Introduction
For centuries, humans have selectively bred livestock to enhance desired traits, a process that has significantly shaped the genomes of domestic animals. In horses, particularly warmblood breeds developed for competitive sports like show-jumping, dressage, and eventing, this selection pressure is expected to have left discernible marks on their genetic makeup. This study delves into the genetic history of four prominent German warmblood breeds—Trakehner, Holsteiner, Hanoverian, and Oldenburger—to identify genomic regions that have undergone selective pressure. By employing a multi-method approach including integrated Haplotype Score (iHS), cross-population Extended Haplotype Homozygosity (xpEHH), and Runs of Homozygosity (ROH), we aim to uncover the genetic underpinnings of athletic performance and breed-specific histories. Understanding these selection signatures offers valuable insights into how breeding programs have influenced equine genomes and can help in identifying genes associated with key performance traits.
Methodology
The research involved a comprehensive genetic analysis of 942 stallions from four German warmblood breeds: Trakehner (N=44), Holsteiner (N=358), Hanoverian (N=319), and Oldenburger (N=221). Blood samples, collected during mandatory health examinations for stallion pre-selection, were genotyped using the Illumina EquineSNP50 BeadChip. After stringent filtering to ensure data quality, 48,410 autosomal single nucleotide polymorphisms (SNPs) were retained for analysis.
To detect signatures of positive selection, three primary methods were utilized:
- Runs of Homozygosity (ROH): This method identifies continuously homozygous segments within the genome, which can indicate regions that have been under selection or are a result of inbreeding. ROH clusters, shared by at least a third of individuals, were analyzed within and across breeds.
- Integrated Haplotype Score (iHS): iHS is designed to detect incomplete selective sweeps within a population by comparing the haplotype structure of ancestral and derived alleles. Significant iHS scores can pinpoint genomic regions where advantageous alleles have rapidly increased in frequency.
- Cross-population Extended Haplotype Homozygosity (xpEHH): xpEHH is powerful for detecting complete selective sweeps by comparing haplotype homozygosity between two populations. It is particularly useful for identifying selection signals that have reached fixation in one breed but are still polymorphic in another.
To determine allele status (ancestral vs. derived), genetic data from domestic donkeys (Equus asinus) was used as an outgroup. Statistical analyses and genomic plotting were performed using specialized software, including R Statistical Software with the REHH package and GCTA. Candidate genes within identified selection signatures were further investigated through functional annotation and enrichment analyses using DAVID and Ensembl’s BioMart tool.
Results
Breed Differentiation and Selection Signatures
A Principal Component Analysis (PCA) of the genotype data revealed distinct clustering patterns among the breeds. The Trakehner breed formed a separate subgroup, reflecting its unique breeding history and genetic distinctiveness. The Holsteiner also clustered apart from the other three breeds, potentially due to their strong historical focus on show-jumping. Hanoverian and Oldenburger breeds showed considerable overlap in the PCA, suggesting shared breeding goals and historical influences, although subsequent xpEHH analyses revealed breed-specific differences.
Across all breeds, ROH analysis identified 37 shared signatures on 16 chromosomes, with some ROH segments being present in up to 43% of the sampled horses. Trakehner horses exhibited the highest number of breed-specific ROHs, likely reflecting their historical population bottleneck.
Across-breed iHS analyses highlighted significant selection signatures on chromosomes 1, 4, and 7. Breed-specific iHS analyses revealed unique signatures for each breed, with Hanoverian and Oldenburger showing particularly similar patterns. xpEHH analyses, comparing each breed against the other three combined, identified numerous breed-specific selection signatures across multiple chromosomes, further underscoring the genetic divergence driven by distinct breeding histories and selection pressures.
Candidate Genes and Functional Pathways
The investigation into regions associated with selection signatures revealed overlaps with known Quantitative Trait Loci (QTL) related to traits such as coat texture, hair density, and sperm count. Enrichment analyses of genes within identified selection regions pointed towards several key biological processes and pathways relevant to equine athleticism and development.
Notably, genes involved in muscle functionality (e.g., TPM1, TMOD2-3, MYO5A, MYO5C) and energy metabolism and growth (e.g., AEBP1, RALGAPA2, IGFBP1, IGFBP3-4) were identified within shared selection signatures. These findings align with the breeding goals focused on athletic performance.
The HOXB gene complex, crucial for embryonic development and patterning, was also highlighted, suggesting selection pressure related to developmental processes. Additionally, genes associated with fertility (e.g., THEGL, ZPBP1-2, TEX14, ZP1, SUN3, and CFAP61) were identified in regions under selection, indicating that reproductive fitness has also been a target of selection, likely driven by natural selection pressures.
Selection signatures were also found in regions containing genes related to pigmentation, such as KITLG, and genes influencing size and conformation, including IGFBP genes, which are associated with growth and height.
Discussion
This study successfully identified genomic regions under selection in four German Warmblood Horse Breeds, revealing both shared evolutionary trajectories and breed-specific genetic adaptations. The distinct clustering in PCA and the varied selection signatures observed across iHS and xpEHH analyses underscore the impact of divergent historical breeding policies and selection focuses on shaping the genomes of these breeds.
The high number of ROHs in Trakehner horses is consistent with their documented population bottleneck, highlighting the lasting genetic impact of such events. The identification of candidate genes involved in muscle function, metabolism, and embryonic development strongly supports the hypothesis that selection for athletic performance has targeted specific genetic pathways. For instance, genes like TPM1 and MYO family members are fundamental to muscle contraction and performance, while IGFBP genes play critical roles in growth regulation, potentially influencing overall size and athletic potential.
Furthermore, the findings related to fertility genes suggest that while artificial selection has driven performance traits, natural selection may have also played a role in maintaining reproductive fitness. The overlap of selection signatures with QTL for traits like sperm count and genes involved in sperm development points to a balance between optimizing for athletic prowess and ensuring reproductive success.
The detected selection signatures around pigmentation-related genes like KITLG also indicate that aesthetic traits have continued to be influenced by selection, reflecting historical preferences for certain coat colors.
Conclusion
This research provides compelling evidence of selection signatures in German warmblood horse breeds, reflecting their shared focus on athletic performance and their distinct breeding histories. The identified candidate genes associated with muscle functionality, energy metabolism, growth, embryonic development, and fertility offer valuable insights into the genetic basis of traits important for sport horses. These findings pave the way for future research aimed at validating these candidate genes and potentially informing breeding strategies to further enhance equine athletic performance and overall health. Further studies integrating detailed phenotyping data with genomic information will be crucial for a comprehensive understanding of the genetic architecture of these complex traits.
References
- Visscher PM, Smith DM, Lee SH, et al. Selection signatures in four German warmblood horse breeds: Tracing breeding history in the modern sport horse. PLoS ONE. 2019;14(4):e0215913.
- Nolte W, Thaller G, Kuehn C. Selection signatures in four German warmblood horse breeds: Tracing breeding history in the modern sport horse. PLoS ONE. 2019;14(4):e0215913.
