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Q&A: Uppsala University's Leif Andersson on Using SNP Chips, Sequencing to ID Horse Gait SNP


leif.jpgName: Leif Andersson

Title: Professor of Functional Genomics, Uppsala University, Sweden

Education: PhD, Swedish University of Agricultural Sciences, 1984

UPPSALA, Sweden — Uppsala University professor Leif Andersson has spent most of his career studying the correlation between genotypes and phenotypes in domesticated animals both to better understand the genetics of particular species and to answer broader biological questions. In recent months, Andersson and colleagues have published studies focused on chickens, pigs, and dogs, but their paper about horses published last week in Nature has drawn considerable attention from both the scientific and horse breeding communities.

Working with researchers from the Swedish University of Agricultural Sciences and other international collaborators, Andersson and colleagues used a combination of SNP arrays and high-throughput sequencing to discover a mutation in a single gene in horses that they claimed is "critical" for the ability to perform ambling gaits and for pacing; and has a major effect on performance in harness racing.

As noted in the study, horses show considerable variation in their patterns of locomotion. The three naturally occurring gaits in all horses are, in order of increasing speed, walk, trot, and gallop. Some horses can use alternate gaits though, typically at intermediate speed, and "gaitedness" is a trait upon which many specialized breeds have been developed. One of these alternate gaits is pace, a two-beat gait in which the horse moves the two legs on the same side of the body in a synchronized, lateral movement, in contrast to the trot, where the diagonal front and hind legs move forward and backward together.

According to the paper, many Icelandic horses — but not all — have the ability to pace, and genetic test scores for pace show high heritability. This interest in the underlying genetic factors of pacing in Icelandic horses led Andersson and colleagues to discover that a single base change in DMRT3 is associated with the ability. Based on that finding, the researchers tested for the mutation in other horses and discovered that it is widespread among those breeds that show alternate gaits like the Tennessee Walking Horse from the USA and the Paso Fino from South America. Moreover, the researchers found that the mutation is common in horses bred for harness racing.

BioArray News discussed these findings and more with Andersson in Uppsala last week. Below is an edited transcript of that interview.

From what I understand, most of your work focuses on animal genetics

We are applying new technologies to study important biological questions. The unique profile for my group is that we have been doing comparative genomics using primarily domestic animals as a model. Most people who work with domestic animals are trained as veterinarians or animal scientists, whereas I am trained as a biologist. We want to use domestic animals as a tool to understand the correlation between genotypic variation and phenotypic variation. For instance, what genetic changes have been critical for transforming a wild animal to a domesticated animal. I think a very beautiful illustration of our approach was published last week in Nature about a mutation that affects the gait in horses.

The thing is that humans have lived together with domestic animals for the last 10,000 years. And during this period we have selected animals to fit certain preferences, for carrying us around, for meat. In this particular case, what must have happened is there was this mutation that occurred at some point, which could potentially have occurred before domestication, but then it was rare, because there was no advantage for a wild horse to have this mutation, but people who use a horse for riding found that horses that carry this mutation are different because they give a smoother ride. It moves its legs a bit different and people liked that, because if you spend a good part of your life on the back of the horse, that's very valuable. So they selected for this trait, and the number of horses with it increased and spread all over the world.

We came across this issue because we wanted to study Icelandic horses where half of the population can pace and the other half of the population cannot pace and then we screened them. In human genetics, if you want to screen for disease, you may take 5,000 patients and 5,000 controls. We took 40 horses that could pace and 30 that could not pace. So it was, of course, a small sample. But we got this beautiful signal on chromosome 23 and were able to nail it down to a single base change in a transcription factor, it's called DMRT3. What we learned is that this transcription factor is expressed in a very specific set of neurons in the spinal cord. And what we discovered was a previously unknown mechanism for controlling movement in vertebrates. If you look at the human genome, there are still large numbers of genes of which we have a poor understanding of the function, and this is one of them. It belongs to a family of transcription factors that plays a role in sex and development, so there is really no clue as to why this one would be important in locomotion.

How exactly did you do identify the mutation?

We screened the horses with a genome-wide association analysis of 50,000 markers using the Illumina EquineSNP50 BeadChip. Then we got the signal on chromosome 23, a very distinct signal, very defined. Instead of targeted sequencing of this particular region, though, we sequenced the whole genome of a horse that could pace and a horse that could not pace, but we knew from the GWAS where to look. We think it is a dominant negative mutation, so we don't know yet if these neurons are gone in those horses who cannot pace, or if they have an altered function. We know that the gene is still expressed in the spinal cord of the horse. And then for the next step, we asked the question, "So where is this mutation found?" Horses are classified as gaited or non-gaited. Gaited horses are those that can take up other gaits, the standard gaits, and then we realized that all gaited horses that we tested have this mutation. You have for instance the Tennessee Walking Horse and the Rocky Mountain Horse in the United States, and the Peruvian Paso and Paso Fino from South America. They all have unusual gaits, and they all carry this mutation. We continued and found that horses selected for harness racing also have this mutation at a high frequency, trotting horses and pacing horses. Another implication is that this mutation inhibits the transition from the trot to the gallop. Because as a horse goes fast, it goes from walk to trot to gallop, but in harness racing, you are not allowed to gallop, so you have to trot at a very high speed. This mutation inhibits this transition, allowing the horse to keep a trot without moving into the gallop. It has a direct industrial application in horse breeding.

After we found this association, we wanted to know why this was the case. There is a stable outside Uppsala where we had 61 Swedish Standardbred horses, which are used for trotting, and some had problems keeping the trot. So we did a blind test. We genotyped all 61 and then we picked up two that were heterozygous for the mutation as the other 59 were homozygous for the mutation. And exactly these two had a problem keeping a trot. In this Nature paper, we have a supplementary figure with a horse showing how it has problems, because when it trots it tends to shift over to the gallop, it doesn't have this clean trot that you need to successfully race.

I will add that in the US, there is the American standard horse that was established around 1850, a famous stallion called the Hambletonian 10. They found it was more convenient to have a trotting horse than a galloping horse, and then this horse began to compete against other horses in races and harness racing. The Swedish population is comprised of some imported American standardbreds crossed with local Swedish breeds creating the Swedish Standardbreds. So when we genotyped the American Standardbred, all that we tested were homozygous mutants. But in Sweden this mutation is not completely fixed. And this is why you find these horses in Sweden that do not completely trot, but the majority is homozygous.

From what I understand, you used the SNP chips and then you did whole-genome sequencing.

We did first the association study and then we identified a 600 kb interval. Then we just sequenced everything using the HiSeq. Why did we not do targeted sequencing? Well, in this case we were sure we could classify them as mutant and wild type, so we took one of each, and it's not that much more expensive to do whole genome than targeted, because you have to order the chip for targeted sequencing. So we did a whole genome and focused on the 600 kb-interval and what mutations differed between these two horses. And then we were able to find this mutation in this transcription factor, that is such an obviously functionally important change, and we were able to verify that this was indeed the causal mutation.

To what extent do you interact with breeding organizations or companies?

There has been a special interest from them. In harness racing in the world, there are two main kinds of horses, the French Trotter and the American Standardbred. They have a different origin, but the thing is that French Trotters often do not have as clean a trot as the American Standardbred. So it may be problematic to breed the French trotters with the American Standardbred because a considerable number will get gait problems. Now, you could use a DNA test and make better decisions in your breeding programs. So I think this raises great opportunities for breeding in the trotting industry, but also for all of the other breeds of horses. As a follow-up study, we are screening 100 horse breeds all over the world, to see which breeds have this particular mutation. A large number of breeds have this, and they could be interested in establishing horses that are particularly useful for some applications and not for others.

Have these organizations that have shown interest adopted newer technologies to do this kind of horse breeding?

Most are doing traditional breeding, but there are companies coming up that would like to provide tests for the horse breeding industry. Our mutation has a very strong association to the phenotype they are selecting on, so for that reason it will be quite widely used because it is directly affecting the phenotype of the horse. But, you know, if you compare it with breeding cattle, that is much more organized by companies, organizations, whereas horse breeding is more like a hobby, some breeding is larger scale, more hands on, but I think they are certainly interested in moving in that direction. What we have found is a mutation with a major effect. There may be many other minor mutations that influence performance of racing horses in general. So, we may see a similar trend in cattle breeding, where they use SNP chips to scan the whole genome for markers for milk production.

Are there any repercussions in terms of how heterogeneous these horses are? Could further breeding for this trait create a bottleneck down the road?

They are not that homogeneous. They retain a certain amount of heterozygosity. It depends on how many breeding individuals you have. In horses, you have to have many, many mares, so that affects the breeding outcome. In Sweden, I think there may be 4,000 mares that are used for breeding in the Swedish Standardbred. But of course the number of stallions used for breeding are much more restricted.

Is this trait inherited from the fathers?

No, you could say it is a recessive autosomal trait because the most favorable genotype is those that are homozygous for the mutation. As I said, the American Standardbreds appear all to be homozygous for this particular mutation but they are segregating at most other loci. An interesting aspect is that we have found that Icelandic horses will pace when they are homozygous for this mutation but the Swedish Standardbred doesn't do pace, it trots despite that most horses are homozygous for the mutation. Thus, the same mutation is associated with two different phenotypes. This mutation has a big effect on gait, but it interacts with its particular background. The thing is that in the US they decided to split the American Standardbred into pacers and trotters. In the 1950s, they started to see that some of these were better pacers than trotters, so they were able to split the population into two. So our next screen will be to look at pacers and trotters to see if we can pick out something that is explaining the difference between them, because there could be another factor in this pathway.

How did you become interested in the horse project to begin with?

The big question in modern biology, and really what the Human Genome Project has been about, is to understand the link between genetic variation and phenotypic variation. In human, it is done to fit a disease to a genetic composition to learn what genes are related and by having this information get better drugs for treating diseases. But in biology you would like to understand evolution. What are the critical differences between a human and a chimpanzee? I think it is almost mission impossible to reveal that, it is too complex a question, we don't have the tools to answer that. What is unique in domestic animals is that over the past 10,000 years we have changed a wild boar into a pig. We have changed a wild bird (the red junglefowl) living in Southeast Asia to a high egg-producing and meat-producing chicken. And this is a unique opportunity to see how genetic variations influence individual characteristics. Horses are a beautiful example. We were able to nail down this key change. This is not to say that gait is controlled by a single gene, but that a single gene has a major effect on gait.

What other projects are you planning?

One of the projects that we are contemplating is to study rabbit domestication. Rabbit is an interesting species because it was domesticated very recently. Cattle and pigs were domesticated about 10,000 years ago, but rabbit was domesticated only about 1,500 years, and we know exactly where it happened, it was in south France, around the year 500. At that time, rabbits were only found on the Iberian Peninsula and in southern France. The Pope at that time made a statement that rabbit meat is not meat, so that you could eat it during Lent. So, we think there are some unique features. It's an interesting event, so we will sequence wild rabbits very carefully to see if there is diversity in the true wild rabbit variation in southern France and on the Iberian Peninsula, and then compare it with stocks of domestic rabbits in an attempt to see what was critical during rabbit domestication.

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