Molecular genetic characterization of Kivircik sheep breed raised in Western Anatolia

Onur Yilmaz, İbrahim Cemal, Orhan Karaca and Nezih Ata

Adnan Menderes University Faculty of Agriculture, Department of Animal Science, Aydin, Turkey.
o-yilmaz@live.com

Abstract

Genetic characterization and diversity of Kıvırcık sheep breed (n=246) reared in three different locations (Aydın, Bandırma and Uşak) were investigated by 21 microsatellite markers recommended by FAO.

The microsatellite markers used in this study showed high levels of polymorphism. A total of 461 alleles were detected in Kıvırcık populations. The mean values of polymorphic information content (PIC=0.84), observed heterozygosity (Ho=0.81) and expected heterozygosity (He=0.85) proved that Kıvırcık populations possess remarkable genetic variability. The mean number of alleles ranged from 11.67 (Bandırma) to 17.05 (Eşme). The results demonstrated that the microsatellite markers that were used were adequately polymorphic, and these markers can be successfully used to investigate genetic diversity in these three populations. Our results showed that within-breed diversity was higher than the between breed diversity. This situation can be seen as a chance in terms of the breeding programs and genetic conservation programs for these breeds.

Key words: genetic diversity, microsatellite, population structure

Introduction

The past century has seen the widespread application of statistical methods to the study of genetic inheritance, especially in farm animals. Recent developments in molecular biology and statistics have opened the possibility of identifying and using genomic variation and major genes for the genetic improvement of livestock. Molecular genetic techniques to identify the genetic structure and diversity in farm animals have shown rapid development in recent years and began to be widely used. Various molecular genetics techniques have been developed to achieve genetic structure and diversity. Microsatellites, which are highly polymorphic and abundant, often found in non-coding regions of the genes, are widely used for paternity and genetic diversity studies.

Microsatellites, which are valuable genetic markers due to their dense distribution in the genome, high variation, co-dominant inheritance, easy genotyping and scored, are used rather widely for the definition of intra and interbreed genetic diversity (Togan et al 2005; Sancristobal et al 2003; Schlötterer 2004; Agaviezor et al 2012; Alvarez et al 2012; Arora et al 2011; Cemal et al 2013; Jyotsana et al 2010; Kusza et al 2011; Lasagna et al 2011; Yilmaz and Karaca 2012; Yilmaz et al 2013, Yilmaz et al 2014).

Turkey hosts a high number of breeds in terms of farm animal species and a high level of diversity thanks to its geographical location as a passage among continents and as a center of domestication of domestic animals such as cattle, sheep and buffalo. Kıvırcık sheep breed is the most important genetic resource for the meat production in Turkey. In addition, this breed has contributed to the formation of many types of sheep such as Türkgeldi, Karacabey Merino and Tahirova sheep, are raised in southern and western provinces in the Marmara and Aegean region as well as Thrace.

This study aims to determine the genetic similarities, differences and the genetic structure of the Kıvırcık sheep breed raised different region in Turkey using microsatellites.

Materials and Methods

Animal material of the study consisted of a total of 246 animals raised at Aydın, Bandırma and Usak (Esme) province.

DNA was extracted from 50 ng of whole blood using the ABM™ Genomic DNA Kit (Applied Biological Materials Inc., Canada) according to the manufacturer’s recommendations. DNA quantity and quality were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA).

Three multiplex groups formed according to fragment size of 21 microsatellite loci selected from the FAO recommended list (2011). Genomic DNA was amplified with these multiplex groups by the Polymerase Chain Reaction (PCR) in accordance with the touchdown PCR technique (Table 1).

Each 25 μl PCR reaction mixture contained dNTP (0.2 mM), MgCl2 (2.0 mM), primers (0.10 μM), 5X PCR buffer, Taq DNA polymerase (1U), 50 ng genomic DNA and nuclease free water. PCR fragments were separated by capillary electrophoresis in the

Analyses of fragments were performed using an automated DNA sequencer (GenomeLab™ GeXP Genetic Analysis System, Beckman Coulter, Inc., USA) and Beckman Coulter GeXP Fragment Analysis Software (Beckman Coulter, Inc., USA)

The polymorphism statistics, Hardy-Weinberg equilibrium and F statistics (Weir and Cockerham 1984) were estimated using GenAlEx (Peakall and Smouse 2006), Fstat ver.2.9.3 (Goudet 2001), POPGENE (Yeh et al 1997) and MEGA 4 (Tamura et al 2007). A bootstrap-supported (1,000 replications) dendrogram was constructed according to Nei's (1972) minimum distances.

The population structures were analyzed by cluster techniques based on the Bayesian approach, using the STRUCTURE 2.1 software ( Pritchard et al 2000). The burn-in and MCMC (Markov Chain Monte Carlo) lengths were 20,000 and 100,000, respectively. The analyses were realized at different K values (2-4). The most appropriate cluster number (cluster-K) was detected using the method (ΔK = m|L''(K)|/s[L(K)]) reported by Evanno et al (2005). The true K was determined using Structure Harvester Web version 0.6.93 (Earl 2012).

Results and Discussion

A total of 461 alleles were detected across the 21 loci investigated (Table 2).

Na=number of alleles, MNa=mean number of alleles, Ne=effective number of alleles, PIC= polymorphic information content, Ho= observed heterozygosity, He= expected heterozygosity, F_IT, F_IS, F_ST =Wright’s F-statistics, D_ST = the diversity between breeds, G _ST =coefficient of gene differentiation for each locus, H_T =Nei’s gene diversity, HWE= Hardy-Weinberg equilibrium, ns: non-significant, ***: P<0.001, **:P<0.01, *: P<0.05

The polymorphism statistics (Na, MNa, Ne, PIC, Ho, and He) obtained from this study were higher than previous research (Grigaliunaite et al 2003; Arora et al 2011; Koban 2004; Stahlberger-Saitbekova et al 2001; Baumung et al 2006; Jyotsana et al 2010; Agaveizor et al 2012; Yılmaz and Karaca 2012; Cemal et al 2013; Yılmaz et al 2013; Pariset et al 2003; Koban 2004; Cerit et al 2004; Mukesh et al 2006; Acar 2010). This situation is an indication that studied microsatellites have quite high polymorphic and genetic variability. It was reported that required He value was range between 0.30 and 0.80 to be used in the genetic diversity studies (Takezaki and Nei 1996). It can be said that used loci enables power of separation even though individuals are close to each other.

It is noteworthy that there is loss heterozygosity in eleven microsatellites when examined obtained F_IS values. Similar findings have been reported in the literature (Paiva et al 2005; Alvarez et al 2012). Although, obtained general mean of F_ST value was higher than those of earlier studies (Cemal et al 2013; Hoda and Marsan, 2012), it was lower than some of these (Mukesh et al 2006; Arora et al 2011).

G_ST value indicates that 4.1% of total genetic variation resulted from the differences between populations; while 95.9% can be explained by the difference between the individuals. General mean of G_ST was lower than the values reported by Agaveizor et al (2012). Obtained D_ST values indicate a low genetic diversity among populations. These findings are an expecting result because this research performed in the same breed raised in a different location. General mean of gene diversity between populations (D_ST) obtained in the present study was lower than the previous literature (Hoda and Marsan 2012). Nei average gene diversity (H_T) for loci ranged from 0.768 to 0.929, with an average value of 0.853. Obtained results from this study in terms of Nei average gene diversity confirmed that these markers were appropriate for measuring genetic variation (Takezaki and Nei 1996). Obtained H_T values were higher than values reported by Ligda et al (2009) and Hoda and Marsan (2012). It is an important indication that the relatively high genetic diversity in studied populations. Eleven loci revealed significant (P < 0.05) deviations from Hardy–Weinberg equilibrium. These results are encountered as a natural result of the selection programs that have been carried out for several years in these populations.

The results of population based evaluation are summarized in Table 3.

Obtained results from the studied populations raised in three different location in terms of number of allele is an indicator of intra-breed diversity. The mean number of allele and He values were higher than the numerous studies implemented in different sheep breeds (Alvarez et al 2012; Qurioz et al 2008; Baumung et al 2006; Grigaliunaite et al 2013; Paiva et al 2005; Pariset et al 2003; Yilmaz et al 2013).

F_IS values, indicated the loss of heterozygosity and that is an important parameter in describing the population characteristics, changed between -0.027 (KIV-E) and 0.060 (KIV-A) in the present study. F_IS values was positive in KIV-B and KIV-A population that indicates general risk of inbreeding. It was found that 16 loci in the KIV-E; 17 loci in the KIV-B and 19 loci in the KIV-A population showed significant deviations from Hardy-Weinberg equilibrium. A total of 165 private alleles were observed in three population. However, only 23 of them in KIV-E and KIV-A population have a frequency higher than 5%. The determined 23 (>5%) private alleles can be said to have the property of being able to determine the populations.

When the dendrogram (Figure 1) was examined, it was found that the KIV-E population was in a separate group from KIV-A and KIV-B population.

Figure 1. Dendrogram based on Nei’s minimum genetic distances of three different Kıvırcık populations (bootstrap resampling methodology (1000 replicates)) (KIV-E: Esme, KIV-B: Bandırma, KIV-A: Aydın).

The results of STRUCTURE analysis containing different numbers of clustering are given in Figure 2.

Figure 2. Estimation of the population structure with different K (K=2, 3 and 4) values (KIV-E: Eşme, KIV-B: Bandırma, KIV-A: Aydın).

The results obtained from structure analysis have been similar to the dendrogram given in Figure 1. STRUCTURE analysis showed that KIV-E population presents quite complicated population structure for loci investigated. At the same time the results obtained by STRUCTURE analysis concordance with the dendrograms genetic similarities between Kıvırcık populations.

For the purpose of presenting the suitable cluster number (K) in structure analysis, the statistical analysis results received with the method which has been stated by Evanno et al (2005) was given in Table 4.

ΔK value, which has been taken from 3 studied Kıvırcık population, shows that the most suitable group number is 2 (K=2). It is noteworthy that the mixed population structure in Kıvırcık according to investigated all loci when the Table 5 examined. This situation are an expecting result because this research performed in the same breed raised in a different location.

Conclusions

• Kıvırcık sheep breed are important storehouse of genetic diversity, have a low level of differentiation and high level of admixture.

• Within-breed diversity was higher than between breed diversity.

• The microsatellite markers used in the study can be successfully used in genetic diversity studies.

Acknowledgements

We acknowledge TÜBİTAK, who supplied animal materials for the study, (Project No: KAMAG-109G017), General Directorate for Agricultural Research and Policies (GDAR), Bandırma Sheep Research Station and Adnan Menderes University Agricultural Biotechnology and Food Safety Application and Research Center (ADÜ-TARBİYOMER) providing laboratory facilities for molecular genetics analyses.

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Received 13 November 2015; Accepted 29 December 2015; Published 1 March 2016

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