Horse Genome Research Articles

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.

Trio-binning of a hinny refines the comparative organization of the horse and donkey X chromosomes and reveals novel species-specific features

We generated single haplotype assemblies from a hinny hybrid which significantly improved the gapless contiguity for horse and donkey autosomal genomes and the X chromosomes. We added over 15 Mb of missing sequence to both X chromosomes, 60 Mb to donkey autosomes and corrected numerous errors in donkey and some in horse reference genomes. We resolved functionally important X-linked repeats: the DXZ4 macrosatellite and ampliconic Equine Testis Specific Transcript Y7 (ETSTY7). We pinpointed the location of the pseudoautosomal boundaries (PAB) and determined the size of the horse (1.8 Mb) and donkey (1.88 Mb) pseudoautosomal regions (PARs). We discovered distinct differences in horse and donkey PABs: a testis-expressed gene, XKR3Y, spans horse PAB with exons1–2 located in Y and exon3 in the X–Y PAR, whereas the donkey XKR3Y is Y-specific. DXZ4 had a similar ~ 8 kb monomer in both species with 10 copies in horse and 20 in donkey. We assigned hundreds of copies of ETSTY7, a sequence horizontally transferred from Parascaris and massively amplified in equids, to horse and donkey X chromosomes and three autosomes. The findings and products contribute to molecular studies of equid biology and advance research on X-linked conditions, sex chromosome regulation and evolution in equids.

Genome-Wide Detection of Copy Number Variations Associated with Miniature Features in Horses.

Copy number variations (CNVs) are crucial structural genomic variants affecting complex traits in humans and livestock animals. The current study was designed to conduct a comprehensive comparative copy number variation analysis among three breeds, Debao (DB), Baise (BS), and Warmblood (WB), with a specific focus on identifying genomic regions associated with miniature features in horses. Using whole-genome next-generation resequencing data, we identified 18,974 CNVs across 31 autosomes. Among the breeds, we found 4279 breed-specific CNV regions (CNVRs). Baise, Debao, and Warmblood displayed 2978, 986, and 895 distinct CNVRs, respectively, with 202 CNVRs shared across all three breeds. After removing duplicates, we obtained 1545 CNVRs from 26 horse genomes. Functional annotation reveals enrichment in biological functions, including antigen processing, cell metabolism, olfactory conduction, and nervous system development. Debao horses have 970 genes overlapping with CNVRs, possibly causing their small size and mountainous adaptations. We also found that the genes GHR, SOX9, and SOX11 may be responsible for the miniature features of the Debao horse by analyzing their overlapping CNVRs. Overall, this study offers valuable insights into the widespread presence of CNVs in the horse genome. The findings contribute to mapping horse CNVs and advance research on unique miniature traits observed in the Debao horse.

A method to generate capture baits for targeted sequencing.

Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Here, we describe a DNA bait synthesis approach for hybridization capture that we call Circular Nucleic acid Enrichment Reagent, or CNER (pronounced 'snare'). The CNER method uses rolling-circle amplification followed by restriction digestion to discretize microgram quantities of hybridization probes. We demonstrate the utility of the CNER method by generating probes for a panel of 23771 known sites of single nucleotide polymorphism in the horse genome. Using these probes, we capture and sequence from a panel of ten ancient horse DNA libraries, comparing CNER capture efficiency to a commercially available approach. With about one million read pairs per sample, CNERs captured more targets (90.5% versus 66.5%) at greater mean depth than an alternative commercial approach.

Genetic variation and domestication of horses revealed by 10 chromosome-level genomes and whole-genome resequencing.

Understanding the genetic variations of the horse (Equus caballus) genome will improve breeding conservation and welfare. However, genetic variations in long segments, such as structural variants (SVs), remain understudied. We de novo assembled 10 chromosome-level three-dimensional horse genomes, each representing a distinct breed, and analysed horse SVs using a multi-assembly approach. Our findings suggest that SVs with the accumulation of mammalian-wide interspersed repeats related to long interspersed nuclear elements might be a horse-specific mechanism to modulate genome-wide gene regulatory networks. We found that olfactory receptors were commonly loss and accumulated deleterious mutations, but no purge of deleterious mutations occurred during horse domestication. We examined the potential effects of SVs on the spatial structure of chromatin via topologically associating domains (TADs). Breed-specific TADs were significantly enriched by breed-specific SVs. We identified 4199 unique breakpoint-resolved novel insertions across all chromosomes that account for 2.84 Mb sequences missing from the reference genome. Several novel insertions might have potential functional consequences, as 519 appeared to reside within 449 gene bodies. These genes are primarily involved in pathogen recognition, innate immune responses and drug metabolism. Moreover, 37 diverse horses were resequenced. Combining this with public data, we analysed 97 horses through a comparative population genomics approach to identify the genetic basis underlying breed characteristics using Thoroughbreds as a case study. We provide new scientific evidence for horse domestication, an understanding of the genetic mechanism underlying the phenotypic evolution of horses, and a comprehensive genetic variation resource for further genetic studies of horses.

42 Introgression within the horse's genome

Introgression is the transfer of genetic material between 2 different species via repeated back crossing of the interspecific hybrid and one of the parent species. These are important events to understand in the study of molecular evolutionary relationships between species. Previous studies have found regions of introgression within the horse genome, likely hundreds of thousands to millions of years ago from a non-caballine equid that are present in current horse populations. Our hypothesis is, these regions are retained among horses because they are doing something functionally beneficial and are increasing the horse's fitness. After identifying these regions with maximum likelihood estimation in a sample Thoroughbred population, the next steps were to analyze the different haplotypes found in modern horse genomes to find what polymorphisms were inherited together. Once identified, the genotypes of 230 Thoroughbred horse genomes were phased with BEAGLE. The positions tagged by BEAGLE were then viewed in Tassel 5 and a linkage disequilibrium plot was created. The phased genotypes were then analyzed, and the different haplotypes were extracted from the variant call file (vcf), taking into consideration the linkage disequilibrium plot and allele frequencies. Three main haplotypes occurred in this sample population: the reference, orTwilight, haplotype, the non-caballine haplotype, and another haplotype of unknown origin. This pipeline presents the number of haplotypes in the sampled population and which polymorphisms are inherited together, which gives us a better understanding of the origin on the introgressed haplotype(s). The short-term goal is to identify which non-caballine equid is the most prominent donor of the non-caballine haplotype. Ultimately, we will develop software to automate this analysis and identify putatively impactful introgressed regions using recently published data from the Equine-FAANG project. Currently, we are finding what the introgressed regions code for in the context of the horse genome, either non-coding RNA, a gene, or a non-coding region, to narrow down the search when putting together phylogenetic trees. Overall, the more we understand introgression, the better we will be able to piece together evolutionary relationships, how they are essential for rapid adaptation historically, and how they impact gene function in modern populations.

Evolutionarily Young African Rhinoceros Gammaretroviruses.

High-throughput sequences were generated from DNA and cDNA from four Southern white rhinoceros (Ceratotherium simum simum) located in the Taronga Western Plain Zoo in Australia. Virome analysis identified reads that were similar to Mus caroli endogenous gammaretrovirus (McERV). Previous analysis of perissodactyl genomes did not recover gammaretroviruses. Our analysis, including the screening of the updated white rhinoceros (Ceratotherium simum) and black rhinoceros (Diceros bicornis) draft genomes identified high-copy orthologous gammaretroviral ERVs. Screening of Asian rhinoceros, extinct rhinoceros, domestic horse, and tapir genomes did not identify related gammaretroviral sequences in these species. The newly identified proviral sequences were designated SimumERV and DicerosERV for the white and black rhinoceros retroviruses, respectively. Two long terminal repeat (LTR) variants (LTR-A and LTR-B) were identified in the black rhinoceros, with different copy numbers associated with each (n = 101 and 373, respectively). Only the LTR-A lineage (n = 467) was found in the white rhinoceros. The African and Asian rhinoceros lineages diverged approximately 16 million years ago. Divergence age estimation of the identified proviruses suggests that the exogenous retroviral ancestor of the African rhinoceros ERVs colonized their genomes within the last 8 million years, a result consistent with the absence of these gammaretroviruses from Asian rhinoceros and other perissodactyls. The black rhinoceros germ line was colonized by two lineages of closely related retroviruses and white rhinoceros by one. Phylogenetic analysis indicates a close evolutionary relationship with ERVs of rodents including sympatric African rats, suggesting a possible African origin of the identified rhinoceros gammaretroviruses. IMPORTANCE Rhinoceros genomes were thought to be devoid of gammaretroviruses, as has been determined for other perissodactyls (horses, tapirs, and rhinoceros). While this may be true of most rhinoceros, the African white and black rhinoceros genomes have been colonized by evolutionarily young gammaretroviruses (SimumERV and DicerosERV for the white and black rhinoceros, respectively). These high-copy endogenous retroviruses (ERVs) may have expanded in multiple waves. The closest relative of SimumERV and DicerosERV is found in rodents, including African endemic species. Restriction of the ERVs to African rhinoceros suggests an African origin for the rhinoceros gammaretroviruses.

Identification of Copy Number Variations in Four Horse Breed Populations in South Korea.

In this study, genome-wide CNVs were identified using a total of 469 horses from four horse populations (Jeju horses, Thoroughbreds, Jeju riding horses, and Hanla horses). We detected a total of 843 CNVRs throughout all autosomes: 281, 30, 301, and 310 CNVRs for Jeju horses, Thoroughbreds, Jeju riding horses, and Hanla horses, respectively. Of the total CNVRs, copy number losses were found to be the most abundant (48.99%), while gains and mixed CNVRs accounted for 41.04% and 9.96% of the total CNVRs, respectively. The length of the CNVRs ranged from 0.39 kb to 2.8 Mb, while approximately 7.2% of the reference horse genome assembly was covered by the total CNVRs. By comparing the CNVRs among the populations, we found a significant portion of the CNVRs (30.13%) overlapped; the highest number of shared CNVRs was between Hanla horses and Jeju riding horses. When compared with the horse CNVRs of previous studies, 26.8% of CNVRs were found to be uniquely detected in this study. The CNVRs were not randomly distributed throughout the genome; in particular, the Equus caballus autosome (ECA) 7 comprised the largest proportion of its genome (16.3%), while ECA 24 comprised the smallest (0.7%). Furthermore, functional analysis was applied to CNVRs that overlapped with genes (genic-CNVRs); these overlapping areas may be potentially associated with the olfactory pathway and nervous system. A racing performance QTL was detected in a CNVR of Thoroughbreds, Jeju riding horses, and Hanla horses, and the CNVR value was mixed for three breeds.

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.

Analysis of ancient and modern horse genomes reveals the critical impact of lncRNA-mediated epigenetic regulation on horse domestication.

Background: The domestication of horses has played critical roles in human civilizations. The excavation of ancient horse DNA provides crucial data for studying horse domestication. Studies of horse domestication can shed light on the general mechanisms of animal domestication. Objective: We wish to explore the gene transcription regulation by long noncoding RNAs (lncRNAs) that influence horse domestication. Methods: First, we assembled the ancient DNA sequences of multiple horses at different times and the genomes of horses, donkeys, and Przewalski horses. Second, we extracted sequences of lncRNA genes shared in ancient horses and sequences of lncRNA genes and the promoter regions of domestication-critical genes shared in modern horses, modern donkeys, and Przewalski horses to form two sample groups. Third, we used the LongTarget program to predict potential regulatory interactions between these lncRNAs and these domestication-critical genes and analyzed the differences between the regulation in ancient/modern horses and between horses/donkeys/Przewalski horses. Fourth, we performed functional enrichment analyses of genes that exhibit differences in epigenetic regulation. Results: First, genes associated with neural crest development and domestication syndrome are important targets of lncRNAs. Second, compared with undomesticated Przewalski horses, more lncRNAs participate in the epigenetic regulation in modern horses and donkeys, suggesting that domestication is linked to more epigenetic regulatory changes. Third, lncRNAs’ potential target genes in modern horses are mainly involved in two functional areas: 1) the nervous system, behavior, and cognition, and 2) muscle, body size, cardiac function, and metabolism. Conclusion: Domestication is linked to substantial epigenetic regulatory changes. Genes associated with neural crest development and domestication syndrome underwent noticeable lncRNA-mediated epigenetic regulation changes during horse domestication.

Analysis of the earliest complete mtDNA genome of a Caribbean colonial horse (Equus caballus) from 16th-century Haiti.

Unlike other European domesticates introduced in the Americas after the European invasion, equids (Equidae) were previously in the Western Hemisphere but were extinct by the late Holocene era. The return of equids to the Americas through the introduction of the domestic horse (Equus caballus) is documented in the historical literature but is not explored fully either archaeologically or genetically. Historical documents suggest that the first domestic horses were brought from the Iberian Peninsula to the Caribbean in the late 15th century CE, but archaeological remains of these early introductions are rare. This paper presents the mitochondrial genome of a late 16th century horse from the Spanish colonial site of Puerto Real (northern Haiti). It represents the earliest complete mitogenome of a post-Columbian domestic horse in the Western Hemisphere offering a unique opportunity to clarify the phylogeographic history of this species in the Americas. Our data supports the hypothesis of an Iberian origin for this early translocated individual and clarifies its phylogenetic relationship with modern breeds in the Americas.

Identification of processed pseudogenes in the genome of Thoroughbred horses: Possibility of gene-doping detection considering the presence of pseudogenes.

Processed pseudogenes, also known as retrocopy genes, are copies of messenger RNAs that have been reverse transcribed into DNA and inserted into the genome. In this study, we identified 62 processed pseudogene candidates as intron-less genes from whole-genome sequencing (WGS) data of Thoroughbred horses using delly structural variation software. The 62 processed pseudogene candidates were confirmed by PCR amplification of intron-less products. A total of 11 processed pseudogenes were confirmed in the genome of all 23 analysed horses, whereas three processed pseudogenes with structures of ATP11B, DPH3 and RPL17 were detected in only one of 115 horses by PCR amplification of intron-less products. Currently, most of the gene doping tests proposed in human and horse sports are adapted PCR-based methods using hydrolysis probes to detect exon/exon junctions in transgenes because the operation is simple and economical. However, when the pseudogene is present in the host genome, the PCR-based methods may have a potential risk of detecting false positives. In this study, because processed pseudogenes that exist less frequently in the horse genome may affect PCR-based transgene detection in gene-doping tests, we propose and demonstrate that PCR amplification and sequencing using primers designed on transgene and promotors and/or polyadenylation signal for gene expression are useful for gene-doping detection as an additional confirmatory test to prevent false positives.

Decoding the Equine Genome: Lessons from ENCODE.

The horse reference genome assemblies, EquCab2.0 and EquCab3.0, have enabled great advancements in the equine genomics field, from tools to novel discoveries. However, significant gaps of knowledge regarding genome function remain, hindering the study of complex traits in horses. In an effort to address these gaps and with inspiration from the Encyclopedia of DNA Elements (ENCODE) project, the equine Functional Annotation of Animal Genome (FAANG) initiative was proposed to bridge the gap between genome and gene expression, providing further insights into functional regulation within the horse genome. Three years after launching the initiative, the equine FAANG group has generated data from more than 400 experiments using over 50 tissues, targeting a variety of regulatory features of the equine genome. In this review, we examine how valuable lessons learned from the ENCODE project informed our decisions in the equine FAANG project. We report the current state of the equine FAANG project and discuss how FAANG can serve as a template for future expansion of functional annotation in the equine genome and be used as a reference for studies of complex traits in horse. A well-annotated reference functional atlas will also help advance equine genetics in the pan-genome and precision medicine era.

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.

Genome collinearity analysis illuminates the evolution of donkey chromosome 1 and horse chromosome 5 in perissodactyls: A comparative study

BackgroundIt is important to resolve the evolutionary history of species genomes as it has affected both genome organization and chromosomal architecture. The rapid innovation in sequencing technologies and the improvement in assembly algorithms have enabled the creation of highly contiguous genomes. DNA Zoo, a global organization dedicated to animal conservation, offers more than 150 chromosome-length genome assemblies. This database has great potential in the comparative genomics field.ResultsUsing the donkey (Equus asinus asinus, EAS) genome provided by DNA Zoo as an example, the scaffold N50 length and Benchmarking Universal Single-Copy Ortholog score reached 95.5 Mb and 91.6%, respectively. We identified the cytogenetic nomenclature, corrected the direction of the chromosome-length sequence of the donkey genome, analyzed the genome-wide chromosomal rearrangements between the donkey and horse, and illustrated the evolution of the donkey chromosome 1 and horse chromosome 5 in perissodactyls.ConclusionsThe donkey genome provided by DNA Zoo has relatively good continuity and integrity. Sequence-based comparative genomic analyses are useful for chromosome evolution research. Several previously published chromosome painting results can be used to identify the cytogenetic nomenclature and correct the direction of the chromosome-length sequence of new assemblies. Compared with the horse genome, the donkey chromosomes 1, 4, 20, and X have several obvious inversions, consistent with the results of previous studies. A 4.8 Mb inverted structure was first discovered in the donkey chromosome 25 and plains zebra chromosome 11. We speculate that the inverted structure and the tandem fusion of horse chromosome 31 and 4 are common features of non-caballine equids, which supports the correctness of the existing Equus phylogeny to an extent.

Ancient horse genomes reveal the timing and extent of dispersals across the Bering Land Bridge.

The Bering Land Bridge (BLB) last connected Eurasia and North America during the Late Pleistocene. Although the BLB would have enabled transfers of terrestrial biota in both directions, it also acted as an ecological filter whose permeability varied considerably over time. Here we explore the possible impacts of this ecological corridor on genetic diversity within, and connectivity among, populations of a once wide-ranging group, the caballine horses (Equus spp.). Using a panel of 187mitochondrial and eight nuclear genomes recovered from present-day and extinct caballine horses sampled across the Holarctic, we found that Eurasian horse populations initially diverged from those in North America, their ancestral continent, around 1.0-0.8million years ago. Subsequent to this split our mitochondrial DNA analysis identified two bidirectional long-range dispersals across the BLB ~875-625 and ~200-50 thousand years ago, during the Middle and Late Pleistocene. Whole genome analysis indicated low levels of gene flow between North American and Eurasian horse populations, which probably occurred as a result of these inferred dispersals. Nonetheless, mitochondrial and nuclear diversity of caballine horse populations retained strong phylogeographical structuring. Our results suggest that barriers to gene flow, currently unidentified but possibly related to habitat distribution across Beringia or ongoing evolutionary divergence, played an important role in shaping the early genetic history of caballine horses, including the ancestors of living horses within Equus ferus.

Horse Clinical Cytogenetics: Recurrent Themes and Novel Findings.

Simple SummaryHorse chromosomes have been studied for veterinary diagnostic purposes for over half a century. The findings show that changes in the chromosome number or structure are among the most common non-infectious causes of decreased fertility, infertility, and developmental abnormalities. Based on large-scale surveys, almost 30% of horses with reproductive or developmental problems have abnormal chromosomes. For a comparison, only 2–5% of horses in the general population have abnormal chromosomes. Most chromosome abnormalities are rare and found in one or a few animals. However, two conditions are recurrent: sterile mares with only one X chromosome, instead of two, and sterile mares with XY male sex chromosomes where the Y has lost the ‘maleness’ gene SRY. The two are signature features of chromosome abnormalities in the horse, being rare or absent in other domestic animals. The progress in horse genome sequencing and the development of molecular tools have improved the depth and quality of diagnostic chromosome analysis, allowing for an understanding of the underlying molecular mechanisms. Nevertheless, cutting-edge genomics tools are not about to entirely replace traditional chromosome analysis, which still is the most straightforward, cost-effective, and fastest approach for the initial evaluation of potential breeding animals and horses with reproductive or developmental disorders.Clinical cytogenetic studies in horses have been ongoing for over half a century and clearly demonstrate that chromosomal disorders are among the most common non-infectious causes of decreased fertility, infertility, and congenital defects. Large-scale cytogenetic surveys show that almost 30% of horses with reproductive or developmental problems have chromosome aberrations, whereas abnormal karyotypes are found in only 2–5% of the general population. Among the many chromosome abnormalities reported in the horse, most are unique or rare. However, all surveys agree that there are two recurrent conditions: X-monosomy and SRY-negative XY male-to-female sex reversal, making up approximately 35% and 11% of all chromosome abnormalities, respectively. The two are signature conditions for the horse and rare or absent in other domestic species. The progress in equine genomics and the development of molecular tools, have qualitatively improved clinical cytogenetics today, allowing for refined characterization of aberrations and understanding the underlying molecular mechanisms. While cutting-edge genomics tools promise further improvements in chromosome analysis, they will not entirely replace traditional cytogenetics, which still is the most straightforward, cost-effective, and fastest approach for the initial evaluation of potential breeding animals and horses with reproductive or developmental disorders.

The equine mononuclear phagocyte system: The relevance of the horse as a model for understanding human innate immunity.

The mononuclear phagocyte system (MPS) is a family of cells of related function that includes bone marrow progenitors, blood monocytes and resident tissue macrophages. Macrophages are effector cells in both innate and acquired immunity. They are a major resident cell population in every organ and their numbers increase in response to proinflammatory stimuli. Their function is highly regulated by a wide range of agonists, including lymphokines, cytokines and products of microorganisms. Macrophage biology has been studied most extensively in mice, yet direct comparisons of rodent and human macrophages have revealed many functional differences. In this review, we provide an overview of the equine MPS, describing the variation in the function and phenotype of macrophages depending on their location and the similarities and differences between the rodent, human and equine immune response. We discuss the use of the horse as a large animal model in which to study macrophage biology and pathological processes shared with humans. Finally, following the recent update to the horse genome, facilitating further comparative analysis of regulated gene expression between the species, we highlight the importance of future transcriptomic macrophage studies in the horse, the findings of which may also be applicable to human as well as veterinary research.

Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer

The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals.Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.

Evaluation of a new variant in the aggrecan gene potentially associated with chondrodysplastic dwarfism in Miniature horses

Chondrodysplastic dwarfism in Miniature horses is an autosomal recessive disorder previously associated with four mutations (D1, D2, D3*, and D4) in the aggrecan (ACAN) gene. The aim of this study was to identify additional variants in the candidate ACAN gene associated with chondrodysplastic dwarfism in Miniature horses. Fifteen dwarf Miniature horses were found to possess only one of the dwarfism-causing variants, and two possessed none of the variants. The ACAN exons (EquCab3.0) of seven dwarf Miniature horses were sequenced. A missense SNP in coding exon 11 (g.95271115A > T, c.6465A > T—RefSeq XM_005602799.2), which resulted in the amino acid substitution p.Leu2155Phe (RefSeq XP_005602856.2), was initially associated with the dwarf phenotype. The variant was tested and found present in 14 dwarf foals as well as one parent of each, and both parents of a dwarf possessing two copies. Genetic testing of 347 phenotypically normal Miniature horses demonstrated that none had more than one of the dwarf alleles or c.6465A > T. However, a study of large breeds revealed the presence of c.6465A > T, which was present in homozygosis in two Mangalarga Marchador horses. We suggest that c.6465A > T as a marker of disequilibrium or complex interactions in the Miniature horse genome could contribute to the associated dwarfism.