2011 Arabian Horse Association Material provided by the AHA Equine Stress, Research and Education Subcommittee on Genetic Disorders

Information contributed by Beth Minnich and Michael Bowling Thank you to Anita Enander, the Institute for the Desert Arabian Horse, for her assistance in preparing this material

Mitochondrial DNA (mtDNA) What Is It and What Does It Tell Us? The Basics Nuclear DNA vs. mtDNA Deoxyribonucleic acid (DNA) is the hereditary material passed from generation to generation, which provides the blueprint for what an individual will look like and how its body systems will function. DNA is made up of chemical bases; hundreds or thousands of base pairs form a gene sequence, which is the basic unit of heredity. Genes provide the instructions (also known as a code) for the production of a variety of proteins; the proteins, in turn, determine how a cell operates and what physical traits will develop.

The most commonly discussed type of DNA is the nuclear DNA (named for its location in the cells nucleus), which is contributed by both parents; the sire and dam each contribute 32 chromosomes that contain this type of DNA. A horse receives half of its nuclear DNA from the sire and the other half from the dam. Each half represents a shuffled recombination of DNA that has been passed down from ancestors through the generations. Because of the DNA recombination which occurs each generation, it is difficult to use nuclear DNA to study lines of descent more distant than direct parentage.

A second type of DNA is the mitochondrial DNA (mtDNA), which is contributed by the dam. Unlike the nuclear DNA, which is contained in the cells nucleus, mtDNA is found in the cells mitochondria. Mitochondria are structures in the cells cytoplasm that convert energy from food into a usable energy form for the cell. Because mtDNA is contributed through the dam line only, it is a powerful tool for tracking ancestry through the tail female line of a pedigree.

In addition to being located in different parts of the cell, nuclear DNA and mtDNA also have different structures. Nuclear DNA molecules are formed as a spiral staircase with each stair composed of DNA bases: adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). Horses have approximately 2.7 billion base pairs of nuclear DNA of which only 2-3% makes up the approximately 20,000 genes contained in the equine genome.

On the other hand, mtDNA is a circular chromosome with about 17,000 base pairs, most of which code for replication activity. In addition, mtDNA does not undergo recombination, so there is no shuffling of DNA as it is passed from generation to generation. Due to a lack of recombination, the mtDNA code is varied only through occasional mutations. When a mutation occurs and is passed along, it becomes a marker of descent. Since mtDNA is passed only from the dam and is inherited completely independent of the nuclear DNA, it allows for the study of tail female lineage back to a common ancestral point.

Table 1: Comparison of nuclear DNA and mtDNA

DNA Type

Location Number of Base Pairs

Contributed By Structure Recombination

Nuclear

On 64 chromosomes (32 pairs) in the cells nucleus

~2.7 billion

Sire and Dam

(each contributes 32 chromosomes)

Linear

Double Helix

Yes

Mitochondrial

(mtDNA)

In mitochondria located in the cells cytoplasm

~17,000

Dam Only

Circular

Double Helix

No

2011 Arabian Horse Association Material provided by the AHA Equine Stress, Research and Education Subcommittee on Genetic Disorders

Information contributed by Beth Minnich and Michael Bowling Thank you to Anita Enander, the Institute for the Desert Arabian Horse, for her assistance in preparing this material

How Can We Use mtDNA and What Does It Tell Us? In the 1990s, the late Dr. Ann Bowling and Michael Bowling conducted research into the dam lines of Arabian horses using mtDNA. Through this research, they identified 27 different haplotypes, which are essentially specific DNA sequences that can be used to genetically distinguish one dam line from another. 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. Studbook research indicates there are approximately 100 dam lines represented in the AHA Registry. However, it is estimated that the 27 identified dam lines in this project account for at least 89% of the horses registered in the US. As such, mtDNA is a tremendously useful tool to help characterize genetic diversity within the breed.

Table 2: Foundation mares and their mtDNA haplotypes Haplotype numbers are sequential numbers assigned by researchers. Mares selected from the mtDNA diversity study of Arabian horses registered by the AHRA. Published in the paper A pedigree-based study of mitochondrial D-loop DNA sequence variation among Arabian horses by A T Bowling, A Del Valle, M Bowling. Animal Genetics, 2000, 31, 1-7.

mtDNA Haplotype

Tail Female Founder

Strain (substrain)

mtDNA Haplotype

Tail Female Founder

Strain (substrain)

A01 Rodania Kehilan (Rodan) A14 Elsissa Hadban (Enzahi)

A01 Wadduda Seglawi (al Abd) A15 Balkis Kehilan (Ajuz)

A01 Noura Maneghi A16 Dafina Kehilan (Krush)

A02 Abeyah Abeyan (Sherak) A17/A28* Dajania Kehilan (Dajani)

A03 Basilisk Seglawi (Jedran) A17 Hadba Hadban (Enzahi)

A03 Reshan Kehilan (Heyfi) A19 Gazella Kehilan (Ajuz)

A04 Sahara Kehilan (Moradi) A19 Murana I No recorded strain

A05 Selma Hamdani (Simri) A20 Nejdme Kehilan (Ajuz)

A06/A01* Urfah Seglawi (al Abd) A21 Zulima Seglawi (al Abd)

A07 Sheha Kehilan (Nowak) A21 Lisa Seglawi (Jedran)

A08 Queen of Sheba Abeyan (Sherak) A22 Werdi Kehilan (Krush)

A09 Zaalee Seglawi (Jedran) A23 Jellabiet Feysul Kehilan (Jellabi)

A10 Ferida Maneghi (Sbaili) A24 Haidee Maneghi (Hedruj)

A11 Ghazieh Seglawi (Jedran) A24 Hamra Johara No recorded strain

A12 El Dahma Dahman (Shahwan) A25 Milordka No recorded strain

A13 Mlecha Kehilan (Dejani) A26 Nuhra Kehilan (Wadnan)

A13 Dahma Dahman (om Amr) A27 Thorayyah Tuwaysan *There is a single mutation difference between the two haplotypes shown for each given dam line, indicating a relatively recent change within the dam lines.

The use of mtDNA has shown some interesting relationships between horses of different strains. For example, Rodania (a Kehilan Rodan), *Wadduda (a Seglawi al Abd) and Noura (a Maneghi) share the same original female ancestor, since they all possess the A01 mtDNA haplotype. Another interesting finding is the A03 haplotype shared by *Reshan (a Kehilan Heyfi) and Basilisk (a Seqlawi Jedran). [see Table 2 for additional examples]

2011 Arabian Horse Association Material provided by the AHA Equine Stress, Research and Education Subcommittee on Genetic Disorders

Information contributed by Beth Minnich and Michael Bowling Thank you to Anita Enander, the Institute for the Desert Arabian Horse, for her assistance in preparing this material

It is important to note that while these horses share the same female ancestry, yet different strains, it does not mean that the strains are wrong. 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 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.

The use of mtDNA analysis has also been incorporated into researching the history of some of the Polish Arabian dam lines. In a study published in 2007, by Iwona Glazewska, et al, mtDNA analysis was used to check the accuracy of the recorded pedigree data of the Polish dam lines. In the analysis of 15 Polish Arabian dam lines, 14 distinct haplotypes were indentified. In the case of two of the dam lines, the mtDNA results were inconsistent with the recorded pedigree data. In one case, the mtDNA resolved a question posed by an earlier researcher who believed there had been a switch in recordation of a mare. The researchers also noted a suggested genetic relationship between some of the dam lines founded by Polish mares of undocumented origin and dam lines established by desert-bred mares.

Another use for mtDNA is to help resolve conflicting information in the historical record, such as that regarding the line of descent from the c1895 mare Bint Yamama, from the stud of the Egyptian Khedive Abbas II and producing for his brother Prince Mohammed Ali. A stallion of this line, Kafifan, sold to Poland in 1924, originally is registered there as Seglawi; studbook references and export certificates from 1930 on assign horses of this family to the [Kuhaylan] Jellabi strain, and Kafifan's registration in later editions of the Polish studbooks is changed to match this. The "RAS History," published in 1948, shows Bint Yamama as a great-grandaughter of the Jellabiet Feysul (of Abbas Pasha), a Kuhaylah Jellabi. The publication in 1986 of Lady Anne Blunt's "Journals and Correspondence" gives us the only contemporary account of the early 20th century horses in Prince Mohammed Ali's possession. Lady Anne describes the Prince's best mare as "the Seqlawieh Yemama (daughter of the old Yemama, dam of Mesaoud)" and adds that her dam was "Yemama owned by the Khedive."

The Bowling research finds the haplotype of contemporary mares descending from Bint Yamama (Khedive) to match that of descendants of Bint Helwa (of Ali Pasha Sherif), tracing in turn to Ghazieh (of Abbas Pasha). Mesaoud's dam is historically assigned to the family of Ghazieh. The Bint Yamama (Khedive) line does not match the haplotype of the line descending from Jellabiet Feysul through Makbula (of Ali Pasha Sherif). The straightforward reading of these results is that Lady Anne's record is correct, and Bint Yamama is out of the dam of Mesaoud.

In addition, the use of mtDNA has assisted in helping to resolve some long standing pedigree questions, such as the case of Domow (*Abu Zeyd x *Wadduda). This mare has been a bit of a mystery because of a color coat incompatibility; she is bay, while both of her recorded parents are chestnut. Using mtDNA testing, Domow and *Wadduda were identified as sharing the same haplotype A01. This haplotype is different from the other possible dam lines representing other mares that could potentially have been the dam of Domow. As such, this information combined with historical records supports the theory that the dam of Domow is actually *Wadduda and the sire of Domow is incorrectly recorded. In addition to the information provided by science, further historical research has provided evidence that *Astraled was in place to be her sire.

2011 Arabian Horse Association Material provided by the AHA Equine Stress, Research and Education Subcommittee on Genetic Disorders

Information contributed by Beth Minnich and Michael Bowling Thank you to Anita Enander, the Institute for the Desert Arabian Horse, for her assistance in preparing this material

Another example of the use of mtDNA involves the potential relationship between the Davenport mare, *Hadba, and the Egyptian mare, Venus both recorded as being of the Hadban Enzahi strain. *Hadba was previously identified as haplotype A17. With samples more recently obtained by the Institute for the Desert Arabian Horse from two branches of the Venus family (Bint Rustem and Samiha), a comparison of the test results show that the two branches from the Venus line share the same haplotype, but it is not the same haplotype as *Hadba. Instead, these samples share the A01 haplotype. So, this is an example of two horses that share the same strain but have different tail female ancestors.

Through the support of organizations such as Al Khamsa, the Davenport Conservancy and the Institute for the Desert Arabian Horse, as well as researchers in the international community, these mtDNA studies have been made possible. As new information becomes available, it builds on the previous work and helps to continue shaping our understanding of the breeds dam lines and provide valuable insight into the breeds history.

Closing Comments (provided by Michael Bowling)

Historically, the Arabian 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. Dam lines in the west have proven to be mostly quite reliable; if the breeding record were randomized, it would not be possible to make sense of the rare examples of switched identities within stud book records. The more dam lines are tested, the better idea we will have of the breed's historical development; this should include the full registered span of the breed, not just those designated "asil" by some.

Reference Materials

1) Bowling AT, Del Valle A, Bowling M. Verification of horse maternal lineage based on derived mitochondrial DNA sequence. J Anim Breed Genet 1998;115:351-6. 2) Bowling AT, Del Valle A, Bowling M. A pedigree-based study of mitochondrial D-loop DNA sequence variation among Arabian horses. Anim Genet 2000;31:1-7. http://www.ncbi.nlm.nih.gov/pubmed/10690354?ordinalpos=1&itool=PPMCLayout.PPMCAppController.PPMCArticlePage.PPMCPubmedRA&linkpos=2 3) Glazewska I, Wysocka A, Gralak B, Prus R, Sell J. A new view on dam lines in Polish Arabian horses based on mtDNA analysis. Genet Sel Evol 2007;39(5):609-19. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682809/?tool=pubmed

http://www.ncbi.nlm.nih.gov/pubmed/10690354?ordinalpos=1&itool=PPMCLayout.PPMCAppController.PPMCArticlePage.PPMCPubmedRA&linkpos=2http://www.ncbi.nlm.nih.gov/pubmed/10690354?ordinalpos=1&itool=PPMCLayout.PPMCAppController.PPMCArticlePage.PPMCPubmedRA&linkpos=2http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682809/?tool=pubmed