http://orcid.org/0000-0002-7450-4311
Abstract
Exposition of variations between breeds is very important for genetic diversity. Determination of this variation is needed to reveal population structure and relationship between populations and planning national breeding and conservation programmes. This study was carried out in 296 animals from 12 different local sheep breeds (Barbarine, Ouled Djellal, Ifilene, Srandi, Darâa, Rembi, Berbere, Taâdmit, Hamra, Sidaou, Tazegzawt and D’men) reared in different regions of Algeria. Fifteen microsatellite markers were used to determine between breed genetic diversity. The population of 12 sheep breeds studied from Algeria exhibited a high number of alleles (24.67) and polymorphic information content (0.90). Observed heterozygosity values were lower than expected for all molecular markers except INRA0123 locus. Obtained GST value from the present study indicated that 1.9% of total genetic variation resulted from the differences between the breeds. The present study supplied important information to understand between breed genetic differences. Moreover, it has provided the opportunity to discuss with previously reported results. In light of these findings, it can be said that studied microsatellite markers can be successfully used to determine genetic diversity and population structure in Algerian sheep breeds.
Keywords:
Introduction
Algeria located in north-west of African continent has a significant traditional background for sheep breeding. It played an important role in meeting community needs such as meat, milk and wool. There are 12 important breeds for the sheep breeding such as Ouled-Djellal, Rembi, Hamra, Berbère, Barbarine, D’man, Sidaou, Tâadmit, Daraa, Ifilen, Srandi and Tazegzawt. These sheep population have been raised commonly in Algeria for disease-tolerant, good productive characteristics and to adapt disease resistance, in arid, steppe and Saharan conditions (Gaouar et al. Citation2011, Citation2014, Citation2015a, Citation2015b, Citation2016a, Citation2016b; Djaout Citation2016).
Native breeds are the primary elements of animal breeding and they have complied with ecological, social and economic conditions of different geographies. These elements have taken shape within the process of thousands of year’s agricultural society throughout history of humanity. Unfortunately, it is reported that some breeds are reported to be in danger of extinction and threatening the genetic diversity of Algerian sheep population as in all over the world as a result of rapidly changing industrialisation, sociological and economic conditions (Chellig Citation1992).
Throughout history, the entire Algerian sheep population has passed through three major classifications based on the phenotypic data, morphological descriptions and breeder’s notifications. The first is described by Couput (Citation1900), which divided the whole population into three groups according to their geographical position and external phenotype, in the second approach, the population is divided into two groups, main and secondary breeds (Chellig Citation1992; Gaouar Citation2002). The final and recent classification based on mapped breeding zones of Algerian sheep population was introduced by Djaout (Citation2016) in which, 12 ovine breeds are phenotypically characterised.
Autosomal microsatellites provide useful and objective information on population structure (Bruford & Wayne Citation1993; Ashley & Dow Citation1994; Arora & Bhatia Citation2004). In addition, these markers provide very useful information to reveal relations between animal populations and level of variation between and within populations (Forbes et al. Citation1995), paternity and kinship analyses (Luikart et al. Citation1999; Schlotterer Citation2004) and linkage analysis (Kappes et al. Citation1997).
There are few studies that are interested in the genetic characterisation of sheep flocks in Algeria. They are all conducted by Gaouar et al. (Citation2014, Citation2015b, Citation2016b) in which they characterise respectively two breeds with six loci of microsatellites, then six sheep breeds by using 30 microsatellites and eight breeds through the use of SNPs. However, the sampling used in these studies is very old dated from 1999 to 2004 in which it does not reflect the actual situation of these breeds furthermore this situation has gradually deteriorated in recent years (Gaouar et al. Citation2015b). Therefore, this present study has come to give an update for the current situation of the genetic structure of the sheep flock in Algeria, in which 12 breeds were investigated (D’men, Hamra, Ouled-Djellal, Rembi, Sidaoun, Tazegzawt, Berber, Barbarine and Taadimit) with three new breeds added (Ifilen, Srandi and Daraa) using 15 autosomal microsatellites.
Materials and methods
Animal material and DNA isolation
The animal material of the study consisted of a total of 296 animals from 12 sheep breeds raised in different location in Algeria (Figure ). The sample sizes of these 12 breeds are presented in Table .
Figure 1. Geographic distribution of sheep breeds studied in the Algerian territory.
Table 1. Origin and sample size of 12 sheep breeds raised in Algeria.
Blood samples were collected from the jugular veins of the animal material using vacutainer tube containing Tri-Potassium Ethylene Diamine Tetra Acetic Acid (K3EDTA). Collected bloods were stored at –20 °C until analyses. The genomic DNA was extracted according to salting out method procedures described by Miller et al. (Citation1988). Afterward, quantification and qualification of DNA were controlled using NanoDrop 2000 (Thermo Scientific, Waltham, MA).
PCR and fragment analysis
This step was carried in molecular genetics laboratory, ADNAN Menders University, Aydin. Fifteen microsatellite markers, labelled with fluorescent dye (D2, D3 and D4), were used according to recommendation of FAO (Citation2011). Two multiplex groups were created according to fragment length of microsatellites (Table ).
Table 2. Details of considered microsatellite loci.
The total volume of the amplification mixture was made to 25 μL. Amplification mixture contained 0.1 μM/each primer, 0.2 mM dNTPs (Applied Biological Materials Inc., Richmond, BC), 2.0 mM MgCl2, 1× PCR buffer, 1 U of Taq DNA polymerase (Applied Biological Materials Inc., Richmond, BC) and ∼50 ng genomic DNA. Genomic DNA was amplified by touchdown PCR technique with the multiplex microsatellite group (Table ).
Table 3. Thermal cycling conditions according to touchdown PCR.
Capillary electrophoresis was used for the separation of the PCR fragments labelled with fluorescent dye in the Beckman Coulter GeXP genetic analyser (Beckman Coulter, Inc., Carlsbad, CA). GenomeLab™ DNA Size Standard Kit 400 was used for determination of fragment size.
Statistical analysis
Number of alleles per locus (Na), mean number of alleles (MNa), effective number of alleles (Ne), polymorphic information content (PIC), observed heterozygosity (Ho), expected heterozygosity (He), average heterozygosity (Ĥ), Hardy–Weinberg equilibrium and null allele frequencies were calculated using GenAlEx 6.5 (New Brunswick, NJ) (Peakall & Smouse Citation2006, Citation2012), POPGENE Version 1.32 (Alberta, Canada) (Yeh et al. Citation1997) and CERVUS 3.0.3 (Bozeman, MT) (Marshall Citation2006; Kalinowski et al. Citation2007). The genetic distance dendrogram for the breed was drawn with MEGA 6 (Tamura et al. Citation2013) and Dendroscope (Huson & Scornavacca Citation2012) software according to Nei’s minimum genetic distance matrix (Nei Citation1972). The bootstrap resampling methodology (1000 replicates) was performed to test the robustness of the dendrogram topology. Wright’s F-statistics (FIT, FIS, FST) (Wright Citation1931; Weir & Cockerham Citation1984) were calculated with POPGENE (Yeh et al. Citation1997). Nei’s gene diversity (HT), diversity between breeds (DST) and coefficient of gene differentiation (GST) values were calculated with FSTAT 2.9.3 (Goudet Citation2001). The factorial correspondence analysis (FCA) is performed to visualise the relationships between individuals from different breeds and to test possible admixtures between the populations. FCA was computed using GENETIX 4.05 (Montpellier, France) (Belkhir et al. Citation2000).
The genetic structure of the populations was investigated using STRUCTURE 2.3.4, (Oxford, UK) (Pritchard et al. Citation2000; Falush et al. Citation2007; Hubisz et al. Citation2009). Analysis was performed with a burn of 20,000 in length, followed by 100,000 Markov chain Monte Carlo iterations for each from K = 2–12, with 20 replicate runs for each K, using independent allele frequencies and an admixture model. Evanno’s method (Evanno et al. Citation2005) was used to identify the appropriate number of clusters using ΔK, based on the rate of change in the log probability of the data. The optimal K values were selected by means of STRUCTURE HARVESTER (Earl & vonHoldt Citation2012).
Results
A total of 367 alleles from 15 microsatellite loci were observed. The number of alleles ranged from 19 (OARCP34) to 35 (OARJMP29), with the average number of alleles amounting to 24.67 and the effective number of alleles was 11.05. Estimated heterozygosity (He) value varied from 7.57 (OARCP34) to 17.3 (BM1818). PIC values were found to be between 0.85 and 0.94. The Ĥ value for all loci studied was 0.86 (Table ).
Table 4. Genetic polymorphism parameters at 15 microsatellite loci in Algerian sheep population.
The lowest and highest expected heterozygosity (He) values were 0.82 (MAF214) and 0.94 (BM1818), respectively. Wright F statistics (FIS, FIT, FST), described the statistically expected level of heterozygosity in the population, were calculated in the present study. Average values of FIS, FIT and FST were 0.047, 0.088 and 0.044, respectively. Obtained overall DST value describing the diversity between breeds was 0.017. General mean of GST value determining gene differentiation coefficient was 0.019. Nei gene diversity (HT) ranged between 0.856 and 0.940. All microsatellite loci deviated from the Hardy–Weinberg equilibrium. The presence of null alleles, defined as non-amplifying alleles, due to mutations at PCR priming sites, causes overestimation of both FST and genetic distance values. The null allele frequencies in the studied microsatellite loci were below 20%. Genetic diversity results according to breed are summarised in Table .
Table 5. Genetic diversity measure according to breed across 15 loci.
The highest and lowest allele number values were seen in OJ (17.80) and DR sheep breeds. HR sheep breed showed the highest values in terms of mean expected heterozygosity. It has been determined that some of the studied loci in the BB, OJ, IL, SR, RB, TD, HR, SD, TG and DM are not in the Hardy–Weinberg equilibrium. On the contrary, all the microsatellite loci in DR sheep breeds were in the Hardy–Weinberg equilibrium. The FIS values, which are an important parameter in defining the population structure and indicating the loss of heterozygosity, ranged between 0.038 (IL) and 0.140 (DR). Although a total of 41 private alleles have been identified in all breeds studied, only four of them have a frequency greater than 5%.
The phylogenetic tree constructed using Nei’s minimum genetic distances obtained from present study is given in Figure . It was seen that there are four different groups considering the dendrogram. The FCA explains a 41.66% of the total variation (Figure ). The first axis explains the 17.27% of the total variation and separates the Hamra, D’man and Sidaou breeds followed by Tazegzawt breed from the rest. The second axes, representing the 13.33% of the total variation, showed the isolation of the breeds Barbarine and Ouled Djellal, while the third one, which represented the 11.08% of the total variation grouped the remaining six breeds together in this last group. These results obtained have been similar to the dendrogram given in Figure .
Figure 2. Dendrogram based on Nei’s minimum genetic distances among 12 breeds (bootstrap resampling methodology (1000 replicates)). BB: Barbarine; OJ: Ouled Djellal; IL: Ifilene; SR: Srandi; DR: Daraa; RB: Rembi; BR: Berbere; TD: Taadmit; HR: Hamra; SD: Sidaou; TG: Tazegzawt; DM: D’men.
Figure 3. The factorial correspondence analysis (FCA) results showing the relationship between 12 breeds. BB: Barbarine; OJ: Ouled Djellal; IL: Ifilene; SR: Srandi; DR: Daraa; RB: Rembi; BR: Berbere; TD: Taadmit; HR: Hamra; SD: Sidaou; TG: Tazegzawt; DM: D’men.
The results of the STRUCTURE analysis containing different numbers of clustering performed to determine the population structure of the studied breeds are given in Figure . For the purpose of presenting, the suitable cluster number (K) in structure analysis results is given in Table .
Figure 4. Estimation of the population structure with different K values (assuming K = 2 and 12). BB: Barbarine; OJ: Ouled djellal; IL: Ifilene; SR: Srandi; DR: Daraa; RB: Rembi; BR: Berbere; TD: Taadmit; HR: Hamra; SD: Sidaou; TG: Tazegzawt; DM: D’men.
Table 6. Nei's genetic identity (above diagonal) and genetic distance (below diagonal).
Table 7. Estimated posterior probabilities [Ln Pr(X|K)] for different numbers of inferred clusters (K) and ΔK statistics.
Discussion
Algerian sheep population increasing in recent years occupy an important position compared to other farm animals. This is because, the products obtained from the sheep such as meat, milk and wool are preferred in most parts of Algeria, and also the main source of income rather than other farm animal industry in the country. The identification of sheep populations has become very important phenomena in Algeria. Thus, molecular genetic identification has been important factors as well as performance records and morphological appraisals for sheep breeding. Thus, the present study was conducted to detect the genetic variability and population structure through microsatellite markers in 12 different sheep breeds raised in Algeria.
It was not possible to compare results obtained from present study with the same sheep breeds, as there were a limited number of molecular genetic studies on the majority of breeds studied. It can be said that the majority of the markers studied were highly polymorphic and all markers showed a significant deviation from the Hardy–Weinberg equilibrium.
Obtained molecular genetic parameters (Na, Ne, PIC and Ĥ) obtained from this study were higher than previous research (Farid et al. Citation2000; Arranz et al. Citation2001; Santos-Silva et al. Citation2008; Jyotsana et al. Citation2010; Al-Barzinji et al. Citation2011; Hoda & Marsan Citation2012; Gaouar et al. Citation2014, Citation2015b, Citation2016a, Citation2016b; Yilmaz et al. Citation2014; Kdidi et al. Citation2015; Othman et al. Citation2016). These results indicated that microsatellites used in the present study have a high confidence to reveal genetic diversity for these breeds. A high level of heterozygosity observed can be explained by the high homogenisation and uncontrolled cross breeding observed in Algeria (Gaouar Citation2002, Citation2009).
It was an important parameter in the study that the FIS and FIT values were considerably lower than the numerous sheep breeds such as Colombian sheep breeds (Ocampo et al. Citation2016), Tunisian native sheep breeds (Ben Sassi-Zaidy et al. Citation2014) and Turkish native sheep breeds (Yilmaz et al. Citation2014), and higher than the values reported by Cemal et al. (Citation2013). It can be said that there is loss of heterozygosity in four microsatellite loci studied considering FIS value is also known as the inbreeding coefficient. It is noteworthy that obtained general mean of FST value is higher than earlier studies (Cemal et al. Citation2013; Ben Sassi-Zaidy et al. Citation2014; Ocampo et al. Citation2016) and lower than others (Sodhi et al. Citation2006; Yilmaz et al. Citation2014). The differences between the previous literature and the present study were mainly due to non-comparative aspects such as studied microsatellite and breed differences.
The mean DST results in our study genotypes were lower when compared to Turkish native sheep breeds for the similar microsatellite loci (Yilmaz et al. Citation2014). These results showed a low genetic diversity between studied sheep populations.
Although the GST values in the present study were lower when compared to Swiss sheep breeds (Stahlberger‐Saitbekova et al. Citation2001), Nigerian native sheep breeds (Agaviezor et al. Citation2012) and Native Turkish sheep breeds (Yilmaz et al. Citation2014) were in fact higher than Albanian sheep breeds (Hoda & Marsan Citation2012). The average GST value obtained from overall loci pointed out that 1.9% of total genetic variation resulted from the differences between the populations. In all other respects, it can be said that 98.1% genetic variation is caused by the difference between individuals.
All studied loci showed a significant deviation from the Hardy–Weinberg Equation. These results may have occurred as a result of a management programme such as controlled mating, some breeding activities for many years to improve the populations studied.
Mean number of alleles, observed (Ho) and expected (He) heterozygosity values obtained in the present study were higher than those of earlier studies (Ben Sassi-Zaidy et al. Citation2014; Gaouar et al. Citation2014, Citation2015b, Citation2016a, Citation2016b; Kdidi et al. Citation2015; Guang-Xin et al. Citation2016; Loukovitis et al. Citation2016; Othman et al. Citation2016). On the other hand, Algerian sheep breeds generally showed a high genetic diversity in comparison with the results obtained by Gaouar et al. (Citation2014, Citation2015b) on the same breeds. These results show that these microsatellites used in Algerian sheep breeds in the present study provide a very high level of information. FIS values were lower than the values reported in Turkish sheep breeds (Yilmaz et al. Citation2014), Tunisian sheep breeds (Kdidi et al. Citation2015) and Moroccan sheep breeds (Gaouar et al. Citation2016a). The low genetic differentiation of Algerian sheep breed displayed here is in concordance with the results obtained by Gaouar et al. (Citation2015a, Citation2015b) with 30 microsatellites. FIS values obtained according to breeds studied indicated loss of heterozygosity. This heterozygosity deficiency was mainly due to subdivision among flocks known as Wahlund effect and uncontrolled cross breeding with not respect to distribution area of each breed by the breeder.
The phylogenetic tree constructed using Nei’s minimum genetic distances among 12 breeds showed four distinct groups. The first group consisted of four breeds (HR, DM, SD and TG) that are closely related respectively in two groups of breeds and both of them clustered together similar to that found by Gaouar et al. (Citation2014) (Table ). While the second group was formed by the BB breed alone. The dendrogram also showed in the third group, a close proximity of three breeds DR to SR followed by IL may be related to their common cradle and geographical distribution (Figure ). The last group included the remaining breeds of TD, BR, RB and OJ breeds (Figure ). The close relationship between these four breeds of the last cluster was probably due to the direct effects of the processes of admixture with OJ breed in their formation (Chellig Citation1992; Gaouar et al. Citation2016b).
Assignment test was carried out using the STRUCTURE programme with the expected number of populations (K) ranging from 2 to 12. The Ln[Pr(X|K)] increased from K = 2 to K = 3. Then it rapidly decreases at K = 4. ΔK value indicated that the most suitable group number was 3 (K = 3)* in the 12 breeds of Algerian sheep breeds studied (). These results are not in agreement with what was found by Gaouar et al. (Citation2015b), that they clustered the Algerian sheep population in six groups using 30 microsatellites. In fact, this situation confirms that in the last years the livestock ovine in Algeria is gradually deteriorated by the effect of the uncontrolled crossbreeding between the sheep breeds and especially the Ouled Djellal breed which is the dominant breed and preferred by the breeders.
Conclusions
Sheep represent the tradition of breeding in Algeria. They constituted the sole income of a third of the Algerian population. The sheep herd with all the local breeds are always classified in a traditional way based on external morphology and production traits. Molecular characterisation using a 15 set of microsatellite markers showed that 12 Algerian sheep breeds are regrouped on three distinct cluster and they have more within breed variation than between breeds genetic variation. But over time this diversity observed has several problems such as restricting the cradles of the breeds for the favourite breed Ouled Djellal. Thus, for the new breeds identified like Ifilen, Srandi and Daraa, despite their small size they indicate a lot of perspective, it would be interesting to complete this work by a study of other parameters of production.
Acknowledgements
The authors acknowledge the Agricultural Biotechnology and Food Safety Application and Research Center (ADÜ-TARBİYOMER) of Adnan Menderes University for providing laboratory facilities for molecular genetics analyses.
Disclosure statement
No potential conflict of interest was reported by the authors.
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