The DMRT3 Gait keeper mutation affects performance of Nordic and Standardbred trotters 1

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1 The DMRT3 Gait keeper mutation affects performance of Nordic and Standardbred trotters 1 K. Jäderkvist,* 2 L. S. Andersson,* 2,3 A. M. Johansson,* T. Árnason, S. Mikko,* S. Eriksson,* L. Andersson,* 3,4 G. Lindgren* 3,4 *Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, SE , Uppsala, Sweden; IHBC AB, Knubbo, SE Morgongåva, Sweden; and Department of Medical Biochemistry and Microbiology, Uppsala University, SE , Uppsala, Sweden ABSTRACT: In a previous study it was shown that a nonsense mutation in the DMRT3 gene alters the pattern of locomotion in horses and that this mutation has a strong positive impact on trotting performance of Standardbreds. One aim of this study was to test if racing performance and trotting technique in the Nordic (Coldblood) trotters are also influenced by the DMRT3 genotype. Another aim was to further investigate the effect of the mutation on performance in Standardbreds, by using a within-family analysis and genotype phenotype correlations in a larger horse material than in the previous study. We genotyped 427 Nordic trotters and 621 Standardbreds for the DMRT3 nonsense mutation and a SNP in strong linkage disequilibrium with it. In Nordic trotters, we show that horses homozygous for the DMRT3 mutation (A) had significantly higher EBV for trotting performance traits than heterozygous (CA) or homozygous wild-type (CC) horses (P = 0.001). Furthermore, AA homozygotes had a higher proportion of victories and top 3 placings than horses heterozygous or homozygous wild-type, when analyzing 4279 performance data for the period 3 to 6 yr of age (P = 0.06 and P = 0.05, respectively). Another finding in the Nordic trotters was that the DMRT3 mutation influenced trotting technique (P = ). Standardbred horses homozygous AA had significantly higher EBV for all traits than horses with at least 1 wild-type allele (CA and CC; P = ). In a within-family analysis of Standardbreds, we found significant differences in several traits (e.g., earnings, P = 0.002; number of entered races, P = 0.004; and fraction of offspring that entered races, P = 0.002) among paternal half-sibs with genotype AA or CA sired by a CA stallion. For most traits, we found significant differences at young ages. For Nordic trotters, most of the results were significant at 3 yr of age but not for the older ages, and for the Standardbreds most of the results for the ages 3 to 5 were significant. For Nordic trotters, the proportion of victories and placings were the only traits that were significant for other ages than 3 yr. Key words: DMRT3, horse, locomotion pattern, racing performance, trotting technique 2014 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas The authors would like to thank the Swedish Trotting Association who supplied the performance data analyzed in the study. We also thank the Nordic trotter trainer that helped us performing the blind study on trotting technique. This study was financially supported by The Swedish Research Council Formas and the Swedish Research Council. 2 Equal contribution. 3 L. S. Andersson, L. Andersson, and G. Lindgren are co-inventors on a patent application concerning commercial testing of the DMRT3 mutation. 4 Corresponding authors: leif.andersson@imbim.uu.se, gabriella. lindgren@slu.se Received March 3, Accepted July 9, INTRODUCTION It was shown that a single base change in DMRT3 has a major effect on the pattern of locomotion in horses (Andersson et al., 2012). The mutation causes a premature stop codon (DMRT3_Ser301STOP) and is expected to result in a truncated DMRT3 protein. The mutation, a change from cytosine (C) to adenine (A), is permissive for performing alternate gaits (pace or ambling gaits) and has a highly significant positive effect on harness racing performance in Standardbreds. Further evidence for a critical role of DMRT3 in co-

2 4280 Jäderkvist et al. ordination of limb movements was obtained by characterization of locomotion and spinal cord function in a Dmrt3 knock-out mouse (Andersson et al., 2012). So far, the mutant A allele has been found to be fixed in both American Standardbred trotters and pacers (Promerová et al., 2014). However, the wild-type allele is still segregating in Swedish Standardbreds with a frequency of about 3%. This is likely due to importation of French trotters having a fairly high frequency of the C allele (Promerová et al., 2014). French trotters are often considered to have difficulty sustaining an even-beat trot at high speed and not to be natural trotters (Andersson et al., 2012). The fact that the mutation was not fixed in Swedish Standardbreds allowed investigation of its influence on racing performance. It was shown that horses homozygous for the mutation had significantly higher EBV for racing performance and increased earned price money than the heterozygotes (Andersson et al., 2012). Frequency of the A allele is 45% in the Nordic (Coldblood) trotters (Promerová et al., 2014). However, the effect of this mutation on performance in this breed has yet to be investigated. The main goal of this study was therefore to test if racing performance and trotting technique are influenced by the DMRT3 genotype in Nordic trotters. We also expanded the analysis of racing performance in Swedish Standardbreds. MATERIALS AND METHODS Horse Material and Phenotypic Measurements Animals. We selected 1,047 horse DNA, blood, or hair samples previously used for parentage control at the Animal Genetics Laboratory, Swedish University of Agricultural Sciences, Uppsala, Sweden. In total, phenotypic information from 427 Nordic trotters (266 males and 161 females) and 621 Standardbreds (385 males and 236 females) born between 1950 and 2010 were included in the study. Racing Performance of Nordic Trotters and Standardbreds. All analyzed competition data were obtained from the years 1992 to Nordic trotters that had competed at least once and were born between 1990 and 2009 were used for the analysis of racing performance traits. We defined different traits based on competition results obtained at different ages: 3 yr of age (161 horses, of which 12 were born outside of Sweden), 4 yr of age (162 horses, of which 11 were born outside of Sweden), 5 yr of age (153 horses, of which 10 were born outside of Sweden), 6 yr of age (136 horses, of which 5 were born outside of Sweden), 3 to 6 yr of age (174 horses, of which 10 were born outside of Sweden), and 10 yr of age (88 horses, of which 5 were born outside of Sweden). For the analysis of EBV we included 352 Nordic horses born between 1980 and All Table 1. Average EBV for Nordic trotters according to DMRT3 genotype (SE in parentheses) Trait/index CC (n = 103) CA (n = 200) AA (n = 49) P Earnings (SEK 1 ) (1.24) (0.84) (1.78) Placings (1.24) (0.84) (1.87) Racing time (1.13) (0.71) (1.55) Start status (1.17) (0.77) (1.63) Total index (1.21) (0.80) (1.71) SEK = Swedish kronor. 2 Total index = 40% racing status and 60% earnings. Swedish trotting horses have breeding values estimated using animal models as described by Árnason (1999). For Standardbreds the model includes effects of genetic base group and combination of sex and birth year. For Nordic (Swedish and Norwegian) trotters the model includes the combined effect of country sex birth year. The genetic evaluation is based on racing status and performance results at the age of 2 to 5 yr for Standardbreds and 3 to 6 yr for Nordic trotters (Árnason, 1999). For the corresponding analysis of direct values in Standardbreds, we used horses that had competed at least once and were born between 1990 and In a similar way, we defined different traits based on competition results obtained at different ages: 3 to 5 yr of age (230 horses, of which 41 were born outside of Sweden) and 10 yr of age (121 horses, of which 29 were born outside of Sweden). For the analysis of EBV we included 465 Standardbred horses born 1971 to Since the number of CC horses was so low, the analyses were made using only 2 genotype groups: horses homozygous AA and horses with at least 1 wild-type allele (CC/CA). For both Nordic trotters and Standardbreds, the direct racing performance values included number of starts, number of victories, proportion of victories, proportion of placings (1 to 3), earned price money, proportion of disqualifications, and racing times. When calculating means and variance for earnings and best racing times we used transformed values, ln(earnings + 1,000) and ln(racing time 68.2; Supplementary Tables 1 and 2), to get normally distributed values (Árnason, 1994). The same transformation is used for these traits in the routine genetic evaluation. For the horses born in Sweden, we had racing performance data both from Sweden and from other countries, while for the horses born outside of Sweden we only had access to performance results from Swedish race tracks. For disqualifications, only results from Swedish race tracks were available for horses born within as well as outside of Sweden. For both Nordic trotters and Standardbreds the performance results were correlated with DMRT3 genotype. We also compared data on racing performance between

3 DMRT3 and trotting performance 4281 Table 2. Average performance results for Nordic trotters at 3 yr of age according to DMRT3 genotype (SE in parentheses) Trait CC (n = 32 34) CA (n = ) AA (n = 25) P Earnings (SEK) 1 54,160 (13,044) 102,700 (14,813) 115,400 (38,280) Earnings transformed (0.28) (0.16) (0.29) 0.03 P lacings 1 3 (frequency) (0.050) (0.027) (0.051) 0.02 V ictories (frequency) (0.034) (0.022) (0.045) Record 1,3 volt start (0.74) (0.38) (0.78) Record volt 4 transformed (0.03) 3.21 (0.01) 3.20 (0.03) SEK = Swedish kronor. For these variables transformed values were used for the statistical calculation. 2 Earnings transformed: ln(earnings + 1,000). 3 Best racing time for 1 km, in seconds. 4 Start method where the horses trot in circles in pairs in a specific pattern to hit the starting line as a group. 5 Record transformed: ln(record 68.2). Nordic horses without the mutation (CC) and horses that had at least 1 mutated allele (AA and CA). Blind Study on Trotting Technique in Nordic Trotters. A blind study on trotting technique in Nordic trotters was performed in cooperation with a professional trainer. Hair samples from 73 Nordic trotters between 2 and 12 yr of age were collected. The trainer was asked to group the horses on his race camp according to their trotting ability. The horses were divided in 3 different groups: horses with a smooth, symmetrical, even-beat trot without rhythmical disruption; horses with difficulties sustaining an even-beat trot at high speed; and horses with a tendency to go towards tölt or pace while trotting but could trot at very high speed once they were trained. The trainer was also asked to judge the horses trotting capacity or potential on a scale from 1 to 5 where 1 was the best. The DMRT3 genotypes were compared with the classification of the horses. Within-Family Analysis in Standardbreds. Among the Standardbred horses tested as heterozygous (CA) for the nonsense mutation, there are a few well-performing horses. One example is a famous stallion that earned more than 20 million Swedish kronor (SEK) on the race track. It is well known that he had some problems with the rhythm and regularity of his trot. The stallion was not as successful in breeding as on the race tracks. He sired many offspring that did not perform well due to problems with their trotting ability. Today, his breeding index is very low compared to the active breeding population. We compared performance data for 92 of his offspring, born between 1986 and 1987, with their DMRT3 genotype. By using horses from the same family the impact of other genetic factors was reduced. Frequency of the Gait Keeper Mutation in DMRT3 over Time. The frequency of the mutated allele is much lower in the Nordic trotter compared to the Standardbreds (Promerová et al., 2014). Hence, to investigate how the frequency of the mutated allele has changed since the 1950s, both Nordic trotters (n = 206) and Standardbreds (n = 283) born between 1950 and 2010 were genotyped. Isolation of DNA and SNP Genotyping Deoxyribonucleic acid was prepared from the hair roots using a standard hair-preparation procedure. One hundred microliters Chelex 100 Resin (Bio-Rad Laboratories, Hercules, CA) and 7 μl of proteinase K (20 mg/ml; Merck KgaA, Darmstadt, Germany) were added to the sample. The mix was incubated at 56 C for 1 h and the proteinase K was inactivated for 10 min at 95 C. For DNA preparation from blood samples, 350 μl of blood was used and isolated by the Qiasymphony instrument (Qiagen, Hilden, Germany). The samples were genotyped for 2 SNP markers: the nonsense mutation in DMRT3 (chromosome 23: 22,999,655 bp) and SNP BIEC located 31,999 bp upstream of DMRT3 (chromosome 23: 22,967,656 bp). These 2 SNP markers are in strong linkage disequilibrium (r 2 = 0.91; Promerová et al., 2014). The marker SNP BIEC was the only marker on SNP50KbeadChip (Illumina, San Diego, CA) that showed significant association with pace in a previous genomewide association study (Andersson et al., 2012). Single nucleotide polymorphism genotyping was performed with the StepOnePlus Real-Time PCR System (Life Technologies [Thermo Fischer Scientific], Waltham, MA) using custom designed TaqMan SNP Genotyping Assays (Applied Biosystems by Life Technologies [Thermo Fischer Scientific]) as previously described (Andersson et al., 2012; Promerová et al., 2014). Statistical Analysis The statistical analyses were performed using the following software: R (R Development Core Team, 2005), PLINK (Purcell et al., 2007), and Simple

4 4282 Jäderkvist et al. Table 3. Average performance results for Nordic trotters 3 to 6 yr of age according to DMRT3 genotype (SE in parentheses) Trait CC (n = 25 39) CA (n = ) AA (n = 12 19) P Earnings (SEK) 1 265,400 (46,053) 362,800 (47,779) 283,900 (79,034) Earnings transformed (0.28) (0.18) (0.42) 0.73 Placings 1 3 (frequency) (0.028) (0.020) (0.051) 0.05 Victories (frequency) (0.018) (0.015) (0.035) 0.06 Record 1,3 auto start (0.78) (0.35) (0.90) Record auto 4 transformed (0.03) 2.95 (0.02) 2.96 (0.05) 0.46 Record 1,3 volt start (0.82) (0.42) (1.27) Record volt 6 transformed (0.03) 3.04 (0.02) 3.05 (0.05) 0.39 Disqualifications (frequency) (0.020) (0.017) (0.031) SEK = Swedish kronor. For these variables transformed values were used for the statistical calculation. 2 Earnings transformed: ln(earnings + 1,000). 3 Best racing time for 1 km, in seconds. 4 Start by car. 5 Record transformed: ln(record 68.2). 6 Start method where the horses trot in circles in pairs in a specific pattern to hit the starting line as a group. Interactive Statistical Analysis (SISA; Quantitative Skills, 2013). For comparing racing performance means of 2 groups (e.g., Standardbreds with genotype AA and CA/CC) the Student s t test was used, for the study on trotting technique in Nordic trotters we used Fisher s exact test, and for comparing the effect of the genotypes we performed a Wald test in PLINK. To quantify the effect of DMRT3 genotype on trotting performance, variance components were estimated using restricted maximum likelihood (REML) with and without DMRT3 genotype in the model. The DMU software (Madsen and Jensen, 2010) was used for the analyses. Pedigrees and trotting performance results (number of starts, proportion of placings, earned price money, and racing time) were obtained from the Swedish Trotting Association database. In total, 330 Standardbreds and 273 Nordic trotters were included in the analyses. The pedigrees were traced back 5 generations and comprised 2,958 Standardbreds and 1,540 Nordic trotters. Animal models were used that included fixed effects of birth year classes and sex of the horses. In addition, the same base models were used but including also the fixed effect of DMRT3 genotype (AA and AC in Standardbreds and AA, AC, and CC in Nordic trotters). The estimated phenotypic variances were compared from models with and without DMRT3 genotype. RESULTS The Effect of DMRT3 on Racing Performance and Breeding Values of Nordic Trotters We found that Nordic trotters that were homozygous AA had significantly higher EBV than horses heterozygous or homozygous wild-type (P = 0.001; Table 1). At the age of 3 yr, AA horses also had the highest percentage of victories and placings (P = and P = 0.02, respectively), they earned most money (P = 0.03), and they had the best time record (P = 0.009; Table 2). However, at older ages (3 to 6 yr of age) they still had the highest percentage of victories and placings (P = 0.06 and P = 0.05, respectively) but for the other traits (e.g., earnings and best racing time), the heterozygous Nordic trotters were just as good or even better than the AA homozygotes (Table 3). Horses with the genotype CC had the lowest means for almost all traits compared to horses heterozygous (CA) or homozygous AA. The number of traits with a significant difference between horses with different genotypes decreased with increasing age (Supplementary Table 1), and for older ages, the differences between the genotypes were lower and few of them were significant. However, even though the results were not significant, they all pointed in the same direction: the genotype CC was the least advantageous for trotting performance, while the difference between CA and AA horses was quite small and varied between traits. When comparing performance results for Nordic trotters with (CA or AA) and without the mutated allele (CC), we found that horses with the mutation had a higher percentage of victories, both for the ages 3 to 6 yr (P = 0.06) and 10 yr of age (P = 0.04; Supplementary Table 3). For the other performance traits, we found that AA and CA horses had better results than CC horses but that the differences were not significant. For example at 10 yr of age, AA and CA horses had earned on average 782,539 SEK compared to 386,722 SEK for the CC horses. When DMRT3 genotype was included in the models for performance traits (number of races, percentage of placings, earnings, earnings per start, and racing time)

5 DMRT3 and trotting performance 4283 Table 4. Number of horses with different DMRT3 genotypes in trotting technique groups for Nordic trotters Trotting technique group CC CA AA Not clean trot Clean trot P ace/tölt tendency; nice technique at high speed P = in REML analyses, the phenotypic variances of the different traits were reduced by 0 to 3.9%. Blind Study on Nordic Trotters The grouping of the horses into 3 classes according to trotting technique agreed well with the 3 genotype classes (Table 4; P = ). The horses with difficulties sustaining an even-beat trot at high speed were predominantly CC homozygotes. Most of the horses that the trainer classified as clean trotters were CA and the horses that had been seen to perform tölt or pace were more often AA. The 22 horses that were misclassified tended to be about 1 yr younger than the rest (3.68 vs. 4.87), but the difference was not statistically significant (P = 0.06). The trainer s classification (scale 1 to 5) also indicated that AA and CA horses had a better harness racing capacity than CC horses (2.96 vs. 3.72; P = 0.06). The Effect of DMRT3 on Racing Performance and Breeding Values of Standardbreds For the Standardbred horses we found significant differences between the 2 genotype groups (CA/CC versus AA) in performance traits for the ages 3 to 5 yr (e.g., earnings, P = 0.02; victories, P = 0.04; number of starts, P = 0.02; best racing time, P = 0.004; and disqualifications, P = 0.02; Table 5). The frequency of homozygous wild-type (CC) Standardbreds is very low and in this study only 3 horses were homozygous CC out of 621 genotyped Standardbreds. The EBV differed significantly between AA horses and horses with at least 1 wild-type allele (CA and CC; Table 6; P = ). For performance of horses at 10 yr of age, the differences were not that large and none of them were significant (Supplementary Table 2). When DMRT3 genotype was included in the models for performance traits (number of races, percentage of placings, earnings, earnings per start, and racing time) in REML analyses, the phenotypic variances of the different traits were reduced by 0.6 to 1.8%. Table 5. Average performance results for Standardbred horses 3 to 5 yr of age according to DMRT3 genotype (SE in parentheses) Trait CA/CC (n = 7 14) AA (n = ) P Earnings (SEK) 1 390,757 (209,665) 1,203,070 (155,043) Earnings (0.78) (0.16) 0.02 transformed 2 Placings (0. 086) (0.016) 0.14 (frequency) V ictories (0.042) (0.012) 0.04 (frequency) No. of starts (3.99) (1.06) 0.02 Record 1,3 auto (0.69) (0.19) start 4 Record auto (0.08) 1.68 (0.03) transformed 5 Record 1, (0.84) (0.14) volt start 6 Record volt (0.09) 2.08 (0.02) 0.18 transformed 5 D isqualifications (frequency) (0.096) (0.012) SEK = Swedish kronor. For these variables transformed values were used for the statistical calculation. 2 Earnings transformed: ln(earnings + 1,000). 3 Best racing time for 1 km, in seconds. 4 Start by car. 5 Record transformed: ln(record 68.2). 6 Start method where the horses trot in circles in pairs in a specific pattern to hit the starting line as a group. Within-Family Analysis of Standardbreds We decided to compare performance data within a large Standardbred half-sib family sired by a heterozygous stallion (CA). Since the great majority of the dams were homozygous AA, we could directly deduce the transmission of C and A alleles to his progeny. The progeny that inherited the A allele earned on average 195,000 SEK while the offspring that inherited the C allele earned on average 85,000 SEK (Table 7). When comparing the racing results at 3 to 5 yr of age, which is the performance data used for breeding value estimations, the horses homozygous AA earned significantly more money (P = ), they entered more races (P = 0.004), and a higher fraction of the offspring did compete (P = 0.002). Table 6. Average EBV for Standardbreds according to DMRT3 genotype (SE in parentheses) Trait/index CA/CC (n = 67) AA (n = 398) P Racing performance 83.0 (1.03) 98.6 (0.76) No. of races 92.0 (0.55) 98.8 (0.34) Racing status 85.4 (0.86) 98.5 (0.53) Total index (0.94) 98.6 (0.68) Total index = 5% number of starts, 75% racing performance (earnings/ start + earnings + percent placings 1 through 3), and 20% racing status.

6 4284 Jäderkvist et al. Table 7. Average performance results for paternal halfsib horses sired by a DMRT3 CA stallion (SE in parentheses) Performance trait CA (n = 41) AA (n = 51) P Earnings (SEK) 1 85,652 (29,931) 195,363 (63,655) Earnings transformed (0.37) 10,34 (0.31) Earnings 3 5 yr 1 (SEK) 20,174 (6,227) 78,618 (17,765) E arnings 3 5 yr 8.39 (0.28) 9.92 (0.28) transformed 2 N o. of entered races 8.71 (1.94) (2.20) (3 5 yr) F raction of offspring entered races (0.056) (0.079) SEK = Swedish kronor. For these variables transformed values were used for the statistical calculation. 2 Earnings transformed: ln(earnings + 1,000). Frequency of the DMRT3 Gait Keeper Mutation over Time In Standardbreds the frequency of the mutant allele has been constant at a frequency of about 98% since the 1950s while for the Nordic trotters the frequency reached over 30% in 1970 and has been quite stable since then (Fig. 1; for more detailed numbers see Supplementary Table 4 and 5). The average frequency of heterozygous Nordic trotters have been around 50% during the last 3 decades and the genotype frequencies did not deviate significantly from Hardy-Weinberg equilibrium. Out of 283 Standardbred horses, there was only 1 horse with the genotype CC (Supplementary Table 4). For Nordic trotters born 1970 and before, samples were only available from approved breeding stallions. That means that the A allele frequencies in 1970 and before were most likely overestimated. DISCUSSION For Standardbred horses the positive effect of the DMRT3 mutation on racing performance is very clear. Horses homozygous for the mutant A allele are faster, they have a cleaner trot, they earn more money, and they win more races. The effect is similar for the Nordic trotters, with the major difference that with increasing age CA horses perform well, or even better in some parameters, than AA horses. There are also differences when it comes to trotting technique. Standardbreds with genotype CA have difficulties keeping a clean, even-beat trot at higher speeds (Andersson et al., 2012), while in this study the CA Nordic trotters had a good trotting technique. In Nordic trotters we found that the mutation had positive effects on performance traits, for example victories and earnings. Horses with the mutation earned more than twice as much money compared with horses homozygous CC when analyzing lifetime competition Figure 1. Frequency of the DMRT3 A allele over time for Standardbreds (SB) and Nordic trotters (CT). data for 10-yr-old horses. However, when it comes to other traits, such as racing time and disqualifications, we did not find any significant differences between horses with or without the DMRT3 stop mutation, even though it was a similar tendency as for victories and placings, that is, that horses with the mutation had better results than horses homozygous CC. For some traits (e.g., proportion of victories and placings) and for EBV, there appeared to be an additive genetic effect of the mutation. Our results for Nordic trotters show that DMRT3 wild-type homozygotes have the poorest performance results, both when it comes to racing time, earnings and victories. However, the difference between CA and AA horses is not as large as for the Standardbreds. The Nordic AA trotters are for most ages the fastest horses, but they are not the ones that earn most money during their career. This may be due to their trotting technique. As showed in this study, CA horses most often have a clean non-pacey trot, which means that they are easy to train and drive and they do not need to be balanced to the same extent as CC and AA horses. Therefore, the Nordic trotters that are AA have the capacity to become very fast but they can be more difficult to train as they may have a tendency to move towards pace. In the blind study on trotting technique, it was found that horses that were not categorized as expected based on their genotype were on average 1 yr younger than the rest. The reason for this misclassification could therefore be because the trainer had less knowledge of these horses as they had not been trained as much as the others. The majority of the significant differences between genotypes for Nordic trotters were obtained from the 3-yr-old horses and for the Standardbreds from 3 to 5 yr of age. A possible reason for this could be that for older ages the environmental effect of training has a greater impact. Another reason could be that the Standardbreds are physically more developed at younger ages and that the Nordic trotters need more time before they have reached their full capacity. Heritabilities of trotting

7 DMRT3 and trotting performance 4285 performance traits recorded at younger ages have been shown to be higher than for performance at older ages. The heritabilities for earnings and best racing time before 6 yr of age have been estimated at 0.31 and 0.34, respectively (Árnason et al., 1989). For earnings and best racing time before 13 yr of age, the estimated heritabilities were 0.23 and 0.26, respectively. Yet another reason for the decreased significance for older ages could be that there were fewer horses with performance data in these age groups. For example, for Nordic trotters at 10 yr of age, we only had 8 AA horses. For the Standardbreds we had many horses that were AA, but since the frequency of the C allele is low we only had 3 CC horses and 11 CA horses with performance data. The differences in EBV between genotypes were very similar for Nordic trotters and Standardbreds; the AA horses had significantly higher values than CA and CC horses. One explanation for that could be that AA horses cannot produce offspring with genotype CC, that is, the ones with the poorest average performance. This will influence the breeding value of the horse, since breeding values takes into account the performance of relatives. The racing times are improved every year and it has been a strong selection on faster horses, especially in Standardbreds (Árnason, 2001). Today Nordic trotters are about 10 to 20 s slower per km than Standardbred trotters. As the overall selection pressure on harness horses is to produce faster horses with an even-beat trot, the racing times may become even lower. In the future, if the Nordic trotters reach kilometer times more similar to the present Standardbred population, we might see a further increase in the frequency of the A allele in this breed. The A allele seems to be more important the faster the horses race and Standardbred horses need to be homozygous for the mutated allele to perform well on the racing track, while in the Nordic trotters it is still enough with only 1 copy of the A allele. Except for DMRT3, there are other genes that influence performance in racing horses, for example, myostatin (MSTN). In Thoroughbreds there have been several studies showing a significant association between the MSTN genotype and preferred racing distance (Binns et al., 2010; Hill et al., 2010a,b). Petersen et al. (2013) showed that an intronic SNP of the MSTN was associated with a higher proportion of fast-contracting muscle fibers (Type 2B) and a lower proportion of the Type 1 fibers (slow-contracting fibers) in Quarter and Paint horses. Genetic variants in many additional genes are likely to influence racing performance in horses. Other factors that determine if a horse will become successful or not are, for example, diseases, training, and conformation. For example, in Norwegian Nordic trotters the conformation was responsible for 12% of the variation in earnings and 10% of the variation in start status (Dolvik and Klemetsdal, 1999). The estimated heritabilities for various performance traits are quite high (e.g., number of starts: 0.18; percentage of places 1 to 3: 0.35; earnings: 0.35; and best time record: 0.38; Árnason et al., 1989), even though performance and trotting ability are complex traits affected by different genes and environment. In this study we showed that including the genotype of DMRT3 in an animal model for the different performance traits gave a reduction in the residual phenotypic variance of between 0 and 3.9%. The reduction was higher for the Nordic trotters than the Standardbreds and one reason for that is that the mutated allele A is almost fixed in the Standardbreds. The positive impact of the mutated A allele on trotting performance is supported by our comparison of offspring with different genotypes from a famous breeding stallion with the CA genotype. Offspring with the genotype AA performed significantly better compared to those that were CA. The frequency of the A allele is very high in Standardbreds (98%) and has been so for quite a long time. The frequency in the Nordic trotter is much lower, and a reasonable explanation for that is that CA horses are favorable when it comes to trotting technique some trainers may prefer horses that have a natural talent for trotting, without any tendency to pace. The DMRT3 mutation is known for its favorable effect on alternative gaits, such as the pace. Therefore, it is very interesting that this mutation also have a strong influence on the trot, even though trot is a diagonal gait, unlike pace, which is a lateral gait. In a previous study by Drevemo et al. (1980) it was found that Standardbred horses showed some gait asymmetries when trotting in high speed. Some of the horses showed a transition gait, which is something in between trot and gallop, and normally it is shown when a horse is forced beyond their trotting capacity. Also it was shown that increased speed might lead to a decreased synchronization of the diagonal motion in trot (Drevemo et al., 1980). The DMRT3 mutation inhibits the transition from the symmetrical gaits, that is, trot or pace, to gallop and it also influences the stride length of the animal, as previously shown on mice (Andersson et al., 2012). It would be very interesting to perform a detailed genetic comparison of American trotters (AA) and pacers (AA), with the aim to identify modifying loci that must exist and explain the difference in gait preference on the same DMRT3 genotype background. Conclusions We show that the gait keeper mutation in DMRT3 has a favorable effect on the performance of Nordic trotters. The effects are similar to those observed in the Standardbreds, although for some traits the CA horses

8 4286 Jäderkvist et al. had as good or even better performance results than AA horses. The CA Nordic trotters also had a good trotting technique in contrast to Standardbreds with genotype CA that have much more problems with keeping an even-beat trot at higher speeds. These results are consistent with the general observation that the phenotypic effect of DMRT3 varies among breeds. The good performance of CA horses is a reasonable explanation for the lower frequency of the mutant allele in the Nordic versus Standardbred trotters. Nordic trotters homozygous AA had the highest percentage of victories and placings, even though the effect was not significant at all ages. They also had the highest EBV, but they were not the ones that earned most money during their entire career. For the majority of the traits analyzed, the Nordic trotters homozygous CC had the least good performance results. 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