The Y chromosome is a valuable genetic marker for studying the origin and influence of paternal lineages in populations. In this study, we conducted Y-chromosomal lineage-tracing in Arabian horses. First, we resolved a Y haplotype phylogeny based on the next generation sequencing data of 157 males from several breeds. Y-chromosomal haplotypes specific for Arabian horses were inferred by genotyping a collection of 145 males representing most Arabian sire lines that are active around the globe. These lines formed three discrete haplogroups, and the same haplogroups were detected in Arabian populations native to the Middle East. The Arabian haplotypes were clearly distinct from the ones detected in Akhal Tekes, Turkoman horses, and the progeny of two Thoroughbred foundation sires. However, a haplotype introduced into the English Thoroughbred by the stallion Byerley Turk (1680), was shared among Arabians, Turkomans, and Akhal Tekes, which opens a discussion about the historic connections between Oriental horse types. Furthermore, we genetically traced Arabian sire line breeding in the Western World over the past 200 years. This confirmed a strong selection for relatively few male lineages and uncovered incongruences to written pedigree records. Overall, we demonstrate how fine-scaled Y-analysis contributes to a better understanding of the historical development of horse breeds.

The Y chromosome carries information about the demography of paternal lineages, and thus, can prove invaluable for retracing both the evolutionary trajectory of wild animals and the breeding history of domesticates. In horses, the Y chromosome shows a limited, but highly informative, sequence diversity, supporting the increasing breeding influence of Oriental lineages during the last 1500 years. Here, we augment the primary horse Y-phylogeny, which is currently mainly based on modern horse breeds of economic interest, with haplotypes (HT) segregating in remote horse populations around the world. We analyze target enriched sequencing data of 5 Mb of the Y chromosome from 76 domestic males, together with 89 whole genome sequenced domestic males and five Przewalski’s horses from previous studies. The resulting phylogeny comprises 153 HTs defined by 2966 variants and offers unprecedented resolution into the history of horse paternal lineages. It reveals the presence of a remarkable number of previously unknown haplogroups in Mongolian horses and insular populations. Phylogenetic placement of HTs retrieved from 163 archaeological specimens further indicates that most of the present-day Y-chromosomal variation evolved after the domestication process that started around 4200 years ago in the Western Eurasian steppes. Our comprehensive phylogeny significantly reduces ascertainment bias and constitutes a robust evolutionary framework for analyzing horse population dynamics and diversity.

Structural rearrangements like copy number variations in the male-specific Y chromosome have been associated with male fertility phenotypes in human and mouse but have been sparsely studied in other mammalian species. Here, we designed digital droplet PCR assays for 7 horse male-specific Y chromosome multicopy genes and SRY and evaluated their absolute copy numbers in 209 normal male horses of 22 breeds, 73 XY horses with disorders of sex development and/or infertility, 5 Przewalski’s horses and 2 kulans. This established baseline copy number for these genes in horses. The TSPY gene showed the highest copy number and was the most copy number variable between individuals and breeds. SRY was a single-copy gene in most horses but had 2–3 copies in some indigenous breeds. Since SRY is flanked by 2 copies of RBMY, their copy number variations were interrelated and may lead to SRY-negative XY disorders of sex development. The Przewalski’s horse and kulan had 1 copy of SRY and RBMY. TSPY and ETSTY2 showed significant copy number variations between cryptorchid and normal males (P < 0.05). No significant copy number variations were observed in subfertile/infertile males. Notably, copy number of TSPY and ETSTY5 differed between successive male generations and between cloned horses, indicating germline and somatic mechanisms for copy number variations. We observed no correlation between male-specific Y chromosome gene copy number variations and male-specific Y chromosome haplotypes. We conclude that the ampliconic male-specific Y chromosome reference assembly has deficiencies and further studies with an improved male-specific Y chromosome assembly are needed to determine selective constraints over horse male-specific Y chromosome gene copy number and their relation to stallion reproduction and male biology.

In horses, demographic patterns are complex due to historical migrations and eventful breeding histories. Particularly puzzling is the ancestry of the North African horse, a founding horse breed, shaped by numerous influences throughout history. A genetic marker particularly suitable to investigate the paternal demographic history of populations is the non-recombining male-specific region of the Y chromosome (MSY). Using a recently established horse MSY haplotype (HT) topology and KASP™ genotyping, we illustrate MSY HT spectra of 119 Barb and Arab-Barb males, collected from the Maghreb region and European subpopulations. All detected HTs belonged to the Crown haplogroup, and the broad MSY spectrum reflects the wide variety of influential stallions throughout the breed’s history. Distinct HTs and regional disparities were characterized and a remarkable number of early introduced lineages were observed. The data indicate recent refinement with Thoroughbred and Arabian patrilines, while 57% of the dataset supports historical migrations between North Africa and the Iberian Peninsula. In the Barb horse, we detected the HT linked to Godolphin Arabian, one of the Thoroughbred founders. Hence, we shed new light on the question of the ancestry of one Thoroughbred patriline. We show the strength of the horse Y chromosome as a genealogical tool, enlighten recent paternal history of North African horses, and set the foundation for future studies on the breed and the formation of conservation breeding programs.

Since their domestication, horses have accompanied mankind, and humans have constantly shaped horses according to their needs through stallion-centered breeding. Consequently, the male-specific portion of the Y chromosome (MSY) is extremely uniform in modern horse breeds. The majority of stallions worldwide carry MSY haplotypes (HT) attributed to an only ~1,500-y-old, so-called, “Crown” haplogroup. The predominance of the Crown in modern horse breeds is thought to represent a footprint of the vast impact of stallions of “Oriental origin” in the past millennium. Here, we report the results of a fine-scaled MSY haplotyping of large datasets of patrilines comprising 1,517 males of 189 modern horse breeds, covering a broad phenotypic and geographic spectrum. We can disentangle the multilayered influence of Oriental stallions over the last few hundred years, exposing the intense linebreeding and the wide-ranging impact of Arabian, English Thoroughbred, and Coldblood sires. Iberian and New World horse breeds contain a wide range of diversified Crown lineages. Their broad HT spectrum illustrates the spread of horses of Oriental origin via the Iberian Peninsula after the Middle Ages, which is commonly referred to as the “Spanish influence.” Our survey also revealed a second major historical dissemination of horses from Western Asia, attributed to the expansion of the Ottoman Empire. Our analysis shows that MSY analysis can uncover the complex history of horse breeds and can be used to establish the paternal ancestry of modern horse breeds.