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Photo illustration of horseA Horse of a Different Color:
Gene Mapping for Better Equine Health

by Alicia P. Gregory

Genes are destiny. DNA holds the secret to life.

"These are phrases that leap to people's minds when you talk about genetics," says Ernest Bailey, an immunogenetics researcher and professor at the Maxwell H. Gluck Equine Research Center at the University of Kentucky. "By creating a gene map, we are not going to unravel every secret. But the reality is that gene mapping is to the next century what vaccines and antibiotics were to this century.

"Vaccines and antibiotics have dramatically improved the health of people and the health of livestock. Research in the next century is going to have an equivalent benefit because of the application of the genomic information we are discovering today," says Bailey, who is leading the horse gene mapping effort at UK.

Photo of Ernest Bailey"A gene map for the horse will allow us to investigate the complex hereditary basis of behavior, performance and disease." --Ernest Bailey

Scientists' efforts to piece together maps of the total genetic code, or genome, for a number of species began in the late 1980s. The U.S. Human Genome Project began in 1990 with the goal of identifying each of the 100,000 genes in human DNA and assigning these genes to specific locations on chromosomes. The National Animal Genome Project, funded by the U.S. Department of Agriculture, began in 1993 to support collaborative efforts among American laboratories working on livestock. In April 1998 this project included, for the first time, support totaling $225,000 over five years to coordinate construction of a horse gene map.

Scientists have made significant progress on gene maps for humans and for the animals we eat or keep as pets: cattle, sheep, pigs, chickens, cats and dogs. The livestock projects began with a business-minded focus on identifying genes that could be manipulated to make leaner meat or thicker wool. Bailey says that although horses do not produce food or fiber they are a significant part of our "agricultural landscape" and contribute heavily to our economy.

To support this contention, he points to a 1996 study commissioned by the American Horse Council Foundation. The study estimates that the horse industry has a $112 billion impact on the nation's gross domestic product and generates 1.4 million full-time jobs. Goods and services produced by the horse industry are valued at $25.3 billion, roughly the same amount as the apparel manufacturing or the motion picture industries.

"A gene map for the horse will allow us to investigate the complex hereditary basis of behavior, performance and disease. But this concept concerns horsemen," Bailey says.

"One of my colleagues made the unfortunate statement awhile ago that in the future horsemen are going to have to understand molecular genetics. That simply isn't true. You don't have to be a mechanic to drive a car," Bailey says. "With this map geneticists are going to be able to give people in the industry better quality information to explain why things happen the way they do and how to avoid problems in the future. We are not going to be developing secrets that are going to destroy the industry."

In fact, Bailey says that much of the concern over the gene map's possible performance applications is unfounded. In March 1998, scientists, horsemen and veterinarians met for a conference focused on "Horse Genomics and the Genetics of Factors Affecting Horse Performance" at the Cold Spring Harbor Laboratory in New York. "One of the revelations for the breeders and veterinarians was that although we are doing these genetic maps, we really don't see a lot of point in going after performance genes," Bailey says. "That is something breeders are doing and have been doing effectively for years, so why should we bother?

"What we are concerned with is that while breeders are selecting for performance traits, these people assume that the related developmental bone diseases will just go away," he says.

Developmental bone diseases affect 10 to 25 percent of horses across a wide range of breeds, decreasing their performance and breeding potential. And often, although breeders are adept at selecting for performance, inherited defects negate those efforts.

"People spend a lot of money on horses. A lot of money is wasted and a lot of suffering is caused because of health problems. Our gene map will save money and create better health for horses," says Bailey.

Mapping the horse around the world
The horse branch of the National Animal Genome Project is part of a larger collaboration which includes laboratories in the United States, the United Kingdom, Australia, New Zealand, Japan, France, Sweden, Norway, Denmark, Germany, the Czech Republic and the Netherlands. In 1994, Bailey and other equine geneticists began to discuss their mutual interest in creating a horse genome map, but funds were a problem. "It wasn't plausible to say we would lead the effort to make a gene map when we didn't have our first dollar," Bailey says.

That same year UK received the first financial boost-a grant from the Grayson Jockey Club Research Foundation. "That grant was really the impetus for us to say, 'Okay, we're in the gene mapping business. We're going to put up the flag in Lexington, let everybody come, and we'll organize a workshop.'"

A second pledge of support from the Dorothy Russell Havemeyer Foundation made the workshop a reality. The Havemeyer Foundation, a small group in New York City that funds workshops on horses, committed to five years of funding for travel and meeting expenses. This money supported the first meeting in Lexington, and subsequent meetings in California and Sweden. Seventy representatives from around the world came to Lexington for this first meeting of the International Equine Gene Mapping Workshop in January 1995.

"Everyone came into this project with different reasons for making the map, but we all agreed that in order to have a successful workshop we had the necessary elements: we have a project that is larger than one laboratory can do, and we will all benefit by not competing," Bailey says. "If we competed to see who could put the most genes on chromosomes, we wouldn't be easily sharing information."

The creed of cooperation established at the first workshop has prompted researchers to circulate abstracts and papers before publication, an activity which required a level of trust some groups were initially reluctant to allow. "Scientists have to be a little competitive, so it took us about two years to achieve the open communication between laboratories we have now. When we find that everyone has different interests for applying the map, we don't mind helping each other because it doesn't take away from our own work."

A plan of attack
The horse genome contains between 60,000 to 80,000 genes-pieces of DNA which code for proteins and enzymes. In order to shed some light on where genes are located, scientists look for markers. "A marker is any measurable inherited difference that shows variation between individuals," Bailey says. The genetic variation scientists use as a marker can be something as small as a change in a single base in the DNA to a whole deleted section. "Ninety-five percent of DNA does not code for proteins. If we find a mutation in that so-called 'junk' DNA, we make that our marker," he says.

Each gene is a segment of the DNA on a chromosome. When the scientists sat down at the Lexington workshop, they realized that before mapping could begin they needed to establish a standard-to identify and number the 64 domestic horse chromosomes.

Photo of Teri LearTeri Lear is a leader in the field of cytogenetics, a branch of biology dealing with the study of heredity from a cellular and genetic perspective.

Teri Lear, a cytogeneticist from UK who works closely with Bailey, was one of the specialists eager to solve this problem. Cytogenetics is a branch of biology dealing with the study of heredity from a cellular and genetic perspective. Lear, one of 10 leading experts in horse cytogenicism in the world, met with her colleagues at the University of California, Davis, in 1995 to address the chromosome problem. "We got together for three days and did nothing but hash out which horse chromosome was which, according to their individual banding patterns, and wrote a description of each chromosome for everyone to use," says Lear, who is currently a research assistant professor at the Gluck Center.

With the chromosome standard in place, Bailey and Lear began the second phase of international mapping: getting samples out to 20 labs all over the world. "We became the coordinating laboratory for this," says Bailey. "DNA from 12 families of horses was sent here, and we distributed 460 blood samples to every laboratory."

A family includes one stallion and between 20 and 60 offspring. The families tested for the gene map were breeds from all over the world, including thoroughbreds, Shetland ponies, quarter horses, and Anglo-Arabs.

"We required each of the laboratories to test at least five markers and to test all of the families for these five markers," Bailey says. Each group of scientists sends their test results to a laboratory outside Paris, France, for analysis and then the data is pooled to create the map.

The tools to do the job
"When I was a student at the University of California, Davis, in the '70s, it was a big deal to discover genetic markers. In fact, my advising professor told me that if I managed to discover and describe three new genetic systems for the horse, I could earn my Ph.D.," Bailey says. "That's a simplification and kind of a joke, but it reflects how difficult this kind of activity was. But with the advent of molecular biology and molecular genetics, technicians can discover 20 or 30 new genetic systems in a year."

The horse genome contains between 60,000 to 80,000 genes-pieces of DNA which code for proteins and enzymes.

When Bailey started doing genetics, researchers could readily detect only about 30 genetic differences in horses. "We took these early markers and tried to use them to explain things like why some animals got sick and others didn't, and why some animals recovered better," he says. "We obviously didn't get very far trying to tie every trait of interest to 30 markers in a sea of 60,000 to 80,000 genes, but that's the point of the map: It allows us to tie genetics to the health traits we're interested in."

Today researchers worldwide have mapped 300 markers. He attributes this advance in part to the workshop and in part to the new technology that has made it safe and easy to map genes.

From the 1970s to the late '80s, scientists relied on a technique called in-situ hybridization (ISH), which used radiation to label DNA. The process took six weeks to map one gene, and if something went wrong the researcher didn't know until six wasted weeks later. With today's fluorescent labeling, these tests can be done overnight. Fluorescent in-situ hybridization (FISH) was first used in 1989, but wasn't widely used until the last five years.

"In FISH, we isolate DNA from the gene we want to study and apply that DNA to a chromosome spread from a horse. We want to know where that particular gene maps on the chromosome," says Lear. "The DNA we're applying is labeled with a fluorescent dye. Wherever the DNA finds homology-corresponding DNA-on the chromosomes, it will stick. We look at it under an ultraviolet light that's attached to the microscope, and we see the fluorescent signal where the gene is located on the chromosomes.

"Another form of this is ZOO-FISH, also known as chromosome painting," she says. "ZOO-FISH is when you place one species' DNA onto the chromosomes of another species. It's sort of a funny little term, but it helps you remember you're working with two different species. This permits us to identify large regions of the chromosomes which have been conserved, over time, between two species."

It's a small world after all
"One of the really great strengths of genomics research is that the genetic organization between species is very similar, so information developed in the human genome project, and information developed in the pig and cattle genomes can be applied to the horse," Bailey says. These genetic similarities allow researchers to predict fairly accurately what will happen before they test certain genes.

The similarities between the human genome and the horse genome were recently illustrated by a Swedish geneticist. By assigning each human chromosome a different pattern, he showed the distribution of human chromosome DNA over the 64 horse chromosomes.

"If you asked me 10 years ago what this would look like, I would have said it would be hash-little stripes all over the chromosomes," says Bailey. "In genetics you learn about evolution and chromosome rearrangements. You don't expect to see broad bands of similarity that have survived intact through evolutionary time."

In fact, though, large sections of DNA were the same in horse and human chromosomes. "All of the DNA from human chromosome eight is located on horse chromosome nine," Bailey says. "And because of that, we can draw information from the human genome and apply it very easily to our studies of the horse."

"People spend a lot of money on horses. A lot of money is wasted and a lot of suffering is caused because of health problems. Our gene map will save money and create better health for horses." --Ernest Bailey

In the late '80s when the other livestock-mapping projects began, Bailey says the horse geneticists knew making a map would be expensive and assumed they'd have to do years of work just to start mapping the horse. At the time, they decided they couldn't afford to drop their other projects.

"Since then, testing for genes has become much cheaper, so it came within our reach to make a map. We were impressed with the very early success other researchers had in mapping genes in cattle, pigs, sheep and chickens so we realized it really would be feasible for us to make a map to study health traits in horses," he says.

"We've basically done in three years what used to take scientists six or seven years to do. In part, we benefited by seeing what mistakes they'd made and by using the basic research they established."

The future of the horse map
The UK gene mapping project is supported by grants from the Grayson Jockey Club, the Morris Animal Foundation and Fares Farm Inc. Issam and Nijad Fares are prominent Lexington thoroughbred breeders known for generously supporting education.

Four full-time faculty and six graduate students are involved in the project. Bailey and Lear's lab includes graduate students Patrick Gallagher and Michelle Mousel. Faculty members Gus Cothran and Kathy Graves of the Equine Blood Typing and Research Laboratory lead a team of other graduate students.

"We've developed 80 markers for gene mapping and there are currently three maps-our workshop map, a map published by Swedish researchers, and a map that is coming out in England. Our markers are forming the background for all three of these," Bailey says. "That's a kudo of some sort because each map is using different sets of markers; but because we developed and shared ours very early in the mapping project, people used them quickly."

Bailey says the future of the horse map lies in collaboration, investigation and application. "We need to promote the map to the horse industry," says Bailey. "We've not done much of that in the last four years because we've not had a product. But now that we have a map, we need to spend some time explaining to people what it is and how we can use it."

"We are dependent upon the cooperation and enthusiasm of horsemen and veterinarians," Lear says. "They provide us with the blood samples from horse families and affected individuals that are the basis of our work."

A second key is more research. "We have the bare minimum for markers. To be honest, a couple of million dollars worth of research across horse labs worldwide would help us flesh out the map," Bailey says. "At this point, they have 2,000 markers on the pig and cattle maps, and those scientists don't believe that's enough. Across the three maps, we have a total of 300 markers. We've got a way to go."

And the final vital step, Bailey says, is applying the map to health problems. "We are ready to demonstrate our worth by tackling and shedding light on some real problems." Mousel and Gallagher in Bailey's lab are tackling two significant equine health issues: "strangles" and swayback.

Mousel, who came to UK this past fall with a master's degree in quantitative genetics from the University of Nebraska, is studying strangles. Streptococcus equi, a bacterial infection that attacks the lymph nodes, causes this disease. Often, the lymph nodes around the horse's windpipe become swollen and restrict breathing. The rasping sound made by these horses has led to the common name of strangles for this infection.

As part of the horse genome project, graduate students Patrick Gallagher and Michelle Mousel are tackling two significant equine health issues: "strangles" and swayback.

"I'm studying the series of genes that deal with the immune system lymphocytes, to see how we can help horses fight this infection," Mousel says. "Infected horses start off with cold-like symptoms, then typically the lymph nodes swell and burst, and the horse either gets better or it doesn't."

Strangles is a widespread, contagious disease. "My horse has had it and when I worked with Dr. Kelly Anderson in Lincoln, Nebraska, strangles went through the group and nearly killed one of the horses," Mousel says.

Gallagher, who has worked in Bailey's lab for more than a year, recently had a paper on his work on DNA markers accepted for publication by the journal Mammalian Genome.

"To some extent, the development of markers is based on the fact that each one is different," Gallagher says. "For this paper, I defined two families, based on areas of similarity in current markers, and I looked at what possible implications these similarities have on the use of markers." Gallagher says geneticists need to think about these similarities as they design experiments to test each genetic marker, to make sure they are using the unique portions, not the shared portions, of DNA.

Gallagher's particular interest in saddlebred horses led him to his current study of lordosis, or swayback, a problem for the American Saddlebred breed. "My dad has owned and shown saddle horses since before I was born," he says. "Swayback appears to be one of the major problems encountered by saddlebred breeders."

Lordosis seems to have a genetic basis, but before Gallagher works to isolate major genes responsible for this disease he is gathering information from horse breeders and trainers on other related factors. "American Saddlebreds are trained to hold their heads up high, which mechanically causes the back, over time, to drop," he says. "That's not genetic and could confound genetic studies."

Research laboratories around the world involved in the horse genome mapping project are beginning studies in health areas ranging from developmental bone and muscle diseases to genetics and allergic conditions.

Today the gene map is a developing information source for equine health researchers, but in the next century, Bailey says, the gene map will be the primary resource. "Scientists investigating the complex genetics of horse health are going to begin all their projects by asking, 'What is the gene for this? Where is it and how is it controlled?' And the map will tell them. We are laying the foundation for the advances of the next century."

These horse chromosomes were taken from a karyotype-a full set of chromosomes arranged according to number, size and shape. The distinct bands, the light and dark stripes, revealed by the karyotype serve as tools to diagnose genetic diseases.