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GENETIC DIVERSITY OF EGYPTIAN ARABIAN HORSES FROM EL-ZAHRAA STUD BASED ON 14 TKY MICROSATELLITE MARKERS

The objectives of this study were, firstly, to conduct genetic characterization of Egyptian Arabian horses based on 14 TKY microsatellite markers, secondly, to investigate the powerfulness of these 14 TKY markers for parentage assignment of Arabian horses. A total of 101 horse samples including (Arabian = 71, Thoroughbred = 19 and Nooitgedacht = 11) were analysed by 14 TKY microsatellite markers. The PCR products were electrophoresed on Genetic analyzer 3500 with the aid of Liz standard. The basic measures of the allele’s size and genetic diversity were computed using bioinformatics software. The polymorphism of the TKY markers across the Arabian population showed moderate values for genetic diversity parameters; number of allele (NA) =8.143, effective number of allele (Ne) = 3.694, observed heterozygosity (HO) = 0.599, expected heterozygosity (HE) = 0.691, polymorphic Information Content (PIC) = 0.636 and Inbreeding coefficient (FIS) = 0.128. The combined probability of exclusion (CPE) value of the 14 TKY microsatellite loci of our Arabian horses was 0.9999. The results from current study confirm the applicability and efficiency of TKY microsatellite panel for evaluating the genetic diversity and parentage assignment of Egyptian Arabian


Introduction
Arabian horse is commonly believed to be one of the oldest and the most influential breed in the world. It is the most valuable and expensive breed as it combines the beauty of the body, fitness, agility, and intelligence as well (1). It is believed to be an immediate progenitor of many horse breeds including the Thoroughbred and Nooitgedacht (2)(3)(4). Nooitgedacht is a South African horse breed developed in 1951 as an attempt to save the endangered Basuto Pony (2). During the early colonial era in Southern Africa, the first

GENETIC DIVERSITY OF EGYPTIAN ARABIAN HORSES FROM EL-ZAHRAA STUD BASED ON 14 TKY MICROSATELLITE MARKERS
imported horses were Arabs, Thoroughbreds, Javan, Persian and Spanish. These breeds had been mixed to form what is called the Cape horse and Basuto pony which later improved to become Nooitgedacht breed. Additionally, the Thoroughbred stallions were introduced to South Africa in the 1900's for the improvement of Basuto pony (2). Thoroughbred is one of the most valuable breed of horse in the world and is used mainly for racing, but is also bred for other riding disciplines (5). Thoroughbred was developed in United Kingdom and the pedigrees of all modern Thoroughbred individuals can be traced to three stallions namely; Darley Arabian, Godolphin Arabian and Byerley Turk which were originally imported into England during 17 th and 18 th centuries. Thoroughbreds are commonly used for crossbreding purposes to create new breeds or to improve existing ones, and have been influential in the creation of various warmblood breeds (6).
Egypt, although not an area of origin, it is considered a focal point for breeding Arabian horses for the past 200 years (7) . El-Zahraa Stud is the biggest Egyptian governmental farm having about 500 horses and is considered the main supplier of Arabian horses for other private farms all over the Egyptian country. Maintaining higher genetic diversity within horses is needed for longterm genetic improvement and to prevent the low performance (8-10) and expression of deleterious recessive genes due to inbreeding effect (11,12). Authorities for Arabian horses registry have adopted parentage testing programs for breed registration, studbook creation, and to maintain the purity of Arabians (7). Moreover, parentage test is important for global certification of our Arabian horses and to avoid breeding violations. In addition, keeping parentage records helps in prevention of animal adulteration during exportation and importation as well as detection of animal thefts. Microsatellites are considered the best markers for evaluation of genetic diversity and parentage testing in different animal species including horses (13,14). Genetic diversity and parentage studies of the Arabian horses reared in Egypt based on International Society of Animal Genetics (ISAG) microsatellite markers are scanty (15,14). Other microsatellite markers called TKY panel was isolated and give sufficient and reliable information for paternity testing and genetic diversity in Thoroughbred horses, which have much less heterozygosity than non-Thoroughbred horses (16). Moreover, TKY microsatellites showed multiple alleles as well as high heterozygosity among Japanese (17,18) and Bhutan (19) horses. Although the use of 12 microsatellite markers of ISAG panel is useful for genetic diversity evaluation, some markers showed few alleles, low heterozygosity and a lower value for probability of exclusion (20,16,21,13). So that the objectives of our study were, firstly, to conduct genetic characterization of Egyptian Arabian horses, secondly, to investigate the powerfulness of these 14 TKY markers for parentage assignment of Egyptian Arabian horses. We believe that this is the first report about the genetic diversity of Egyptian Arabian horse using TKY microsatellite panel.

Sample collection
A total of 101 horse samples representing three different populations were examined, including 71 Arabian horse samples collected from El-Zahraa stud, Egyptian Agricultural Organization (EAO), Cairo, Egypt. In addition to 30 Equine DNA samples were brought for the ISAG Horse Comparison Test (HCT) 2017 and 2019 including 19 samples of Thoroughbred and 11 samples of Nooitgedacht breeds. The source of ISAG samples 2017 and 2019 are South Africa and Germany respectively. These thirty samples were used as a reference samples to compare and standardize allele sizes of TKY Panel and to represent a group of non-Arabian breeds.

DNA Extraction and Microsatellite Analysis
Total DNA from the hair follicles of the Arabian horses were extracted using EZ-10 Spin Genomic DNA Minipreps purification kit following the manufacturer's protocol. A total of 14 TKY microsatellite markers (TKY287, TKY294, TKY  297, TKY301, TKY312, TKY321, TKY325, TKY333,  TKY 337, TKY341, TKY343, TKY344, TKY374 and TKY394) specific to equine were used in this study (16). TKY287, TKY321, TKY343, and TKY344 markers were labeled with FAM dye while TKY297, TKY301, TKY312, TKY337, and TKY374 markers were labeled with HEX dye. Moreover, TKY325, TKY333, TKY341, and TKY394 markers were labeled with NED dye and finally, TKY394 marker was labeled with PET dye. These four dyes allow the amplification of the 14 markers in one multiplex PCR reaction without any overlapping between the markers. TKY Panel was adjusted by using known reference samples brought for the ISAG Horse Comparison Test 2017 and 2019. Alleles were designated with alphabetical symbols from the above allele sizes.

Marker polymorphisms and populations diversity
The total numbers of alleles were 115, 88 and 78 for Arabian, Nooitgedacht and Thoroughbred, respectively. The comparison and standardization for alleles size and frequency of the 14 TKY loci in the Arabian and the two reference horse populations (Nooitgedacht and Thoroughbred) was shown in the  Table 1). The frequency of the five alleles of locus TKY337 in the Arabian and in the two reference populations (Nooitgedacht and Thoroughbred) was shown in Figure 1. Allele 177 bp recorded very high frequency    (19).
The high mean of N A and lower mean of N e and the positive mean for F IS in addition to 12 out of 14 loci showed significant deviation from HWE might indicate that there was non-random mating among our Arabian horses in El-Zahraa stud and a selection program favoring some morphological characters might be practiced on this population. TKY337 locus recorded the lowest values for all diversity indices and significantly deviated from HWE and showed the highest and positive value for F IS (0.330) in our Arabian horses. Moreover, only one allele (177 bp) out of the five reported alleles of the TKY337 locus recorded a very high frequency as shown in Figure 1. This might be attributed to that allele (177 bp) might be under some morphological or beauty related traits of (0.704) in Arabian comparing to Nooitgedacht (0.227) and Thoroughbred (0.211) populations. The parentage assignment of the Arabian population using a combination of the 14 loci was very successful indicated by their high power of exclusion. The combined probability of exclusion (CPE) was 0.9999 in case of excluding putative parent pair. The highest PE value was reported for TKY394 while the lowest for TKY337 loci.

Marker polymorphisms and populations diversity
This study presents the first description of genetic characterization of Egyptian Arabian horses based upon the TKY microsatellites panel. The number of alleles (N A ) and the frequency distribution of these alleles (N e ) in any population could tell us how informative a locus is. The number of alleles (N A ) of our Arabian population was higher than that of Thoroughbred breed of Tozaki study (16) for the most of studied TKY markers except for TKY301 and TKY325 loci (16). The means for H O (0.599) and H E (0.691) of our Arabian horses were higher than selective interest in our Arabian population in El-Zahraa stud. In respect to population genetic diversity, the Arabian population showed high inbreeding level indicated by the higher and positive value of F IS (0.129).
The CPE value of the 14 TKY microsatellite loci of our Arabian horses was 0.9999 which is higher than the required value by the International Stud Book Committee (0.9995), So the 14 TKY microsatellites are suitable for evaluation of genetic diversity not only for Thoroughbred populations (16), but also for our Egyptian Arabian horses. In this study, seven microsatellite loci (TKY287, TKY312, TKY325, TKY333, TKY341, TKY343, TKY374 and TKY394) had high PIC values (>0.600). High level of CPE (>0.9999) can be achieved using only six of the 14 loci (TKY287, TKY325, TKY333, TKY343, TKY344 and TKY394) as shown in (Figure 2), which makes these six markers highly valuable for parentage testing in Arabian horses. Ten microsatellite loci at least was suggested to be used for achieving maximum exclusion in horses (25), but our results showed that fewer loci can achieve relatively high power of exclusion, similar result was recorded by Sereno et al. (26). Two markers, TKY301 and TKY337 were found to have a PIC value lower than 0.500 for the studied Arabian population. As these two markers are considered less informative, they can easily be excluded from parentage testing for the Arabian horses with no significant loss of exclusion power. and should be analyzed in further studies in different Arabian horse populations to test if it is linked to any morphological traits or not. Finally, the high F IS (0.129) of Arabian horses in El-Zahraa stud should be corrected by modifying mating system through avoiding excessive use of certain sires. The data presented in our study will provide the horse breeders with an effective tool for confirming parentage and lineages. Genetic diversity analysis will also generate valuable data necessary for the conservation of this valuable horse breed. Ključne besede: arabski konji; genska raznolikost; mikrosatelit; markerji TKY