because these two SNPs are in very strong linkage disequilibrium. We show that the DMRT3 mutation is present in 68 of the 141 genotyped horse breeds at a frequency ranging from 1% to 100%. We also show that the mutation is not limited to a geographical area, but is found worldwide. The breeds with a high frequency of the stop mutation (>50%) are either classified as gaited or bred for harness racing.

Fine-mapping and mutation analysis of TRPM1: a candidate gene for leopard complex (LP) spotting and congenital stationary night blindness in horses.

Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Leopard Complex and CSNB phenotype in horses. Re-sequencing of the gene and associated splice sites within the 105 624 bp genomic region of TRPM1 led to the discovery of 18 SNPs. Most of the SNPs did not have a predictive value for the presence of LP. However, one SNP (ECA1:108,249,293 C>T) found within intron 11 had a strong (P < 0.0005), but not complete, association with LP and CSNB and thus is a good marker but unlikely to be causative. To further localize the association, 70 SNPs spanning over two Mb including the TRPM1 gene were genotyped in 192 horses from three different breeds segregating for LP. A single 173 kb haplotype associated with LP and CSNB (ECA1: 108,197,355- 108,370,150) was identified. Illumina sequencing of 300 kb surrounding this haplotype revealed 57 SNP variants. Based on their localization within expressed sequences or regions of high sequence conservation across mammals, six of these SNPs were considered to be the most likely candidate mutations. While the precise function of TRPM1 remains to be elucidated, this work solidifies its functional role in both pigmentation and night vision. Further, this work has identified several potential regulatory elements of the TRPM1 gene that should be investigated further in this and other species.

The Crest phenotype in chicken is associated with ectopic expression of HOXC8 in cranial skin

The Crest phenotype is characterised by a tuft of elongated feathers atop the head. A similar phenotype is also seen in several wild bird species. Crest shows an autosomal incompletely dominant mode of inheritance and is associated with cerebral hernia. Here we show, using linkage analysis and genome-wide association, that Crest is located on the E22C19W28 linkage group and that it shows complete association to the HOXC-cluster on this chromosome. Expression analysis of tissues from Crested and non-crested chickens, representing 26 different breeds, revealed that HOXC8, but not HOXC12 or HOXC13, showed ectopic expression in cranial skin during embryonic development. We propose that Crest is caused by a cis-acting regulatory mutation underlying the ectopic expression of HOXC8. However, the identification of the causative mutation(s) has to await until a method becomes available for assembling this chromosomal region. Crest is unfortunately located in a genomic region that has so far defied all attempts to establish a contiguous sequence.

Copy number expansion of the STX17 duplication in melanoma tissue from Grey horses

BackgroundGreying with age in horses is an autosomal dominant trait, associated with loss of hair pigmentation, melanoma and vitiligo-like depigmentation. We recently identified a 4.6 kb duplication in STX17 to be associated with the phenotype. The aims of this study were to investigate if the duplication in Grey horses shows copy number variation and to exclude that any other polymorphism is uniquely associated with the Grey mutation.ResultsWe found little evidence for copy number expansion of the duplicated sequence in blood DNA from Grey horses. In contrast, clear evidence for copy number expansions was indicated in five out of eight tested melanoma tissues or melanoma cell lines. A tendency of a higher copy number in aggressive tumours was also found. Massively parallel resequencing of the ~350 kb Grey haplotype did not reveal any additional mutations perfectly associated with the phenotype, confirming the duplication as the true causative mutation. We identified three SNP alleles that were present in a subset of Grey haplotypes within the 350 kb region that shows complete linkage disequilibrium with the causative mutation. Thus, these three nucleotide substitutions must have occurred subsequent to the duplication, consistent with our interpretation that the Grey mutation arose more than 2,000 years before present.ConclusionsThese results suggest that the mutation acts as a melanoma-driving regulatory element. The elucidation of the mechanistic features of the duplication will be of considerable interest for the characterization of these horse melanomas as well as for the field of human melanoma research.

Regulatory mutations in TBX3 disrupt asymmetric hair pigmentation that underlies Dun camouflage color in horses

Dun is a wild-type coat color in horses characterized by pigment dilution with a striking pattern of dark areas termed primitive markings. Here we show that pigment dilution in Dun horses is due to radially asymmetric deposition of pigment in the growing hair caused by localized expression of the T-box 3 (TBX3) transcription factor in hair follicles, which in turn determines the distribution of hair follicle melanocytes. Most domestic horses are non-dun, a more intensely pigmented phenotype caused by regulatory mutations impairing TBX3 expression in the hair follicle, resulting in a more circumferential distribution of melanocytes and pigment granules in individual hairs. We identified two different alleles (non-dun1 and non-dun2) causing non-dun color. non-dun2 is a recently derived allele, whereas the Dun and non-dun1 alleles are found in ancient horse DNA, demonstrating that this polymorphism predates horse domestication. These findings uncover a new developmental role for T-box genes and new aspects of hair follicle biology and pigmentation.

Sonic Hedgehog-Signalling Patterns the Developing Chicken Comb as Revealed by Exploration of the Pea- comb Mutation

The genetic basis and mechanisms behind the morphological variation observed throughout the animal kingdom is still relatively unknown. In the present work we have focused on the establishment of the chicken comb-morphology by exploring the Pea-comb mutant. The wild-type single-comb is reduced in size and distorted in the Pea-comb mutant. Pea-comb is formed by a lateral expansion of the central comb anlage into three ridges and is caused by a mutation in SOX5, which induces ectopic expression of the SOX5 transcription factor in mesenchyme under the developing comb. Analysis of differential gene expression identified decreased Sonic hedgehog (SHH) receptor expression in Pea-comb mesenchyme. By experimentally blocking SHH with cyclopamine, the wild-type single-comb was transformed into a Pea-comb-like phenotype. The results show that the patterning of the chicken comb is under the control of SHH and suggest that ectopic SOX5 expression in the Pea-comb change the response of mesenchyme to SHH signalling with altered comb morphogenesis as a result. A role for the mesenchyme during comb morphogenesis is further supported by the recent finding that another comb-mutant (Rose-comb), is caused by ectopic expression of a transcription factor in comb mesenchyme. The present study does not only give knowledge about how the chicken comb is formed, it also adds to our understanding how mutations or genetic polymorphisms may contribute to inherited variations in the human face.