Knowing where, why, and how genes and athletic prowess intersect in a racehorse has long been the goal of countless researchers, veterinarians, breeders, trainers, and owners [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38]. While great strides in this area have recently been made for gallop racing horses, similar advancements for harness racing horses have been limited [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. Using the NSCT, the current study explored the genetic background for athletic prowess in a harness racing horse by performing GWA analyses and functional classification for three traits associated with harness racing success. These analyses resulted in a total of 32 SNPs of interest with 9 demonstrating genome-wide significance and 13 residing in genes. Subsequent functional classifications went on to provide further support of the complexity of harness racing success with several candidate genes involved in neurological, metabolic, and musculoskeletal regulation identified. Since a gene can be declared as a candidate gene if at least 1 out of the 4 following characteristics are present: 1) the gene has a known physiological role in the phenotype of interest, 2) the gene affects the trait in question based on studies of knockouts, mutations, or transgenics in other species, 3) The gene is preferentially expressed in organs related to the quantitative trait, or 4) the gene is preferentially expressed during developmental stages related to the phenotype, a large fraction of the genes identified in the current study can plausibly be considered as candidate genes [56]. As a result, the following discussion prioritizes genes that contained variants with significant or suggestive associations with our traits of interest. The rationale for this prioritization is simply that for associated variants that reside outside of annotated genes, it is in general more difficult to determine which gene(s) the variants act on.

Glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B)

Two genome-wide significant SNPs associated with career earnings were located in the GRIN2B gene, a gene also identified in a previous study exploring pacing ability in Icelandic horses [57]. The gene has been shown to be involved in neural regulations in humans and laboratory species with mutations in the gene having been associated with neurodevelopmental disorders [58,59,60]. Considered to be an important factor for learning and memory, one can only speculate as to its association with career earnings in a harness racing horse. However, horses with a greater capacity to learn and adapt to the highly variable nuances of harness racing would conceivably be more likely to achieve racing success. On the other hand, the gene’s association with attention-deficit/hyperactivity disorder in humans suggests that perhaps certain horses lack the ability to focus on racing and training, thereby preventing or at least hindering their racing performance [61].

ATPase copper transporting beta (ATP7B)

A single suggestive genome-wide significant SNP associated with best time was located in the ATP7B gene on ECA17. The gene encodes a protein that functions as a monomer, exporting copper out of cells. Excess copper can cause serious toxicity with the process of excess copper disposal relying heavily on ATP7B [62, 63]. Over 500 mutations have been identified in the gene, 380 of which are considered to be disease causing mutations [64]. Elevated levels of copper in the body often result in muscle stiffness with acute muscle stiffness prior to a race having the potential to affect individual performance and chronic muscle stiffness likely to impact a horse’s conditioning, trainability, and overall capacity for speed [62, 63].

Potassium channel regulator (KCNRG)

The KCNRG gene on ECA17 was also identified by two SNPs demonstrating suggestive genome-wide significance for best time. The gene encodes a protein which regulates the activity of voltage-gated potassium channels with a study using songbirds suggesting potassium channels to be lineage specific [64, 65]. The same study also revealed that apart from broad expression in the brain a subset of potassium channel genes are selectively expressed, with the authors hypothesizing that the KCNRG gene may be associated with learning [65]. Although no previous studies of racehorse performance have specifically identified KCNRG as important for racing success, a large conserved haplotype on ECA17 has been advocated to have selective importance in Thoroughbreds and closely related breeds [4, 66].

Also of note is the role voltage-gate potassium channels play in Hyperkalemic periodic paralysis (HYPP), a genetic disorder predominantly seen in Quarter Horses. The condition, caused by a mutation in the sodium voltage-gated channel alpha subunit 4 (SCN4A) gene, manifests intermittently with clinical signs ranging from muscle fasciculation to signs of paresis [67,68,69,70]. Hyperkalemia, a term used to describe abnormally high levels of potassium in the blood, is often seen during or immediately after an attack. Voltage-gated potassium channels are thought to remain open, allowing continual potassium efflux, thereby promoting an open sodium channel configuration. As a result, an HYPP attack can be triggered or an already occurring attack can increase in severity [67, 70,71,72]. Horses with HYPP also tend to possess hypertrophic muscles; however, they have been reported as having a reduced tolerance to exercise with relatively more lactate being produced during exercise [73, 74].

Phosphatidylinositol-4-phosphate 5-kinase type 1 beta (PIP5K1B)

A single suggestive genome-wide significant SNP associated with best time was also located in the PIP5K1B gene on ECA23. Three widely expressed isoforms of PIP5K1 are responsible for the regulation of the major pools of cellular phosphatidylinostitols in mammalian tissues, with PIP5K1B negatively regulated in response to oxidative stress [75, 76]. Neurite outgrowth, a critical process for neuronal development, has also been shown as negatively regulated by PIP5K1A [77]. Since the current study is the first to suggest an association between PIP5K1B and racing success, understanding the roles PIP5K1 isoforms have on a horse’s capacity for speed remains a task for future studies. However, genes that influence cell differentiation processes, such as endocytosis, assuredly contribute in some way or another to the physical limitations and overall performance of any racehorse.

Dedicator of cytokinesis 8 (DOCK8)

Perhaps the most obvious candidate gene for harness racing success in the current study was DOCK8. Five suggestive genome-wide significant SNPs indicated the importance of DOCK8 to a horse’s best time, with 4 of the SNPs located in the gene. Mutations in DOCK8 result in a form of hyper-IgE syndrome; however, loss or mutations of DOCK8 have also been associated with intelligence and motor retardation [78,79,80,81,82,83]. While the importance of intelligence in a racehorse has been briefly discussed above, in the case of DOCK8 the significance of the gene may lie with its link to motor skills. DOCK8 is not only located on ECA23, the same chromosome as DMRT3, but multiple studies have hypothesized some sort of commonality or overlap between DMRT-(1,2,3) gene effects and DOCK8 [22, 82, 84]. Despite the established association between DMRT3 and harness racing performance, additional research of DMRT3 in horses strongly suggest that the mutation is unlikely to be the single cause of gaiting ability [22, 53, 85,86,87,88]. Therefore, it is conceivable that DOCK8 also significantly contributes to gaiting ability, ultimately playing some role in a harness racing horse’s propensity to exhibit speed at trot or pace.

Phosphodiesterase 3A (PDE3A)

The protein encoded by the PDE3A gene, a gene on ECA6 in which a single genome-wide significant SNP associated with career earnings is located, plays a critical role in cardiovascular function [89,90,91]. The encoded protein regulates vascular smooth muscle contraction and relaxation and has been linked to familial hypertension, cardiovascular disease, and fertility [89,90,91,92]. Healthy cardiovascular function is important for racing success as the act of racing undeniably requires a higher than resting-level of oxygen to support the horse’s increased muscle activity. A mutation in the PDE3A gene that ultimately alters cardiovascular function could potentially prevent a horse from meeting the higher metabolic demands of racing, thus decreasing his/her chances of winning and limiting his/her career earnings. On the contrary, an advantageous mutation in the gene could allow some horses to perform at an even greater cardiovascular level, increasing their likelihood of winning races and earning more prize money.

Inositol polyphosphate-5-phosphatase D (INPP5D) & SRY-box 5 (SOX5)

Also identified by single genome-wide significant SNPs on ECA6 were the INPP5D and the SOX5 genes. The INPP5D gene is an important regulator of immune cell signaling, while the SOX5 gene is involved in embryonic development and has been associated with multiple human diseases and disorders [93,94,95,96,97,98,99]. Moreover, both genes have been suggested as important in B cell activity indicating that their association with career earnings in the current study may be rooted in the immune response of a horse [95, 100]. However, mutations in SOX5 have also been theorized to disrupt neuronal development and function [101, 102].

Other candidate genes

Regions on ECA1, ECA7, and ECA16 have also previously been described as important for endurance performance traits, while regions on ECA14 and ECA18 associated with gallop racing in other studies do not appear to play a significant role in harness racing [4,5,6,7,8, 10,11,12,13,14,15, 19,20,21, 39, 66]. This likely suggest a greater demand for endurance in harness racing compared to gallop racing and is perhaps a sign of the different physiological demands for speed in trot versus speed in gallop. Candidate genes for harness racing success in the current study were also identified on ECA1, ECA2, ECA6, ECA7, ECA16, ECA17, ECA23, ECA25, ECA28, ECA29, and ECA31. However, it is important to note that the MAF threshold applied in the current study is slightly lower than is generally accepted. Although this may have inadvertently resulted in some SNP associations being simply by chance, it is also plausible that the lower MAF threshold allowed for the capture of candidate genes/regions that are perhaps the difference between an elite horse and a very, very good horse. Racing performance is undoubtedly complex and the unique history of the NSCT, being a blend of draught horse and racehorse, means that rare variants cannot be ruled out purely because they are rare – particularly when one considers the rarity of an elite racehorse. While not all candidate variants/genes are discussed above, the results of the current analyses clearly suggest that different molecular and cellular events mediate adaptive processes in the neuromusculoskeletal system in response to exercise. High intensity exercise (e.g. racing) is known to be associated with significant physiological adaptations in the neuromuscular system in equine athletes with prolonged and intense exercise potentially resulting in oxidative damage to cellular constituents [103]. Moreover, the importance of the central nervous system (CNS) as a critical “central governing” factor in sporting performance has been previously documented in endurance horses with exercise shown to induce several biological processes that regulate neurological functions that help to maintain good mental health [104]. Our results add to this line of thought, providing further evidence that genes involved in neural regulations (e.g. GRIN2B) likely play an important role in controlling the fundamental biological processes underlying adaptation to equine athletic performance.