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Volume 24: Issue 3 Dec. 2015


Warmest Wishes in this coming Holidays

CAMPOBELLO PERUVIANSDeer Park, WA IS PROUD TO PRESENT THREE FULL SISTERS BORN AND RAISED AT THE RANCH WHO ARE RECIPIENTS OF U.S. NATIONAL HONORS! CBP JAZMIN •  2013 Best Gaited Horse of Show •  2014 & 2015 Canadian National Ch. of Ch. Breeding Mare •  2014 NAPHA Region 1 High Point Horse CBP SERAFINA •  1999 Canadian National Ch. of Ch. Breeding Mare •  2000 AAOBPPH National High Point Gait Horse

CBP INDEPENDENCIA •  2009 BC & Alberta Regional Ch. of Ch. Breeding Mare •  2012 NAPHA Region 1 High Point Horse Horses Available for Sale – Training Available Contact us at 509-276-2735 or 509-703-0727

In This Issue Paso Peruano Europa Gait Keeper Mutation

BDS Carmelita 2012 Bay Filly RDLF Aro de Luna/RJM Mi Lucia. Beautiful 3 year old filly, AV Sol de Paijan. Currently in bozal training. Preparing for junior horse US$5,000.00

BDS Comandante 2010 Chestnut Stallion *Caporal/BDS No Me Olvides 2014, 1st year of showing, Ch of Ch’s Breeding Stallion & Ch of Ch’s Performance Stallion. Overall winner for the intermediate rider. US$10,000.00

BDS Santuaria 2011 Bay Mare

BDS Valiente 2009 Chestnut Gelding Currently in four reins, this *Caporal/SRO Marie mare has a lot to offer, great Multiple Ch of Ch Luxury Gelding mind, very willing, great for in US and Canada, Best Gait and show or trail. 2015 Canadian 1st Place US National Am. Regional 1st place Bozal Performance Gelding Performance. US$12,000 US$7,500 RDLF Aro de Luna x *Capilla

BDS Chubasco 2009 Liver Chestnut Gelding *Caporal/BDS Oro Pesa flashy, show and trail experience. US$8,000

BDS Sabrosada 2011 Chestnut Mare *Caporal/BDS Sabrosa Beautiful elegant mare started in bit training, above average gait. Some show & trail experience. Wonderful mind. US$7,500

Owner: Ben & Dori Sawatzky Managed By: Shannon & Cindy Zaitsoff Phone: (250) 558-4743 Email: Website: Vernon B.C CANADA

BDS Vencedora 2010 Chestnut Mare *Caporal/Luna Nova Show stopping gait, quiet temperament, all around versatile show mare, royal bloodlines. Her dam is US National Laureada Breeding Mare, her Sire, US national champion Get of Sire. US$15,000.00 BDS Cacique 2010 Chestnut Gelding *Caporal/*Capilla. Beautiful arrogant & strong. Finished in the bit. US$9,500

BDS Basilio 2012 Chestnut Gelding RDLF Aro de Luna x BDS Oro Pesa

Chestnut gelding, 15HH Started in the bit, heart of Gold! Ideal horse for Novice or Junior. US$5,000

Message From the President

Dar PHAC Members,

I am honored to serve as your president this year. For those whom I do not know personally, I will give an introduction: My husband Dale and I maintain a small breeding herd of Peruvian horses near Calgary Alberta. We both are also employed full time o the farm. Our first love is trail riding, but we are also active in showing. I have been on the Board of Directors of the PHAC for a number of years. The Peruvian Horse Association is in a strong financial position and holds its own in comparison with any registry of Peruvian horses worldwide. We have been fortunate to have good leadership, and importantly, good members who remain active with their horses and with the organization. My message to you today, is that each and every one of you is critical to the future of the PHAC. I invite you to communicate with me or another member of the Board, and let us know what is important to you. Are you interested in events where you can compete against other breeds and promote Peruvian horses? Would you like an enhanced trail rider mileage program? Would you be willing to volunteer at a show or at an exhibition? Your participation can make a very positive impact. Please enjoy a healthy and happy holiday season with your family, horses, and friends.

Mimi usk-Downey

PHAC Board of Directors Mimi Busk-Downey, President Box 449 Acme, AB T0M 0A0 Phone (403) 546-4331 Email: Kristin Elridge Vice-President

7431 Cokedale Rd.,

Sedro Woolley, WA 99284 phone: 360-856-9963 email:

Sherri Rosia, Secretary R.R. 1 Cochrane, AB T4C 1A1 Phone (403) 932-7032 Email Rick Cones, Treasurer

6027 VLA (Veteran) Road
 Chase, BC   
 V0E 1M0, Canada
 Phone:  250-679-2624 email: Cathy Taggart, Director 2004 Kelsey Rd., Armstrong, B.C. V0E 1B0 (250) 546-3704 email:


Gus McCollister, Exec. Secretary General Delivery Lyalta, AB T0J 1Y0 Phone (403) 935-4435 Fax (403) 935-4774 Email: Lynn Moker, Exec. Treasurer

RR1Red Deer, AB T4N 5E1 phone(403)-343-2814
 email: AWARDS COMMITTEE: Sherri Rosia Mimi Busk-Downey Shannon Zaitsoff




Box 207 Armstrong, BC V0E 1B0 Don Noltner, President Phone: (250) 832-1188 Email: Website:

PERUVIAN HORSE CLUB OF ALBERTA 11003 Oakfield Dr.SW. Calgary,AB T2W 3H3 Grant Mckinney, President

Email: Website:


740325 Sideroad 10, RR #3, Holland Centre,Ont. N0H 1R0 Louis Fialllos, President phone:(416) 576-5847 Email: Website: PERUVIAN ENTHUSIASTS & RECREATIONAL RIDERS UNLIMITED c/o Mimi Busk Downey, Secretary Box 449 Acme, AB T0M 0A0 Phone (403) 546-4331 Email:



! ! !

Peruvian Horse Association of Canada!! ! New Membership Application Name_______________________________________________________ Ranch Name__________________________________________________ Address______________________________________________________ Town_______________________Province______Postal_______________ Telephone________________________Email________________________ !!Owner-Breeder $45.00!


!!Aficionado $15.00!

Aficionado memberships are non-voting and do not require ownership of a Peruvian Horse. Owner-Breeder members receive the member rates for registration services. I / We qualify as Owner-Breeder members through the ownership of the following horse registered with the Peruvian Horse Association of Canada: Name______________________________________Reg. #_______________________! ! !"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"!"

The above application is for new members. Current members are billed directly by the CLRC, OTTAWA. Important: Current members please use the CLRC form with remittance OR quote CLRC ID number on this form here: ________________________

Make all cheques or money orders payable to: PHAC Mail to: Canadian Livestock Records Corporation (CLRC) 2417 Holly Lane, Ottawa, Ontario K1V 0M7 " "

" "

Peruvian Paso Horse Breeding & Training Center Located in Rocky Mountain House, AB - 150 acres near the Clearwater River

Horses for sale • Stallions at Stud • Visitors Welcome Training for 2016 season

JOE & KIM SHERIDAN • 403.510.8090

The European Championship – Paso Peruano Europa By Mimi Busk-Downey I had the pleasure of judging the Paso Peruano Europa championship in Aegidienberg, Germany, August 2014. The show was held over 4 days, at a large facility famous for gaited horses. Icelandic and Peruvian Horse are well known there and the stud farm is famous for having developed the Aegidienberger, a cross of the two breeds that is very popular in the area. This is pronounced ‘’Ah-gideon-burger.’’ The show was two years in planning, and exhibitors brought horses from as far away as Denmark, Austria, and southern Germany. Day One morning began with a number of dressage tests. These are well attended both with entries and spectators, and are held in a covered dressage arena with soft footing. The show secretary brought his laptop and acted as the scribe, entering the scores live into a spreadsheet so that when each class finished, the scores and placings were announced. There was a dressage judge, and he took the lead in the dressage classes, with the two of us collaborating on the scores for each maneuver. Day One in the afternoon, an Endurance Ride was held of 15 or 25 km, (exhibitors could choose), on a groomed trail. There was a guide / monitor with the group of riders – herself a judge of gaited horses and mounted on an Icelandic Horse. There were vet checks performed on the horses. Day 2 in the morning, the stallion and mare tests were held. This is judged by the State Judge (all breed) with collaboration from the Peruvian Horse Judge. This consists of the horses performing a Prueba of 30 minutes in gait on a hard track. After 10 minutes they must stop in front of the veterinarian and judges, dismount, then remain holding the horse for a few minutes while the heart rate is checked. The rider then must mount unassisted and the horse is judged on manners. The remainder of the 20 minutes is performed in gait. Every part of the competition is scored with regard to various criteria, including the character of the horse as observed. In order to receive a certification from the state, a horse must perform a dressage test, the stallion test, an endurance ride, a conformation class and the official gait class test, achieving a minimum score on each. These are called ‘’stallion tests’’ but mares are allowed to do them, and for both genders, their value as breeding stock is enhanced through completion. These tests are done for all breeds in Germany and the

standard is recognized by horseman as a measure of excellence. There are different levels of the test and Level A certificate must be completed before a Level B may be attempted. The choice of the Dressage Test and endurance ride determines which certificate can be achieved, but for each attempt all the elements must be completed within one four-day show. In most of the competition there were student judges. The Dressage judge who had the lead in the dressage tests switched to student judging during conformation and gait classes on the same day. Then for the main show, I had a student judge for Peruvian Horses who is a judge for Icelandic and Aegidienberger horses. She also acted as performed ring steward. On Days 3 and 4, classes found at most Peruvian horse shows were held. Merito Zootecnico classes were mandatory for every horse that was trying for a certificate and every horse that wished to qualify for a breeding championship. If a horse missed his conformation class he could not enter his ‘’Under Saddle In Bit’’ breeding class. The Zootecnico classes were scored in each area, so horses are not only placed but they receive a number score for each criteria. For example, legs, body, neck and head from the side, breed recognition (Type), way of going (at a walk) and character (the horse is approachable, well behaved). The aggregate score then carried forward to the breeding class! In other words, if the horse scored a 7 in conformation overall, then in the breeding class he would have a 7 in his conformation column and that could not be adjusted. So if conformation is 35% of the Breeding Class, he would have to be much better in gait and brio to achieve a first place over a horse that scored a 9 in conformation. I talked to the show secretary about the scoring system later, and pointed out that the show rules state that the horses are judged in comparison to one another and placed, *then* a score is applied. It does not exactly line up with the way the sheets are actually handled in the classes. It was important to score as I judged, so as not to use up ten minutes doing it at the end of each class. So the function of the ring steward was critical, because she could help with quick tallies to make sure the first place horse was first in the scoring and that the ordinal positions matched with the scores. The secretary is the son of a long-time breeder and he agrees with me that the horses must be placed in their order first, and that to judge primarily by scores can lead to the high placing of horses that a good overall and may miss horses that are extraordinary. Like all horses, extraordinary horses have faults. Horses with extraordinary positives that are hard to come by must be rewarded, and they should not be placed lower due to an aesthetic failing which does not affect their function. The conformation and performance classes were held on grass, but the breeding and gait classes were on a hard track covered in small crushed rock. Most of the horses wore shoes or boots. Shoes must be same all around and must not be designed to influence the way of going. There are many different gait classes, including classes where the judge has no control over the speed of the exhibitors – they are allowed to show the horse as they wish and the judging must be done on that basis. In the first of those gait classes, most horses were being shown very fast, and then the announcer called for an extended gait! Some had obviously worked hard to achieve a true singlefoot – it looked like they had sprouted wings! But several horses began to hop or attempt to gallop. The placings of horses have to be explained by the judge, so I took the opportunity to comment that in the paso classes, the gait must be a four beat evenly spaced gait without hopping or going to three beats, even when extended. The rest of the classes were shown somewhat slower, although in one class the most excellent fast horse did win, and later showed to also be excellent in a slow gait (much to my surprise). The best gaited horse of show was a horse that was very locked-in gait, loose, smooth, had excellent thread and very precise. There are a few classes for three gaited where a canter is called. My observation was

they were similar to the all-gaited shows I have judged, in that it is rare to see a three-beat canter performed correctly, and more rare still to see s horse with the talent to do that and good paso llano. In general showing format, there were many differences from North America. The horses have numbers, but the names of horses and riders are on the judging sheets and also announced when each one enters the class. I asked about his, saying that in North America the judge is not allowed this information. The response was that this way, it was fair, because all of the exhibitors get to be known, and called by name, rather than just the ones the judge knows. They also want to recognize and celebrate every entry, and the announcer would make ‘’color commentary’’ during the judging such as ‘’Mimi, are you finding these two horses similar? They are halfbrothers!’’ They do not expect the judges to be influenced by having more information. Another difference is that the show schedule is on time, with times published for each class. So if a spectator wants to come for a certain breeding class, scheduled for 2:15 PM, that class will not start early. Breaks occurred whenever we were ahead of schedule. There were less classes in a day than in North American shows. A two-hour lunch was standard, and the show finished every day at an hour that made it possible for exhibitors to care for their horses and eat a relaxed dinner at a restaurant. For most classes, the horses can be shown in any saddle and bridle but must use a Peruvian bit. A bozal, 4 reins, or bitless bridle is allowed in most classes. In breeding division classes the horses must wear full Peruvian gear including the guarnición. Attire is traditional in the breeding classes, and to match the saddle in all others, or whites can be worn, including white jodhpurs. However, ponchos are worn in nearly all classes even with other types of saddles, which adds some uniformity to the look. North American rules allow the use of other saddles but exhibitors rarely do that. In Germany, there were many saddles used including the Iberian saddles, dressage saddles, and Icelandic saddles. The show was enjoyable, interesting, with wonderful enthusiasm and camaraderie among the exhibitors. They congratulated each other and were genuinely happy to see another’s success. On Saturday night there was a group dinner, presentation of sale horses, and evening fun classes such as champagne. Then we were treated to demonstrations by exhibitors on natural horsemanship, liberty work, and bridle-less riding. To top off the night, there was an amazing Fire Dance by a talented young woman who happens to also be on the board of directors. With the arena in darkness, she lit torches and juggled them to loud rock music! It was an unforgettable performance that wowed the crowd. I know what dedication it takes to plan a show, especially with an evening performance. When I realized that performances were mostly done by the organizers themselves, it underscored the afición of this group of owners and their love for their horses. !

Selling Peruvian tack. Complete saddle outfit. Wide tree. $800 Burgundy bridle set. Almost new. $500 Call Dorothy Blain Located in Duncan BC. 778-422-1543

“A ride through the sacred valley of the Inca, from Cusco to Machu Picchu, on a Peruvian Paso is a haunting, spiritual experience. The dramatic setting in the Andes features sheer cliffs and lush valleys.” - National Geographic, top ten horseback rides in the world Are you ready to challenge yourself for the ride of a lifetime? This highly specialized tour is not for everyone. This exclusive tour is limited to six riders and is not an experience for the casual rider. Much of the riding is in the Andes Mountains, and portions are somewhat challenging, therefore requiring energetic horses and intermediate to advanced riding skills. The welfare of the horses is paramount to Perol Chico, our riding tour operator, and there is a strictly enforced rider weight limit of 85 kg (190 lb). Only two spots left! April 9 – 10, 2016 – Lima April 11 – 12 – Cusco April 13 – 16 – Incredible horseback riding in the Sacred Valley April 17 – Rest and day tour in Cusco April 18 – 20 – More incredible trail riding April 21 – take the amazing train ride to visit Macchu Picchu April 22 – 24 – Lima and the Peruvian Horse National Show Visit our website for details

Facilitating personal growth through experiencing the gentleness, beauty, majesty and wisdom of the horse. I learned so much from my experience with you, Colorado and my man Ricky! I’ve made many changes, taken numerous strides outside of my box and feel I am a better person for doing so. – CO Jocelyn is a certified life and executive coach, and is also certified in the Equine Gestalt Coaching Method ®. Contact by phone at (403) 601-2500 or visit the website at

“Where the trail horses are show ready” Jocelyn Hastie is the founder of Celestina Ranch. The first foal out of her breeding program, RJT Colorado Real++, was born in 1994. This team won many Photo by Michele King Championships until his retirement from the show ring in 2006. Partners for life, Photography he now assists Jocelyn in her equine facilitated coaching and personal development program ( She continues to compete in the show ring and loves to share this remarkable breed with the public, participating in Horse Haven at the Calgary Stampede for the last twenty years.

Contact Jocelyn by phone at (403) 601-2500 or visit our websites at or

Box 214, Armstrong BC V0E 1B0

PHCBC Board of Directors for 2016   

President    Vice President           Treasure                                         Directors at Large  Don Noltner   Tracy Brown                 John McMillan                     Rob Sjodin                         Deb Cones  Salmon Arm, BC  Aldergrove, BC             Armstrong, BC                     Salmon Arm, BC               Chase, BC       (250)835‐8472  (604)626‐0011             (250)546‐6621                     (250)832‐1188                 (250)679‐2624                          


Activities coming up next year: Plans are being put together for our annual Clinic for 2016 Stay tuned on our website for updates WILD WEST CLASSIC PHCA & PHCBC DOUBLE REGIONAL SHOW June 3-5 2016 ARMSTRONG, BC Honourable Judge: PHCA, TBA Honourable Judge: PHCBC, TBA The Interior Gaited Horse Show June 11-12, 2016. Approved points for Peruvian Horses Contact John McMillan,

PHCBC Memberships are due January 1st 2016 Membership for 2016 will stay at $30.00 for full & $15.00 for Aficionado’s Our new mailing address is Box 214 Armstrong, BC V0E1B0 Go to our website for membership form and to read The Paso Llano PHCBC’S Newsletter


doi: 10.1111/age.12120

Worldwide frequency distribution of the ‘Gait keeper’ mutation in the DMRT3 gene M. Promerov! a*1, L. S. Andersson†1, R. Juras‡, M. C. T. Penedo§, M. Reissmann¶, T. Tozaki**, y***†††, R. Bellone††, S. Dunner‡‡, P. Ho"r!ın§§, F. Imsland*, P. Imsland¶¶, S. Mikko†, D. Modr! K. H. Roed‡‡‡, D. Schwochow†, J. L. Vega-Pla§§§, H. Mehrabani-Yeganeh¶¶¶, N. Yousefi-Mashouf¶¶¶, E. G. Cothran‡, G. Lindgren† and L. Andersson*† *Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden. †Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden. ‡Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4458, USA. §Veterinary Genetics Laboratory, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. ¶Breeding Biology and Molecular Genetics, Faculty of Agriculture and Horticulture, Humboldt University, 10115 Berlin, Germany. **Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya, Tochigi 320-0851, Japan. ††Department of Biology, University of Tampa, Tampa 33606, USA. ‡‡ Veterinary Faculty, Universidad Complutense de Madrid, 28040 Madrid, Spain. §§Institute of Animal Genetics, CEITEC, University of Veterinary and Pharmaceutical sciences, 612 42 Brno, Czech Republic. ¶¶Menntaskolinn vid Hamrahlid, Hamrahlid 10, 105 Reykjavı´k, Iceland. ***Department of Pathology and Parasitology, Faculty of Veterinary Medicine, CEITEC, University of Veterinary and Pharmaceutical sciences, 612 42 Brno, Czech Republic. †††Institute of Parasitology, Biology Centre of Academy of Sciences of the Czech " ! Republic, v.v.i., 612 42 Cesk e Bud" ejovice, Czech Republic. ‡‡‡Department of Basic Sciences & Aquatic Medicine, Norwegian School of !n Aplicada, Cr!ıa Caballar de las Fuerzas Armadas, 14080 Cordoba, Veterinary Science, N-0033 Oslo, Norway. §§§Laboratorio de Investigacio Spain. ¶¶¶Department of Animal Science, University of Tehran, 54500 Tehran, Iran.


For centuries, domestic horses have represented an important means of transport and served as working and companion animals. Although their role in transportation is less important today, many horse breeds are still subject to intense selection based on their pattern of locomotion. A striking example of such a selected trait is the ability of a horse to perform additional gaits other than the common walk, trot and gallop. Those could be fourbeat ambling gaits, which are particularly smooth and comfortable for the rider, or pace, used mainly in racing. Gaited horse breeds occur around the globe, suggesting that gaitedness is an old trait, selected for in many breeds. A recent study discovered that a nonsense mutation in DMRT3 has a major impact on gaitedness in horses and is present at a high frequency in gaited breeds and in horses bred for harness racing. Here, we report a study of the worldwide distribution of this mutation. We genotyped 4396 horses representing 141 horse breeds for the DMRT3 stop mutation. More than half (2749) of these horses also were genotyped for a SNP situated 32 kb upstream of the DMRT3 nonsense mutation because these two SNPs are in very strong linkage disequilibrium. We show that the DMRT3 mutation is present in 68 of the 141 genotyped horse breeds at a frequency ranging from 1% to 100%. We also show that the mutation is not limited to a geographical area, but is found worldwide. The breeds with a high frequency of the stop mutation (>50%) are either classified as gaited or bred for harness racing. Keywords ambling, domestication, horse, locomotion, pace, running walk

Introduction Address for corrrespondence L. Andersson, Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-75123 Uppsala, Sweden. E-mail: 1

Equal contribution.

Accepted for publication 19 November 2013


Few animals have been of such great value to humans as horses when it comes to means of transportation. All over the world, horses have been used for everyday transportation, in military settings, cattle herding and agricultural power, pulling carriages and carts, pleasure riding or racing. Over the centuries, horse populations and breeds

© 2014 Stichting International Foundation for Animal Genetics, 45, 274–282

The ‘Gait keeper’ mutation in horses have been shaped by humans based on the different purposes for which the animals were used. For instance, in breeds used for riding over long distances, as those used by nomads and cattle herders, it was important that the horse moved in a way comfortable for the rider. Later, these traits also were valued for trail riding as well as show performances. In horses used for harness racing, breeders selected for the ability of a horse to trot or pace at high speed. The ambling gaits are particularly comfortable for the rider, and horse breeds exhibiting these gaits are referred to as gaited. Some gaited horses also are able to perform pacing, which is a lateral two-beat gait. The ambling gaits have a four-beat rhythm with the footfall pattern of a walk, but often reaching speeds comparable to, or even exceeding, the trot without any aerial phase typical of the faster gaits (Robilliard et al. 2007). Ambling gaits are classified into numerous types, nearly as many as there are gaited breeds. Gaits are often characteristic of a particular breed (for a kinematic study see Nicodemus & Clayton 2003). Some horses are not able to learn a desired gait, others require extensive training, whereas some have a natural talent for ambling gaits. In some breeds, mostly from the New World, such as the Puerto Rican Paso Fino or the Tennessee Walking Horse, very strong selection has been applied, and basically all individuals of the breed are naturally gaited. In other breeds, such as the American Saddlebred or Mangalarga Marchador, both types of performances are utilised; thus, a fraction of the population is gaited and the rest cannot perform the ambling gait(s). By contrast, in many breeds, such as horses bred for show jumping or high-speed gallop racing, a pacing or ambling phenotype is considered inferior, and these traits have been strongly selected against. Until recently, the genetic basis and the mode of inheritance of gaitedness were unknown. However, one of the founders of genetics, William Bateson, proposed as early as 1907 based on breeding records that pacing may be a recessive trait in horses (Bateson 1907). A recent study in fact demonstrated that a single-base substitution in the double-sex and mab-3-related transcription factor 3 (DMRT3) gene has a major impact on the ability of a horse to pace and amble (Andersson et al. 2012), revealing the strong genetic basis for this trait. The mutation (cytosine to adenine), initially discovered in Icelandic horses, causes a premature stop codon and thus a truncation of the DMRT3 protein (Andersson et al. 2012). The mutation (DMRT3_ Ser301STOP) also was denoted Gait keeper due to the strong effect on the pattern of locomotion. DMRT3 is one of the eight known genes that are homologous to the doublesex gene of Drosophila and the mab-3 gene of Caenorhabditis elegans (Raymond et al. 1998). These genes encode transcription factors with a DM DNA-binding domain. The most well-studied members of this gene family have an established role in control of sex differentiation, but the expression of some members of the family in tissues other than

gonads suggests other functions (Hong et al. 2007). In fact, DMRT3 is expressed in neuronal cells, and detailed studies in mice showed that it is expressed in inhibitory interneurons with projecting ipsi- and contralateral axons that make direct synaptic connections to motor neurons present in a specific region of the spinal cord (Andersson et al. 2012). Furthermore, the characterisation of Dmrt3 knockout mice confirmed that the DMRT3 protein plays a pivotal role for coordinating limb movements (Andersson et al. 2012). Screening several horse breeds for the DMRT3 mutation revealed that the occurrence of this mutation is dichotomous with an allele frequency of nearly 100% in six tested gaited breeds and zero in seven tested non-gaited breeds and in the Przewalski’s horse (Andersson et al. 2012). In addition to the strong association with ambling gaits, this mutation was shown to occur in breeds used for harness racing, pacers as well as trotters. The mutated allele was shown to be associated with superior racing performance in Swedish Standardbred trotters and to be fixed in American Standardbred trotters and pacers (Andersson et al. 2012). This finding suggests a more complex effect of the DMRT3 nonsense mutation on locomotion in horses by extending the range of speed at which horses keep a symmetrical gait, such as trot or pace, instead of switching to the asymmetric gallop, the ancestrally preferred gait at high speeds. In this study, we genotyped the DMRT3 stop mutation in additional horse breeds with wide geographical distribution. Our goal was (i) to investigate in which parts of the world the mutation occurs, (ii) to investigate whether the distribution of the mutation follows the distribution pattern of gaited horses in a wider sample set than previously studied and (iii) to test whether the mutation is strictly restricted to gaited breeds or might occur also in breeds considered non-gaited.

Material and methods Sample collection We obtained (i) extracted DNA from the Animal Genetics Laboratory, SLU, Sweden, and (ii) DNA, hair or blood samples collected by collaborators, provided by horse owners or archived at the Veterinary Genetics Laboratory, University of California, Davis, USA. DNA from blood samples was extracted using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instruction. DNA from hair samples was extracted using standard hairpreparation methods. Details of breeds and sample sizes are provided in Table 1. A total number of 4396 horses representing 141 horse breeds were included in the study.

SNP genotyping, PCR and Sanger sequencing of the DMRT3 coding region The DMRT3_Ser301STOP mutation (chr23: 22 999 655 bp) and a SNP 32 kb upstream of it (BIEC2_620109, chr23:

© 2014 Stichting International Foundation for Animal Genetics, 45, 274–282



Promerov" a et al. Table 1 Genotype and allele frequencies of the DMRT3_Ser301STOP mutation in different horse breeds. Breeds are sorted based on gaitedness (where this information was available) and alphabetical order of the breed names. Genotype

Frequency of A allele (%)


Country of origin1



American Paso Fino Campolina Colombian Paso Fino Cretan Horse Florida cracker Hokkaido Horse Icelandic Horse4 Kentucky Mountain Saddle Horse4 Missouri Fox Trotter4 Peneia (Pinia) Peruvian Paso4 Puerto Rican Paso Fino4 Rocky Mountain Horse4 Tennessee Walking Horse4 American Curly American Saddlebred Appaloosa Asturcon Basuto Pony Boer Pony Brazilian Criollo Colombian Trocha Pura Colombian Criollo Trocha y Galope Faeroe Pony Kirgiz Lewitzer Mangalarga Mangalarga Marchador Marajo Marwari Miniature Horse Mongolian Morgan Newfoundland Nooitgedacht Pindos Pura Raza Galega Rhodes Shackleford Banks Sierra Tarahumara Spanish Mustang Spanish Pure Breed Turkmen Welsh Cob Venezuelan Criollo Akhal-Teke Altai Arabian4

USA Brazil Colombia Crete, Greece USA Japan Iceland USA

34 18 80 66 24 48 219 25

31 15 75 63 15 28 117 21

3 2 1 3 2 13 93 4

0 1 4 0 7 7 9 0

95.6 88.9 94.4 97.7 66.7 71.9 74.7 92.0

42 17 22 78 27 54 45 89 20 24 30 20 21 67 4

42 16 22 77 27 54 0 6 1 0 0 0 0 2 0

0 1 0 1 0 0 15 37 3 0 4 6 1 10 2

0 0 0 0 0 0 30 46 16 24 26 14 20 55 2

100.0 97.1 100.0 99.4 100.0 100.0 16.7 27.5 12.5 0.0 6.7 15.0 2.4 10.4 25.0

21 31 20 14 22 14 9 109 134 50 26 14 15 3 6 41 18 15 46 20 11 21 43 25 69

0 7 0 0 5 0 0 1 2 1 0 1 1 1 1 1 2 0 0 0 0 0 0 0 0

6 11 3 2 10 2 0 4 8 12 6 4 5 1 3 7 3 3 1 4 0 7 0 4 0

15 13 17 12 7 12 9 104 124 37 20 9 9 1 2 33 13 12 45 16 11 14 43 21 69

14.3 40.3 7.5 7.1 45.5 7.1 0.0 2.8 4.5 14.0 11.5 21.4 23.3 50.0 41.7 11.0 19.4 10.0 1.1 10.0 0.0 16.7 0.0 8.0 0.0

39 15 15 15 1 3 14

0 0 0 0 0 0 0

0 0 0 0 0 0 0

39 15 15 15 1 3 14

0.0 0.0 0.0 0.0 0.0 0.0 0.0

Ardennes4 Barb Black Forest Chestnut Breton British Riding Pony Camargue Canadian

USA Greece Peru Puerto Rico USA USA USA USA USA Spain South Africa South Africa Brazil Colombia Colombia Faeroe Islands Kyrgyzstan Germany Brazil Brazil Brazil India USA Mongolia USA Canada South Africa Greece Spain Greece USA Mexico USA Spain Iran Great Britain Venezuela Turkmenistan Russia (West Siberia) Middle East, Syria, Iran Belgium North Africa Germany France Great Britain France Canada



Chi-square values for HWE test2


0.07 3.45 62.27*** 0.04 15.84*** 5.23* 3.27 0.19

Gaited Gaited Gaited Gaited Gaited Gaited Gaited Gaited

NA 0.02 NA 0.00 NA NA 1.80 0.16 1.98 NA 0.15 0.62 0.01 2.74 NA

Gaited – foxtrot, pace Gaited Gaited – paso Gaited – paso Gaited – singlefoot, rack Gaited – running walk Some – foxtrot Some – rack, slow gait Some Some? Some? Some Some Some – trocha Some – trocha

0.58 2.14 0.13 0.08 0.15 0.08 NA 10.78** 12.23*** 0.00 0.44 0.32 0.07 NA NA 0.65 3.94* 0.19 0.01 0.25 NA 0.84 NA 0.19 NA

€lt, pace Some – to Some – pace Some – pace Some – marcha Some – marcha Some Some – revaal Some – pace Some Some – singlefoot Some Some Some Some Some Some Some Some Some? Some – pace Some – pace Some Not gaited Not gaited Not gaited


Not Not Not Not Not Not Not

– paso – marcha – paso – singlefoot €lt, pace – to – singlefoot

gaited gaited gaited gaited gaited gaited gaited

© 2014 Stichting International Foundation for Animal Genetics, 45, 274–282

The ‘Gait keeper’ mutation in horses Table 1 (continued)


Frequency of A allele (%)

Chi-square values for HWE test2



Country of origin1

Caspian Clydesdale Colombian Criollo Trote y Galope Comtois Connemara Pony Czech Warmblood Dafur Pony Dartmoor Pony Dølahest Don D€ ulmen Pony Exmoor Fell Pony Fjord French Saddle Horse Friesian Galiceno German Riding Horse German Riding Pony Gotland Pony4 Haflinger Hucul

Iran Great Britain Colombia

52 17 35

1 0 1

7 0 8

44 17 26

8.7 0.0 14.3

1.15 NA 0.16

Not gaited Not gaited Not gaited

France Ireland Czech Republic Sudan Great Britain Norway Russia Germany Great Britain Great Britain Norway France Netherlands Mexico and Spain Germany Germany Sweden Austria Czech Republic and Poland Hungary Russia (Caucasus) Sudan Czech Republic Japan Czech Republic Denmark Poland Iran Russia (Western Siberia) Latvia Germany Slovenia and Austria Spain Portugal Great Britain Norway Austria Sweden USA France Poland Spain Asia USA Germany Russia Hungary Great Britain Great Britain Portugal Germany Great Britain Indonesia Sweden Sweden

6 35 19 14 18 16 16 7 27 15 27 19 15 26 11 10 29 18 23

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

6 35 19 14 18 16 16 7 27 15 27 19 15 26 11 10 29 18 23

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not

gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited

11 10 10 19 24 37 25 28 17 11 3 20 8 10 19 42 30 18 34 49 35 14 10 27 104 4 14 19 55 19 16 3 19 6 2 64

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 6 0 0 0 8 2 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0

11 10 10 19 24 37 25 28 15 11 3 20 8 10 19 35 30 18 34 41 33 14 10 27 100 4 14 19 55 19 16 3 19 6 2 64

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.9 0.0 0.0 0.0 0.0 0.0 0.0 9.5 0.0 0.0 0.0 8.2 2.9 0.0 0.0 0.0 2.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0


Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not

gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited gaited

Hungarian Coldblood Kabardian Khartoum Pony Kinsky Horse Kiso Kladruby5 Knabstrup Konik (Polish Primitive) Kurd Kuznet Horse Latvian Liebenthaler Lipizzaner Losino Lusitano New Forest Nordland Noriker North Swedish Horse4 Paint Percheron Polish Heavy Horse Potoka Przewalski’s Horse4 Quarter Horse Rhineland Heavy Draught Russian Riding Horse Shagya Arabian Shetland Pony4 Shire Sorraia South German Coldblood Suffolk Sumba Swedish Riding Pony Swedish Warmblood4



© 2014 Stichting International Foundation for Animal Genetics, 45, 274–282





Promerov" a et al. Table 1 (continued)

Genotype Breeds

Country of origin1




Taishu Thessalian Thoroughbred4 Timor Pony Tinker Trakehner Waler Welsh Mountain Pony Welsh Pony Yakut Yamut Zemaitukai Coldblood Trotter French Trotter4 German Trotter Hackney Pony Orlov Trotter Standardbred Pacer4 Standardbred Trotter4 Standardbred Trotter4 Bashkir Pony Cheju Israeli Local Jordanian North Sudan Pony Retuertas Tushuri Cxeni (Tushuri Horse) Viatka

Japan Greece Great Britain Indonesia (Timor) Great Britain Germany Australia Great Britain Great Britain Russia (Siberia) Iran Lithuania Sweden France Germany England Russia USA Sweden USA Russia South Korea Israel Jordan Sudan Spain Georgia Russia

24 10 55 6 11 34 15 16 54 25 17 15 306 59 9 35 5 40 270 57 29 14 9 5 9 10 15 2

0 1 0 0 0 0 0 0 0 0 0 0 50 36 4 1 0 40 253 57 0 0 0 0 0 0 4 0

0 3 0 0 0 0 0 3 8 0 1 0 176 20 2 1 2 0 17 0 1 4 1 1 0 2 6 0

CC 24 6 55 6 11 34 15 13 46 25 16 15 80 3 3 33 3 0 0 0 28 10 8 4 9 8 5 2

Frequency of A allele (%)

Chi-square values for HWE test2


0.0 25.0 0.0 0.0 0.0 0.0 0.0 9.4 7.4 0.0 2.9 0.0 45.1 78.0 55.6 4.3 20.0 100.0 96.9 100.0 1.7 14.3 5.6 10.0 0.0 10.0 46.7 0.0

NA NA NA NA NA NA NA 0.17 0.35 NA 0.02 NA 7.98** 0.01 NA 14.87*** NA NA 0.29 NA 0.01 0.39 NA NA NA NA 0.58 NA

Not gaited Not gaited Not gaited Not gaited Not gaited Not gaited Not gaited Not gaited Not gaited Not gaited Not gaited Not gaited Harness – some pace Harness Harness – some pace Harness Harness – some pace Harness – all pace Harness – some pace Harness Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown


Country of origin refers to origin of the breed, not necessarily the origin of the samples. Chi-square values for tests of Hardy–Weinberg equilibrium (HWE) are given in breeds where applicable; degrees of freedom = 1. 3 Summary of information on the degree and type of gaitedness that we have been able to collect based on breed descriptions. Gaited, the breed is generally considered gaited; some, a fraction of the horses of this breed are gaited, whereas others are not; not gaited, gaited horses are generally not observed for this breed; harness, breed used for harness racing; unknown, information on gait not found. Gait descriptions: lateral gaits, two beats: pace; lateral gaits, four beats (denoted in parenthesis are differences in speed, rhythm, liquidity of movement as specified by breed specific names): €lt; diagonal gaits, four beats: foxtrot, paso gaits (fino, corto, largo, llano, marcha), rack, revaal, running walk, slow gaits (stepping pace, singlefoot), to trocha, marcha batida; ? means that gait classification is uncertain. 4 All or some of the horses in this breed were included in the previous study by Andersson et al. (2012). 5 The material included 23 Black Kladruby and 14 Grey Kladruby horses. *P < 0.05; **P < 0.01; ***P < 0.001. 2

22 967 656 bp) were genotyped; the latter SNP was the only SNP that showed a statistically significant association with the ability to pace in the previous genome-wide association analysis (Andersson et al. 2012). The DMRT3_Ser301STOP mutation was genotyped using a custom-designed TaqMan SNP Genotyping Assay (Applied Biosystems) as previously described (Andersson et al. 2012). The BIEC2_620109 SNP was also genotyped with a TaqMan SNP Genotyping Assay that included the following reagents: forward primer, 5′-GCAAAGTGCAGAAATAGTCTTTTGGA-3′; reverse primer, 5′-CACTCTTTTGGAATGGTTCACATTAAGG-3′; wildtype probe (FAM), 5′- TAGTGCAAACGGTACGTT-3′ and mutant probe (VIC), 5′-AAATAGTGCAAACAGTACGTT-3′. The amplification was carried out in a final volume of 5 ll, with 2.5 ll of Genotyping Master Mix (Applied Biosystems),

0.125 ll Genotyping Assay, 0.875 ll deionised water and 1.5 ll DNA. Thermal cycling consisted of 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s and 60 °C for one min. For individuals genotyped in Uppsala, where TaqMan genotyping did not yield any results for the stop mutation (n = 39), and for some individuals, where it was deemed interesting to investigate whether they might possess any other DMRT3 mutation, the DMRT3 coding region was PCR-amplified and Sanger-sequenced as previously described (Andersson et al. 2012).

Data analysis Sequences were edited using Codon Code Aligner (CodonCode Corporation). Pairwise linkage disequilibrium between

© 2014 Stichting International Foundation for Animal Genetics, 45, 274–282

The ‘Gait keeper’ mutation in horses

Icelandic Horse Faeroe Pony


Miniature Horse Appaloosa Missouri FT American PF Spanish Mustang American Curly Quarter Horse Standardbred P&T Sierra Tarahumara

American Saddlebred

CC Trocha y Galope Peruvian Paso

N<20 20≤N≤50

Tushuri Cxeni

Morgan Kentucky MSH Shackleford Banks Rocky MH Paint Florida Cracker Tennessee WH Puerto Rican PF

Colombian PF CC Trote y Galope


Orlov Tro!er Bashkir Pony


Turkmen Yamut Kurd



Coldblood Tro!er

Venezuelan Criollo

Colombian TP

Mangalarga Marchador

Marajo New Forest


Welsh P&MP



Standardbred Tro!er

Hackney Pony

Brazilian Criollo


Hokkaido Horse


Israeli Local Jordanian Caspian

Boer Pony Nooitgedacht

German Tro!er French Tro!er Basuto Pony

Percheron Retuertas


Spanish PB

Peneia Cretan Horse

Thessalian Rhodes

Figure 1 Frequency distribution of the DMRT3 gait-altering mutation for breeds with at least five samples tested. The sizes of the pie-charts reflect sample size. Only breeds for which the mutation was observed are shown. The following abbreviations are used in the figure: CC, Colombian Criollo; FT, Fox Trotter; MH, Mountain Horse; MSH, Mountain Saddle Horse; PB, Pure Breed; PF, Paso Fino; P&MP, Pony and Mountain Pony; P&T, Pacer and Trotter; TP, Trocha Pura; WH, Walking Horse.

the DMRT3_Ser301STOP and BIEC2_620109 was tested in ARLEQUIN (Excoffier et al. 2005) using 1000 permutations. PHASE implemented in DNASP v5 (Librado & Rozas 2009) was used to reconstruct the gametic phase of the two SNPs, with default settings. Hardy–Weinberg equilibrium (HWE) for the DMRT3_Ser301STOP SNP was tested for in breeds that displayed both wild-type and mutant alleles and where sample size was at least 14 individuals (53 breeds). Allele frequencies were visualised as pie-chart graphs in Microsoft EXCEL with the background of the world map created in PANMAP (Diepenbroek et al. 2000).

Results We generated DMRT3_Ser301STOP genotypes for a total of 4396 individuals belonging to 141 breeds (Table 1, Fig. 1). The number of samples per breed varied from one (British Riding Pony) to 306 (Swedish Coldblood Trotter), with an average of 31 samples per breed. The mutation was observed in horses from 68 breeds. Within these breeds, the frequency of the mutant allele (A) varied from 1.1% (Spanish Pure Breed, n = 46) to complete fixation in some North and South American gaited breeds (Table 1, Fig. 1). Nine of the tested breeds showed significant deviations from HWE (Table 1). A subset of 2749 horses also was genotyped for SNP BIEC2_620109, which is located about 32 kb upstream of DMRT3_Ser301STOP (Andersson et al. 2012). The two SNPs are significantly linked according to the pairwise linkage disequilibrium test in ARLEQUIN (v2 = 5314, df = 2, P < 0.0001). The genotype frequencies at these two SNPs were used to estimate the haplotype frequencies using PHASE

Table 2 Estimated haplotype frequencies for the DMRT3_Ser301STOP mutation (C>A) and the closely linked SNP BIEC2_620109 (C>T) in a total of 2749 horses across different breeds.

Haplotypes Wild type at both loci Mutant 620109 and wild-type DMRT3 Mutant 620109 and mutant DMRT3 Wild-type 620109 and mutant DMRT3

BIEC2_ 620109






3237 109

0.589 0.020









n, number of chromosomes.

software (Table 2; Phase probabilities for all cases 1.00). This analysis revealed two predominant haplotypes across all breeds. C-C (i.e. the reference allele at both SNP loci) was the most common haplotype (59%; Table 2). T-A was the other abundant haplotype (39%), with the non-reference allele (T) at the BIEC2_620109 SNP and the DMRT3 nonsense mutation (A). The third most common haplotype (2%) was T-C, with the non-reference allele (T) at the BIEC2_620109 SNP and the reference allele (C) at DMRT3. The simplest interpretation of these data is that T-C represents the ancestral haplotype on which the DMRT3 nonsense mutation arose and that the T-A haplotype has increased in frequency because of strong positive selection for the gait mutation. This interpretation is supported by the observation that the T-C haplotype was observed in 16 breeds that lack the DMRT3 nonsense mutation (Table S1), whereas no breed displayed only the DMRT3 nonsense mutation in the absence of the BIEC2_620109 SNP

© 2014 Stichting International Foundation for Animal Genetics, 45, 274–282



Promerov" a et al. non-reference (T) allele. The fourth haplotype (C-A) was found in the heterozygous state in only 19 horses, representing six breeds, and of 5498 haplotypes in total (0.3%, Table 2, Table S1). This haplotype has most likely originated through recombination between the C-C and T-A haplotypes.

Discussion The present study has demonstrated that the DMRT3 gaitaltering nonsense mutation has a worldwide distribution. It was detected in 68 of 141 breeds included in the study. The very strong linkage disequilibrium between this polymorphism and the closely linked BIEC2_620109 SNP across all tested breeds implies that this mutation arose once and has spread across the world by positive selection. The positive selection is most certainly related to the appreciation of the smooth ride offered by ambling gaits as well as the increased performance capacity of mutant horses when raced in gaits other than gallop. The mutation is abundant in all breeds classified as gaited, for instance the Hokkaido Horse and the Icelandic Horse, and in horses used for harness racing. However, the mutation also occurs at a low frequency in many other breeds not classified as gaited, such as the Welsh and New Forest Ponies from the UK. The presence of the DMRT3 mutation in breeds that are not generally classified as gaited might be explained by a preference for ambling gaits in the past, by historical crossing with gaited breeds or by the mutation having segregated in the ancestral population prior to breed formation. Cross-breeding has been used frequently to improve vigour of breeds that were nearly extinct and suffered from inbreeding, or to introduce a desired conformation, character or colour trait. One such example might be the Israeli Local horse, which has been influenced by the Tennessee Walking Horse amongst other breeds (Hendricks 1995) and in which we observed the mutation at a frequency of 5.6%. The Miniature Horse is a breed with an influence of many different breeds in the past, and in our data set, the DMRT3 mutation occurs at a frequency of 3.1% (Table 1). Similarly, the Bashkir Pony (allele frequency 1.7%) is known to have been cross-bred to, amongst others, trotters (Hendricks 1995). If gaitedness was not disadvantageous in such populations, one can surmise that there was little or no selection applied to eliminate it, and thus, the mutation could persist in the population at low frequency. Similarly, little or no selection would be applied if the phenotypic effect of the mutation is not obvious, as may be the case in the heterozygous state. It will be of considerable interest to study genotype– phenotype relationships between DMRT3 mutant and wildtype horses from such ‘non-gaited’ breeds. We observed a significant deviation from expected Hardy–Weinberg proportions in nine of the breeds included in this study, which is more than expected by chance. There

are many possible reasons why significant deviations from HWE may occur, including non-random mating, population substructure, selection and cross-breeding, but may also be due to genotyping errors. We are convinced that the deviation from HWE in this study is not due to genotyping errors but rather reflects selection and non-random mating given that the Gait keeper mutation has a major effect on a crucial trait in the domestic horse. All gaited horse breeds that we tested carry the DMRT3_Ser301STOP mutation, and many of them at a very high frequency or in complete fixation. However, there are exceptions where individual horses claimed to be gaited have tested negative. The best documented cases concern Icelandic horses for which at least some horses homozygous C-C for the wild-type allele can amble (t€ olt) to a variable extent, but are unable to pace. Another mutation in the coding sequence of DMRT3 has been excluded for these individuals, which leaves the possibility of (i) a high-impact DMRT3 mutation outside the coding sequence, (ii) a high-impact mutation affecting another gene important to regulation of locomotion or (iii) a polygenic effect. There has been strong selection over many generations in the Icelandic Horse for the ability to amble with perfect smoothness. It is possible that this strong selection pressure led to enrichment for mutations promoting ambling capacity so that even horses that are homozygous wild type at DMRT3 can amble, with good training and riding. Thus, an important topic for future research is to screen for additional loci affecting gaitedness in horses. In fact, the discovery of the DMRT3 mutation in horses shows that the domestic horse is probably the best animal model available to study the genetic control of locomotion because of the very long history of selection for different types of locomotion (speed in gallop, trot and pace, high jumping and dressage). It has long been discussed whether gaitedness is a trait that the domestic horse inherited from its ancestors or acquired during the process of domestication. In support of the former hypothesis, a study on footfall patterns of three individuals of Hipparion (dated to 3.5 Myr ago) found that they were moving at a running walk, equivalent to the t€ olt of the Icelandic Horses (Renders 1984). However, gaitedness in domestic horses must be a derived trait given that it is so strongly associated with the DMRT3_Ser301STOP mutation. It is clear that this is a recent, derived mutation because all tested Przewalski’s Horses, a close relative to the wild ancestor of the domestic horse, were homozygous wild type at DMRT3. We also extracted sequence data from two recently published genome sequences of ancient horses (Orlando et al. 2013), and only the wild-type sequence was found. Furthermore, the mutation disrupts the DMRT3 coding sequence that is otherwise maintained in all vertebrates for which sequence information is available (Andersson et al. 2012). Our current characterisation of the haplotype associated with the DMRT3_Ser301STOP mutation does not have the

© 2014 Stichting International Foundation for Animal Genetics, 45, 274–282

The ‘Gait keeper’ mutation in horses resolution required to firmly conclude when and where the mutation arose. This will require re-sequencing of the 440 kb region showing strong linkage disequilibrium with the gait mutation (Andersson et al. 2012) to identify diagnostic polymorphisms that were present on the ancestral haplotype or that have occurred on the mutant haplotype subsequent to the causal mutations. The present study provides an excellent screening for horses to be used in such a detailed haplotype analysis. Tracing how humans have spread the DMRT3 mutation across the globe using modern samples is challenging because the frequency of the DMRT3 mutation is strongly influenced by the preferred use of horses. For instance, Icelandic horses originate primarily from Scandinavian horses brought by the settlers in the 9th century, and the Icelandic sagas strongly indicate that horses with the ability to pace were present on Iceland 1100 years ago (!Islendingasagna! utg! afan 1946). However, we did not find the DMRT3 mutation in contemporary horses representing breeds originating from Scandinavia (e.g. Fjord, Gotland Pony and North Swedish Horse), with the exception of Coldblood Trotters used for harness racing. A possible explanation is that the way horses are used in Scandinavia has changed since the 9th century when Iceland was settled and that Scandinavian horses were for a long period of time more extensively used as draught horses in agriculture and forestry, which was not the case on Iceland. A general trend in our analysis across breeds is that the DMRT3 mutation is rare or absent in draught horse breeds. Furthermore, the DMRT3 mutation is very common in horse breeds from both South and North America, particularly in the USA, as proved by several of the American breeds showing complete fixation of the mutant allele (Fig. 1, Table 1). All American breeds are derived from horses originating from Europe, particularly the Iberian Peninsula (Rodero et al. 1992; Vega-Pla et al. 2005; Luis et al. 2006). Although we observed the mutation in Spanish horses, it was present at a very low frequency. Nevertheless, we did not have any samples of the extinct jennet type, which might have been the type of horse first taken to the Western Hemisphere by the early colonisers and which was known to be gaited (Hendricks 1995). Thus, a possible reason for the low frequency of the gait mutation in modern Spanish horses is that ambling is considered a negative trait in most Spanish breeds because the use of horses has changed since the time Spanish conquistadores arrived in the Americas. To conclude, the extensive worldwide sampling shows that the DMRT3 Gait keeper mutation is not restricted to a geographical range but has spread across the world. In general, it follows the distribution of breeds classified as gaited and breeds used for harness racing, but it also appears at various frequencies in other breeds. It is still unclear where the mutation arose. A large-scale analysis of the whole haplotype should shed more light on this intriguing question.

Acknowledgements We most sincerely thank all sample contributors: Jennifer Leonard (Retuertas), Yekrun Horse Industry Development Co. Ltd. (horses from Iran), Dr. T. Ohnuma (Hokkaido Horses), Lutfi Musa (Sudanese breeds), Sonia Zakizadeh (Yamut and Turkmen), Sergey Knyasev (Altai and Kuznet Horse), Ines Wemken (Barb horses), Julia McCann (American Saddlebred Horses) and Mario Calcagno (Marwari). We are grateful to Dr. Deb Bennett for help with gait classification, Reto Burri for discussions and Markus S€ allmanAlm!en for extracting published sequence data from ancient horses. The study was financially supported by the Knut and Alice Wallenberg Foundation.

Conflict of interest Leif Andersson, Lisa Andersson and Gabriella Lindgren are co-inventors on a patent application concerning commercial testing of the DMRT3 mutation.

References Andersson L.S., Larhammar M., Memic F. et al. (2012) Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice. Nature 488, 642–6. Bateson W. (1907) Trotting and pacing: dominant and recessive? Science 26, 908. Diepenbroek M., Grobe H. & Sieger R. (2000) PANMAP, http://www. Excoffier L., Laval G. & Schneider S. (2005) ARLEQUIN (version 3.0): an integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1, 47–50. Hendricks B. (1995) International Encyclopedia of Horse Breeds. University of Oklahoma Press, Norman, OK, USA. Hong C.S., Park B.Y. & Saint-Jeannet J.P. (2007) The function of Dmrt genes in vertebrate development: it is not just about sex. Developmental Biology 310, 1–9. !Islendingasagna! utg! afan (1946) !Islendinga s€ogur. Hrappseyjarprent hf. Reykjav!ık, Iceland. Librado P. & Rozas J. (2009) DNASP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–2. Luis C., Bastos-Silveira C., Cothran E.G. & Oom M.D. (2006) Iberian origin of New World horse breeds. Journal of Heredity 97, 107–13. Nicodemus M.C. & Clayton H.M. (2003) Temporal variables of fourbeat, stepping gaits of gaited horses. Applied Animal Behaviour Science 80, 133–42. Orlando L., Ginolhac A., Zhang G. et al. (2013) Recalibrating Equus evolution using the genome sequence of an early Middle Pleistocene horse. Nature 499, 74–8. Raymond C.S., Shamu C.E., Shen M.M., Seifert K.J., Hirsch B., Hodgkin J. & Zarkower D. (1998) Evidence for evolutionary conservation of sex-determining genes. Nature 391, 691–5. Renders E. (1984) The gait of Hipparion sp. from fossil footprints in Laetoli, Tanzania. Nature 308, 179–81. Robilliard J.J., Pfau T. & Wilson A.M. (2007) Gait characterisation and classification in horses. Journal of Experimental Biology 210, 187–97.

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