by Beth Minnich, with Michael Bowling

Sahanad Preservation Farms

PRESERVING ALL FIVE AL KHAMSA LINES

BREEDING and EDUCATING THE NEXT GENERATION ABOUT SAHANAD’S LEGACY

STALLIONS AT STUD

MonaLisas Majesty (Black Lightning X Aziza Mona Lisa) 1998 Homozygous Black AK/Sahanad Frozen Semen only

SCID, CA, & LFS Clear/OAAM Carrier

Sir Ravenn Mone (Ravenwood Zaim X Lightnings Lilly) 2002 Black AK/Sahanad Cooled or Frozen Semen

SCID, CA, LFS & OAAM Clear

Available by Private Treaty

Sir Ravenn Mone

Aziza Mona Elisa with Aziza Kamona Halisa

Special thanks to Sahanad enthusiast Anita Enander on her contribution to ensure Sir Ravenn Mone can be utilized via Cooled/Frozen Semen

Sahanad Preservation is forever indebted to Trucilla Enz-McLaughlin and her family for producing Sahanad’s get that are the foundation of our preservation efforts.

Patty Conklin Cedar Hill Missouri sahanadpreservation@gmail.com

WE ARE TWO DEDICATED WOMEN PRESERVING SAHANAD’S BLOODLINES ESTABLISHED 1994 AND 2006 WITH 44 YEARS COMBINED EXPERIENCE

Siggi Saha

Sarah Clower Moorefield West Virginia sahanadpreservationfarm@gmail.com

STALLIONS AT STUD

OPA Yowel (Black Lightning X HPF Basantaa) 2002 Homozygous Black AK/Sahanad

SCID, CA, LFS & OAAM Clear

RMR Hanad (Black Lightning X Sahanadah) 2003 Black AK/Sahanad

SCID, CA, & LFS Clear/OAAM Carrier

Siggi Saha (Prince Lycoris X JEN Beauty A Saha) 2016 Grey AK/Sahanad & Davenport

SCID, CA, LFS & OAAM Clear

Storrmee Shahmonee (Sir Ravenn Mone X Storrmee Shahleah) 2018 Homozygous Black AK/Sahanad

SCID, CA, LFS & OAAM Clear

Available by Private Treaty Currently Live Cover Only (Ask about the possibility of Shipped Semen!)

FOALS EXPECTED IN 2022

Visit our site often for updates www.sahanadpreservation.com OR visit us on Facebook at SAHANAD PRESERVATION FARMS and the SAHANAD PRESERVATION GROUP (a general page featuring all types of articles of interest)

Phosphorus 2021 Colt by Siggi Saha

An Overview of Y Chromosome Ancestry in Al Khamsa Foundation Sire Lines

by Beth Minnich with Michael Bowling

Author’s note: As this article was in final preparation, a paper from the lab of Dr. Barbara Wallner (Remer, et al.: “Y-Chromosomal Insights into Breeding History and Sire Line Genealogies of Arabian Horses”) was published in Genes. Using finescaled MSY haplotyping, this new work provides further detail on Arabian breed haplotypes, along with refining the crown haplotype structure to better understand the historical development of horse breeds. To maintain the intended focus of this article on Arabian breed Y chromosome haplogroups and introduce results of Al Khamsa foundation sire lines which have been tested, the most relevant findings from the paper have been incorporated (along with some other highlights). Readers interested in the broader scope of this research are encouraged to read the publication for a full update on the status of this project. https://www.mdpi.com/2073-4425/13/2/229 (open access) Additionally, though some of the figures initially selected for this article are no longer current, they are helpful visuals - so a decision was made to leave them in place (these figures are noted). Figures from the above-mentioned paper have also been incorporated.

Equine Y chromosome research offers new perspectives on the fascinating history of the horse through study of the origin and influence of paternal lineages. Utilizing this information, we can gain a deeper understanding of the development of the Arabian horse, helping to connect the threads of culture, history, and genetics. With these threads, a more detailed tapestry can be woven depicting the origins of the breed and its Bedouin bred ancestors, to help guide preservation efforts of this celebrated animal. This article will focus on Arabian breed Y chromosome haplogroups and introduce results of Al Khamsa foundation sire lines, which have been tested.

The Y Chromosome as a Storyteller With application of genomic tools, natural mutations can be used to trace ancestry of individuals as these mutations are passed in the DNA from one population to another over time. The Y chromosome is paternally inherited, making the male-specific, non-recombining part of this chromosome (MSY) ideal for tracking genetic history through the tail male line. The same principle applies to mitochondrial DNA (mtDNA), that is inherited maternally and used for tracking genetic history through the tail female line [see Figure 1]. Genetic variations on the MSY can be classified into ‘haplotypes’ and ‘haplogroups’. Haplotype (HT) is short for ‘haploid genotype’ and this is a group of linked DNA sequences on one chromosome which are inherited together. A haplogroup (HG) is a group of closely related haplotypes.

Through identification of haplotypes and haplogroups, a horse Y chromosome phylogenetic tree can be created to view relationships between the haplotypes in different groupings and breeds. As overviewed in “Y Chromosome Ancestry in the Arabian Horse” (September 2021, Khamsat)1, most modern horse breeds form a haplogroup designated the ‘crown haplogroup’. The crown group contains three branches (clades), identified as A, H, and T,2 and it is along the A and T branches we will travel, as we continue the journey to further understand the history of the Arabian horse [see Figure 2 & 3].

**Figure 2. Horse MSY Phylogenetic Tree, 2019. The three clearly separated crown group clades are represented in pink (H), green (A) and yellow/red (T). The Przewalski’s horses are shown in brown. Blueish clades correspond to early splitting Asian samples (O), Northern European breeds (N and I) and other autochthonous Asian samples (M, Y and J).

Source: Modified from Felkel, et al. The horse Y chromosome as an informative marker for tracing sire lines. Sci Rep. 2019 Apr 15;9(1):6095. http://creativecommons.org/licenses/by/4.0/ **see author’s note at beginning of article

“Those horses in North America that can reasonably be assumed to descend entirely from Bedouin Arabian horses bred by horse-breeding Bedouin tribes of the deserts of the Arabian Peninsula without admixture from sources unacceptable to Al Khamsa.” - Al Khamsa Arabian Horse Definition

Figure 3: Latest Crown Haplotype Tree based on Next Generation Sequencing Data HTs are indicated as circles, with circle size being proportional to frequency. The 14 crown haplogroups are indicated in the outer circle, with the breeds listed beside them. Blue HTs were detected in Arabians, and light blue HTs were detected in a horse that traced back to an imported Arabian in the paternal lineage. The HTs of the three founders of the English Thoroughbred (Tb-oB1, Tb-oB31, and Tb-d) are marked with red lines. Source: Modified from Remer, et al. Y-Chromosomal Insights into Breeding History and Sire Line Genealogies of Arabian Horses. Genes 2022, 13(2), 229.

A Tale of Two (Now Three) Arabian Breed Haplogroups

As we begin to study Y chromosome haplotypes found in Al Khamsa Arabians, understanding how the haplogroup classifications were developed is important. In previous studies, Arabian horses were roughly grouped into the haplogroups Ao and Ta, with some outliers in Tb.2 Haplogroups Ao and Ta have been considered typical for the Arabian breed. Recent data now splits Ao into several groups, of which Ao-aA and Ao-aD, along with Ta, are characteristic of Arabian ancestry.3 All three of these haplogroups are also represented in Al Khamsa Arabian horses.

Although haplogroup Tb-oB1* has not been found in Al Khamsa Arabians, it has been found in Arabians (including sampling from Syria) and is shared among Arabians, Turcomans and Akhal-Tekes.3 [see Figure 4] This raises questions about the historic connections between Oriental horse types and scenarios that could have led to the widespread distribution of this haplogroup. As noted in the recent publication from the Wallner group, TboB1* could be of Turcoman origin. But an additional scenario is also offered for consideration. “Together with the other Arabian HTs (Ao-aA, Ao-aD, and Ta), Tb-oB1* could have been typical of autochthonous Arabian horses from the Nejd highlands. Selection and genetic drift could have led to haplotype frequency differences among different strains and areas. In this scenario, Tb-oB1* may have been distributed from the Arabian plateau during the migrations of the Bedouins.” With continued research, hopefully more pieces to this puzzle will fit into place.

Figure 4. MSY Haplotypes in globally active Arabian lines, Middle Eastern Arabians, and other breeds. Haplogroup (bold) and haplotype distribution in breed or breed groups in absolute numbers (N = total number). [results from sampling in Syria and Iran highlighted in brown box] Source: Modified from Remer, et al. Y-Chromosomal Insights into Breeding History and Sire Line Genealogies of Arabian Horses. Genes 2022, 13(2), 229.

The Ao grouping was named for initially genotyped Original Arabians. At the time researchers selected the designation ‘Ao’, it appeared to be specific for Arabians and was the only haplogroup found in the Arabian breed. However, as sample numbers increased, some Arabian horses were found to cluster at the root of the T branch of the crown group, but distinctly separate from the other T sub-branches.4 This discovery resulted in creation of a new haplogroup, designated by researchers as Ta (T-Arabian), being assigned to this group of horses [see sidebar “The T Doesn’t Stand for Turcoman”].

While existence of different haplogroups in the breed can initially appear counterintuitive, it is to be expected and can be explained by differences in desert source population substructures and buying expeditions to the region collectively known as ‘Arabia’. Variations to consider include timing, location, tribes visited, horses available, and selection goals of expedition patrons. Further, given the diversity of mtDNA found in Arabian horses from cradle countries5, having just one foundation sire line (i.e., only one haplogroup) would be surprising.

The T Doesn’t Stand for Turcoman

Word selection can have tremendous impact on interpretation of information and the resulting narratives developed. An example is the discovery of the Ta haplogroup in the Arabian breed. Although Ta has not been found in any breed other than Arabians so far, speculation has been raised, by some, that horses with a Ta haplotype are actually of Turcoman ancestry. To further understand this idea, some background is useful. • The T clade in the horse Y chromosome phylogenetic tree was initially detected in ‘Thoroughbred influenced’ breeds, hence the selection of the letter ‘T’ by researchers to identify this branch. As sampling expanded, new discoveries were made and ‘non-Thoroughbred influenced’ breeds were identified in the T grouping. Researchers opted to keep the original

‘T designation’ for the main branch, but naming conventions were modified for the T sub-branches, to reflect ‘T’ was no longer limited to ‘Thoroughbred influenced’ breeds. o The T clade has now five sub-branches, Ta (T-Arabian), Tu (T-Ubiquitous, previously listed as T-Unknown), Tb-d (Thoroughbred-Darley), Tb-o (Thoroughbred-Other), and Tk (T-Kladruber). The Ta, Tu, Tb, and Tk sub-branches are distinctly separate. • In a 2017 publication by Dr. Barbara Wallner, findings were announced indicating the Thoroughbred founding sires (the

Darley Arabian, Byerley Turk and Godolphin Arabian) are likely of Turcoman origin, not Arabian4. Their haplotypes all cluster into the Tb clade. This paper also described a separate discovery, identifying some Arabian horses found to cluster at the root of the T branch, but not into the Tb clade (this would become the Ta haplogroup in later studies). [see “Y Chromosome Ancestry in the Arabian Horse” (September 2021, Khamsat) for further information]. Continued on next page.

As seen in Figure 3, the Akhal-Teke (a breed considered a remnant of the extinct Turcoman horse) appears in the bottom left of the network within the Tb clade, close to the Thoroughbred. As such, it is tempting to assume the Ta branch comes from a

Turcoman origin. However, the relatedness of these groups is not limited to one group acting as a ‘founder’, with the other being a ‘descendent; rather the relatedness can reflect ‘shared ancestry’. In other words, Arabian and Turcoman horses may share common tail male ancestry far back in time. • The ancestors of Arabian, Turcoman, North African, and Iberian horses formed the crown group ancestral pool. Given the human history of the region, gene flow between horse populations would be expected. As such, the concept of shared ancestry is central to this discussion and could be the key for reconciling DNA findings with the historical record. For interpreting deep ancestry data, it is also helpful to view early ancestral stock as ‘landraces’ rather than ‘breeds’. It is understandable why the letter ‘T’ subliminally suggests Turcoman. In hindsight, had different naming conventions been used for the branches (e.g., numbers or symbols instead of letters), there would not be a subconscious association between specific letters and breeds. But given the nomenclature in use, clarification is important that use of ‘T’ is not an abbreviation for Turcoman. Of note from the recently published work from the Wallner group, the Ta haplogroup is represented in Arabian horses from Syrian populations (with no recently reintroduced occidental lines); further supporting Ta as a genuine Arabian breed haplogroup.3 [see Figure 4]

It’s All in the Variants

As mentioned previously, the Ao-aA, Ao-aD, and Ta haplotypes are considered typical for the Arabian breed. The most recent common ancestor (MRCA) of the crown group, that contains the A, T (and H) clades, is estimated at around 500 CE with the Ta branch split estimated at around 800 CE. Branching is a result of naturally occurring mutations and the specific genetic variants possessed by a horse determines its haplotype.

The first step in identifying an Arabian horse’s haplotype involves testing the DNA sequence it carries at markers rA and rX, to roughly assign the sample to clade A (rX) or T (rA) (these are the clades relevant to the Arabian breed) [see Figure 5]. From there, additional markers are typed, and the results determine the specific haplotype. The mutations defining haplogroups Ao-aA, Ao-aD, and Ta, as well as the haplotypes within those groups, are shown in Tables 1, 2, and 3. 3

Figure 5. Simplified Haplotype Network for Ao-aA, Ao-aD, and Ta Haplogroups with Genetic Variants Indicated on Branches [key variants shown in red] Source: Modified from Remer, et al. Y-Chromosomal Insights into Breeding History and Sire Line Genealogies of Arabian Horses. Genes 2022, 13(2), 229.

Al Khamsa Foundation Sire Lines

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 Table 4 and Figure 6]. With this information, we can further 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 the origins of the Arabian horse.

Table 4: Haplogroup Assignments for Al Khamsa Foundation Sire Lines Tested, as of January 20222,3,6,7

Note: Some samples do not cluster in agreement with most samples of their recorded sire line. These outliers may be the result of sampling errors, errors in record keeping, or incorrect ancestry (e.g., a horse swap in the pedigree).

Figure 6. MSY Haplotypes (HTs) in Arabian sire lines. Genotyping results from 145 males. Foundation sires are shown for each HT, with the number of samples for each line in parenthesis. Circle size proportional to frequency. HTs/foundation sires that are only active in breeds other than Arabians are given in light blue. Source: Remer, et al. Y-Chromosomal Insights into Breeding History and Sire Line Genealogies of Arabian Horses. Genes 2022, 13(2), 229.

Identity in the Age of Ancestral DNA

An interesting dichotomy arises between culture and genetics when attempting to determine breed identity of historical horses. While DNA is informative, it has limitations and is not the entire story. To go beyond DNA testing and expand the scope of this discussion — several questions emerge. In the age of genomic testing, what makes a historical breed identity ‘authentic’? How does DNA data influence development of narratives regarding horse breed history? What are the implications, especially for a breed identity directly tied to the culture of the people who developed it? [see “Genetic Diversity & Complex Ancestry in the Arabian Horse” (May 2021, Khamsat) for additional context]. 8

The Arabian horse is intimately connected to the physical environment of its native region and the culture of the Bedouin Arab horse breeders. Arab Bedouin tribes defined the ‘Arabian horse’ based on a framework steeped in their cultural values, with the Bedouin notion of authenticity and the standards of rasan (strain) and the marbat at the core of what makes a horse ‘an Arabian’. As such, breed origins must be viewed within these parameters and not from a Western idea of ‘purity of blood’. Most notably, from a Bedouin perspective the notion of breed purity is more cultural than biological. With the use of DNA there are tremendous opportunities to gather information to help in connecting the past to the present. However, the charismatic desert-Arabian horse should first and foremost be appreciated through the lens of the environment that shaped its development and the culture of its original custodian.

“Any definition of the Arab breed must comply with the cultural values ascribed to it by its original custodians, the Arab Bedouin tribes of Arabia.” – The Arabian Horse Manifesto

Although Y chromosome research is specific to the tail male line in a pedigree, fine scaled MSY haplotyping is a powerful tool for use to enrich our understanding of the complexity and richness of the Arabian breed’s captivating history and delve further into haplotypes found in the Al Khamsa population. Keeping in mind ‘discovery’, by its nature, can yield unexpected results; some of the findings from this research will be controversial. However good science should lead to more questions and as we learn more about the background of the Arabian horse, further questions will surface. Even with its twists and turns, this journey of discovery will grow our understanding and appreciation of the origins of the Arabian horse, as well as other breeds. Using these genomic tools, breeders can access valuable information to assist in guiding preservation efforts of the Al Khamsa Arabian horse. Acknowledgements:

Thank you to Dr. Barbara Wallner for her assistance and gratitude to Scott Benjamin for his advice and inspiration. References: 1. Minnich and Bowling. Y Chromosome Ancestry in the Arabian Horse. Khamsat, September 2021. 2. Felkel, et al. The horse Y chromosome as an informative marker for tracing sire lines. Sci Rep. 2019 Apr 15;9(1):6095. 3. Remer, et al. Y-Chromosomal Insights into Breeding History and Sire Line Genealogies of Arabian Horses. Genes 2022, 13(2), 229. 4. Wallner, et al. Y Chromosome Uncovers the Recent Oriental Origin of Modern Stallions. Curr Biol. 2017 Jul 10;27(13):2029-2035. 5. Khanshour and Cothran. Maternal phylogenetic relationships and genetic variation among Arabian horse populations using whole mitochondrial DNA D-loop sequencing. BMC GENET. 2013 Sep 13;14:83. 6. Michaelis, Eva. 2019 Master’s Thesis: Analysis of Arabian stallion lines with Y chromosomal markers. University of Veterinary Medicine Vienna. 7. Author personal correspondence with Dr. Barbara Wallner. 8. Minnich and Bowling. Genetic Diversity & Complex Ancestry in the Arabian Horse. Khamsat, May 2021. 8. The Arabian Horse Manifesto, https://arabianhorsemanifesto.org/

Juvenile Idiopathic Epilepsy in the Arabian Horse

Ongoing Study at the University of Florida

Funding Research to Support the Arabian Horse

The Brooks Equine Genetics Lab is working to identify the genetic basis of Juvenile Idiopathic Epilepsy (JIE) in the Arabian Horse. JIE is a seizure condition that can affect very young horses. Medication can be used to help control or lessen the severity and duration of seizures — but there can be complications associated with seizures, including head injury and aspiration pneumonia. Identifying the genetic cause(s) of JIE will provide breeders with information to assist in avoiding production of affected foals. Data from this study suggests JIE is not a single gene trait, which may explain the wide range of severity exhibited by foals with this condition. Dr. Samantha Brooks and her research lab are evaluating several candidate genomic regions of interest. DNA samples are sought from horses previously diagnosed with JIE, as well as horses who have had JIE offspring. It will take a community effort to find the answer and develop a genetic test. All sample submissions are confidential, so identity of owners or horses will not be released; should you decide to participate. For more information please contact the Brooks Equine Genetics Lab at: phone (352) 273-8080 or email: equinegenetics@ifas.ufl.edu This research project is financially supported, in part, by the Arabian Horse Foundation. Donate Today

thearabianhorsefoundation.org