Genetic diversity and distances of Albanian local sheep breeds using microsatellite markers

A Hoda, P Dobi and G Hyka

Department of Animal Production, Faculty of Agriculture and Environment, Agricultural University of Tirana, (AUT), Albania
hodanila@yahoo.com

Abstract

Sheep are considered as an important livestock species in Albania. An analysis of six microsatellite loci in 124 unrelated individuals was performed to define the genetic structure and variability of four local sheep breeds: “Bardhoka”, “Ruda”, “Recka” and “Shkodrane”. 31 individuals were selected per each breed. Allele diversity, observed heterozygosity, expected heterozygosit, G_ST estimates, D_S genetic distance were calculated. Wright F-statistics, (F_IS, F_IT and F_ST) values were estimated.

A total of 98 alleles were found. The average number of alleles per locus was 8.15. Within breeds, the mean number of alleles ranged from 7.7 in Bardhoka to 8.8 in Shkodrane. Mean expected heterozygosity (He) ranged from 0.755 in Bardhoka to 0.781 in Shkodrane. Allelic richness varied from 7.63 in Bardhoka to 8.71 in Shkodrane. Inbreeding for all population is rather high F_IS =0.061 ranging from -0.011 in Ruda to 0.087 in Shkodrane. The genetic differentiation, mean F_sT was 0.106 and demonstrated that 89.4% of total genetic variation is due to genetic differentiation within each population. In the neighbor –joining tree based on D_S distance, Bardhoka and Shkodrane were grouped together, than Ruda and finally Recka. 

The Principal Component Analysis (PCA) analysis supported the conclusion suggested by dendrogram based on D_S distance

Key words: genetic distance, genetic diversity, indigenous breeds, microsatellite, sheep

Introduction

Sheep is one of the most important livestock species in Albania. There are four indigenous sheep breeds: Bardhoka, Ruda, Shkodrane and Recka. Bardhoka breed is classified in long tail group. Its origin is from north/northeast of Albania, as well as in Western part of Kosovo. Ruda is triple production breed with half-fine wool. This breed is part of Cigaya family, regarding the wool quality. It is originated and widespread in north-eastern part of Albania. The Shkodrane sheep breed belongs to the long tail group. The area of origin is north Albania. Recka breed is part of Cakel group. It is located all over the Albanian territory, especially on mountain areas of Central and South Albania. There were applied crossing between Bardhoka and Shkodrane, known as Baca, in order to improve the milk production, but no crossbreeding with Recka. Also, more than 30 years ago are introduced Cigaya rams for breeding Recka, in order to improve this breed. Selection programs in sheep are less advanced and systematic then in other livestock. Lack of parentage control and breed purity has facilitated a continuous gene flow. Because artificial insemination with frozen sperm is rarely practiced, this gene flow is limited by distance and geography. On the other hand, local management may have led to genetic isolation, which reduces the effective population size.

Polymorphic DNA markers are very useful in assessment of genetic diversity within and between breeds. Microsatellite is widely used as genetic markers for the analysis of genetic variability within and between breeds due to their high number, distribution throughout the genome and the efficacy of genotyping.

Recently, they are used for the study of diversity in European sheep breeds (Álvarez et al 2004, Arranz et al 1998,  Diez-Tascón et al 2000, Farid et al 2000, Pariset et al 2003, Rendo et al 2004, Stahlberger-Saitbekova et al 2001, Tapio et al 2005, Tapio et al 2003, Peter et al 2007, Cinkulov et al 2008).

For long time the genetic diversity of indigenous sheep breed has been evaluated on the basis of biochemical methods. In this study that diversity has been characterized four local sheep breeds using 6 microsatellite markers. The objective was to characterize the relationships among the breeds, by estimating genetic distances from microsatellite markers.

Materials and methods 

Sample collection and microsatellite markers

Blood samples of 124 unrelated animals representing 4 different Albanian sheep breeds were analyzed. For each breed were sampled 31 individuals. There were selected maximum three unrelated individuals (two females and one male) per flock based on the information provided by the farmer. Sampling was carried out from an average of 11 flocks per breed, in the traditional rearing area of each breed. There were selected only the farms with pure breed animals. The breeds are marginally farmed and indigenous: Bardhoka (Bar), Ruda (Rud), Shkodrane (Sko) and Recka (Rck) (Figure 1, Table 1). A total of 6 microsatellite markers were analyzed: BM8125, MAF65, OarCP34, OarFCB304, OarHH47, and OarVH72.

Statistical analyses

Allele frequencies and tests of genotype frequencies for deviation from Hardy-Weinberg equilibrium (HWE) were carried out using exact tests of the GENEPOP V.1.2 package (Raymond and Rousset 2001), performing a probability test using Markov chain Monte Carlo simulation (dememorization 10,000, batches 1000, iterations per batch 5,000). Significant levels were calculated per locus, per population, and over all loci and populations combined. Genetix version 4.05.2 (http://www.genetix.univ-montp2.fr/genetix/genetix.htm) was used for measuring genetic diversity within population, by the calculation of observed heterozygosity (H_O) and mean unbiased estimates of gene diversity (H_e), (Nei 1978), mean number of alleles (MNA) per locus, the number of private alleles (PA, alleles found in only one breed). The program FSTAT (http://www2.unil.ch/popgen/softwares/fstat.htm) was used for the calculation of corrected allele diversity (allelic richness). F-statistics is widely used to characterize population genetic structure. These statistics allow the partition of genetic diversity/heterozygosity within and among populations. F_IS measures the heterozygote deficit within populations, F_ST among populations, and F_IT the global deficit of heterozygotes. Therefore, F-statistic parameters, (F_IT, F_IS, and F_ST) were estimated in the form of F, q and f respective estimators of these parameters proposed by Weir and Cockerham (1984). These were computed using FSTAT program (Goudet 1995). The significance was tested by 1000 permutations (Goudet 1995).

There were calculated two genetic distances from allele frequencies using GENETIX software, Nei genetic distance (D_A) that is useful in case of closely related populations, where the genetic drift is the primary factor of genetic differentiation. It was calculated also standard genetic distance of Nei (1972) (D_S). D_S distance is used for the construction of Neighbor-Joining consensus tree (Saitou and Nei 1987) with Phylip package (http//evolution.genetics.washington.edu/phylip.html). Bootstrap (1000 replicates) resampling was performed to test the robustness of the dendrogram topology.

PCA using microsatellite data was performed using XLSTAT program (Angresti 1990; Saporta 1991)

Results 

Microsatellite loci and genetic variability

The allele and genotype frequencies of six microsatellite loci were determined in 4 Albanian sheep breeds. In table 2 are summarized the estimates of various measures of diversity at the breed level. All the markers were polymorphic.

The total number of alleles and allele size for each locus are presented in Table 3.

A total of 98 alleles were detected over all loci in 124 individuals. The average number of alleles varied from 9 (BM8125 and OARVH7) to 24 (OARFCB). All markers showed more than 5 alleles. Observed heterozygosity (H_O) per locus ranged from 0.664 (OARFCB) to 0.835 (OARVH7). There were found some breed specific alleles. Frequencies of private alleles for Bardhoka and Shkodrane were <5% and OARFCB304¹⁷⁹ for Ruda had the frequency of 8.1%. In Recka the frequency of breed specific alleles was higher than 5%. In total 1 from 24 locus-population comparisons revealed significant departures (p>0.05) from Hardy-Weinberg proportions.

The polymorphism information content (PIC) values for all loci ranged from 0.743 (BM8125) to 0.956 (OarHH4). The PIC values are very high, indicating that these loci are suitable for genetic studies of sheep breeds.

At individual loci, F_IS estimates over all sheep breeds ranged between -0.036 (locus BM8125) and 0.11 (locus OarHH4), (Table 4).

On average, breeds had a 3.4% significant deficit of heterozygotes (p<0.001), whereas the total population had a 13.5% significant deficit of heterozygotes (p<0.001). To the observed deficit contribute only the markers Oarfcb and OarHH4.

The different estimates of genetic differentiation (F_ST and G_ST) with F_IS and F_IT are shown in table 2. F_ST values of genetic differentiation and G_ST values of breed differentiation were similar. The average genetic differentiation between all breeds was 10.6% significantly different from zero (p<0.001). F_ST values indicated that about 11% (p<0.001) of the total genetic variation was explained by a population difference and 89% correspond to the differences among individuals. The overall G_ST value was 0.104. That indicates that 10.4% of the total genetic diversity was observed among populations while 99.6% was within population.

The mean genetic differentiation within population (H_S) was 0.783 and in total populations (H_T) was 0.851. The lowest value showed BM8125 (0.73 and 0.769 respectively) and highest at OarVH7 (0.856 and 0.877 respectively).

Genetic variation within and among breeds

Allelic richness (AR) ranged from 7.63 in Bardhoka to 8.71 in Shkodrane. The mean expected heterozygosity (H_E) ranged from 0.755 in Bardhoka to 0.781 in Recka. Observed heterozygosity (H_O) varied from 0.713 in Shkodrane to 0.796 in Ruda. Mean values of H_E and H_O, overall loci and breeds were 0.769 and 0.755 respectively. The mean number of alleles per locus (MNA) varied from 7.7 in Bardhoka to 8.8 in Shkodrane. Positive values of F_IS per locus and per breed indicate inbreeding in all breeds, except of Ruda. F_IS values ranged from -0.011 in Ruda to 0.087 in Shkodrane.

Genetic distances

Nei’ genetic distance D_A ranged from 0.067 between Ruda and Shkodrane to 1.807 between Shkodrane and Recka (Table 5).

The highest D_S value was observed between Shkodrane and Recka (0.204) and the lowest was between Bardhoka and Shkodrane. A NJ topology tree based on D_S genetic distance relating 4 sheep populations studied is presented in figure 2.

The number at the nodes is values for 1000 bootstrap resampling of the typed loci. The bootstrap values obtained at the nodes are high (>50%) and suggest that the robustness of the tree is high. The phylogenetic tree showed the grouping of Bardhoka and Shkodrane. The most separated, appeared Recka.

In the scattergram (figure 3), Bardhoka and Shkodrane exhibited a close relationship, forming the same group, thus supporting the conclusions suggested by the dendrograme based on Nei’s distance.

From both analyses Bardhoka and Shkodrane breeds formed the closest group.

Discussion 

This study investigated the allele frequencies and genotype distributions of 6 microsatellite loci in 124 individuals of local sheep breeds. The genetic analysis of 4 Albanian local sheep breeds with 6 microsatellite markers showed high gene diversity. The microsatellite loci for genetic diversity studies should have more than four alleles. The microsatellite markers showed a high genetic polymorphism. The total number of alleles per locus in the present study ranged from 9 to 24. The high number of alleles for each locus, as well as the high PIC values, suggested that all the markers used were appropriate to analyze diversity in Albanian local sheep breeds. The mean number of alleles per breed is a good indicator of genetic variation within populations. Gene diversity for each breed ranged from 0.75 in Bardhoka to 0.78 in Recka. According to Takezaki and Nei (1996), a marker can be useful in measuring genetic variation if the average heterozygosity ranges from 0.3 to 0.8 in the population. This confirmed that these markers were appropriate for measuring genetic variation. These breeds are characterized by a local distribution, small population size, living in extensive condition, grazing in the poor pastures, linked to traditional farming system. This may be the explanation for the high degree of genetic variability.

The high value of heterozygosity may be result of application of empiric selection and different breeding objectives, as well as application of uncontrolled natural mating. The heterozygosity values observed in Albanian sheep breeds are higher to other sheep breeds: 0.68 in French Mutton Merino by Diez Tascon et al (2000); 0.70 in Muzzafaranagri sheep by Arora and Bhatia (2004); 0.71 in Baltic sheep breeds by Tapio et al (2005); 0.67 in Nali and Chokla sheep breeds by Sodhi et al (2006); 0.69 in Magra sheep by Arora and Bhatia (2006).

The genetic differentiation (F_ST) among the local goat breeds in this study is 10.6%. The F_ST values obtained in this study suggested a moderate genetic differentiation between Albanian sheep breeds. The values of genetic differentiation are higher than those reported by other authors. The genetic differentiation were 5.7% between 57 European and Middle Eastern sheep breeds (Peter et al 2007), 5.2% between West Balkan pramenka sheep types (Cinkulov et al 2008), 8% between finish sheep breed, (Tapio et al 2003), 8.8% between Baltic sheep breeds (Tapio et al 2005), or 8.5% (Forbes et al 1995). In all these cases the number of markers was much higher than in our case. It is clear that 89.4% of genetic variation corresponds to differences among individuals and 10.6% is result of differences among breeds.

In the present study F_IS=0.035, F_IT=0.137 and F_ST=0.106. The values of F_IS are positive except of Ruda. The cause for the loss of heterosigotes may be the mating between the relatives. Only Bardhoka breed showed 2 of 6 loci with a significant deficit of heterozygotes, therefore the mating between relatives is not likely. The excess of homozygotes could be due to locus under selection, or presence of “null alleles” that may lead to a falce observation of homozygotes. A very feasible explanation for the defficit of heterozygotes is the presence of Wahlund effect (population substructure within the breed). Since sampling is carried out in 11 flock per breed, therefore it is possible the presence of genetic structure.

The genetic differentiation (10.6%) may be as result of inbreeding and of random genetic drift. The moderate values of F_ST, is most likely attributed to genetic drift between the investigated breed. There is no herd book since 1990 and maybe there is some admixture of breeds. In Albania are applied crossing between Bardhoka and Shkodrane, known as Baca, in order to improve the milk production. This may be one explanation for the smallest distance between Bardhoka and Shkodrane and their grouping, revealed by phylogenetic analysis and PCA. Recka showed always the largest genetic distance with other breeds. There is no crossbreeding with Recka. Also, more than 30 years ago are introduced Cigaya rams for breeding Recka, in order to improve this breed. These can explain why Recka behave like a separate breed.

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Received 5 March 2009; Accepted 15 May 2009; Published 1 June 2009

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