Investigation of genetic relationships among Hamra and Béni-Ighil sheep breeds based on microsatellite markers
Suheil Samir Bachir GAOUAR1,2* , Samia KDIDI3,4 , Nacéra TABET AOUEL 1 , R AÏTYAHIA 5 , Nadjet BOUSHABA 1, Miloud AOUISSAT 6 , Nathira SAIDI-MEHTAR 1.
1
-Laboratoire de Génétique Moléculaire et Cellulaire, Université des sciences technique d’Oran
(USTO), Oran 31000, Algérie. 2
-Département de biologie, Faculté des sciences de la nature, de la terre et de l’univers,
Université Aboubakr Belkaid, Tlemcen 13000, Algérie. 3
- Livestock & Wildlife Laboratory, Arid Lands Institute, 4119 Medenine Tunisia,
4
- Laboratory of Genetics, Immunology and Human Pathology, Faculty of Sciences, Tunis-El
Manar University, 2092 Tunisia. 5
- Département de génétique moléculaire appliqué, Faculté des sciences, Université des sciences
technique d’Oran (USTO), Oran 31000, Algérie. 6
- Institut téchnique de l’élevage (ITElv) d’Aïn El-Hadjar 20100 Saïda, Algérie.
Corresponding author: *
[email protected]
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Abstract:
The knowledge of the genetic relationship and admixture among historical populations having kinship relation is crucial for conservation efforts. The aim of this study was to analyse the genetic diversity of Béni-Ighil (Moroccan sheep breed, n= 50) and Hamra (Algerian sheep breed, n=35) using 6 microsatellites. Blood samples from 85 individuals belonging to the aforementioned breeds were obtained and genotyped. The mean number of alleles per locus was 12.17. Moreover, the mean proportion of private alleles was 0.027. Observed heterozygosity average over loci was 0.672±0.179. The estimated within-population inbreeding FIS was significant in studied breeds. The Reynold’s genetic distance (DR) and the number of migrants Nm values were respectively 0.044 and 5.21, indicating a relatively high gene flow among breeds.
Key words: Béni-Ighil, Genetic diversity, Hamra, Microsatellites, Sheep
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Introduction
The North African region, which includes Morocco, Algeria, Tunisia, Libya and Egypt, raises more than 100 million sheep (FAOstat 2012), and is ranked among the top sheep production areas in the world (FAO, 1981). In Morocco and Algeria sheep farming is an important economic and social activity contributing to 38 % (Fellah-trade 2011) and 54% (Nedjraoui 2006) of the total red meat production, respectively. There are, respectively in Algeria and Morocco, more than twenty one million (ONS 2009), and seventeen (Maroccan Ministry of Agriculture 2005) million sheep heads. These two neighbouring countries show an important sheep diversity. Indeed, seven (Ouled Djellal, Rembi, Hamra or Béni-Ighil, Berbère, Barbarin, D’men and Sidahou) and six (Timahdite, Sardi, Béni-Ighil, Boujaâd, Beni Ahsen and D’man) breeds are known respectively in Algeria and Morocco. The Algerian Hamra sheep breed also named Béni-Ighil which represents 0.31% (Algerian Ministry of Agriculture, 2003) of total sheep number is known as originated in eastern Morocco where it derived from the Moroccan Béni-Ighil (11.42%). The breed is primarily localized in the Western steppes close to the Moroccan border showed a significant census decrease last years due to a massive cross breeding with Ouled-Djellal by the stockbreeders leading to its replacement in many areas. Hamra is small size sheep known for its high meat organoleptic qualities as sheep of Oranie, and is distinguished from the other breeds by a head and legs dark chestnut tending towards the red, the wool being white with guard hair going to brown the russet-red (Chellig 1992). The Béni-Ighil Moroccan breed wich represents 11.42% (Moroccan ministry of Agriculture, 2005) resembles all points with the Algerian ovine Hamra. The geographic separation of these two populations would go up at a few decades (Chellig 1992).
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Several studies of genetic relationships were carried out in sheep. These studies provided useful information related to genetic divergence between populations using microsatellites markers (Tapio et al., 2010, Tolone et al., 2012). Identification of agreement between subjective classifications (phenotypic traits) and genetic information based on molecular markers could help breeders to prioritize the breeds in terms of phylogenetic distinctness and variability values, and to implement rational decisions for conservation. Genetic characterization and relationships among Hamra and Moroccan Béni-Ighil sheep breeds are poorly studied, and no molecular studies using microsatellite markers was published so far. Thus, the objective of the present work is to assess the genetic diversity and the relationship between these breeds by using microsatellites.
Materials and methods
Samples
The samples were collected from Hamra Algerian sheep breed (n=35) and the Moroccan BéniIghil (n=50). Genomic DNA was extracted using salting out method as described by (Miller et al., 1988).
PCR amplification and genotyping
Six bovine microsatellite loci were amplified (table1). The microsatellites were amplified by PCR by using marked with different fluorophores. The PCR reaction included 10 mM Tris-HCl Buffer, 0.25 units of polymerase Taq, 1.5 mM MgCl2, 250 µM dNTPs, 10 pM of primers and 200 ng of DNA. The PCR thermal profile started with an initial denaturation step at 94°C (5
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min) followed by 30 cycles of 30 s at 94°C for DNA denaturation, 30 s for primer annealing at 55°C and 30 s at 72°C for primer extension followed by a final extension at 72 °C for 15 min. PCR products were analysed by denaturing poly-acrylamide gel electrophoresis using an automated ABI 373 DNA sequencer (Applied biosystems, CA, USA).
Statistical analysis The results were analyzed using GENESCANTM 6.7.2 Ver. 3.0 program (Applied bio systems, USA). Cervus v. 3.0.3. (Kalinowski et al., 2007) software was used to analyze the number of alleles, expected heterozygosity corrected for sampling bias, observed heterozygosity and polymorphic information content (PIC). Genepop v.4 (Raymond and Rousset 1995) software was utilized to calculate the exact test for Hardy–Weinberg equilibrium and the Linkage disequilibrium test between markers. We used Arlequin 3.5.1.2 (Excoffier et al., 2005) to determinate the FST values for pairwise comparisons of the breeds and their significance level for genetic differentiation, and for the analysis of molecular variance (AMOVA) to evaluate genetic structure of the populations. Parameters of genetic differentiation (F statistics), mean number of alleles across populations, observed, average expected (non-biased) and average observed heterozygosities were calculated using Genetix 4.05.2 software (Belkhir and Borsa 1998).
Results and discussion
Diversity of populations The total number of alleles was 73. The highest number of allele was detected in OarCP 49 loci (k=20). However, the lower was showed in OarHH56 loci (k=6). The mean number of alleles per locus was 12.17. These results seem similar to that observed in Nigerian breeds (Agaviezor
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et al., 2012) but higher than that showed in South Africa Meatmaster sheep breed (Peters et al., 2010). A higher values were observed in some European sheep breeds (Dalvit et al., 2008, Kusza et al., 2009). All markers were highly informative (PIC>50) with PIC values ranging from 0.59 for ILSTS 05 to 0.88 for OarCP 49 (table2). The two populations showed significant deviations (p < 0.01) from Hardy–Weinberg proportions when considering all loci, which would reflect a subdivision population structure (Wahlund effect), isolation, founder effects, genetic drift and different selection pressures conducted. Expected (He) and observed (HO) heterozygosities for each breed are given in table 2. HO average over loci was 0.672±0.179, whereas He was 0.761±0.118, being He < Ho in all breeds. The lowest value of observed heterozygosity is noticed in hamra (0.67). Wright’s FIS coefficient are 0.098 in Hamra and 0.135 in Béni-Ighil breed (Table 2). The estimated FIS was significant in the two studied breeds. These values are lowest than that found in Indian and Nigerian sheep breeds (Dixit et al., 2010, Agaviezor et al., 2012). Hamra FIS is similar to that showed for the Italian Gentile di Puglia sheep breed (d’Angelo et al., 2008). Hight Fis value was observed in breeds that did not undergone to a selection program. Structure of Populations The mean proportion of private alleles was 0.027, being the number of immigrant after correction for size 4.519, indicating a relatively high gene flow among breeds. Similar frequencies of private alleles were observed in some European and Asian sheep breeds (Luikart et al., 2011, Tolone et al., 2012). The global FIT was 0.16 (0.101 - 0.219) at 95% confidence interval and after 1000 bootstraps. In the overall population the homozygote excess (FIT) was caused mainly by a significant
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homozygote excess within breeds FIS 0.119 (0.047 - 0.200) and partially by the genetic differentiation among breeds FST 0.045 (0.0142 - 0.0791). The AMOVA analysis revealed that 86.4% (p
