Unlocking Horse Y Chromosome Diversity

G C A T T A C G G C A T genes Review Unlocking Horse Y Chromosome Diversity Irene Cardinali 1, * , Andrea Giontella 2, * , Anna Tommasi 3 , Maurizio Silvestrelli 2 and Hovirag Lancioni 1 1 2 3 * Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy Department of Veterinary Medicine, University of Perugia, 06126 Perugia, Italy Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, 27100 Pavia, Italy Correspondence: [email protected] (I.C.); [email protected] (A.G.) Abstract: The present equine genetic variation mirrors the deep influence of intensive breeding pro‑ grams during the last 200 years. Here, we provide a comprehensive current state of knowledge on the trends and prospects on the variation in the equine male‑specific region of the Y chromosome (MSY), which was assembled for the first time in 2018. In comparison with the other 12 mammalian species, horses are now the most represented, with 56 documented MSY genes. However, in contrast to the high variability in mitochondrial DNA observed in many horse breeds from different geographic areas, modern horse populations demonstrate extremely low genetic Y‑chromosome diversity. The selective pressures employed by breeders using pedigree data (which are not always error‑free) as a predictive tool represent the main cause of this lack of variation in the Y‑chromosome. Neverthe‑ less, the detailed phylogenies obtained by recent fine‑scaled Y‑chromosomal genotyping in many horse breeds worldwide have contributed to addressing the genealogical, forensic, and population questions leading to the reappraisal of the Y‑chromosome as a powerful genetic marker to avoid the loss of biodiversity as a result of selective breeding practices, and to better understand the historical development of horse breeds. Keywords: horse Y‑chromosome variation; horse genetic diversity; uniparental genetic markers; worldwide horse breeds; horse MSY phylogeny Citation: Cardinali, I.; Giontella, A.; Tommasi, A.; Silvestrelli, M.; Lancioni, H. Unlocking Horse Y Chromosome Diversity. Genes 2022, 13, 2272. https://doi.org/10.3390/ genes13122272 Academic Editor: Chunjiang Zhao Received: 22 October 2022 Accepted: 30 November 2022 Published: 2 December 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil‑ iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction The horse (Equus caballus) is one of the domestic species to have played an important role in the development of human society. Over the centuries, their domestication necessarily resulted in a strong pressure in the selection of individuals and breeds [1–4]. The adaptation of horses to the human niche led to the current genetic variation [5,6] carried by the domestic horses that left the Western Eurasian steppes at the beginning of the second millennium BC and moved towards Eurasia, thus almost entirely replacing the wild populations [7]. Methodological and bioinformatics tools have recently been developed, allowing for increased accuracy in the analysis of high‑throughput genomes, and over last decades, the equine research community has aimed to reconstruct the evolutionary paths that can still be detected in their genomes [8]. Genetic evidence has pinpointed multiple horse domestication events occurring across Eurasia 5000–6000 years ago [2,9–16]. After this evidence emerged, the history of the horse domestication process was revised. Further‑ more, the analysis of the horse remains from the Botai site, deeply described by Outram and colleagues [13], identified this area as the cradle of extant Przewalski’s horses’ an‑ cestors [17], while modern horses have been domesticated in a more Western centre: the lower Volga‑Don region [7]. The first appearance of the ancestor of all modern horses is dated back to 4200 years ago [18]. Since then, both the disappearance of the earliest domes‑ tic lineages and the emergence of the modern ones occurred, with an increasing genetic variability that remained constant during the last 4000 years until it significantly dropped in the last ≈250 years [19]. These modern horse lineages rapidly spread across Eurasia, colonizing a region from central Anatolia to central Russia, completely replacing almost Genes 2022, 13, 2272. https://doi.org/10.3390/genes13122272 https://www.mdpi.com/journal/genes Genes 2022, 13, 2272 2 of 16 all other local populations about 4000 years ago [18]. The genetic profile of these colo‑ nizing horses was found in the archaeological remains buried in Sintashta kurgans in the West‑Eurasian steppe [20,21]. The improvement of livestock breeds usually involves inbreeding to select individu‑ als with favorable traits [22]. Recently, many studies have been focused on the loss of bio‑ diversity [23–25] or the increase in deleterious genotypes [26] caused by such inbreeding (as reviewed in [7]). In the last 200 years, the inbreeding practice led to the phenotypical expression at homozygous sites of deleterious variants [27], as highlighted when the co‑ efficients are calculated in order to evaluate the genetic diversity among different horse breeds [28–78]. Inbreeding has caused reduced fertility and survival among offspring of related individuals, resulting in a decline in fitness [79] and the emergence of disadvan‑ tageous traits [27,80]. The increased knowledge about inbreeding depression and the ge‑ netic structure of breeds [81,82] have allowed breeders to select horses by avoiding mating closely related individuals [83]. The risk of losing genetic diversity, resulting in more uniform populations with highly specialized traits, is especially evident in those breeds that are under strong human se‑ lection [26]; thus, it is recommended that breeders use the less intensive practice of line breeding and ensure a certain extent of variation among horse breeds [84]. As a result of intensive breeding programs, domestic horse populations changed along with the hu‑ man development, above all in the last 200 years [19], as highlighted by most research focused on autosomal loci or maternally inherited mitochondrial DNA (mtDNA) (as re‑ viewed in [85]). 2. The Horse Genome In 1995, the international consortium of the Horse Genome Project was established to enhance knowledge about the evolutionary history and inherited traits of domestic horses, supported by many funding organizations. The project’s team of researchers decoded the diploid genome of a domestic horse named Twilight, an English Thoroughbred female racehorse, and found that the genome is distributed over 31 pairs of autosomes, the X chromosomes and the mitochondrial genome. The RefSeq genome records for E. caballus were annotated by an automated pipeline (NCBI Eukaryotic Genome Annotation Pipeline) on the only two high‑quality genome as‑ semblies for equids: EquCab2.0 (accession number: GCF_000002305.2) [86] and EquCab3.0 (accession number: GCF_002863925.1) [87]. The first reference sequence (EquCab2.0) was obtained by performing a Sanger se‑ quencing with a 6.8‑fold genomic coverage and including about 315,000 BAC clones from a library collected from Twilight’s half‑brother, Bravo [88], and partial sequences from seven horses belonging to different breeds. Over a million of SNPs were identified and used to perform molecular, evolutionary and clinical studies on horses [89]. As EquCab2.0 contained many gaps, the genome of Twilight was re‑sequenced and assembled in 2018 using high‑throughput sequencing technologies, thus resulting in the new reference genome: EquCab3.0. The new assembly contains 3771 gaps comprising 9 Mb (0.34% of the genome) with a scaffold N50 of 86 Mb (Table 1) [87,90]. Horse Y Chromosome Sequencing and Comparison with Humans and Other Mammals However, the resulting horse reference genome was still incomplete as it was based on the genome of a female horse; that is, the analysis of the horse Y chromosome was lack‑ ing. During last ten years, many efforts have been made to produce Y chromosomal DNA data for E. caballus [16,91,92]. Until 2018, Janečka and colleagues published the assembly of 9.5 Mb based on the sequencing of the Y chromosome from the thoroughbred stallion Bravo, thus providing the first comprehensive assembly of the male‑specific region of the Y chromosome (MSY) (accession number: MH341179) [93]. Genes 2022, 13, 2272 3 of 16 Table 1. Genome information for reference and representative genomes of horse (EquCab3.0; GCF_002863925.1). Type Name RefSeq INSDC Size (Mb) GC% Protein rRNA tRNA Other RNA Gene Pseudogene Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr ‑ Un 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 X MT ‑ NC_009144.3 NC_009145.3 NC_009146.3 NC_009147.3 NC_009148.3 NC_009149.3 NC_009150.3 NC_009151.3 NC_009152.3 NC_009153.3 NC_009154.3 NC_009155.3 NC_009156.3 NC_009157.3 NC_009158.3 NC_009159.3 NC_009160.3 NC_009161.3 NC_009162.3 NC_009163.3 NC_009164.3 NC_009165.3 NC_009166.3 NC_009167.3 NC_009168.3 NC_009169.3 NC_009170.3 NC_009171.3 NC_009172.3 NC_009173.3 NC_009174.3 NC_009175.3 NC_001640.1 ‑ CM009148.1 CM009149.1 CM009150.1 CM009151.1 CM009152.1 CM009153.1 CM009154.1 CM009155.1 CM009156.1 CM009157.1 CM009158.1 CM009159.1 CM009160.1 CM009161.1 CM009162.1 CM009163.1 CM009164.1 CM009165.1 CM009166.1 CM009167.1 CM009168.1 CM009169.1 CM009170.1 CM009171.1 CM009172.1 CM009173.1 CM009174.1 CM009175.1 CM009176.1 CM009177.1 CM009178.1 CM009179.1 ‑ ‑ 188.26 121.35 121.35 109.46 96.76 87.23 100.79 97.56 85.79 85.16 61.68 36.99 43.78 94.6 92.85 88.96 80.72 82.64 62.68 65.34 58.98 50.93 55.56 48.29 40.28 43.15 40.25 47.35 34.78 31.4 26 128.21 0.02 97.81 41.7 42.2 41.0 39.8 40.9 41.5 42.7 42.7 39.9 41.7 45.6 45.0 47.0 40.6 41.3 42.0 38.6 39.2 39.6 41.4 40.3 45.2 40.4 43.2 45.2 39.8 39.5 42.7 40.4 40.3 41.2 39.3 42.0 44.5 4764 329 2541 2013 2951 2868 3422 2246 1378 3009 3163 1767 2034 2016 1917 2309 1019 1489 1272 1866 101 1501 850 1257 1656 698 763 1174 676 527 446 2087 13 908 4 ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑ 2 8 30 ‑ 9 17 69 2 5 6 10 4 35 9 26 8 6 5 5 4 2 196 2 1 1 2 2 ‑ 1 3 1 1 1 3 22 8 1219 790 666 566 779 603 698 734 491 660 687 350 426 538 598 548 321 488 328 489 264 391 476 509 336 197 196 378 213 195 130 514 ‑ 618 2591 1559 1333 1133 1566 1431 1985 1168 753 1637 1568 1212 965 1041 1011 996 569 685 627 1271 584 779 544 740 775 403 356 587 342 305 242 1206 37 1169 193 92 110 100 97 113 222 90 53 167 62 231 35 73 58 52 62 53 62 106 61 26 52 48 60 34 23 26 50 30 25 134 ‑ 247 Several special features set the Y chromosome apart from the rest of genome: its male‑ limited transmission, the absence of recombination, abundance of Y‑specific repetitive se‑ quences, degeneration of Y‑linked genes during evolution, acquisition of autosomal genes, and accumulation and functional cluster of “testis genes” for maleness and reproduction. The recent advent of new molecular tools in genomics shed light on the biological and med‑ ical relevance of the Y chromosome and helped answer specific biological queries about the roles of the Y chromosome in testis determination, spermatogenesis and beyond the reproductive tract, with a large implications on health and disease [94]. The human Y chromosome was sequenced in 2003 [95], followed by the Y chromo‑ somes of chimpanzee, mouse and rhesus macaque [96–98]. To date, additional Y chromo‑ some genes have been mapped and/or functionally analysed in many other species, from insects [99] to carnivores [100] and cattle [101,102], and to a limited extent for other domes‑ ticated species [103,104], often targeting specific questions. Comprehensive comparative genomic analyses of the Y chromosomes of multiple mammalian species have demonstrated that, despite their shared ancestry in terms of evo‑ lutionary history, mammalian Y chromosomes display enormous variation in size, gene content, and structural complexity among species. Several unique features of the Y chro‑ mosome, such as the opportunities for crossing over restricted to the pseudoautosomal re‑ gions, the functional specialization for spermatogenesis and the high degree of sequence amplification of repetitive DNA, have contributed to this wide variation [94]. Nevertheless, the species studied until now showed a progressive genetic decay in the MSY’s diversity resulting in deletions and gene losses that collectively decimated the Y chromosome [105]. In horses, even though the stallion fertility has prime importance in breeding management, very little is known about the complexity of the equine Y chromosome’s structure and its genetic degeneration. Genomic analyses recently showed several autosomal loci and mutations significantly associated with stallion fertility [106], and abnormalities in a number of X‑ and/or Y chro‑ Genes 2022, 13, 2272 4 of 16 mosomes have been shown to be causes for aberrant sexual development [107]. As pre‑ viously reviewed [83], among all the above mutations associated with infertility, only the deletion in the sex‑determining region (SRY) leads to infertility in horses, which was found in the 25% of horses with chromosomal aberrations, but it was not detected in other species [108]. All other alterations and rearrangements known to be associated with horse infertility are ascribed to loci not localized in the Y chromosome. Currently, the growing deep sequencing and SNP genotyping array is likely to increase the number and complex‑ ity of chromosomal syndromes associated with infertility detected in horses, especially those related to Y‑chromosomal abnormalities, where the complex genetic architecture is still understudied [82,109]. The exclusion of the Y chromosome from genomic analyses may previously have been justifiable, based on the assumption that it was a genetic wasteland, a junkyard, a barren chromosome [110]. Nowadays, the increase in published studies on the topic makes clear the potential of analyses focused on the variation in the Y chromosome for identifying individuals with greater susceptibility to disease and for forensic analysis and paternity testing. As argued by Chowdhary and other researchers, “Finally, the Y chromosome got some stardom and was not just referred to as the sex‑determining chromosome” [111,112]. Nevertheless, the horse Y chromosome remains the most understudied chromosome in the equine genome. It is comparable in size to the smallest equine autosomes, around 40–50 million base pairs (Mbp). A very limited effort has been made to date to develop a gene map for this chromosome and understand its structure and organization [113]. In 2018, Janečka and colleagues generated the first comprehensive assembly and func‑ tional annotation of the euchromatic male‑specific region of the Y chromosome (eMSY). They demonstrated its dynamic nature and provided a reference sequence useful in im‑ proving our knowledge about the male equine development and fertility [93]. In a radial plot denoting the presence or absence of a gene on the Y chromosome, Janečka and collabo‑ rators recorded 88 unique genes and transcripts and compared them across 13 mammalian species (12 from Eutheria and one from Metatheria). The horse was the most represented with 56 documented genes (56% of MSY), among which there are five pseudoautosomal genes and no pseudogenes, followed by human and pig, with published evidence for 38% of the MSY in these species (Figure 1 and Table S1). Figure 1. Rate of MSY gene data for 13 mammalian species available in 2018 when the horse Y chro‑ mosome assembly was released. Details and comparison were described in [93]. Each bar represents the amount of MSY coding genes, pseudogenes and transcripts known (or not) in horses and other eutherian species with sequenced or partially sequenced MSYs. See also Table S1. Genes 2022, 13, 2272 5 of 16 The conservation of equine MSY was at higher level [93] than that previously ob‑ served in primates [96,97]; indeed, even though the divergence time between horse and donkey [114] is comparable to that between human and chimpanzee [96], they show sig‑ nificantly different evolutionary rates in the MSY. Horse and donkey retain the same single‑ copy MSY and multi‑copy sequences [93,113], while more than 30% of human and chim‑ panzee MSY genes are not homologous and differ in gene content [96]. 3. The Debate about Stallion Pedigrees In contrast to the high mtDNA variability reported in previous studies [2,8,9,115,116], which was already present immediately after their domestication [17,19,117], the Y chromo‑ some shows a very low level of genetic polymorphism in modern horse populations [91,118]. Despite a large diversity of domestic male founders contributing to their early domestica‑ tion [117], the Y chromosome variability considerably decreased in the last 200 years because of selective pressures and the reduction in the stallion population size operated by breed‑ ers [19]. It is known that Thoroughbreds in particular were widely used in the development of many breeds to obtain a less bulky and lighter horse more suitable for riding [119]. Due to the intensive selection of stallions, the establishment of a studbook‑mediated sire line represents the main cause of this lack of Y‑chromosome variation. The importation of stallions used to improve autochthonous breeds started from the Arabian region, where the modern legacy is represented only by few foundation sires, thus confirming the strong sex bias in horse breeding [26]. In any case, there are significant differences among the quality of breeding information depending on the breed. It is common knowledge that pedigree has been used in breeding programs as a pre‑ dictive tool for the unique and reliable identification of individual organisms in breeding management and conservation since to the late 18th century [120]. Pedigree data were al‑ ready employed to analyse the population’s structure and identify factors that affected the genetic variability of horses [121]. The pedigrees of Arabian Horses, English Thoroughbreds and some European breeds [61,70,75,77,84,111,122] have been deeply described in the dedicated studbooks, showing a notable influence of Arabian, Spanish and Thoroughbred stallions. However, the information included in studbooks is not always error free, and genetic analyses are essential for an accurate characterization of patrilines in a breed [77]. Indeed, for horses, as for many other species, it is crucial to know the animal’s founders; thus, today, the accu‑ racy of pedigrees recorded in modern horse studbook/registries is based on the parentage verification [123]. Through the molecular analysis of DNA markers, it is possible to depict the genetic vari‑ ation of a breed and allow for the identification of errors in the pedigree registration [124,125]. These analyses could ensure the accuracy of studbook data and prevent the loss of genetic diversity, thus representing a crucial factor for the preservation and management of indige‑ nous breeds. In any case, the results of molecular analysis depend on the sample received from individuals which could tamper with the material to have a specific result [123]. As many horse breeds were improved with the Arabian Horse, this breed was subject to accu‑ rate molecular analyses. The examination of nearly 400 Arabians and related breeds revealed extensive outcrossing to the English Thoroughbred, even though the pedigrees reported an entirely Arabian ancestry [126]. This is only one example of the frauds that move around the horse pedigrees with economic and race‑related motivations. Nevertheless, the genomes of these horses unveiled a particular genetic signature probably representing the common ancestor of both Arabian and English Thoroughbred horses. However, no sampled mod‑ ern population has provided answers for the places and times of origin of the ancestral population [126]. The Y‑chromosome sequence data date back the origin of modern Ara‑ bian horses above all to crosses of mares native to the British Isles with three oriental stallions (Godolphin Arabian, Darley Arabian and Byerly Turk), although Thoroughbreds seems to share ancestry with Akhal‑teke horse, the remnant of the Turkoman horse [91]. Furthermore, the contribution from these three stallions to the genome of an English Thor‑ Genes 2022, 13, 2272 6 of 16 oughbred is probably overestimated [127], and even if Arabian and English Thoroughbred breeds share a common ancestry that has not been deepened, there is a small proportion of Arabian DNA in the modern Thoroughbred [126,128]. The identification of the prohibited outcrossing (a practice of crossing between different breeds made to introduce distantly related genetic material into a breeding line) in most of Arabian samples created many controversies among breeders and buyers [123]. Thus, they cannot count on the pedigree of all modern horses to be accurate, and the matter does not concern the qualities of the animal, but rather the violation of breed definitions and the tampering of registries. Ge‑ netic tools could rapidly improve the accuracy of studbooks and registries, tell the history of each horse and also acquire a cultural significance only if based on the integrity of all subjects involved in the equine industry [123]. 4. The Horse Y Chromosome’s Variation Until recent times, the main problem with the horse paternal marker was the lack of sequence polymorphisms [129], and only a few patrilines were suggested in horse domestication [1,130,131]. When the first microsatellite sequences for the equine MSY were detected [130,132] and used to analyse the variability among horse breeds from different continents, no vari‑ ants were individuated in modern breeds, except for two alleles detected in one polymor‑ phic site (YA16) of native Chinese horses [133]. Later, the sequencing of 4 kb of Y chro‑ mosome DNA derived from nine ancient remains (one 2800‑year‑old domesticated horse and eight wild horses) retrieved in permafrost sites of Siberia and North America, and the comparison with the known Y‑chromosomal polymorphisms in modern domestic and Przewalski’s horses, allowed for the identification of 28 segregating sites and eight differ‑ ent haplotypes, thus confirming a genetic variability among horses before domestication also in the paternal line [134] and stimulating new research in this field. In 2017, Librado and colleagues analysed the genomes of 14 horses lived from Bronze to Iron Ages, con‑ firming the presence of a large genetic diversity in the early stages of domestication [117]. Recently, the presence of the most ancient paternal lines in Chinese native horses was con‑ firmed through the analysis of 16 Y‑chromosomal microsatellite markers, thus reporting for the first time seven novel microsatellite loci [135], in addition to those previously de‑ tected [130,132,136]. Among them, four were highly polymorphic only in Chinese local populations and the analysis of the genetic relationships between all the 268 male horses analysed allowed for the identification of 19 Y‑chromosomal haplotypes. Three indigenous breeds (Debao pony, Guizhou and Jinjiang) living in the isolated regions of southwestern China, showed haplotypes distant from those arbored by other Chinese indigenous and in‑ troduced horses, thus suggesting a highly paternal diversity preserved in this geographic area [135]. In the last twenty years, many modern horse breeds from different geographic areas were analysed for the Y chromosome by employing different sequencing technolo‑ gies (Table S2). In some cases, the MSY from modern horse breeds was compared with the results from Przewalski’s horse [52,91,111,133,136], which is considered the feral descendant of the domesticated Botai horse [17] and has been thoroughly investigated; as a result, a certain number of variable sites were individuated, and a horse Y‑chromosomal phylogeny was generated [84,91,111]. In 2013, Wallner and colleagues sequenced Y‑chromosomal BAC clones to obtain a systematic screen for horse Y‑chromosomal variants, and described, for the first time, the relationships among the identified haplotypes [92]. They observed a strong influence from the Near East, with the description of six MSY haplotypes for modern horses and two addi‑ tional haplotypes from the Przewalski’s horse [137]. Among these, three haplotypes (HTs) were particularly common: HT1 represents the ancestral haplotype and was distributed across almost all breeds analysed; HT2 was frequent among all breeds, except for those from Northern Europe and the Iberian Peninsula; HT3 was present above all in the En‑ glish Thoroughbred [92]. All the remaining haplotypes arose from HT1 and were found Genes 2022, 13, 2272 7 of 16 only in Northern European breeds. The widespread presence of HT1 and HT2 is probably due to the Oriental wave, as Arabian horses carrying these MSY haplotypes were imported into Central European studs, while the frequency of HT3 in modern horses is attributable to the intensive use of English Thoroughbred in the improvement of other horse breeds. The SNPs and indels identifying the different haplotypes were further investigated in 42 breeds from different areas of Europe, Asia and America [136] and in 13 Chinese in‑ digenous [138], 3 Sardinian [139] and 1 Kazach [140] horse breeds (Figure 2 and Table S3). Figure 2. Geographic distribution of Y‑chromosome haplotypes (classification as in [92,138]) among 76 modern horse breeds from different geographic areas. Further details are reported in Table S3. HT1 is the most represented haplotype in the Eurasian context, followed by HT2, which is also particularly frequent among American horse breeds. In particular, differently from the European and North American breeds [92,136], almost half of Chinese horses did not show a direct link between the SNP T/A and the deletion in np 10,594 for the HT3 [138]. The same low variability in the other modern European breeds was observed in three local breeds from Sardinia (Italy), each showing one distinctive haplotype (HT1, HT2 and HT3) frequency: 50% of HT1 in the Sarcidano breed, 75% of HT2 in the Giara breed and 58% of HT3 in the Sardinian Anglo‑Arab breed [139]. These haplotype distributions highlight the replacement of autochthonous Y chromosomes with the import of stallions belonging to three paternal lines (ancestral line, Neapolitan/Oriental and Thoroughbred wave) for the improvement of native breeds. Contrary to these findings, Chinese indigenous horse populations seem to have preserved their genetic diversity because they have not been subjected to intensive selection, thus presenting a particular genetic pattern and unique MSY haplotype variants [133,135,138,141,142]. A recent study focused on a large sample of Chinese horses filled the known gap in the worldwide context by adding information on the Asian populations not previously analysed [142]. The screening of single‑nucleotide polymorphisms (SNPs), copy number variants (CNVs) and allele‑specific CNVs showed a deep genetic distance between Chinese indigenous horse populations and those from Europe and the Middle East. Two microsatellites investigated in Hucul and Mongolian horses (YP9) and in a Shetland pony (YN04) [136], in addition to the analyses of Y‑chromosome‑specific high‑resolution hap‑ Genes 2022, 13, 2272 8 of 16 lotyping, have revealed even more variation among European domestic horses [91] and the SNP at locus YA16 in the Yakutian Horse [143,144], thus confirming the Y variability within this breed and the preservation of the autochthonous variation [143]. In 2017, Wallner and colleagues partially assembled Y‑linked regions and generated a horse MSY reference sequence of 2491 high‑quality single‑copy contigs, covering a length of 1.46 Mb [91] of the 15 Mb‑spanning euchromatic part of the horse MSY [113,145]. Then, they mapped the whole‑genome data of 52 male domestic horses from 21 breeds, a Prze‑ walski’s horse and a donkey, to detect their MSY variants and build a phylogenetic tree using the Przewalski’s horse and donkey as outgroups [91]. They observed that almost all MSY haplotypes of modern equine breeds clustered together in a predominant group, called “crown group” (A, L, S and T haplogroups—HGs) whose most recent common an‑ cestor originated about 1000–2000 years ago from Oriental founder stallions [143]. This group separated from Northern European horses around 1300 years ago (haplogroup N, found in Norwegian Fjord Horse, Swedish Coldblood horse and Shetland Pony) and from the sister clade I (found in the Icelandic horses) around 1000 years ago [91]. However, all modern European breeds clustered together in the crown group and exhibited an in‑ fluence of Oriental stallions with the MSY ancestry belonging to the Original Arabian and Turkoman lineages, with the latter profoundly influencing English Thoroughbred stal‑ lions (Darley Arabian, Byerley Turk and Godolphin Arabian) to which the sub‑branches of haplogroup T have been attributed [91,144]. Then, another 211 variants and 58 haplo‑ types were described by the analysis of 5.8 Mb of MSY in 130 domestic horses and nine Przewalski’s horses, confirming the clear separation between the crown group (found in Central and South European, North American and most East Asian modern horses) and the non‑crown haplotypes (found in Przewalski’s horses and some North European and Asian breeds) [111]. The major MSY clades that belong to the crown group are A (first described in an Arabian Horse), H (first described in a Spanish Horse), L (first described in Lipizzan) and T (first described in Thoroughbreds), while the non‑crown group is mostly represented by I (found in Icelandic Horse), J (in Jeju Horse), N (typical of North European breeds), O (found in Mongolian Horse), P (in Przewalski’s horse), and Y (found in Yakutian Horse) clades [84,91,111]. In order to define if a sample belong to the crown group, two key variants (rAY and rAX) have to be tested; then, through the analysis of rA, rW and fYR variants, it is possible to cluster horses into one of the three major crown clades (T, A or H, respectively) [84]. To summarize, among the 169 worldwide horse breeds analysed until now for the Y chromosome (Table S2), 46 were classified into haplogroups [84,91,111,143] (Table S4). As reported in Figure 3, 38 breeds belong to the crown group, seven to the non‑crown group and only one Asiatic breed (Yakutian Horse) represents both (Figure 3 and Tables S4 and S5). In a worldwide context, the non‑crown group was detected in Icelandic Horse, Jeju Pony, Mongolian Horse, North Swedish Draft, Norwegian Fjord Horse, Przewalski’s Horse and Shetland Pony (Figure 3), which, similar to many other native breeds, carry specific breed variants that were not replaced by the Arabian and Turkoman lineages [84]. Due to the large use of Arabian stallions in the improvement of local horse breeds, the recent study by Remer and colleagues also focused on the breeding history of these horses [84]. In the last two centuries, the so‑called “Arabian wave” has profoundly influ‑ enced the selective breeding practices of European horses [19], but no genetic signatures were found in the English Thoroughbred’s MSY ancestry [126]. A particular genetic vari‑ ability was found among the Arabian stallions from the Middle East [29,84,126,146], thus suggesting an origin from this area for this breed. Recent studies focusing on ancient samples tried to describe the times and modes of the loss of horse MSY variation, and different scenarios were proposed. The correla‑ tion between the genetic admixtures observed in European populations and the spread of the Yamnaya culture from the Pontic‑Caspian steppe [21] initially suggested this area as the centre of horse domestication [91]. The loss of horse Y chromosome diversity origi‑ nated from there, with one MSY lineage that gradually replaced all the other [118], except Genes 2022, 13, 2272 9 of 16 for the lineage found only among Yakutian horses [143,144,147] and a quite marked ge‑ netic diversity observed today in other Asian breeds [38,140,144,148]. In 2018, Wutke and colleagues conducted an analysis on the MSY polymorphic sites of 96 European ancient stallions dated back from Copper and Bronze Age to the Middle Ages, declaring that the loss of different Y chromosome lineages in modern horses is due to an artificial selection started in the Iron Age and not to a founder or demographic effect [118]. The reduction in horse Y chromosome diversity over time was stated in a study focused on about 1500 MSY polymorphic sites of 105 ancient stallions dated back from the Upper Palaeolithic to Early Modern periods, showing that the genetic diversity of paternal lines decreased during the last 2000 years [19]. In 2021, Librado and colleagues replaced this scenario showing a high genetic diversity before the spread across Eurasia, with horses migrating from the lower Volga‑Don region [21] and preserving a quite constant Y chromosome diversity during the last 4000 years, until an important decrease started ≈250 years ago with the intensive breeding programs operated by breeders [19]. Figure 3. Worldwide distribution of crown and non‑crown haplogroups and frequencies of each Y chromosome HG among the 49 breeds analysed in [84,91,111,143]. Geographic origin, breed codes, frequencies and proportion of “crown” and ”non‑crown” groups for each breed are reported in Tables S4 and S5. 5. Conclusions and Perspectives The highly repetitive structure of Y chromosome makes its sequencing and assembly very difficult [149], but the longstanding debates about origin, spread and genetic variabil‑ ity of domestic horses and the abundant studies focused on the female counterpart have al‑ lowed researchers to uncover the evolutionary processes that affected the paternal lineages. To enhance knowledge about the evolutionary history and inherited traits of domestic horses, the analysis of high‑throughput genomes led to the production of two high‑quality genome assemblies for equids (EquCab2.0 and EquCab3.0) [86,87] and the first comprehen‑ Genes 2022, 13, 2272 10 of 16 sive assembly of the MSY [93], representing one of the most complete MSYs for eutherian mammals and filling a gap in the horse genome reference sequence. These results provided an important model for the research focused on stallion biology. Therefore, the recent devel‑ opment in the fine‑scaled analysis of the horse Y chromosome has contributed to tracing patri‑ lines and pedigrees [38,91,111,141,143,148] and has provided different scenarios for the time and causes of the loss of Y chromosome diversity [7,19,118]. The discovery of new variants and a better understanding of the pathways through with domestication occurred were possi‑ ble through the analysis of modern [91–93,111,136,138,144] and ancient [7,19,21,118,150,151] horse populations. Despite further investigation still being needed, MSY variation represents a powerful lineage and pedigree tracer crucial for strengthening horse management and is an evaluable genetic marker that contributes to avoiding further loss of biodiversity and under‑ standing the historic development of breeds. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13122272/s1, Table S1: Rate of available MSY gene data for 13 mammalian species analysed in [97]; Table S2: Information about worldwide modern horse breeds investigated for various loci of the Y Chromosome through different sequencing techniques; Table S3: Summary of horses from 76 modern breeds classified in Y‑chromosome haplotypes (HT and CHT) as reported in [144,146], respectively; Table S4: Geographic origin and haplogroup clas‑ sification for the 429 samples phylogenetically analysed in [81,91,92,111]; Table S5: Haplogroup frequencies and “crown” and “non‑crown” proportions for the 46 modern horse breeds analysed in [81,91,92,111]. 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