Horse Lineage Research Articles

Optimization of Autosomal STR Markers for Equine Genotyping Using Multiplex PCR

The investigation of horse lineage was of paramount importance in the registration of different breeds, trade, and formulation of studbooks. The pioneering technique of DNA fingerprinting emerged as the first highly responsive method reliant on DNA for individual identification and the examination of genetic affiliations. Microsatellites were a valuable tool for analyzing the genetic variations present among different horse breeds. The International Society for Animal Genetics (ISAG) has endorsed a set of 17 specific Short Tandem Repeats (STRs) for the equine identification, although these can be quite expensive to obtain through commercially available multiplex kits. Objective: To determine five autosomal STR markers (HMS6, HMS7, ASB23, VHL20, and LEX14) were optimized using multiplex PCR for equine genotyping. Methods: DNA was extracted from a Thoroughbred horse blood sample via an organic extraction method. Sensitivity analysis determined the optimal PCR concentration. Genotyping was performed on the ABI PRISM® 3100XL, and data were processed with Gene Mapper ID 3.2v software. Results: The optimal conditions for multiplex PCR of HMS6, HMS7, ASB23, VHL20, and LEX14 primers were 60°C annealing temperature, 3ng DNA concentration and 6μM primer concentration. A 12.5μL PCR reaction volume was recommended for cost efficiency. Conclusions: The results of this research have the potential to create a cost-effective, regionally produced multiplex PCR kit. This kit would be designed for analyzing parentage lineage within the Equine family in Pakistan, incorporating ISAG-recommended markers: VHL20, HMS6, HMS7, ASB23, and additionally LEX14. It could significantly streamline the import and export of horses in Pakistan.

Widespread horse-based mobility arose around 2200 bce in Eurasia

Horses revolutionized human history with fast mobility1. However, the timeline between their domestication and their widespread integration as a means of transport remains contentious2–4. Here we assemble a collection of 475 ancient horse genomes to assess the period when these animals were first reshaped by human agency in Eurasia. We find that reproductive control of the modern domestic lineage emerged around 2200 bce, through close-kin mating and shortened generation times. Reproductive control emerged following a severe domestication bottleneck starting no earlier than approximately 2700 bce, and coincided with a sudden expansion across Eurasia that ultimately resulted in the replacement of nearly every local horse lineage. This expansion marked the rise of widespread horse-based mobility in human history, which refutes the commonly held narrative of large horse herds accompanying the massive migration of steppe peoples across Europe around 3000 bce and earlier3,5. Finally, we detect significantly shortened generation times at Botai around 3500 bce, a settlement from central Asia associated with corrals and a subsistence economy centred on horses6,7. This supports local horse husbandry before the rise of modern domestic bloodlines.

Horse domestication as a multi-centered, multi-stage process: Botai and the role of specialized Eneolithic horse pastoralism in the development of human-equine relationships

For over a decade there has been general, but not universal, consensus that the earliest known evidence for horse husbandry was at Eneolithic Botai, Kazakhstan, circa 3,500 BCE. Recent ancient genomic analyses, however, indicate that Botai is not the source of modern domestic horse stock (DOM2 lineage), but is instead related to the Przewalski clade of horses. DOM2 appears to instead to have emerged in early Bronze Age (mid 3rd Mill. BCE) in the Pontic-Caspian steppe, and spread quickly replacing other horse lineages after approximately 2,000 BCE. Whilst the specific evidence for earlier husbandry at Botai is not diminished by this evidence, it has broken the consensus regarding the early stages of horse domestication, with some now viewing it as a later event. This paper argues that domestication is rarely an event, but instead a process that is ongoing. The case is made for a “prey pathway” initial phase of domestication in multiple localities during the Eneolithic, which was based around local subsistence pastoralist niche construction. This took different forms due to the presence or absence of ruminant domestic stock in the Eastern European or Central Asian steppes, respectively. Whilst “push” factors likely played a part in the development of horse specialist pastoralism at Botai, it is suggested that “pull” factors accelerated the spread of DOM2 lineages, replacing others, in the later Bronze Age. The DOM2 spread was principally driven, not by local subsistence needs, but wider social, economic and military desirability of equestrianism. The long-term process of horse domestication continues in modernity with major breed changes caused first by the post-medieval agricultural revolution and, more currently, the desire for sporting achievement.

A Satellite-Free Centromere in Equus przewalskii Chromosome 10.

In mammals, centromeres are epigenetically specified by the histone H3 variant CENP-A and are typically associated with satellite DNA. We previously described the first example of a natural satellite-free centromere on Equus caballus chromosome 11 (ECA11) and, subsequently, on several chromosomes in other species of the genus Equus. We discovered that these satellite-free neocentromeres arose recently during evolution through centromere repositioning and/or chromosomal fusion, after inactivation of the ancestral centromere, where, in many cases, blocks of satellite sequences were maintained. Here, we investigated by FISH the chromosomal distribution of satellite DNA families in Equus przewalskii (EPR), demonstrating a good degree of conservation of the localization of the major horse satellite families 37cen and 2PI with the domestic horse. Moreover, we demonstrated, by ChIP-seq, that 37cen is the satellite bound by CENP-A and that the centromere of EPR10, the ortholog of ECA11, is devoid of satellite sequences. Our results confirm that these two species are closely related and that the event of centromere repositioning which gave rise to EPR10/ECA11 centromeres occurred in the common ancestor, before the separation of the two horse lineages.

Whole-genome identification of transposable elements reveals the equine repetitive element insertion polymorphism in Chinese horses.

Transposable elements (TEs) are diverse, abundant, and complicated in genomes. They not only can drive the genome evolution process but can also act as special resources for adaptation. However, little is known about the evolutionary processes that shaped horses. In this work, 126 horse assemblages involved in most horse breeds in China were used to investigate the patterns of TE variation for the first time. By using RepeatMasker and melt software, we found that the horse-specific short interspersed repetitive elements family, equine repetitive elements (ERE1), exhibited polymorphisms in horse genomes. Phylogenetic analysis based on these ERE1 loci (minor allele frequency ≥0.05) revealed three major horse groups, namely, those in northern China, southern China, and Qinghai-Tibetan, which mirrors the result determined by SNPs to some extent. The present ERE1 family emerged ~0.26 to 1.77 Mya ago, with an activity peak at ~0.49 Mya, which matches the early stage of the horse lineage and decreases after the divergence of Equus caballus and Equus ferus przewalskii. To detect the functional ERE1(s) associated with adaptation, locus-specific branch length, genome-wide association study, and absolute allele frequency difference analyses were conducted and resulted in two common protein-coding genes annotated by candidate ERE1s. They were clustered into the vascular smooth muscle contraction (p=0.01, EDNRA) and apelin signalling pathways (p = 0.02, NRF1). Notably, ERE1 insertion into the EDNRA gene showed a higher association with adaptation among southern China horses and other horses in 15 populations and 451 individuals (p = 4.55 e-8). Our results provide a comprehensive understanding of TE variations to analyse the phylogenetic relationships and traits relevant to adaptive evolution in horses.

Genetic Diversity and Structure of the Main Danubian Horse Paternal Genealogical Lineages Based on Microsatellite Genotyping

Simple SummaryThe Danubian horse was created on the former Klementina stud farm near Pleven to satisfy the demands of the Bulgarian Army for light draft horses and to improve the working capacity of the local horse population. The privatization of the Klementina stud farm in the late 1990s and the lack of economic activity have led to a sharp reduction in the number of mares and stallions and their sale to private owners. At present, only six of the main paternal lines which participated in the creation of the Danubian horse breed are preserved: Zdravko, NONIUS XVII-30, Torpedo, Lider, Kalifa, and Hrabar. This is the first study on the genetic and population diversity of the Danubian horse paternal lines based on microsatellite markers (STRs). The results showed that the studied Danubian horse population was characterized by a high level of genetic diversity with a medium value of 0.84. The fixation index (FST) was 0.08 for all studied markers, which is indicative of the low genetic differentiation of the Danubian horse population. Our analysis also confirmed the low level of inbreeding and heterozygous deficiency among the animals selected from the six paternal lineages of the Danubian horse. The present research could be helpful for the development of breeding and conservation programs for the Danubian horse, as well as for making informed decisions on the management of paternal lines.The Danubian horse, together with the Pleven and the Eastern Bulgarian horse breeds, is one of the modern breeds in Bulgaria. The objective of this study was to compare the genetic structure and genetic diversity of six paternal genealogical lineages of the Danubian horse breed (Zdravko, NONIUS XVII-30, Torpedo, Lider, Kalifa, and Hrabar). In total, 166 individuals from the six genealogical lines were investigated, based on 15 STR markers (short tandem repeats, also known as microsatellites). In total, 184 alleles were found in the six populations, using 15 microsatellite loci. The mean number of alleles, the effective number of alleles, and the polymorphic information content (PIC) values per locus were 12.28, 9.48, and 0.73, respectively. In a comparison of the allelic diversity among sire lineages, the highest genetic diversity (Na) was observed in Lider and Kalifa (14.60 ± 0.21), while the lowest value of this parameter was observed in the Zdravko lineage 4.20 ± 0.35. The largest genetic diversity was found in loci HMS3 and HMS7, with 13 alleles, and the smallest polymorphism was noted for the locus ASB17, with 10 alleles. The level of observed heterozygosity was in the range of 0.65 ± 0.069 for the Zdravko lineage to 0.93 ± 0.01 for the Torpedo lineage. The expected heterozygosity level range was from 0.57 ± 0.048 to 0.91 ± 0.01 for all horse lineages. Structure analysis revealed three main gene pools in the study population. The first pool included the Zdravko lineage; the second had the NONIUS XVII-30, Torpedo, Lider, and Kalifa lineages; and the third defined the Hrabar lineage, which was significantly differentiated from the other genealogical lineages.

Organization for Breeding the Horse Lineage during the Second Constitutional Era: The Case of Islah-ı Nesl-i Feres Cemiyeti

Bu makale, II. Meşrutiyet sonrasında 11 Nisan 1912’de iyi cins at yetiştirmek, at yarışları düzenlemek, biniciliği geliştirmek amacıyla padişahın himayesinde kurulan Islah-ı Nesl-i Feres Cemiyeti’nin faaliyetlerine odaklanmıştır. Osmanlı Devleti’nde atın çekim gücü ve taşıma kapasitesi, tarımsal ekonomi ve savunma açısından hayati bir öneme sahipti. Ancak XIX. yüzyılın ilk yarısından itibaren ihtiyaçlara göre at neslinin ıslahı ve çoğaltılması hususunda bazı sıkıntılar mevcuttu. Zira tımar sisteminin bozulması ve Yeniçeri Ocağı’nın kaldırılması at yetiştiriciliğine zarar vermişti. Askeri ve tarımsal ihtiyaçları karşılamak için modern haralar kurulsa da bu çaba yeterli olmadı. Arz sıkıntısı ve satın alma düzenlemeleri, Osmanlı ordusunun lojistiğini tehdit eder boyuta ulaştı. Bu nedenle Osmanlı Hükümeti, Avrupalı devletlerde olduğu gibi nitelikli atlarda yerli bir pazarın yaratılmasına yönelik politikalar üretmeye başladı. Hükümet, at cinsinin ıslahını sağlayan vasıtalardan birisi olan at yarışlarını düzenlemek için 1912’de Islah-ı Nesl-i Feres Cemiyeti’ni kurdu.

The origins and spread of domestic horses from the Western Eurasian steppes

Domestication of horses fundamentally transformed long-range mobility and warfare1. However, modern domesticated breeds do not descend from the earliest domestic horse lineage associated with archaeological evidence of bridling, milking and corralling2–4 at Botai, Central Asia around 3500 bc3. Other longstanding candidate regions for horse domestication, such as Iberia5 and Anatolia6, have also recently been challenged. Thus, the genetic, geographic and temporal origins of modern domestic horses have remained unknown. Here we pinpoint the Western Eurasian steppes, especially the lower Volga-Don region, as the homeland of modern domestic horses. Furthermore, we map the population changes accompanying domestication from 273 ancient horse genomes. This reveals that modern domestic horses ultimately replaced almost all other local populations as they expanded rapidly across Eurasia from about 2000 bc, synchronously with equestrian material culture, including Sintashta spoke-wheeled chariots. We find that equestrianism involved strong selection for critical locomotor and behavioural adaptations at the GSDMC and ZFPM1 genes. Our results reject the commonly held association7 between horseback riding and the massive expansion of Yamnaya steppe pastoralists into Europe around 3000 bc8,9 driving the spread of Indo-European languages10. This contrasts with the scenario in Asia where Indo-Iranian languages, chariots and horses spread together, following the early second millennium bc Sintashta culture11,12.

Horses were not domesticated in Anatolia

Archaeology The domestication of the horse was one of the most consequential innovations in human history and led to profound changes in the economic, political, and social structures of prehistoric Eurasia. However, when, where, and how many times horses were domesticated remains an open question. Anatolia has been proposed as an early center of domestication because of the long history of exploitation of equids there. Using advanced paleogenomic methods, Guimaraes et al. tested this hypothesis on archaeological samples of equids from 14 sites from central Anatolia across a temporal span from about 8000 to 1000 BCE. They determined that the ancient wild horses of the region were never domesticated and identified the introduction of nonlocal and already domesticated horse lineages around 2000 BCE. Sci. Adv. 10.1126/sciadv.abb0030 (2020).

EPAS1 Gain-of-Function Mutation Contributes to High-Altitude Adaptation in Tibetan Horses.

High altitude represents some of the most extreme environments worldwide. The genetic changes underlying adaptation to such environments have been recently identified in multiple animals but remain unknown in horses. Here, we sequence the complete genome of 138 domestic horses encompassing a whole altitudinal range across China to uncover the genetic basis for adaptation to high-altitude hypoxia. Our genome data set includes 65 lowland animals across ten Chinese native breeds, 61 horses living at least 3,300 m above sea level across seven locations along Qinghai-Tibetan Plateau, as well as 7 Thoroughbred and 5 Przewalski’s horses added for comparison. We find that Tibetan horses do not descend from Przewalski’s horses but were most likely introduced from a distinct horse lineage, following the emergence of pastoral nomadism in Northwestern China ∼3,700 years ago. We identify that the endothelial PAS domain protein 1 gene (EPAS1, also HIF2A) shows the strongest signature for positive selection in the Tibetan horse genome. Two missense mutations at this locus appear strongly associated with blood physiological parameters facilitating blood circulation as well as oxygen transportation and consumption in hypoxic conditions. Functional validation through protein mutagenesis shows that these mutations increase EPAS1 stability and its hetero dimerization affinity to ARNT (HIF1B). Our study demonstrates that missense mutations in the EPAS1 gene provided key evolutionary molecular adaptation to Tibetan horses living in high-altitude hypoxic environments. It reveals possible targets for genomic selection programs aimed at increasing hypoxia tolerance in livestock and provides a textbook example of evolutionary convergence across independent mammal lineages.

Horse Paleogenomes and Human–Animal Interactions in Prehistory

A new analysis of paleogenomic data from 278 ancient horses (Fages et al. Cellhttp://doi.org/10.1016/j.cell.2019.03.049) finds that this animal - crucially important to many ancient and contemporary human societies for subsistence, transportation, conflict, and more - was domesticated in at least two different regions, but with the geographic and cultural origins of the modern domestic horse lineage remaining unknown. By tracing ancient horse population movements and inferring the spatiotemporal trajectories of phenotypic adaptations, this study provides fresh perspectives on past human group interactions and activities.

Ancient genomes shed light on dark horses.

A study of ancient horse genomes, described in Cell, reveals the existence of two now-extinct horse lineages and shows that modern breeding practices reduced genetic diversity in horses.

Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series

SummaryHorse domestication revolutionized warfare and accelerated travel, trade, and the geographic expansion of languages. Here, we present the largest DNA time series for a non-human organism to date, including genome-scale data from 149 ancient animals and 129 ancient genomes (≥1-fold coverage), 87 of which are new. This extensive dataset allows us to assess the modern legacy of past equestrian civilizations. We find that two extinct horse lineages existed during early domestication, one at the far western (Iberia) and the other at the far eastern range (Siberia) of Eurasia. None of these contributed significantly to modern diversity. We show that the influence of Persian-related horse lineages increased following the Islamic conquests in Europe and Asia. Multiple alleles associated with elite-racing, including at the MSTN “speed gene,” only rose in popularity within the last millennium. Finally, the development of modern breeding impacted genetic diversity more dramatically than the previous millennia of human management.

Mechanics of evolutionary digit reduction in fossil horses (Equidae).

Digit reduction is a major trend that characterizes horse evolution, but its causes and consequences have rarely been quantitatively tested. Using beam analysis on fossilized centre metapodials, we tested how locomotor bone stresses changed with digit reduction and increasing body size across the horse lineage. Internal bone geometry was captured from 13 fossil horse genera that covered the breadth of the equid phylogeny and the spectrum of digit reduction and body sizes, from Hyracotherium to Equus To account for the load-bearing role of side digits, a novel, continuous measure of digit reduction was also established-toe reduction index (TRI). Our results show that without accounting for side digits, three-toed horses as late as Parahippus would have experienced physiologically untenable bone stresses. Conversely, when side digits are modelled as load-bearing, species at the base of the horse radiation through Equus probably maintained a similar safety factor to fracture stress. We conclude that the centre metapodial compensated for evolutionary digit reduction and body mass increases by becoming more resistant to bending through substantial positive allometry in internal geometry. These results lend support to two historical hypotheses: that increasing body mass selected for a single, robust metapodial rather than several smaller ones; and that, as horse limbs became elongated, the cost of inertia from the side toes outweighed their utility for stabilization or load-bearing.

Y Chromosome Uncovers the Recent Oriental Origin of Modern Stallions

The Y chromosome directly reflects male genealogies, but the extremely low Y chromosome sequence diversity in horses has prevented the reconstruction of stallion genealogies [1, 2]. Here, we resolve the first Y chromosome genealogy of modern horses by screening 1.46 Mb of the male-specific region of the Y chromosome (MSY) in 52 horses from 21 breeds. Based on highly accurate pedigree data, we estimated the de novo mutation rate of the horse MSY and showed that various modern horse Y chromosome lineages split much later than the domestication of the species. Apart from few private northern European haplotypes, all modern horse breeds clustered together in a roughly 700-year-old haplogroup that was transmitted to Europe by the import of Oriental stallions. The Oriental horse group consisted of two major subclades: the Original Arabian lineage and the Turkoman horse lineage. We show that the English Thoroughbred MSY was derived from the Turkoman lineage and that English Thoroughbred sires are largely responsible for the predominance of this haplotype in modern horses.

Diversidad de haplotipos del complejo principal de histocompatibilidad en equinos de la raza Árabe de la República Argentina

La diversidad de los genes del complejo principal de histocompatibilidad (MHC) es clave en su función en la presentación de antígenos en el sistema inmune. La tipificación indirecta basada en microsatélites (STR) del MHC aporta información de la variabilidad genética y de la estructura poblacional y es una estrategia para conocer procesos selectivos y evolutivos. Con el fin de caracterizar una población de caballos de raza Árabe, se identificaron los haplotipos del MHC sobre la base de tres microsatélites (UM011, DRB2-STR2 y COR112) abarcando una región de ~1Mpb. El ADN genómico se extrajo de sangre y pelo de 30 caballos de cinco haras de la provincia de Buenos Aires y los STRs se amplificaron con cebadores fluorescentes para tipificar en secuenciador automático. Seestimaron parámetros poblacionales y de diversidad y la detección de haplotipos se realizó mediante análisis de segregación y con el programa de reconstrucción de haplotipos PHASE. Se reconocieron 24 haplotipos; entre ellos, 12 se verificaron por segregación en los registros de pedigree del StudBook. Los resultados obtenidos demostraron la existencia de ligamiento con alelos conocidos de genes de clase II del equine leukocyte antigen (ELA) y la cantidad de haplotipos identificada permitió expandir la valoración de la diversidad del MHC equino. Esta metodología constituye una herramienta de utilidad y un método alternativo para la tipificación del MHC en linajes de caballos y en estudios poblacionales relacionados con la inmunidad.

Complete mitochondrial genome sequences of Korean native horse from Jeju Island: uncovering the spatio-temporal dynamics.

The Korean native horse (Jeju horse) is one of the most important animals in Korean historical, cultural, and economical viewpoints. In the early 1980s, the Jeju horse was close to extinction. The aim of this study is to explore the phylogenomics of Korean native horse focusing on spatio-temporal dynamics. We determined complete mitochondrial genome sequences for the first Korean native (n = 6) and additional Mongolian (n = 2) horses. Those sequences were analyzed together with 143 published ones using Bayesian coalescent approach as well as three different phylogenetic analysis methods, Bayesian inference, maximum likelihood, and neighbor-joining methods. The phylogenomic trees revealed that the Korean native horses had multiple origins and clustered together with some horses from four European and one Middle Eastern breeds. Our phylogenomic analyses also supported that there was no apparent association between breed or geographic location and the evolution of global horses. Time of the most recent common ancestor of the Korean native horse was approximately 13,200-63,200 years, which was much younger than 0.696 My of modern horses. Additionally, our results showed that all global horse lineages including Korean native horse existed prior to their domestication events occurred in about 6000-10,000 years ago. This is the first study on phylogenomics of the Korean native horse focusing on spatio-temporal dynamics. Our findings increase our understanding of the domestication history of the Korean native horses, and could provide useful information for horse conservation projects as well as for horse genomics, emergence, and the geographical distribution.

Genome-wide evolutionary and functional analysis of the Equine Repetitive Element 1: an insertion in the myostatin promoter affects gene expression.

BackgroundIn mammals, an important source of genomic variation is insertion polymorphism of retrotransposons. These may acquire a functional role when inserted inside genes or in their proximity. The aim of this work was to carry out a genome wide analysis of ERE1 retrotransposons in the horse and to analyze insertion polymorphism in relation to evolution and function. The effect of an ERE1 insertion in the promoter of the myostatin gene, which is involved in muscle development, was also investigated.ResultsIn the horse population, the fraction of ERE1 polymorphic loci is related to the degree of similarity to their consensus sequence. Through the analysis of ERE1 conservation in seven equid species, we established that the level of identity to their consensus is indicative of evolutionary age of insertion. The position of ERE1s relative to genes suggests that some elements have acquired a functional role. Reporter gene assays showed that the ERE1 insertion within the horse myostatin promoter affects gene expression. The frequency of this variant promoter correlates with sport aptitude and racing performance.ConclusionsSequence conservation and insertion polymorphism of ERE1 elements are related to the time of their appearance in the horse lineage, therefore, ERE1s are a useful tool for evolutionary and population studies. Our results suggest that the ERE1 insertion at the myostatin locus has been unwittingly selected by breeders to obtain horses with specific racing abilities. Although a complex combination of environmental and genetic factors contributes to athletic performance, breeding schemes may take into account ERE1 insertion polymorphism at the myostatin promoter.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0281-1) contains supplementary material, which is available to authorized users.

Hereditary equine regional dermal asthenia (HERDA) in Quarter Horses: A review of clinical signs, genetics and research

SummaryHereditary equine regional dermal asthenia (HERDA) results from a genetic mutation which affects the skin and other tissues of Quarter Horses and horses with Quarter Horse lineage. The disease HERDA has an autosomal recessive mode of inheritance and has become a significant concern in the Quarter Horse industry due to the high frequency of heterozygote carriers. Affected homozygous horses appear normal at birth; however, within the first 2 years of life they usually acquire loose, hyperextensible skin and wounds which result in disfiguring scars either spontaneously or from minor trauma. Some severely affected horses also develop haematomas and seromas. Consequently, most affected horses are subjected to euthanasia at an early age. No treatment options other than palliative therapy currently exist. As part of a five panel test (http://www.aqha.com/News/News-Articles/2013/April/04292013-Genetic-Testing.aspx) the American Quarter Horse Association presently requires DNA testing for HERDA on all breeding stallions. There are currently no restrictions on registration of horses heterozygous or homozygous for the HERDA mutation. Due to the autosomal recessive nature of the disease, Quarter Horse mares and horses of all breeds from HERDA‐associated bloodlines should also be tested.

Characterization of an Equine α-S2-Casein Variant Due to a 1.3 kb Deletion Spanning Two Coding Exons.

The production and consumption of mare’s milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows’ milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage.