This study aimed at screening genetic diversity and differentiation
in four horse breeds raised in Tunisia, the Barb, Arab-Barb, Arabian, and
English Thoroughbred breeds. A total of 200 blood samples (50 for each breed)
were collected from the jugular veins of animals, and genomic DNA was
extracted. The analysis of the genetic structure was carried out using a
panel of 16 microsatellite loci. Results showed that all studied
microsatellite markers were highly polymorphic in all breeds. Overall, a
total of 147 alleles were detected using the 16 microsatellite loci. The
average number of alleles per locus was 7.52 (0.49), 7.35 (0.54), 6.3 (0.44),
and 6 (0.38) for the Arab-Barb, Barb, Arabian, and English Thoroughbred
breeds, respectively. The observed heterozygosities ranged from 0.63 (0.03)
in the English Thoroughbred to 0.72 in the Arab-Barb breeds, whereas the
expected heterozygosities were between 0.68 (0.02) in the English
Thoroughbred and 0.73 in the Barb breeds. All
Biological diversity or biodiversity refers to variability in the hereditary characteristics in a species. The highest genetic diversity level, for a species or a population, offers the opportunity for animals to challenge harsh environmental conditions and cope over with climate change and global warming. The worldwide breeds or populations' sizes went through continuous fluctuations that mirror environmental variation and demographic population growth (Willi et al., 2006; Andrew et al., 2011).
Recent statistics of the Food and Agriculture Organization (FAO) reported that the populations of numerous domestic animals, especially horses, are in steady decline, with some already extinct, thereby affecting both interbreed (decline in the actual number of equine breeds themselves) and intra-breed (decline in the number of individuals) diversities (FAO, 2011). Horses' population sizes of the Tunisian breeds account for around 26 000 heads, of which 14 000, 6000, 5000, and 1000 are Arab-Barb, Barb, Arabian, and English Thoroughbred breeds, respectively. There are also 40 000 male and female mules (FNARC, 2015). These animals are generally used in the fantasia (traditional exhibition of horsemanship performed during cultural festivals), as well as in the equestrian sports. Appropriate conservation and sustainable management programs for the Tunisian equine breeds need comprehensive information about their genetic diversity and populations' structures. Concurrently, a high genetic diversity may indicate a genetic diversity hot spot, a tool for targeting conservation efforts in livestock species (Freeman et al., 2006; Khanshour et al., 2013).
Native horse breeds in Tunisia have been playing key roles through history in agriculture, transportation, and leisure activities. These breeds are now facing major constraints due to harsh climatic conditions and poor management practices. Haddad (2015) reported that the first studbook for Tunisian equine breeds was established in 20 June 1896. There were a few studies on the genetic diversity in the Tunisian horse breeds. The first study aimed at assessing autochthonous Tunisian horse's genetic diversity and was published in 2014 (Haddad et al., 2014; Jemmali et al., 2015). These authors attempted to characterize genetic relationships within and among local breeds (Barb, Arab-Barb and Mogod Pony). A poor genetic diversity was revealed. In the present time, there are attempts to start breeding programs for horses. Pedigree and performance recording are underway. Artificial insemination is a common practice in HARAS of FNARC in the north of Tunisia. Cross breeding is also used to upgrade breeds and to satisfy breeders' demand. There are, however, no conservation programs for equine breeds.
Microsatellite markers have been widely used to assess genetic variability for different horse breeds (Karima et al., 2011). Genetic diversity studies within and among horse breeds analyzed using microsatellites have been conducted on French breeds (Moureaux et al., 1995), Polish breeds (Zabek et al., 2005), Austrian breeds (Druml et al., 2007), American breeds (Tryon et al., 2009), and Algerian breeds (Berber et al., 2014).
The objective of the study was to characterize the molecular structure, microsatellite polymorphism, and genetic distances among breeds and to draw the phylogenic tree for horse breeds in Tunisia.
A total of 200 blood samples were taken and tested from the four Tunisian horse breeds (Barb: BA; Arab-Barb: AB; Arabian: AR; and English Thoroughbred: PS). To ensure that each sample was representative of the respective population, a strict sampling strategy was employed and studbook was consulted. The origins and familial relationships of individual animals were considered and 50 samples were taken for each breed.
Analyzed animals were chosen according genealogical information and familiar relationship. Only individuals with different ancestral ascendance were sampled. In order to choose most genetic variability contributors, for each breed, male and female were randomly equally represented.
Genomic DNA was amplified using 16 microsatellite loci (Table 1). All analyzed individuals were registered in the Tunisian breed's studbook. Approximately 5 mL of veins blood per animal was collected aseptically in tubes containing ethylenediaminetetraacetic acid (EDTA, 0.5 mM, pH 8.0).
Genomic DNA was extracted from total blood using Purelink
Microsatellite sequences and length size.
Amplification of used microsatellites included an initial denaturation at
95
Collected molecular data were edited for possible genotyping errors due to
null alleles, short allele dominance, typographic errors, and the scoring of
stutter peaks. Genetic diversity within breeds, genetic variation, and
relationships among breeds were assessed using different softwares. GenAlex 6.2
was used to calculate gene diversity indices for each breed population
(Khanshour et al., 2013). Genetix software (version 4.04) was used to screen
allelic frequencies and the number of alleles per locus. Observed
heterozygosity (Ho), expected heterozygosity (He), and unbiased expected
heterozygosity (UHe) were calculated across loci and populations (Berber et
al., 2014) and the effective number of migrants per generation (
Structure software (Pritchard et al., 2000) was used to analyze the genetic
structure of sampled animal. The best
Allelic frequencies by population with graph over loci.
All loci microsatellites used in this study were amplified successfully in analyzed individuals for the four breeds (Arab-Barb, Barb, Arabian, and English Thoroughbred). Collected data showed no evidence for null alleles or scoring error considering all screened samples, although there were differences among tested samples with regard to presence or absence of alleles and their frequencies. All tested microsatellites were polymorphic in all populations. A total number of 147 different alleles were found across the 16 amplified loci. Allele frequency for analyzed data varied from 0.00 to 0.77. The highest allelic frequency was observed for HTG7 (127 bp) with 0.77 and 0.72 for the Barb and Arab-Barb breeds, respectively. Arabian and English Thoroughbred breeds had the highest frequencies for HMS1 (177 bp) and HMS2 (226 bp) with 0.58 and 0.64, respectively (Fig. 1).
The number of effective alleles per locus indicates low or high genetic
polymorphism richness of the used markers. The latter number ranged from 3.20
to 3.78. This parameter was 3.78 (0.31), 3.78 (0.30), 3.41 (0.25), and
3.20 (0.22) in the Arab-Barb, Barb, Arabian and English Thoroughbred,
respectively. Although values of the number of effective alleles in studied
breeds were in comparable ranges, there is additional morphological
variability (muzzle, nose, tail, mane, etc.) that can be
detected by other parameters such as mean numbers of alleles per locus,
observed heterozygosity, expected heterozygosity, and
The number of alleles per locus (Na) varied between 6 (HTG6, AHT6 and HMS1) and 13 (ASB23 and ASB17) with a mean of 9.31 (2.40) alleles. In Barb and Arab-Barb breeds, comparable mean values were found for observed heterozygosity, expected heterozygosity, and unbiased expected heterozygosity. These values were 0.7 (0.03), 0.73 (0.03), and 0.73 (0.03), respectively. For Arabian and English Thoroughbred breeds, respective values for Ho were respectively 0.65 (0.04) and 0.64 (0.03), for He they were 0.69 (0.02) and 0.68 (0.02), and for UHe they were 0.68 (0.02) and 0.68 (0.01). In all analyzed breeds we find Ho lower than He. When the observed heterozygosity is smaller than expected, it is because the mating has deviated from random mating-related individuals. Results of this study are similar to those reported by Solis et al. (2005), Khanshour et al. (2013) and Berber et al. (2014). Furthermore, Canon et al. (2000), Tozaki et al. (2003), Behl et al. (2007) and Kusza et al. (2013) reported 6, 5.8, 5.2 and 6.6 as mean numbers of alleles per locus. However, Haddad et al. (2014) obtained 4.23 as a mean value in Barb and Tunisian Mogod Pony horse breeds.
Allelic frequency and heterozygosity for analyzed Tunisian breeds.
AB: Arab-Barb; BA: Barb; AR: Arabian; PS: English Thoroughbred; Np: private alleles.
Principal component analysis for the Tunisian horse breeds.
The mean of the coefficient of inbreeding (
Mean
The
Wright (1978) reported that the
The numbers of effective migrants (
In total, 147 alleles were tested for the Hardy–Weinberg equilibrium (HWE)
for all breeds. Significant (
The first three axes performed on allelic frequencies explain 63.48 % of total inertia. Those first three axes explain 25.45, 23.39, and 14.64 %, respectively. The special distribution indicates high similarities between Barb and Arab-Barb breeds. A significant difference was observed between the Arabian and English Thoroughbred breeds (Fig. 2).
Factorial correspondence analysis for the Tunisian horse breeds.
The neighbor-joining dendrogram including the four studied Tunisian breeds. AB: Arab-Barb (pop1); BA: Barb (pop2); AR: Arabian (pop3); and PS: English Thoroughbred (pop4).
Graphical representation of membership of 200 individuals from AB: Arab-Barb (1); BA: Barb (2); AR: Arabian (3); and PS: English Thoroughbred (4).
The factorial correspondence analysis as shown in Fig. 3 clearly differentiates the Barb, Arabian, and English Thoroughbred breeds. The Barb and Arab-Barb breeds were clustered together. The Arabian and the Arab-Barb breeds were also grouped together on another cluster with minor similarity compared to the first cluster. Berber et al. (2014) reported genetic proximity of both Barb and Arab-Barb breeds. These results corroborate those advanced by Ouragh et al. (1994). The neighbor-joining clustering approach and the factorial correspondence analysis were used as efficient tools that give precise information on breed relationships (Figs. 3 and 4). Genetic distances, the factorial correspondence, and principal coordinate analyses showed that the significant amount of genetic variation is within population.
Estimated individual proportions of membership in each breed are represented by one cluster color. The Arab-Barb (AB) and Barb (BA) breeds are clustered together. The English Thoroughbred (PS) breed was separated from the rest of populations. There are relative similarities between the Arab-Barb (AB) and the Arabian (AR) breed.
This paper highlights the genetic structure of the Tunisian horse breeds. There is a genetic differentiation between Tunisian Barb and Arab-Barb horses and other breeds. The Barb and Arab-Barb appeared to be genetically similar and considered as the same group. This is can be explained by the continuous gene flow between both breeds. Furthermore, the significant amount of genetic variation was within populations. These results may help in the implementation of conservation programs of Tunisian horse breeds and enhance efforts to improve preserving revealed genetic diversity.
The data used in the study can be obtained by email (Mezir Haddad, mezirhaddad@yahoo.fr) from the Fondation Nationale d'Amélioration de la Race Chevaline Sidi Thabet.
The authors declare that they have no conflict of interest.