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Small Ruminant Research 144 (2016) 23–27

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Estimating population structure and genetic diversity of five Moroccan sheep breeds by microsatellite markers Samir Bachir Souheil Gaouar a,b,∗,1 , Samia Kdidi c,d,1 , Lahoussine Ouragh e a

Department of Biology, Aboubakr Belkaid Tlemcen University, Tlemcen 13000, Algeria Molecular and Cellular Laboratory (USTOM), University of Sciences and Technology, Mohamed Boudiaf, Oran 31000, Algeria Livestock and Wildlife Laboratory, Arid Lands Institute Medenine, 4119 Medenine, Tunisie d Laboratory of Genetics, Immunology and Human Pathology, Faculty of Sciences, Tunis-El Manar University, Tunis 2092, Tunisie e Institut Agronomique et Vétérinaire Hassan II, Rabat 10101, Morocco b c

a r t i c l e

i n f o

Article history: Received 26 January 2016 Received in revised form 7 May 2016 Accepted 27 July 2016 Available online 28 July 2016 Keywords: Genetic diversity Genetic differentiation Microsatellites Moroccan sheep breeds

a b s t r a c t Investigating the genetic variability and structure of Moroccan sheep breeds will reveal crucial information for the conservation and management of this population. This study used 22 microsatellite markers to assess the genetic diversity among and within five Moroccan sheep breeds: Sardi (N = 35), Boujaad (N = 31), Timadhite (N = 35), Beni Guil (N = 35), and D’man Morroco (N = 35). In the whole sample, a total of 299 alleles were detected. The five breeds showed a relatively high level of gene diversity ranging between 0.725 (D’man Morocco) and 0.764 (Timahdite). The Analysis of Molecular Variance (AMOVA) indicated that variability among populations contributed only 3.64% of the observed genetic diversity. Wilcoxon tests of excess heterozygosity under the two-phase model (TPM) did not provide strong evidence for recent bottlenecks in the five studied breeds. Unrooted neighbour joining (NJ) tree for the modified Cavalli-Sforza chord distance (DA ), pairwise multilocus estimates of an effective number of migrants (Nm) and the Bayesian clustering method cohesively revealed poor structure of genetic variation among breeds. Our results also show that in spite of the high level of phenotypic diversity in the Moroccan sheep breeds, the past breeding strategies could lead to genetic admixture occurring as a result of relatively high gene-flow among the breeds. © 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction The economic importance of sheep production is increasing in North African countries. In Morocco, ovine meat production exceeded 35% of total red meat (Boujenane, 2006), and the population size of sheep was more than 17 million animals in total (26% males and 74% females) in 2009. Complex topography of Morocco offers diversified ecological conditions and climate types, which influenced notably sheep breed distribution (Fig. S1(a)). Successful adaptation to a particular set of environmental conditions allowed acquiring specific characteristics that vary from breed to breed. More than 10 sheep breeds have been reported in this country (Boujenane, 2005). The main breeds were Sardi, Boujaâd, Timadhite, Beni Guil and D’men. They are thin-tailed and mainly dual-purpose animals with meat being the most essential prod-

∗ Corresponding author at: Department of Biology, Aboubakr Belkaid Tlemcen University, Tlemcen, 13000, Algeria. E-mail address: [email protected] (S.B.S. Gaouar). 1 These authors contributed equally in the work.

uct. The wool remains the next desirable product; Milk is often produced only for family use (Bourfia, 1989). The Sardi breed is the preferred sheep in several social and religious celebrations, and this is led to its special phenotype named “Chatbi” which is characterized by a neat whiteness an open spiral-shaped horns. Bourfia (1989) reported that high demand for the Sardi could play a crucial role in increasing the area of the Sardi breed. According to Boujenane et al. (1995), Beni Guil breed includes Beni Guil, Harcha, Tounsint and Zoulay varieties, and it is adapted to different climate conditions. Timadhite breed was the result of a crossing between Berbère and Tadla Breeds, BeniGuil breed contributed also to the development of the Timahdit breed (Bourfia, 1989). Boujaâd sheep breed is with high growth rate and good conformation. D’man breed inhabits the oases of southern Morocco and the Wadi Saorua Valley of south Algeria and was known as the most prolific sheep. Different breeding strategies have been adopted in different regions of Morocco for improving wool production, quality and body weight in sheep (Boujenane et al., 1995, 2005; Boujenane, 2012a,b). Furthermore, the need to exploit the highly productive species (e.g. cattle instead of Beni Ahsen sheep breed) and breeds, some of less

http://dx.doi.org/10.1016/j.smallrumres.2016.07.021 0921-4488/© 2016 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).

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Table 1 Diversity of studied breeds of sheep obtained from the analysis of 22 microsatellite loci. Breed

N

NF

Nf

An

Ae

PA

Ar

Ho (SD)

He (SD)

FIS (IC 95%)f

Tim Sar Bou D’ma Ben

35 35 31 35 35

23 13 16 26 16

7 8 6 6 4

9.18 8.73 8.41 8.82 8.96

4.65 4.07 4.45 4.04 4.78

0.60 0.64 0.66 0.70 0.70

6.67 6.15 6.54 6.31 6.55

0.724 (0.035) 0.695 (0.036) 0.626 (0.038) 0.609 (0.037) 0.656 (0.028)

0.764 (0.021) 0.731 (0.021) 0.748 (0.024) 0.725 (0.022) 0.742 (0.028)

0.054* (0.00006–0.07754) 0.050* (−.01209–0.07970) 0.165* (0.10419–0.18932) 0.163* (0.09078–0.20126) 0.132* (0.06841–0.16282)

Tim: Timahdite. Sar: Sardi. Bou: Boujaad. D’ma: D’man Morocco. Ben: BeniGuil. N: number of individuals. NF : number of females, Nf : number of flocks. An: mean number of alleles in each population. Ae: average number of effective alleles per locus. PA: frequence of private alleles per breed. Ar: Allelic Richness (rarefacted). HO : observed heterozigosity. He : unbiased heterozygozity. f 10,000 Bootstrap over FIS by population, IC 95% = confidence interval at 95%. * Significant p-values (p < 0.01).

productive local breeds have been neglected; others have been crossed with foreigner breeds or even replaced by more productive breeds. As a result, Beni Ahsen and Atlas Mountain breeds, and perhaps the Beni Guil, have decreased tremendously in numbers (Boujenane, 2005). Molecular Genetic studies have been conducted on different breeds of sheep worldwide (Tapio et al., 2010; Calvo et al., 2011; Tolone et al., 2012; Ciani et al., 2013; Kunene et al., 2014). Northwest Africa is a major hotspot of sheep diversity, but little is known about the genetic structure of ovine populations in this region. Here, we use microsatellite DNA markers to examine patterns of genetic diversity and differentiation among Moroccan sheep breeds. Specifically, we ask: (i) Is there evidence for bottlenecks in these breeds and (ii) What are the patterns of genetic diversity within vs. among breeds in Morocco? 2. Materials and methods 2.1.Samples and amplification In the present study, a total of 171 blood samples of sheep were collected from Morroco. Samples came from 5 native breeds (Fig. S1(a)):Sardi (N = 35), Boujaâd (N = 31), Timadhite (N = 35), Beni Guil (N = 35), and D’men (N = 35). Sampling was carried out in 2006, and was obtained from different flocks. Information about relatedness between animals was obtained from breeders and farmers when pedigree data is not available and unrelated animals were taken per flock. The genomic DNA purification from blood was performed according to the Salting out protocol (Miller et al., 1988). The 171 samples were genotyped at 22microsatellite loci (Table S1). Moreover, a sample of 30 Merino de Rambouillet sheep breed was used as an outgroup for tree topology. The microsatellite analysis and detection of amplified products were performed as described in Gaouar et al. (2015). 2.1. Data analysis Significant deviations from Hardy-Weinberg equilibrium expectations were evaluated by Fisher’s exact tests, with unbiased P-values (10,000 dememorizations, 100 batches, 1000 iterations per batch) as implemented in GENEPOP 3.4 program (Raymond and Rousset, 1995). We used GENALEX 6.1 (Peakall and Smouse, 2006) to estimate, in the whole sample, mean number of alleles (MNA) and effective allele number (Ne) by locus, and for each breed, mean number of alleles (An), observed and expected unbiased heterozygosity (Ho and He respectively) and average number of effective alleles per locus (Ae). Rarefaction approach as developed in HP-RARE (Kalinowski, 2005) was used for allelic and private allelic richness estimation. We calculated for each locus number of individuals typed (N), number of alleles (Na) observed (Ho ) and expected unbiased (He ) using CERVUS 3.0.3 (Marshall et al., 1998). GENETIX 4.05 (Belkhir and Borsa, 1996–2004) was used to determinate the FST values for pairwise comparisons of the breeds, to compute Wright’s inbreeding estimator (FIS ; Weir

and Cockerham, 1984) and to assess FIS significance using 1000 random permutations of alleles in each breed. To test for bottlenecks, we used the program BOTTLENECK version 1.2 (Cornuet and Luikart, 1996) utilizing the Wilcoxon test for heterozygote excess, as well as the two-phase model (TPM) recommended by Piry et al. (1999) and Peery et al. (2012). The significance of fixation indices determined using permutation tests (1000 permutations) and the estimation of the variance within and between breeds, and analysis of molecular variance (AMOVA) were performed with ARLEQUIN 3.5.1.2 (Excoffier et al., 2005). The population structure was analysed by cluster techniques with the software STRUCTURE 2.3.4 (Pritchard et al., 2000) with K ranging from 1 to 8. The most probable K was determined using STRUCTURE HARVESTER Web version 0.6.93 (Earl, 2012). Modified Cavalli-Sforza chord distance (DA ; Nei et al., 1983) were estimated using POPULATIONS v 1.2.32 (Langella, 1999) and dendrograms were constructed according to the neighbour-joining algorithm, with the French sheep breed Merino de Rambouillet as an outgroup (data not published). Tree topology was constructed using POPULATIONS v 1.2.32, and the reliability of each node was estimated by 1000 resampling of the data. The unrooted tree was viewed in FIGTREE version 1.4 (http:// tree.bio.ed.ac.uk/software/figtree/). To assess the genetic relationship of Algerian (data from Gaouar et al., 2015) sheep breeds, a standard Nei genetic distance (Ds ; Nei, 1972) matrix between all pairs of populations was calculated using French Merino de Rambouillet sheep breed as outgroup in the program POPULATIONS v 1.2.32 (Langella, 1999). Bootstrap values were calculated over all loci and neighbour-joining (NJ) data were exported to FIGTREE program version 1.4 (http://tree.bio.ed.ac.uk/software/figtree/) for graphing. 3. Results and discussion Genetic diversity statistics are summarised in Table S1. Twentytwo microsatellite markers resulted in a total number of 299 alleles. Twenty-one markers showed high informative (Polymorphic Information Content (PIC) > 0.5), only one locus (OarAE129) showed moderate PIC (0.25 < PIC < 0.5). Numbers of alleles per locus ranged from 6 (BM182andOarAE129) to 23 (HUJ616). The result of HPRARE program revealed a low number of private alleles (between 0.60 and 0.70 per breed) as well as medium allele richness (between 6.15 and 6.67). Allelic richness values were lower than those observed in Turkish sheep breeds (9.40–13.75; Yilmaz et al., 2014) and Tunisian sheep breeds (8.08–8.94; Sassi-Zaidy et al., 2014), but higher than those reported in Algerian sheep breeds (4.97–6.16; Gaouar et al., 2015). The mean value of He (Table 1) was between 0.725 (Moroccan D’man) and 0.764 (Timahdite), and that of Ho between 0.609 (D’man Morocco) and 0.724 (Timahdite). Previous studies on D’man breeds showed similar values to D’man breed of Algeria (Ho = 0.64; He = 0.68; Gaouar et al., 2015) and D’man breed of Tunisia (Ho = 0.716; He = 0.815; Sassi-Zaidy et al., 2014). The gene

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Fig. 1. Unrooted neighbour joining (NJ) tree for the modified Cavalli-Sforza chord distance (DA , Nei et al., 1983) for the sampled sheep breeds of Morocco. Bootstrapping support is given at the nodes and indicates the percentage of trees constructed from 1000 bootstrapped distance matrices that shared that node.

Fig. 2. Graphic assignment for a three cluster (K = 3) assessment of the five Moroccan sheep breeds using STRUCTURE program. (a) colored barplots show the weighted assignment of individuals belonging to the investigated sheep breeds to each K. (b) Triangle plot displays average admixture of these five breeds.

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Table 2 Pairwise genetic differentiation between breeds estimated from 22 microsatellite loci. Values above diagonal represent Nm (number of migrants) and values below diagonal FST . Bold values of FST indicate significance (P < 0.05).

Timahdite Sardi Boujaad D’man Marocco BeniGuil

Timahdite

Sardi

Boujaad

D’man Marocco

BeniGuil

– 0.027 0.023 0.030 0.030

9.85 – 0.036 0.052 0.043

14.25 8.23 – 0.037 0.044

10.13 5.14 8.16 – 0.065

7.03 4.78 5.30 4.12 –

diversity observed for Moroccan sheep breeds was moderate. This diversity is distinctive trait of traditional populations (Edea et al., 2013). It may notably be the result of the lack of artificial selection pressure and high level of genetic admixture in the breeds. Fifteen of the 22 microsatellites showed significant (p < 0.05) departures from the Hardy-Weinberg equilibrium in the whole population. However, when considering breeds separately, several markers per breed were in Hardy–Weinberg disequilibrium (p < 0.05). The number of these markers varied between 5 loci in Timahdite to 12 loci in Boujaad and D’man Morocco. All the studied breeds showed a highly significant departure (p < 0.01) from Hardy Weinberg equilibrium when considering all loci. This departure and the high positive mean values of FIS (Table 1) may indicate the existence of heterozygote deficiencies, which could be the result of an uncontrolled mating between breeds. The global FIT and its confidence interval at 95% after 1000 bootstraps was 0.109 (0.056–0.169). In the overall population the homozygote excess (FIT ) was caused mainly by a homozygote excess within breeds (FIS = 0.142 (0.090–0.200)) and partially by the low genetic differentiation among breeds (FST = 0.036 (0.026–0.049)). This FST value (3.6%), though significant, was low. It could be attributed to genetic admixture of the breeds and absence of selection process. Furthermore, these results could be interpreted to indicate common ancestry of these breeds. The Analysis of Molecular Variance (AMOVA) indicated that most of the genetic diversity occurred within individuals (85.83%) while the variability among populations and among individuals within populations contributed 3.64% and 10.53%, respectively, to the observed genetic diversity (Table S2). The differentiation value observed in this study was similar to values reported in six Algerian sheep breeds (FST = 3.8%; Gaouar et al., 2015) using 30 microsatellite markers and six Tunisian sheep breeds in a study using 17 microsatellite markers (FST = 3%; Sassi-Zaidy et al., 2014). However, it was higher than reported value (FST = 1.7%)for four Tunisian sheep breeds in a study using 30 microsatellite markers (Kdidi et al., 2015). Higher differentiation values, which ranged between 0.049 and 0.121, have been previously reported in the literature for some breed of Spanich (Arranz et al., 2001; Álvarez et al., 2004; Calvo et al., 2011), Portuguese(Santos-Silva et al., 2008), Nigerian (Agaviezor et al., 2012) and Turkish sheep (Yilmaz et al., 2014). The genetic differentiation (FST ) and gene flow between pairs of breeds are shown in Table 2. The FST values ranged from 0.023 (Timahdite – Boujaad pair) to 0.065 (D’man Morocco – Beni Guil pair). The pairwise exact tests of differentiation among the five breeds showed significant comparisons after 1000 permutations. The gene flow occurring between populations in each generation varied between 4.12 (D’man Morocco – Beni Guil pair) and 14.25 (Timahdite – Boujaad pair). Number of migrants after correction for size (Nm) across all studied breeds was 4.13 indicating the presence of considerable gene flow among them (Nm > 1). Gene flow plays an effective role in decreasing the genetic differentiation between the breeds, especially between those raised within the same or close geographical area. Timahdite breed has high rate of gene flow with the other sheep breeds (Table 2) which may be the result of uncon-

trolled mating due to close geographical location of this breed to other breeds (Fig. S1(a)). None of the breeds showed significant bottlenecks based on the Wilcoxon test under two-phase model (TPM which allows multiple-step mutations). The topology of the Neighbour Joining tree (Fig. 1), built on DA (Nei et al., 1983) genetic distances, clearly highlighted the genetic differentiation of Beni Guil breed (average DA = 0.156) in comparison with other breeds. The topology was in agreement with the geographical location of the studied sheep breeds. In order to compare these five breeds with the Algerian (6 sheep breeds (Fig. S1(a)); Gaouar et al., 2015) and French (data not published) sheep breeds, Nei’s standard genetic distance (Ds; Nei, 1972) was estimated for each pair of populations. The neighbour-joining tree built from Ds was rooted using the Merinos de Rambouillet as out-group (Fig. S1(b)) The NJ tree clearly displayed the genetic differentiation of the French sheep breed (average Ds = 0.986) in comparison with Algerian and Moroccan sheep breeds which were closer. The tree topology obtained agreed with the results of Gaouar et al. (2015) for the Algerian Ouled Djelel, Rembi and Taadmit sheep breeds as shown by low genetic differentiation. Based on the results of the STRUCTURE analysis, the most likely value of K was 3 indicating that the 5 Moroccan sheep breeds included in this study can be assigned to three clusters. The proportions of each breed that contributed to each of the three clusters are shown in Fig. 2(a). Beni Guil, Boujaad and D’man Morocco appear as three genetically distinct groups. D’man Morocco and Sardi belonged to the same cluster. However, Timahdite remains the less differentiated breed. In the triangle plot (Fig. 2(b)), D’man Morocco and Sardi sheep breed occupied the same corner which may be explained by their common origin. Bourfia (1989) and Boujenane et al. (1995) reported that Berbère, Boujaad and Beni Guil sheep breeds contributed to the development of Timahdite sheep breed that showed large distribution in the triangle plot. 4. Conclusions Microsatellite loci analyses revealed that most of the investigated Moroccan sheep breeds were not genetically well differentiated. The level of genetic diversity could be attributed to lack of artificial selection pressure and high level of gene flow among breeds typical of traditional breeding systems. The data generated by this study may be useful for breeding programs and conservation plans. Conflict of interest statement The authors declare that there is no conflicts of interest. Acknowledgements The authors thank all the subjects who contributed samples. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.smallrumres. 2016.07.021. References Álvarez, I., Royo, L.J., Fernández, I., Gutiérrez, J.P., Gómez, E., Goyache, F., 2004. Genetic relationships and admixture among sheep breeds from Northern Spain assessed using microsatellites. J. Anim. Sci. 82, 2246–2252. Agaviezor, B.O., Peters, S.O., Adefenwa, M.A., Yakubu, A., Adebambo, O.A., Ozoje, M.O., Ikeobi, C.O.N., Wheto, M., Ajayi, O.O., Amusan, S.A., Ekundayo, O.J., Sanni, T.M., Okpeku, M., Onasanya, G.O., De Donato, M., Ilori, B.M., Kizilkaya, K.,

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