Genetic analysis of Greek sheep breeds using microsatellite markers for setting conservation priorities

Small Ruminant Research 83 (2009) 42–48

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Genetic analysis of Greek sheep breeds using microsatellite markers for setting conservation priorities Ch. Ligda a,∗ , J. Altarayrah b,1 , A. Georgoudis b , the ECONOGENE Consortium2 a b

National Agricultural Research Foundation, Agricultural Research Centre of Northern Greece, Thessaloniki, Greece Aristotle University of Thessaloniki, School of Agriculture, Department of Animal Production, Thessaloniki, Greece

a r t i c l e

i n f o

Article history: Received 21 June 2008 Received in revised form 7 April 2009 Accepted 8 April 2009 Available online 18 May 2009 Keywords: Microsatellites Genetic diversity Phylogenetic tree Genetic differentiation Dairy sheep breeds

a b s t r a c t Ten Greek sheep breeds were analysed at 28 microsatellite markers in order to estimate their genetic diversity and differentiation. This study aims to provide information on the genetic structure of the breeds analysed and the ancestral populations, and give indications and proposals for the conservation strategies. The breeds included were the local sheep breeds raised in different regions of Greece. In total, 310 animals were sampled. Non-biased average expected heterozygosity ranged from 0.68 ± 0.134 (Skopelos breed) to 0.76 ± 0.103 (Karagouniko breed) with an average of 0.74, while the average observed heterozygosity ranged from 0.626 ± 0.132 (Skopelos) to 0.74 ± 0.135 (Kefallenias). Estimates of inbreeding coefficient (Fis) were significant for all breeds studied, except for Kefallenias and Lesvos breeds (P < 0.05). The results of the phylogenetic relationships are in accordance with the geographical location of the breeds, the history of the origin of the breeds and the breeding practices. The phylogenetic tree showed three groupings according to the bootstrapping values. Correspondence analysis showed the isolation of the Skopelos breed and the grouping of Sfakia and Anogeiano breeds in a separate cluster. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Sheep and goat breeding is one of the most widespread agricultural sectors of Greece, and the most important livestock sector, according to their share in the animal production (about 43%) and in the total agricultural production (about 13%). A total of 78% of sheep and 91% of goats are raised under low input production system in the mountainous and marginal regions of the country. The sheep population presents high variation in morphology and production characteristics and shows excellent adaptability to the local environment (Hatziminaoglou, 2001).

∗ Corresponding author. Tel.: +30 2310 47 11 10; fax: +30 2310 47 12 09. E-mail address: [email protected] (Ch. Ligda). 1 Current address: Faculty of Agriculture and Sciences, Jerash Private University, Jerash, Jordan. 2 http://www.econogene.eu. 0921-4488/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2009.04.002

Sheep genetic resources in Greece were formed under the impact of different processes. The geographical isolation, the genetic drift, selection and crossbreeding, and the transhumance of population, contributed to the current picture of sheep breeds and varieties in Greece, where the main feature is the presence of high percentage of crossbred animals, resulting from uncontrolled crossbreeding within the local population during the recent past (Hatziminaoglou, 2001). A number of research works have been published in relation to the genetic diversity of Greek sheep and goat breeds based on blood polymorphic systems (Koutsouli, 1996; Gourgoulis and Rogdakis, 2000) and microsatellites (Bizelis et al., 2007; Koutsouli et al., 2007). The present study deals with the sheep genetic diversity in Greece for the first time to such an extent, covering 10 local sheep breeds analysed in 28 microsatellites. Furthermore, the sampling was performed on flocks covering the whole breeding region of each breed. In Table 1, the breeding region, the population

Ch. Ligda et al. / Small Ruminant Research 83 (2009) 42–48

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Table 1 The breeds studied, their breeding regions and demographic trendsa . Breed

Breeding region

Population size

Ne

F

Ne

F

Population trend

Anogeiano Kalarritiko Karagouniko Kefallenias Kymi Lesvos Orino Peliou Sfakia Skopelos

Anogeia, other regions in Crete island Tzoumerka mountains–Plains of Thessaly Plains of Thessaly Kefallonia island Around Kymi (Euboia island) In Lesvos and Limnos islands Mountains of Epirus and West Macedonia In Magnesia. Pilio mountains In Crete island–Sfakia Islands of Skopelos and Skiathos and in Magnisia

4500 5350 180000 40000 559 180000 15000 2462 60000 1824

838 670 23200 5154 117 23200 1933 494 7733 368

0.06 0.07 0.002 0.010 0.43 0.002 0.026 0.10 0.006 0.13

586.6 469 16240 3607.8 81.9 16240 1353.1 345.8 5413.1 257.6

0.09 0.11 0.003 0.01 0.61 0.003 0.04 0.14 0.01 0.19

Increase Stable Increase Stable Decrease Stable Decrease Stable Stable Stable

a

From Georgoudis et al. (2006).

size, trends, Ne and F are presented and also the corrected Ne and F according to the model proposed by Santiago and Caballero (1995), using the simplified formula as Ne× 0.7. Microsatellite markers have been extensively used in studies of the genetic characterisation of sheep breeds (Arranz et al., 2001; Rendo et al., 2004; Peter et al., 2007; Dalvit et al., 2008; Santos-Silva et al., 2008). Their high mutation rate, the abundance, the distribution throughout the genome, the neutrality, the co-dominance nature and easy automation of analytical procedures permit the estimation of within- and between-breed genetic diversity (Cannon et al., 2001; FAO, 2007). To promote the use of common markers, FAO is proposing an updated, ranked list of microsatellite loci for the major livestock species (FAO, 2007). These 10 sheep breeds of Greece among breeds of the countries of the European Union and the Middle East were studied in the frame of the ECONOGENE project, funded under the 5th Framework for Research, which aimed at the identification of the sheep and goat populations of high conservation priority and the mapping of the priorities, the investigation of the socioeconomic conditions of the breeding regions and the perspectives of development and the definition of proposals for specific measures and activities for the sustainable conservation and management of the local genetic resources (Ajmone-Marsan et al., 2005). This article is part of the above-mentioned project and aimed to explore genetic diversity within and between the sheep breeds and to determine genetic relationships among them. More specifically, the results will provide information on the genetic diversity and the phylogenetic relationships of the sheep breeds analysed, in order to have some indications on the current status of the diversity of sheep breeds in Greece and make suggestions for the development of the conservation strategy.

the breeds Kymi and Skopelos, while the breeds Karagouniko, Lesvos and Sfakia were included in the study as local breeds with economic importance as these are widely used in Greece. The DNA of 31 animals of each of the 10 breeds were analysed for 28 microsatellite markers. The samples were taken from 11 flocks (except from Kefallenias breed, where the samples came from nine flocks). In each flock, three unrelated animals were sampled. Thirty-one bovine, ovine and caprine microsatellite markers were used. These markers were: BM1329, BM1824, BM8125, DYMS1, HUJ616, ILSTS005, ILSTS011, ILSTS028, INRA63, MAF33, MAF65, MAF70, MAF209, MAF214, McM140, McM527, OarAE129, OarCP34, OarCP38, OarFCB20, OarFCB128, OarFCB193, OarFCB193, OarFCB304, OarHH47, OarJMP29, OarJMP58, OarVH72, SR-CRSP-1, SR-CRSP-5 and SR-CRSP-9. The markers covered all autosomal chromosomes besides OAR8, OAR21, OAR22 and OAR23. DNA extraction was performed following the protocol of Montgomerry and Sise (1990). Primer sequences, size ranges, multiplexing information and PCR protocols of the markers are available from the FAO’s website (http://dad.fao.org/en/Home.htm) (FAO, 2004) which comprises a list of markers ranked by typing efficiency, as used within the ECONOGENE project. Genotyping was performed on ABI Prism 377 (Department of Animal Breeding and Genetics, Justus-Liebig University, Giessen) and ABI 3100 (School of Biosciences, Cardiff University and School of Biological Science, University of East Anglia, Norwich) semi automated DNA analysers using standard methodologies (Peter et al., 2007). 2.2. Statistical analysis FSTAT software (Goudet, 2002) was used to estimate the following parameters: number of alleles per locus and per population, allele frequencies per population and overall populations, unbiased gene diversity per population and locus and Fis per locus and population. In order to evaluate the significance of the Fis values (HW testing), permutation tests

2. Materials and methods 2.1. Samples The breeds included in this study are local sheep breeds raised in different regions in Greece and were the following: Anogeiano, Kalarritiko, Karagouniko, Kefallenias, Kymi, Lesvos, Orino, Peliou, Sfakia and Skopelos. The distribution of the breeds is presented in Fig. 1 and the demographic information in Table 1. The choice of the breeds was based on their importance for the development of mountainous and marginal areas, such as the breeds Orino, Kalarritiko, Peliou, Anogeiano and Kefallenias. Furthermore, breeds were chosen because of their small population size, such as

Fig. 1. Geographical distribution of the sampled breeds.

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Ch. Ligda et al. / Small Ruminant Research 83 (2009) 42–48

Table 2 Total number of alleles (TNA), mean number of alleles (MNA) and Nei’s estimation of heterozygosity of each microsatellite averaged over all populations. Locus

TNA

MNA

Ho

Hs

Ht

Gst

Gst

Gis

BM1329 BM8125 DYMS1 HUJ616 SRCRSP1 OARCP34 OARCP38 OARFCB128 OARHH47 OARVH72 MAF65 INRA63 OARFCB20 OARJMP58 OARJMP29 OARFCB193 BM1824 MAF70 MAF209 OARFCB304 SRCRSP9 ILSTS5 ILSTS11 ILSTS28 MAF214 MAF33 MCM140 OARFCB226

10 12 15 22 12 13 10 11 17 8 11 19 16 15 21 18 7 23 14 18 10 12 9 17 25 12 14 15

6.2 7.3 9.6 9.8 6.5 7 5.6 6.9 10.7 7.3 7.1 10.7 10 9.6 10.7 9 5.3 12.5 9 9.5 5.6 5.7 5.9 9.4 9.5 7.7 9.5 10

0.642 0.674 0.699 0.716 0.642 0.729 0.487 0.581 0.751 0.742 0.710 0.771 0.835 0.706 0.758 0.581 0.739 0.797 0.771 0.697 0.481 0.621 0.712 0.771 0.641 0.735 0.774 0.732

0.685 0.721 0.754 0.778 0.686 0.760 0.588 0.774 0.827 0.795 0.747 0.797 0.859 0.740 0.815 0.589 0.762 0.834 0.797 0.692 0.515 0.632 0.737 0.811 0.726 0.713 0.822 0.780

0.692 0.762 0.774 0.820 0.712 0.795 0.601 0.804 0.848 0.820 0.782 0.827 0.873 0.776 0.848 0.612 0.767 0.861 0.827 0.714 0.524 0.647 0.773 0.840 0.736 0.733 0.841 0.800

0.011 0.054 0.025 0.051 0.036 0.045 0.021 0.037 0.025 0.030 0.045 0.035 0.016 0.046 0.038 0.038 0.007 0.031 0.037 0.032 0.017 0.024 0.046 0.034 0.014 0.027 0.022 0.026

0.012 0.059 0.028 0.056 0.040 0.050 0.023 0.041 0.027 0.034 0.050 0.039 0.018 0.051 0.043 0.042 0.007 0.034 0.041 0.035 0.018 0.027 0.051 0.038 0.015 0.030 0.025 0.029

0.062 0.065 0.073 0.080 0.065 0.040 0.172 0.250 0.093 0.067 0.050 0.033 0.027 0.046 0.070 0.014 0.030 0.045 0.032 −0.007 0.067 0.017 0.035 0.049 0.116 −0.031 0.058 0.061

Overall

14.5

0.696

0.741

0.765

0.031

0.035

0.060

8.34

were carried out by permuting the alleles within populations over all loci in each breed. The hypothesis of heterozygosis deficit was assumed in these tests. Observed, average expected (non-biased) and average observed heterozygosity were calculated, with their standard errors for each population averaging over all loci using Genetix (v. 4.05) (Belkhir et al., 2000). In order to define the degree of differentiation among the populations studied, the following parameters were estimated using the FSTAT software (Goudet, 2002): Nei’s statistics per population and overall, Wright’s fixation index (Fst) and the number of effective migrants (Nem). The PHYLIP 3.62 software (Felsentein, 2004) was used to build the phylogenetic tree, based on the Reynold’s genetic distance, using the neighbour-joining method (Saitou and Nei, 1987). Correspondence analysis was used to investigate further the differentiation of the breeds, taking into consideration a likely occurring of admixture between some of the populations. For the correspondence analysis, the statistical package GENETIX 4.05 (Belkhir et al., 2000) was used. The genetic structure of the populations was investigated using the software STRUCTURE (Pritchard et al., 2000). The software infers the number of populations into which the analysed genotypes can be divided, by estimating the natural logarithm of the probability that a given genotype X is part of a given population K: ln Pr(X | K). In order to choose the appropriate number of inferred clusters (K) we performed nine runs, fitting K from 2 to 9 and at K = 12. All runs used a burn-in period of 100 000 iterations and a period of data collection of 100 000 iterations.

3. Results and discussion 3.1. Microsatellite analysis A total of 406 alleles were detected in the 28 loci studied. The number of alleles per locus ranged from 7 (BM1824) to 25 (MAF214) with a global mean of 14.5 ± 4.65 (Table 2). The majority of the markers were highly polymorphic, with detected alleles between 10 and 19, while in four loci

(HUJ616, OARJMP29, MAF70 and MAF214) more than 20 alleles were detected. Only in three markers (OARVH72, BM1824 and ILSTS11) less than 10 alleles were detected. The mean He across loci was 0.74 ± 0.10, while the mean Ho was 0.69 ± 0.11. The highest He was observed in locus OARFCB20 (0.86) and the lowest in SRCRSP9 (0.52). The same loci showed also the highest (0.84) and lowest (0.48) Ho, respectively. In total, 47 from the 280 locus per breed combinations revealed significant (P < 0.05) deviations from Hardy–Weinberg equilibrium. OARFCB128 and OARCP38 showed the greater deviations, where six and four populations, respectively, were not in equilibrium. The number of alleles observed in each breed per locus ranged from 3 to 15. Skopelos showed the lowest values concerning the number of observed alleles (three) and mean number of alleles (6.36). The maximum number of observed alleles per locus was found in Kalarritiko and Orino (15). Gene diversity in each breed over all microsatellite loci ranged as following: Anogeiano 0.428–0.886; Kalarritiko 0.576–0.899; Karagouniko 0.485–0.894; Kefallenias 0.513–0.868; Kymi 0.508–0.893; Lesvos 0.482–0.895; Orino 0.481–0.863; Peliou 0.415–0.873; Sfakia 0.363–0.886 and Skopelos 0.257–0.847. The lowest value of gene diversity (0.257 of OARFCB193 locus) over all populations and over all loci was observed in Skopelos breed, while the highest (0.899 of MAF70 locus) in Kalarritiko. The observed heterozygosity values in the 10 breeds studied, overall loci were: Anogeiano 0.290–0.935; Kalarritiko 0.387–1.000; Karagouniko 0.322–0.903; Kefalenias 0.419–0.903; Kymi 0.161–0.871; Lesvos 0.451–0.935; Orino

Ch. Ligda et al. / Small Ruminant Research 83 (2009) 42–48 Table 3 Mean number of alleles (MNA), average expected (He) observed (Ho) heterozygosity and Fis estimates per breed across the 28 loci. Breed

MNA

He

Anogeiano Kalarritiko Karagouniko Kefallenias Kymi Lesvos Orino Peliou Sfakia Skopelos

8.61 8.54 9.04 8.43 8.54 8.25 9.29 8.29 8.11 6.36

0.738 0.759 0.760 0.743 0.756 0.751 0.750 0.739 0.722 0.680

Ho ± ± ± ± ± ± ± ± ± ±

0.099ab 0.084a 0.103a 0.102a 0.096a 0.104a 0.084a 0.106a 0.111b 0.134b

0.717 0.695 0.726 0.740 0.665 0.735 0.703 0.672 0.684 0.626

Fis ± ± ± ± ± ± ± ± ± ±

0.144ab 0.14ab 0.144ab 0.135a 0.144bc 0.141a 0.145 ab 0.102abc 0.122abc 0.132c

0.03* 0.086*** 0.046** 0.003 0.123*** 0.021 0.063*** 0.091*** 0.054*** 0.081***

Similar letters indicate that there were not statistically significant differences (P < 0.05). *P < 0.05, **P < 0.01 and ***P < 0.001 after Bonferroni corrections, based on 5600 randomizations using FSTAT software. Hypothesis tested was the heterozygosis deficit.

0.387–0.903; Peliou 0.354–0.838; Sfakia 0.387–0.903 and Skopelos 0.225–0.838. In Table 3, the average non-biased expected and the observed heterozygosity per population are presented. The average non-biased expected heterozygosity ranged from 0.68 ± 0.134 (in Skopelos) to 0.76 ± 0.103 (in Karagouniko) with a mean value of 0.74, while the average observed heterozygosity ranged from 0.63 ± 0.132 (in Skopelos breed) to 0.74 ± 0.135 (in Kefallenias) and averaged 0.696. Similar results are presented by Arranz et al. (2001), where the expected heterozygosity in five Spanish sheep breeds (Churra, Latxa, Rasa-Aragonesa, Castellana and Merinos), ranged from 0.63 at BM1258 locus to 0.86 at MAF70 locus, with an average value of 0.77. In a study of the phylogenetic relationship between Swiss sheep breeds, analysing 31 microsatellites, the average expected heterozygosity was estimated between 0.60 and 0.71 (Stahlberger-Saitbekova et al., 2001), while higher estimates are reported, ranging from 0.68 to 0.77 for the coarse-wool sheep breeds in Portugal (Santos-Silva et al., 2008), from 0.73 to 0.79 for the Spanish Latxa, Carranzana, Sasi Ardi, Navarra, Castellana and Rasa Aragonesa (Rendo et al., 2004) and from 0.73 to 0.82 in Alpine breeds located in Germany, Italy and Slovenia (Dalvit et al., 2008). In Diez-Tascon et al. (2000), where certain Merino sheep populations were studied, the observed heterozygosity ranged from 0.679 to 0.763, while for the Alpine breeds ranged from 0.578 to 0.728 (Dalvit et al., 2008) and for the coarse-wool sheep breeds of Portugal ranged from 0.65 to 0.72 (Santos-Silva et al., 2008). Relatively higher estimates (from 0.696 to 0.784) are referred by Rendo et al. (2004) for six sheep breeds of the North Iberian peninsula, while Arranz et al. (1998) report higher estimates for the Spanish Churra, Latxa, Manchega, Rasa-Aragonesa and Merinos, ranging from 0.713 in Latxa sheep to 0.771 in Merinos. Hardy–Weinberg testing was carried out in order to evaluate the significance of inbreeding occurring in each locus in each population and overall loci in each population. Coefficient of inbreeding, Fis, was estimated for each locus and overall loci in each population according to Wright (1965). The Fis values differed widely between loci in each population. The values per locus in each population

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ranged from −0.207 to 0.322 in Anogeiano; −0.175 to 0.399 in Kalarritiko; −0.146 to 0.599 in Karagouniko; −0.155 to 0.238 in Kefallenias; −0.017 to 0.728 in Kymi; −0.094 to 0.238 in Lesvos; −0.165 to 0.501 in Orino; −0.195 to 0.283 in Peliou; −0.125 to 0.374 in Sfakia; and from −0.102 to 0.268 in Skopelos. The highest values were found in Karagouniko (0.599 in OARFCB128) and Kymi (0.728 in OARCP38). The highest inbreeding coefficient was observed in Kymi breed, which may be due to the bottleneck effect, as the population size has been decreased drastically to few hundreds individuals in the last 20 years. Estimates of inbreeding coefficient per population were significant (P < 0.05) except in Kefallenias and Lesvos breeds (Fis = 0.003, P < 0.435; and Fis = 0.021, P < 0.107, respectively) (Table 3). Koutsouli et al. (2007) used 12 microsatellites and reported higher estimated of inbreeding for the Lesvos breed (0.131), but this was probably due to the sampling of the animals (four flocks). The estimated inbreeding coefficients Fis are within the literature ranges. Diez-Tascon et al. (2000) reported Fis values between 0.023 and 0.096 in related Merinos populations, while higher values, ranging from 0.052 to 0.113 were reported by Santos-Silva et al. (2008). 3.2. Genetic differentiation Nei’s F-statistics (Nei, 1987) were calculated for each microsatellite marker (Table 2). The ranges of the values of Ho, Hs, Ht, Gst, Gst and Gis were as follows: Ho: 0.481–0.835; Hs: 0.515–0.859; Ht: 0.524–0.873; Gst: 0.007–0.054; Gst : 0.007–0.059 and Gis: −0.031 to 0.250. The Nei’s coefficient of differentiation, Gst, was generally low for all the loci and also when averaged over all the breeds and loci (0.031). The low value of Gst may indicate that these breeds are not differentiated enough and that they may have a common history and breeding practices. The high gene flow between the breeds may explain the relative high heterozygosity observed in the breeds under study. Thus, a small part of the variability at the 28 microsatellite loci analysed was ascribed to the between-breed variability, while most of the variability (0.969) was observed within breeds. Higher estimates of genetic differentiation are reported in the literature. Stahlberger-Saitbekova et al. (2001) in a study of Swiss sheep breeds report a value of Gst equal to 0.17, while Forbes et al. (1995) estimated an Fst value of 0.085, analysing Awassi, Romney, Merinos and Bighorn. Arranz et al. (2001) in the study of differentiation of five Spanish breeds report an Fst at 7%. The Fst values and Nem for the pairs of the examined breeds are presented in Table 4. The highest degree of genetic differentiation for the pairs of the 10 breeds was observed between the Skopelos breed and all the others, which ranged from 7% to 8%. The lowest values were found between the breeds Anogeiano and Sfakia, Lesvos and Orino, Lesvos and Karagouniko, and Karagouniko and Orino. The Fst estimates ranged from 0.007 between Orino and Karagouniko to 0.078 between Skopelos and Sfakia. The gene flow between populations ranged from 2.95 (between Skopelos and Sfakia) to 23.61 (between Anogeiano and Sfakia) with an average of 9.78. The Skopelos breed showed

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Table 4 Fst estimates (above the diagonal) and Nem (below the diagonal) per pair of breeds. Breed

Anogeiano

Kalarritiko

Karagouniko

Kefallenias

Kymi

Lesvos

Orino

Peliou

Sfakia

Skopelos

Anogeiano Kalarritiko Karagouniko Kefallenias Kymi Lesvos Orino Peliou Sfakia Skopelos

– 6.07 8.99 7.52 5.81 8.10 12.28 6.44 23.61 3.41

0.039 – 14.38 9.77 9.67 8.79 18.85 12.52 5.45 3.06

0.027 0.017 – 12.19 10.09 21.88 31.90 16.69 7.01 3.43

0.032 0.024 0.020 – 7.17 7.83 17.47 8.52 6.53 3.05

0.041 0.025 0.024 0.033 – 10.38 13.75 7.87 5.24 3.35

0.029 0.027 0.011 0.030 0.023 – 19.00 10.90 7.71 3.47

0.019 0.013 0.007 0.014 0.017 0.012 – 14.73 9.00 3.95

0.037 0.019 0.014 0.028 0.030 0.022 0.016 – 6.03 3.41

0.010 0.043 0.034 0.036 0.045 0.031 0.027 0.039 – 2.95

0.068 0.075 0.067 0.075 0.069 0.067 0.059 0.068 0.078 –

the highest values of pair-wise Fst and the lowest values of Nem with other breeds, indicating that this population has maintained an important genetic isolation from all the other breeds. The phylogenetic tree based on the Reynold’s genetic distance showed three groupings according to the high bootstrapping values. The first group consisted of the Anogeiano and Sfakia breeds with a high bootstrapping value of 1000, while the second group was formed by the Skopelos breed alone with a bootstrap value of 415 out of 1000. The third group included the remaining breeds of Kefallenias, Orino, Karagouniko, Lesvos, Kymi, Peliou and Kalarritiko, with low bootstrap values (lower than 443 out of 1000) (Fig. 2). The close relationship between the seven breeds of the third cluster was probably due to the effects of the processes of admixture. According to the correspondence analysis the first three components explain the 48.60% of the total variation (Fig. 3). The first axis explains the 19.62% of the total variation and separates the Skopelos breed from the rest. The second axes, representing the 16.70% of the total variation, showed the isolation of the breeds Anogeiano and Sfakia, while the third one, which represented the 12.30% of the total variation grouped the remaining seven breeds together. In this last group, the Kymi breed is differentiated

from the rest of the breeds. The results of the correspondence analysis further supported the previous findings, based on the Fst and Nem, which also indicated the isolation of the Skopelos breed. Clustering obtained by STRUCTURE reflects this situation as the Skopelos breed was the first to diverge from all the other breeds in a separate cluster or ancestral population (K = 2). According to the STRUCTURE analysis, the best value of ln Pr(X | K) was obtained for K = 7 (−30558.5). In Table 5, the proportion of membership of each of the 10 sheep populations in the seventh clusters is presented. For K = 7, the following clusters were formed: Anogeiano and Sfakia; Kymi; Kefallenias; Lesvos; Skopelos; Peliou, and the last cluster, where the individuals from the Kalarritiko breed were assigned. Individuals from the Karagouniko breed were assigned to the clusters of Kalarritiko, Lesvos and Peliou, while individuals from the Orino breed were assigned to Kalarritiko and Kefallenias clusters (Fig. 4). The presence of common ancestors in these breeds is supported with the available information on the history and the followed breeding strategies. Karagouniko and Lesvos breeds are widely use for the upgrading of the mountain populations, while the Orino, Kalarritiko and Kefallenias breeds belong to the mountain breeds of North West Greece, and crosses between them used to be a common practice.

Fig. 2. Genetic relationships among the 10 sheep breeds using RD genetic distance.

Ch. Ligda et al. / Small Ruminant Research 83 (2009) 42–48

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Fig. 3. Results of the correspondence analysis for the 10 sheep breeds. Table 5 Proportion of membership of each of the 10 sheep populations in the seventh clusters using STRUCTURE software (>50% in bold). Breed/cluster

1

2

3

4

5

6

7

Anogeiano Kalaritiko Karagouniko Kefellenias Kymi Lesvos Orino Peliou Sfakia Skopelos

0.789 0.012 0.044 0.047 0.016 0.043 0.078 0.022 0.845 0.020

0.025 0.505 0.306 0.119 0.061 0.034 0.203 0.079 0.030 0.019

0.054 0.135 0.065 0.054 0.668 0.084 0.154 0.034 0.026 0.015

0.062 0.183 0.148 0.597 0.051 0.057 0.251 0.171 0.031 0.054

0.021 0.044 0.223 0.096 0.127 0.682 0.141 0.107 0.042 0.013

0.018 0.012 0.013 0.012 0.016 0.019 0.027 0.021 0.009 0.858

0.031 0.111 0.200 0.077 0.061 0.081 0.147 0.567 0.018 0.020

In general, the analysed breeds present sufficient levels of genetic diversity. Skopelos breed presented the lowest mean number of alleles and the lowest mean heterozygosity. All breeds show heterozygosity deficit, apart from Kefallenias and Lesvos breeds. All results showed the isolation of the Skopelos breed, which has been developed in the island of Skopelos and originates from the Chalkidiki breed, which is extinct

(Hatziminaoglou, 2001; Hatziminaoglou et al., 1996). Moreover, the clustering of the two breeds of the island of Crete, Anogeiano and Sfakia, together was evident. The differentiation of the Kymi breed, although originates from the Skopelos breed, is probably due to the ‘founder effect’ on the initial selection of the individuals of the Skopelos breed that formed the Kymi breed. The allele frequencies were further differentiated during the evolution of the

Fig. 4. Results of the structure analysis for K = 7.

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population of the Kymi breed. Moreover, the high rate of inbreeding, the low Ne and the decreasing trends, together with the ageing of the farmers and the lack of successors and the fact that all farms are concentrated in a small geographical region, increase the degree of endangerment of the breed and set the Kymi breed as a high priority for conservation (Georgoudis et al., 2006). Koutsouli (1996), in her study of the genetic structure of sheep breeds using blood polymorphisms, reports the separate branch of the Skopelos and Kymi breeds as well. However, the group of breeds used was different from the one in the present study and also the samples originated from experimental flocks, and therefore the results of the two studies cannot be fully compared. The Lesvos breed, although an island breed, had a distinct development from other island breeds. The breed, due to its productivity and ability to exploit the poor vegetation, has been used extensively for the upgrading of the sheep population of the mainland of Greece in different time periods (Hatziminaoglou, 2001). For this reason, Lesvos is clustered together with the Karagouniko breed and the mountain breeds (Kalarritiko, Kefallenias, Orino and Peliou), as showed the correspondence analysis. Karagouniko breed, as already mentioned, is a widely distributed breed that is used for the upgrading of numerous sheep populations. The results of the analysis are in accordance with the geographical location of the breeds, the history of the origin of the breeds and the breeding practices. This information provides an overview of sheep genetic diversity in Greece and could be used to support the conservation activities, mainly in marginal agricultural areas in order to assist the conservation decisions. Genetic diversity, as presented in this study, can be considered as an initial guide for conservation decisions, as well as other aspects of non-profit values, should be taken into consideration, such as specific characteristics and the importance of the breed in the region to cultural, historical and environmental reasons. Acknowledgements The present study was a part of the ECONOGENE project funded by the European Union within the QUALITY OF LIFE FRAMEWORK programme (QLK5-CT2001-02461). The content of the paper does not necessarily represent the views of the Commission or its services. The authors thank the directors and all the personnel of the Genetic Improvement Centers of Athens, Ioannina and Karditsa for their significant support on the collection of blood samples. References Ajmone-Marsan, P., Econogene Consortium, 2005. Overview of ECONOGENE, a European project that integrates genetics, socio-economics and geo-statistics for the sustainable conservation of sheep and goat genetic resources. In: International Workshop, The role of Biotechnology, Torino, 5–7 March 2005. Arranz, J.J., Bayon, Y., Primitivo, F.S., 1998. Genetic relationships among Spanish sheep using microsatellites. Anim. Genet. 29, 435–440.

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