by Beth Minnich, with Michael Bowling

What Does the DNA Say?

Using Genomic Tools in Preservation Breeding by Beth Minnich & Michael Bowling

Preservation breeding represents the interface of history, genetics, and biology with the practical side of horse breeding. Due in large part to the Human Genome Project, the past 30 years has brought significant advancements in the fields of genetics and genomics. Using knowledge and technology garnered from this landmark project, the first map of the horse genome was completed in 2007, with the third version (EquCab3.0) released in 2018. Along with research devoted to simple and complex diseases, color, and athletic performance, growing areas of interest include investigation of genetic diversity, breed ancestry, mtDNA, and the Y chromosome.

Genomic tools have been in use in the Arabian horse community for decades; with the test for SCID available since 1997 and DNA profiling (including parent verification) being part of Arabian horse registration since 2002. Prior to the implementation of DNA profiling, bloodtyping of stallions was initiated in the 1970’s and parent verification via bloodtyping started with 1991 foals. With the rate at which research is expanding and the increased speed of test development, updates from the lab are frequently announced. At times, the information can be overwhelming and confusing, not to mention controversial. This can be especially true as owners work to understand how the current ‘science’ impacts their horses and breeding programs. When the research has implications for the selection of breeding stock, especially where small population closed groups are involved, it becomes crucial to discuss and understand this work. purchasing horses and determining breeding selections. Inaccurate pedigrees can significantly impact these decisions by erroneously assigning individuals to incorrect lineages (or even breed identification), resulting in identification errors, inaccurate estimates of inbreeding and relatedness, and misguided decision making. The process of DNA profiling and parent verification helps ensure accuracy at registration, resulting in reliable identification and pedigrees for use by owners and breeders. • DNA profiling, currently done with microsatellite markers, is a unique form of genetic identification. DNA profiles provide a base for conducting parent verification for registration purposes. These ‘DNA fingerprints’ also provide a method to confirm identity of a horse should a question arise. A simple comparison between the profiles of samples will show if the profiles belong to the same horse. This is especially valuable for a horse sold without registration papers or pedigree information. With DNA profiles included in horse records, accurate identification is possible to obtain the identity of an unknown horse or clarify a questionable identity. o In addition to mandatory DNA profiling and parent verification for registration, the Arabian Horse Association also provides a DNA test service to assist in identifying a horse with an ‘unknown identity’. If the horse was previously registered (with a DNA profile on file), the test result will indicate there is a profile match, and the horse can be identified through its associated registration record. While a successful identification does not guarantee an owner transfer regarding registration papers (there are still paperwork requirements), gaining the identity of an ‘unknown horse’ can help with keeping horses in the Al Khamsa breeding pool. If there is no profile match, it means the horse is unregistered (or possibly it is an aged horse registered prior to the DNA requirement). The ability to reunite a horse with its registered identity emphasizes the importance of registration and its value for maintaining a horse’s identity and worth. All Al Khamsa horses should be registered, to help safeguard this vital group of Arabian horses. (Note: if there is no profile match, the process ends – as AHA will not use the DNA profile of an unmatched horse to try to determine parentage). • Parentage testing identifies individuals who, due to a specific combination of genetic markers (alleles), could qualify as a parent for a specific offspring. The parent verification process

Breeders can benefit from these scientific studies by gaining a more general understanding of what the sciences does and does not tell us and incorporating genomic tools into their breeding programs.

DNA Profiling and Parent Verification

DNA testing for individual profiling and parent verification is a familiar procedure for owners and breeders registering horses with the Arabian Horse Association. Although this requirement has been in place for decades, it is an example of a technology that plays an important role in supporting preservation breeding programs. Breeders rely heavily on pedigrees to inform decision making for

is based on the principle of exclusion. This means if a DNA profile of an offspring is compared to the profiles of its reported sire and dam, and a match cannot be made to one or both parents, the parent(s) is/are excluded. However, if matches are made (at each DNA marker evaluated), then the parent(s) is/ are said to qualify. For registration purposes, an offspring must qualify to both parents - any exclusion results in a registration disqualification. o Parentage testing is highly reliable, with exclusions being 100% accurate and most parentage qualifications having an accuracy >99% when both parents are included. However, this accuracy can decrease when the potential parents are part of a group of closely related animals. This is due to the potential of an animal closely related to an actual parent possessing markers that make it appear to be the correct parent. In complicated cases, the laboratory can test with additional DNA markers to assist with determining parentage. o For some interesting case history. Over the years, there have been several Arabian horse registration cancellations due to

DNA or parentage disqualification. One of the most unusual cases involves the cancellation of Half-Arabian registration papers for two competitive cutting horses who were determined to have no Arabian parent.

Genetic Disorder Testing

Currently, there are >25 equine genetic disorders that have DNA tests available, including several associated with the Arabian horse (Cerebellar Abiotrophy, Lavender Foal Syndrome, Occipitoatlantoaxial Malformation, and Severe Combined Immunodeficiency). The mutations for these disorders have been found in a wide range of lines within the Arabian breed, including Al Khamsa lineages. From a historical perspective, a primary reason why these mutations have persisted in the gene pool is that popular ancestral horses were carriers. Because many of these horses were desirable individuals and therefore regularly used for breeding, these mutations have continued to be passed along through the generations. An important consideration regarding a recessive disorder is the mutant gene is not the disease; it is simply 1 of 20,000 genes in a horse’s genome and is not expressed in the single state. In practical terms, the negative impact of these genetic disorders comes from the production of affected foals, and not simply from the existence of the mutations.

While these mutations have a lengthy history within the Arabian breed, modern technology provides an opportunity to avoid the production of affected foals with testing, along with selective mating strategies to prevent breeding carriers together. [See Figure 1]

Although a primary goal in breed wide management of genetic disorders is the production of zero affected foals, the breeding of Arabian horses is not just about test results. Decision making also needs to consider the best ways to produce quality Arabian horses, while maintaining as much genetic diversity as possible within the breed. The overall goal of incorporating testing into the decisionmaking process for breeding is to produce quality without tragedy; it is not to automatically remove horses from the breeding shed.

Another option available to help manage these disorders and maintain as many lines as possible is the use of suitable clear offspring of carrier horses, when available. This combines the best of both worlds, in that desirable lines can continue to be used through clear offspring. Within the Arabian breed, and particularly within the Al Khamsa community, there needs to be a preservation and continuation of lines, not an exclusion or removal of them. For once these lines are gone, they are gone forever and cannot be replaced. Genetic Diversity

Charles Craver wrote: “Preservation breeding means setting a boundary around treasured areas of past breeding. Each is kept as a separate entry, as a capsule of Arabian breeding that does not change with time as far as essentials are concerned. These separate, distinct capsules are not isolates in Arabian breeding. They contribute on a continuing basis to the larger Arabian breeding scene as effective elements of current activity. Some Preservation horses attain the highest honors in their own right. It is surprising how many others turn up as parents and grandparents of successful horses. Preservation breeding is not a matter of being a hermit or operating a zoo for antique types of Arabian horses.”

Within the realm of Al Khamsa, the goal of preservation breeding is to maintain the traits, characteristics, and hereditary factors which are representative of the horse of Bedouin Arabia – in short, to preserve genes for the future. This preservation effort goes beyond the Al Khamsa community though. Since a breed is the sum total of all its individual horses, preservation breeding carries the unspoken assumption that the ‘preserved’ genes will benefit the breed as a whole. While at the same time, preservation breeding emphasizes that a breed must not be viewed as the average of all its ‘random mating’ individuals; in order to preserve we must identify and try to understand the differing strands of its makeup. • Inbreeding Inbreeding is an old and powerful tool which has been in use in agriculture for thousands of years. In fact, it is a part of any closed breeding group; the basis comes from taking related animals for use as breeding stock. Inbreeding is an important tool that has up and down sides. It can be a useful tool to concentrate the genes of a superior ancestor, fix a desired type, and increase prepotency. However, excessive inbreeding comes with a cost that can manifest as a less robust immune system, reduced reproductive efficiency, reduced growth, and increased incidence of genetic diseases.

Within the Arabian breed, research shows evidence of relatively high inbreeding within some individuals, especially in the Straight Egyptian subgroup — which had a group mean inbreeding coefficient (F) of 30%. This is an important finding, since the study notes the level of inbreeding “may be reaching levels sufficient to impact animal health.” In addition, because inbreeding drives genes to fixation and can lead to the loss of alleles from the population, one goal of preservationist planning should be to minimize the average degree of inbreeding. Inbreeding is not an end in itself. Another important point is homozygosity does not equal ‘breed purity’. In fact, this skewed concept of ‘purity’ is directly at odds with horses in the Middle East maintaining higher levels of historical genetic diversity. • Relatedness and Heterozygosity/Homozygosity – Pedigree vs. Genome

Breeders have long been accustomed to assessing relatedness through pedigree evaluation. But pedigree is only an estimate and is often not nearly as accurate as once thought. Although we know half of an individual’s DNA physically comes from each parent, what is in the background of that DNA is not always the same. Even with the existence of elegant mathematical equations, Mother Nature can add in some twists. Due to natural distribution, on average full siblings share a 50% (.5) relationship. However, any two individuals can vary in the degree of sharing, depending on which alleles they received from their sire and dam. The actual genomic relationship of full siblings is represented by a bell curve; with some horses showing the expected .5 (50%) and others showing, for example, .4 (40%) or .6 (60%). [See Figure 2] The wider that curve, the greater the lack of accuracy when using a pedigree estimate. numbers for each horse are needed, instead of using a general trend over the herd average.

As shown by data from a study by Dr. Mohamed Al Abri, et al., pedigree estimates can greatly under- or over-estimate actual inbreeding values based on genomic measures. Another example from this study shows full siblings with the same pedigree inbreeding values can have different genomic inbreeding values and levels of homozygosity. [See Figure 3 and Table 1]

Arabian horse breeders, especially those interested in preservation of Al Khamsa horses, want to maintain rare alleles and heterozygosity in unique groups, both in the Middle East and abroad. The remarkable genetic diversity found in cradle countries suggests these geographically defined populations may contain valuable archives of ancient ancestry. [See Genetic Diversity & Complex Ancestry in the Arabian Horse. Khamsat, May 2021 for further context.] To enable conservation efforts and management of inbreeding in at-risk subgroups, more information on genomic diversity in breed subgroups is needed. Since genetic diversity is the basis of the genetic health of an individual or breed, having inbreeding and hetero / homozygosity values is useful. Commercial testing is now available which provides calculations of ‘F value’ (measure of inbreeding) and ‘heterozygosity’ (measure of the proportion of loci which have different alleles). Higher heterozygosity means more genetic variation and lower homozygosity (measure of the proportion of loci which have two identical alleles). With this information in hand, breeders can utilize genomic data, along with pedigree, conformation, and health as selection criteria when making breeding decisions.

mtDNA

Depth of pedigree (the number of generations available to evaluate) can also significantly impact the degree of accuracy of using pedigree as the basis for estimating homozygosity. If these measures are going to be used to guide breeding decisions and assist in keeping inbreeding levels in check, a better method is needed for calculating. Although there is a relationship between genomic measures and pedigree estimates, breeding decisions are made for individual horses; breeders do not work with herd averages when planning specific matings. As such, the individual

Mitochondrial DNA (mtDNA) is inherited maternally and used for tracking genetic history through the tail female line. Numerous studies have found there is a great deal of mtDNA variation in the horse, consistent with the interpretation that many wild dam lines contributed to the early domestic population. mtDNA haplotypes are specific DNA sequences that can be used to genetically distinguish one dam line from another; making this a tremendously useful tool to help characterize genetic diversity within the breed. Over the past 30 years, 28 haplotypes representing over 115

tested maternal lineages have been identified. This research has identified several foundation mares that share the same haplotype, which means they share a historical common female ancestor (several hundred to several thousand years ago). Other lines are unique, which means to date there have been no other foundation lines that match.

A central finding from mtDNA research is some families recorded with different strains have the same mtDNA. While others, recorded as of the same strain, show substantial mtDNA differences. Even though horses in this first category share the same female ancestry, yet different strains, it does not mean the strains are wrong. What this finding strongly implies is strains, as they have come down to us, descend from multiple foundation mares and were sampled from a pre-existing population without pedigree records.

The tradition of using strain names is something developed by the Bedouins and passed down through the generations. In looking back to when the strain naming system was originally developed, it suggests that the Bedouin had a group of mares of unknown maternal relationships, and strain names were given based on ownership or some special individual characteristic of a famous mare. With this in mind, a strain does not automatically indicate a ‘unique’ beginning. So, it is logical and quite possible that two horses can have different strains, but still share common maternal line ancestors. There are also lines recorded of the same strain which have different mtDNA haplotypes, meaning different ancestors. This implies that a strain could be founded on a group of mares, perhaps in a specific human family ownership. So, the concept of strain name was not something tied to a unique biologic origin, but rather was influenced by the customs of the Bedouin. mtDNA analysis has also been used to help resolve conflicting information in the historical record (e.g., Bint Yamama), as well as long standing pedigree questions (e.g., Domow). mtDNA research has also identified cases where separate branches of the same foundation line show distinct haplotypes (e.g., Urfah dam line – Saleefy branch is different from the Sheria and Rhua branches). When reconciling the information that has come out of these cases (and others like them), it is important to remember that knowing about the mtDNA has not changed anything about the horses. All it has changed is an aspect of what we know about them, specifically about the biological dam lines as opposed to (or at least separate from) what the oral traditions and written records say about them.

Historically, the Arabian horse community has placed considerable emphasis on the female lines of pedigrees, based on the desert tradition of tracing descent through matrilineal strains; studying mitochondrial DNA gives students and breeders a handle on the biological reality of these dam lines. Western science is not calling into question the accuracy of tribal strain designations; what we have a chance to do is reconstruct the history of strain development and compare the biology with the oral tradition.

The information obtained from mtDNA testing provides key information about individual tail female lines, along with furthering knowledge about the breed’s historical development and structure.

Y Chromosome

The Y chromosome is paternally inherited, making the malespecific, non-recombining part of this chromosome (MSY) ideal for tracking genetic history through the tail male line. To date, 20 Al Khamsa foundation sire lines have been tested; all clustered into one of the Arabian defined haplogroups (Ao-aA, AoaD, or Ta). [See An Overview of Y Chromosome Ancestry in Al Khamsa Foundation Sire Lines. Khamsat. March 2022 for details] With commercial Y chromosome testing becoming accessible in the near future, further information will be available to study individuals of each haplotype, seeking insight into any potential connections between haplotypes and tribal sources or geographic regions. As more lines are tested, we will continue to gain a deeper understanding of individual tail male lines, as well as structure and origins of the Arabian horse.

Using the Genomic Toolkit Wisely

Biological advances provide access to another set of information, another angle from which to view individual horses and breed history. We can expect such information both to answer old questions and to raise new ones. Even with its twists and turns, this journey of discovery will grow our understanding and appreciation of the Arabian horse. Using these genomic tools, breeders can access valuable information to assist in guiding preservation efforts of the Al Khamsa Arabian horse.

While individual breeders will always have their preferences for which horses to breed, it is hoped breeding decisions will be informed and educated. The use of genomic tools, whether testing for genetic disorders or inbreeding measures, are tools to be used to assist in making informed breeding decisions; nothing more and nothing less. For breeders, the information gained from these genomic studies can be used to assist in guiding decisions regarding their breeding programs and the preservation of rare bloodlines. For the breed, the value of these genomic tools is their use by breeders to assist in making decisions that will maintain the quality, integrity, and health of the Arabian horse. Acknowledgements

Thank you to Dr. Samantha Brooks for her assistance and gratitude to Scott Benjamin for his advice and inspiration.

References: 1. Cosgrove, EJ, et al. “Genome Diversity and the Origin of the Arabian Horse.” Sci Rep. 2020 Jun 16;10(1):9702. 2. Al Abri, M, et al. “Application of Genomic Estimation Methods of Inbreeding and Population Structure in an Arabian Horse Herd.” Journal of Heredity, 2017, 361–368. 3. Minnich and Bowling. “Genetic Diversity & Complex Ancestry in the Arabian Horse.” Khamsat, May 2021. 4. Minnich and Bowling. “Mitochondrial DNA (mtDNA) – What Is It and What Does It Tell Us?” AHA Equine Stress, Research and Education Committee. 2011. 5. Almarzook, S, et al. “Diversity of mitochondrial DNA in three Arabian horse strains.” Appl Genet. 2017 May;58(2):273-276. 6. Al Khamsa Arabians III. Ed. by Al Khamsa, Inc. 2008 Edition, Al Khamsa, Inc. 7. Minnich and Bowling. “An Overview of Y Chromosome Ancestry in Al Khamsa Foundation Sire Lines.” Khamsat. March 2022.

Funding Research to Support the Arabian Horse

Donate Today

thearabianhorsefoundation.org

Like Mother, Like Daughter!

Summer Blush WA 1996 black mare (Persuasion x Modesty Blaise)

Davenport Arabians

Visit us and enjoy horses and the beautiful Rocky Mountains Windsor Arabians

Mary Buser Gills PO Box 882560 Steamboat Springs, CO 80488 mgills327@ gmail.com 970.846.3747

Gypsy Moth WA 2006 black mare (Barbican CF x Summer Blush WA)

Chef Al Khamsa and QCD

Chef Al wants you to know that if you are over 70 you can make a tax free gift from your IRA as a Qualified Charitable Distribution (QCD). The transfer must go directly from your IRA to Al Khamsa. Your IRA custodian has an easy form for you to use. The amount of your gift is excluded from your gross income and it counts toward your required minimum distribution. A Charitable Remainder Trust may be another way to give to Al Khamsa that can provide you with tax advantages. Helping Al Khamsa may end up helping you at tax time. Check it out with your accountant. If you want to donate the old fashioned way as well, you can designate your donation specifically for the general fund, the endowment, the AERC Fund or give to the Preservation Task Force by pressing this button on our website

or mailing your check to the Business Office 470 Folsom-Jonesville Road Jonesville, KY 41052