Mitotyping Project
By Loren Bolinger 9/14/2005, Wednesday, September 14, 2005

Unlike previous scientific inquiries, my first impulse was to present our project directly to the typical Thoroughbred breeders across the USA and internationally. The Internet and several breeders' e-mail discussion groups proved useful to query a wide range of breeders directly at minimum cost. I was curious as to the interest of other Thoroughbred breeders to my passion and obsession. I also thought that it would be more likely to pick up a wider range of unusual matrilines, scientifically interesting, and suitable candidates for sampling. Some of the matrilines are rare or obscure and more likely to be found in the hinterlands. These rare matrilines may have important relevance to our project.

One, very satisfying observation I've made, is the surprising numbers of horse breeders sophisticated about maternal lineages, mitochondrial inheritance, the Lowe family numbers of their horses, and the potential of my mitotyping project to not only accurately resolve known identity problems, discover unknown, longstanding mis- identifications, but also for its application by horse breeders to future selective matings. All horse breeders passionately want to improve their stock. Accurate identification of lineage is crucial to many selective breeding schemes.

The response to my mtDNA inquiry has been warmly rewarding to me through the very large volume of e-mail and overall enthusiasm of horse breeders internationally. I had planned to place small advertisements in industry publications to get a wider range of samples, but now must wait additional funding.

I have received tremendous grassroots response to what some might regard as a rather obscure, technical inquiry. I can assure the Jockey Club that my informal and anecdotal survey reveals that a majority of Thoroughbred breeders are, in fact, tremendously interested in the potential of mitochondrial research, more accurate identification of maternal lineages and therefore, more accurate identification of most all Thoroughbreds in the current population to the current limits of modern scientific methods. The biological knowledge and advances in scientific technologies are accelerating at a mind-spinning rate - new discoveries coming almost day-by-day. We are in a new golden age of genomic discovery. It would be unfair to the breed we love to not take advantage of these advances to preserve and conserve the Thoroughbred horse. For the most part, we still use archaic selective breeding practices at a time when the biological sciences are making major contributions the health and welfare of humans and many species of domesticated and wild plants and animals. If the knowledge learned from mitotyping could be used to reduce the needless wastage and injury, minimize genetic defects and diseases, conserve the breed for the future, while improving selective mating techniques that produce the modern Thoroughbred racehorse, the effort and expense would be well worth it. To my mind, it is a win - win situation.

I strongly believe that ultimate responsibility for the identification of the Thoroughbred lies with the keepers of the General Stud Book, such as the Jockey Club and other international keepers of the studbooks in their countries. They have the existing recorded data, the authority, and the resources. There are an unknown number of undiscovered haplotypes in lineages of the current Thoroughbred population. One of my intentions is to attempt to discover as many of the unknown haplotypes as my means allow; the purpose being to make known the entire number of surviving haplotypes comprising the modern Thoroughbred breed. I believe this is an important project and I hope that my tiny mtDNA study will inspire and encourage the appropriate authorities to create and fund an international project to identify and verify all Thoroughbreds to the limits of our technologies as far back as possible.

Yours truly,
Loren

Thoroughbred horses as unique research subjects:
An Ideal Model for Studies of Inheritance and
Selective Mating
By Loren Bolinger

I have been a Thoroughbred horse breeder for over 36 years in New Mexico. I have a passion for selective mating, historical research, and improvement of the breed. I believe that improvement to the breed can be made by breeders through selective mating. Many horse breeders since the founding of the breed have had an anecdotal belief in maternal inheritance. In 1894, Charles Bruce Lowe classified and ranked Thoroughbreds by matrilineal lineage and each family's success at elite races. His was also the first widely publicized [among Thoroughbred horse breeders], systemic attempt to document and prove maternal inheritance and the validity of certain forms of selective mating. During the 1960's biologists discovered the importance of the mitochondrial genome and established incontrovertible proof of the second system of biological inheritance - maternal inheritance through DNA located in the cytoplasm of the ovum. Mitotyping is the term used for methods that identify the true matriline of the individual under examination. Errors in Thoroughbred parentage classification of many historical individuals' lineages have propagated through entire branches of their family trees to the present GSB, and even when known, have never been possible to verify and corrected.

Now that there are accurate, reproducible, and cost effective techniques, it is especially important to breeders to verify and correct matrilineal lineages. Any sort of reasonable attempt at breed improvement through selective mating depends on accurate lineages.

Goals include reconnection of the lost American, Australian Colonial, Argentinean families with their English roots [and other unidentified members of the current Thoroughbred breeding population]. Another goal is to untangle, separate, and correct the misidentified, incorrectly recorded English lineages that occurred primarily in the 1800's. Yet another goal is to assess the connection between mitochondrial inheritance and the expression of performance traits.

Dr. Phillip W. Danielson, University of Denver and Dustin Mark Gilbert, our research assistant, are skilled and highly trained in DNA forensics. Dr. Danielson conducts training seminars for law enforcement and investigative personnel in crime scene procedures. Their protocols and procedures are accurate, rigorous, comprehensive, and reproducible. The mitotyping we use produces cutting edge accuracy and reproducibility [better than 99.9 per cent accuracy]. They have trained me in mtDNA collection, data collection, and fieldwork. Our DNA technology is far cheaper, faster, and more accurate. We are using the same procedures used in criminal forensics. Our lab is currently processing about 35 - 50 samples per week with only one research assistant.

1. Our laboratory procedures used for processing buccal swabs, blood, and hair samples incorporates duplicate methods used by the US Jockey Club for parentage verification and registration. Our hope is to eventually gain access to the DNA repository of the Jockey Club to continue this important research.

2. Our procedures and protocols are compatible with those used by Dr. Emmeline Hill reported in Animal Genetics, August 2002 [pp. 287-294]. We have verified and reproduced results as reported in "History and Integrity of Thoroughbred Dam Lines Revealed in Equine mtDNA Variation."

Why the Thoroughbred
The Thoroughbred horse represents an unusual research opportunity with potentially far reaching implications for all species. Nearly all plants and animals chosen for research are relatively randomly mated, wild mated, mated without human-caused selection, or mated with selection for simple or single gene traits. Often, inappropriate "selfing" or other radical selection practices are used in the laboratory and often on strains of animal models completely divorced from the real world.

In contrast, among all domesticated animals, the Thoroughbred is mankind's longest lived and most successful, selective-mating experiment. It has had a relatively consistent goal over approximately three hundred fifty to four hundred years - the test of the winning post. That the selective mating strategies have been successful can be assessed by the physical changes in the organism since its founder stock. The phenotypes of the modern day Thoroughbred, evolved over about 350 - 400 years, can also be compared against those of other breeds of horses. This particular breed, developed through specialized selective mating, performance testing, and culling, is unique among all domesticated animals.

It is generally agreed that multi-factorial genes (and possibly) maternal inheritance control traits involved with elite performance. Research into inheritance of multi-factorial traits has been extremely difficult and complex. Traits inherited through the mitochondrial genome are not well understood. The Thoroughbred brings a unique genetic background that is better suited to the unraveling of the conundrum of understanding complex traits. It is unique in the organization, selection, and culling of its lineages and individuals. Historically, all forms of selective mating experiments have been tried.

The large body of Thoroughbred horse breeders is highly motivated to improve horse breeding techniques yet for the most part biological research has ignored the Thoroughbred. The horse among domesticated animals has had a far stronger economic impact today than the proportionate research dollars spent on its behalf would indicate. The North American Thoroughbred industry has an economic impact as reported in the Wednesday, June 29, 2005 electronic edition of the Thoroughbred Times of $39.2 billion , an economic impact study in Australia revealed the contribution of the Australian Horse Industry was estimated at 6.3 billion in 2001 , and in Great Britain, in 1999 [most recent year], it was estimated that the economic impact of the British horse industry was about 2.5 billion, second largest economic activity in the British countryside only to farming , yet Thoroughbred breeders have received little attention from the biological sciences. We are basically still using crude, eighteenth- century selection practices and/or many breeders are governed by non- genetic issues such as commercial appeal, fad, and fissionability. [The element of uncertainty added by misclassification of lineages should be unacceptable to horse breeders intent on breeding the very best individuals possible incorporating selective-mating methods with correction, verification, and finally accuracy of lineages using scientific objectivity from the genomic era. An unacceptable variable can be easily eliminate by modern biological science.]

Current Progress
Dr. Emmeline Hill [et al 2002] and Dr. Phillip Danielson [Gilbert, Bolinger et al 2005 preliminary] have shown that mitochondrial DNA analysis is an easy, cost effective, accurate, and reproducible way to identify and characterize horses by their families or maternal lineages.

Analysis by both groups [above] has shown large discrepancies in the GSB [General Stud Book] classifications. Most errors seem to have arisen from misclassification of horses very early in the bloodlines of the Thoroughbred.

There seems to be far fewer founding mares [matriarchs] than originally believed due to convergence of many families into one haplotype and the fact that diverging families seem to result from misclassification rather than multiple founding mares or mutations.

Lastly, the founding mare population was more than likely small due to the very limited and controlled breeding by the early English founder breeders in the initial stages of the species' development.

With our limited, current database, our first goal is to continually gather more data, especially on families that have not yet been sequenced.

The next step while we continue to enlarge our database would be to begin tracking down American, Australian, Argentinean, and other lost bloodlines and connecting them back to their English ancestors.

Extremely valuable horses are being created with 19th century selection methods. Far too much wastage and injuries occur, and needless, catastrophic breakdowns occur. In the amazing explosion of the current Genomic Era, it seems relevant to employ biological discoveries in breed improvement, conservation, humane treatment, and preservation of the Thoroughbred. The possibilities exist that significant progress can be made to reduce unsoundness and wastage in the Thoroughbred through breakthroughs in scientific selective mating.

We wish to understand the contribution toward elite athletic performance that comes through maternal inheritance - the mitochondrial DNA contained in the cytoplasm that is inherited only through the tail-female lineage. In order to accomplish this objective, the accuracy of the lineages must be first restored or corrected. Until this is done, selective mating schemes are useless. If I didn't have the help of a top genetic laboratory and one of the best forensic geneticists in the United States, Dr. Phillip Danielson, this project would never have been possible.

My personal aim is to disclose the pure research, hopefully, if it has any merit at all, published in a scientific journal and on the TBHeritage website, like the other matriline discoveries. The goal is to be able to produce practical results that breeders can apply, not ivory tower research that only gathers dust and cobwebs and only satisfies intellectual curiosity. I'm a pragmatist, I wholeheartedly believe in maternal inheritance with respect to performance traits, have put my limited resources where my mouth is, and incorporated my beliefs in my breeding program. I am satisfied [at my level of participation in the horse breeding business] the results I am getting. It would be very satisfying to have biological evidence to back up my passion for breeding horses.

Yours truly,
Loren