J. Anim. Breed. Genet. ISSN 0931-2668
ORIGINAL ARTICLE
The genetic diversity and structure of 18 sheep breeds exposed to isolation and selection c1, J. Ramljak2, S. Ivankovic1, B. Mioc2, A. Ivankovic2, V. Pavic2, M. Brka3, C. Veit-Kensch4 & M. Curkovi I. Medugorac4 1 2 3 4
Faculty of Agriculture and Food Technology, University in Mostar, Mostar, Bosnia and Herzegovina Faculty of Agriculture, University of Zagreb, Zagreb, Croatia Faculty of Agriculture and Food Sciences, University of Sarajevo, Sarajevo, Bosnia and Herzegovina Faculty of Veterinary Medicine, The Ludwig-Maximilians-University Munich, Munich, Germany
Summary
Keywords Agriculture; conservation priority; genetic diversity; genetic structure; phylogeny; sheep; sustainable breeding. Correspondence I. Medugorac, Faculty of Veterinary Medicine, The Ludwig-Maximilians-University Munich, Veterinaerstr. 13, 80539 Munich, Germany. Tel: +49 89 2180 3310; Fax: +49 89 2180 99 3310; E-mail:
[email protected] Received: 20 January 2014; accepted: 10 March 2015
The phylogenetic layout of the genotyped (30 microsatellite) 18 sheep breeds in this study demands and provides the opportunity to evaluate both neutral and adaptive components of genetic diversity in a naturally and artificially selected and subdivided sheep population. Seven Pramenka strains from Bosnia and Herzegovina and Croatia characterized by a very low intensity of artificial selection, preserved the highest neutral genetic variability. Eight central and north-western European breeds under considerable artificial isolation and selection preserved the lowest genetic variability. Only combinations of various phylogenetic parameters offer a reasonable explanation for underlying evolutionary forces working in the investigated island and mainland sheep breeds under variable natural and artificial selection. More than 60% of total genetic, diversity was allocated to virtually unselected Pramenka strains, and an additional 25% to native moderately selected Graue Gehoernte Heidschnucke and intensively selected Ostfriesische Milchschafe. Some economically very important breeds and strains did not contribute to a pool with maximal genetic diversity, while they play an important role in the cultural heritage of respective countries.
Introduction The number of extinct sheep breeds throughout the world is 180, of which 82% or 148 breeds are in Europe and Caucasus (FAO 2007). If we consider that sheep production is the third main branch of livestock production in Europe, the relevance of genetic variation for its maintenance and development is of great importance. Genetic diversity in livestock populations can be preserved in effectively and numerically large populations with geographically widespread flocks due to large neutral variability. On the other hand, strongly selected, well adapted, effectively small and closed populations can contribute to the conservation of the adaptive component of genetic diversity. © 2015 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 133 (2016) 71–80
Autochthonous breeds provide both options: a large genetic pool because of lower selection pressure and adaptive traits as a result of a long history of traditional husbandry under local environmental conditions (Ramljak et al. 2011; Tapio et al. 2005). A large proportion is endangered or facing extinction due to crossing and replacing with the most popular commercial breeds more suited to the current practice of intensive livestock production. In Croatia, the sheep population size is estimated at around 630 000 individuals (80% are autochthonous breeds), but only 7.1% are recorded with an established herd-book and monitoring of genetic and production data (CAA 2012). From nine autochthonous breeds, seven are listed as at risk according to FAO doi:10.1111/jbg.12160
c et al. M. Curkovi
The genetic diversity of 18 sheep breeds
guidelines (FAO 2007). Most of these sheep breeds or strains are not characterized genetically and only four breeds from Croatia (Dalmatian, Cres, Krk, and Travnik) have been characterized based on phenotype and production traits in recent years (Mioc et al. 2004; c et al. 2009). For some Pavic et al. 1999, 2006; Siri island breeds, there is sufficient pedigree information to estimate genetic parameters, like effective population size (Ne), that is the Ne of the Krk Island sheep and Cres Island sheep is estimated at 26.34 and 122.76, respectively. In contrast, there is no reliable information concerning phenotype, production and population size of the progressively decreasing sheep population of the Kornati archipelago (KOR). Consequently, KOR is not recognized as an autochthonous Croatian sheep breed by the Government. Direct communication with owners confirmed strong population decline and revealed that there are only about 100 individuals on the islands; therefore, it is a critically endangered strain according to FAO recommendations (FAO 2007). For Bosnian and Herzegovian (BiH) sheep breeds, there are even less reliable records on population size, threat status or established herd-book and supervision by the government bodies. Breeding objectives vary considerably across breeders of the same strain. Almost all sheep strains in BiH belong to the Pramenka meta-population. Pramenka is an indigenous sheep breed divided into various strains throughout the entire Balkans (Cinkulov et al. 2008) and can be identified by its open fleece form of coarse-copped tuft of wool. The strains differ mainly in fleece type (coarse, less coarse, wavy), colour of fleece, head and legs, frequency of polled rams and horned ewes, tail length, body size and milk yield. Although each mountain and region supports its own strain of Pramenka and are predominantly named according to their breeding region, these strains are poorly differentiated. Furthermore, Pramenka are characterized by seasonal migration, which supports the admixture of neighbouring strains. Pramenka strains are characterized by excellent adaptation to harsh climatic conditions and their resistance to disease is thought to be exceptional but has not been investigated objectively. These sheep are traditionally bred in extensive husbandry, on large pastures without supplemented feed in highland areas. Pramenka strains are mainly bred for lamb meat and milk, which is processed to cheese. Lambs are slaughtered at a body weight of about 20 kg, dressing percentage is approximately 50%, but the meat is a gastronomic product with esteemed quality and taste, especially from lambs raised in highland and karst areas. Average milk production of the 72
Pramenka strains is from 100 to 180 litres (Mioc et al. 2011). The wool is low quality and is used for blankets and carpets. The Croatian Government initiated a national breeding program for breeding and selection of sheep in 1999, and in 2011, a new breeding program was established and implemented. In BiH, the Pramenka strains are currently not under controlled mating schemes or selection means supervised by the government or breeders association bodies. The mainland meta-population of Pramenka strains in Croatia and BiH exist in completely opposing demographic circumstances to neighbouring insular sheep breeds. The three Croatian insular sheep breeds, Creska (CRS), Kornatska (KOR) and Krcka (KRK), represent separate breeds due to their geographical isolation. There is no possibility to exchange individuals across islands except by human involvement. In clear contrast to this, animals of commercial breeds typically fulfil some phenotypic standards defined by breeding organizations and are subjected to recording (pedigree and phenotypes), artificial selection and assortative mating. These purebred breeds are administratively isolated from each other (in population genetic sense), even if they occupied the same geographic area or were in the same mews. Further to artificial isolation, some commercial breeds are artificially selected for different exterior and/or performance characteristics leading to genetic differentiation. In this study, we sampled and genotyped sheep breeds and strains from all three demographic groups: strains of Pramenka meta-population from BiH and Croatia, island breeds from Croatia and commercial breeds from central and north-western Europe. Due to population genetic constraints and the threat of extinction, this study has several objectives: (i) to genetically characterize and provide baseline information on BiH and Croatian sheep breeds at a molecular level, (ii) to elucidate genetic relationships among and between breeds and strains from the two states, (iii) to compare genetic diversity within and between three demographic groups with different levels of natural and artificial isolation, as well as selection, (iv) to provide factual evidence for conservation priorities and management options related to breeding strategies, in order to protect farm animal genetic resources.
Materials and methods Breeds, strains and environmental factors
A total of 814 random blood samples were obtained from 18 sheep breeds in Europe (Figure 1). Breed © 2015 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 133 (2016) 71–80
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The genetic diversity of 18 sheep breeds
Figure 1 Geographic distribution of the 18 investigated sheep breeds. Countries of origin are marked with official country code and coloured different shades of grey (HR – Croatia, BiH – Bosnia and Herzegovina, DE – Germany, NL – Netherlands, GB – United Kingdom of Great Britain and Northern Ireland).
name, code, origin, number and utilization of breeds are given in Table 1. Seven of these subpopulations Sjenicka (SJE), Stolacka (STO), Privorska (PRV), Kupreska (KUP), Travnicka sampled in BiH (TRV1) Travnicka sampled in Croatia (TRV2), and Dalmatian (DAL) belong to the group of Pramenka sheep, and are, therefore, Pramenka strains rather than breeds. These seven strains are characterized by the absence of coordinated artificial selection and flock movement along the more extensive travel routes. The three Croatian insular sheep subpopulations, Kornatska (KOR), Creska (CRS) and Krcka (KRK), represent separate breeds characterized by natural isolation and relatively low artificial selection pressure (Table 1, Figure S1). As KOR sheep are predominantly left to their own devices, especially during the tourist season in the summer months, exposure to natural selection is particularly strong in this breed (Figure S1). Eight breeds, Braunes Bergschaf (German Brown Mountain, BBS), Merinolandschaf (MLS), Coburger Fuchsschaf (Coburger Fox Sheep, CFS), Graue Gehoernte Heidschnucke (Grey Horned Heath Sheep, GGH), Schwarzk€ opfiges Fleischschaf (German Blackheaded Mutton, SFS), Ostfriesische Milchschafe (East Friesian Milk Sheep, OFS), Texel (TXL) and Suffolk (SUF), were sampled in Germany but two of the breeds originate in the Netherlands (TXL) and England (SUF) (see Figure 1). Excluding OFS, these central and north-western European sheep breeds are mainly bred for meat production, although their impact on vegetation management is not negligible. All eight are well managed, artificially selected and © 2015 Blackwell Verlag GmbH
• J. Anim. Breed. Genet. 133 (2016) 71–80
isolated by breeding organizations. While interventional husbandry strategies such as providing high-quality feeds, artificial housing or disease management helping to mitigate natural selection pressures, artificial selection pressure is high but varies from breed to breed. The environmental parameters considered were geographical and climatological factors provided by appropriate meteorological observation stations close to sampling area (Table 3). For each breed, averages of climatic parameters for the last ten years were taken in accordance with the geographical origin (latitude and longitude). According to data availability, climatic parameters were averaged from several weather stations (Table 3). In case of Croatian and BiH sheep breeds, climatic parameters were collected from only one or two weather stations. Although TRV2 was sampled in Croatia, climatic parameters were taken from the place of origin (Travnik in BiH). The same applies to TEX and SUF sampled in Germany. Microsatellite amplifications
Thirty microsatellites (Table S1) were used for the evaluation of genetic diversity. The DNA was extracted from 30 ll of whole blood with the QIAmp DNA Blood Mini Kit (Qiagen, Valencia, USA) according to the manufacturer’s instructions. Amplified PCR products were analysed with ABI Prism 310 and 377 DNA Sequencer (Foster City, CA, USA). Fragment analysis was carried out using ABI GENESCAN and ABI GENOTYPER software (Foster City, CA, USA). All 814 73
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The genetic diversity of 18 sheep breeds
Table 1 Description of investigated breeds and strains. Original breed name, short description of sample and breed origin, abbreviation (Abr) used in text and figures and number of samples (n) No
Original breed name
Breed origin and sampling area
Abr.
Purpose
n
1 2 3 4 5 6 7
Sjenicka pramenka Stolacka pramenka Privorska pramenka Kupreska pramenka Travnicka Pramenka Travnicka Pramenka Dalmatinska Pramenka
SJE STO PRV KUP TRV1 TRV2 DAL
Meat-milk-wool Meat-milk-wool Meat-milk-wool Meat-milk-wool Meat-milk-wool Meat-(milk-wool) Meat-(milk-wool)
46 54 34 45 46 48 42
8 9 10 11
Kornatska sheep Cres Island sheep Krk Island sheep Braunes Bergschaf
KOR CRS KRK BBS
Meat Meat-milk-wool Meat-milk-wool Meat-wool
54 43 50 50
12
Merinolandschaf
MLS
Meat-wool
50
13 14
Coburger Fuchsschaf Graue Gehoernte Heidschnucke €pfiges Schwarzko Fleischschaf Ostfriesische Milchschafe Texel Suffolk
Most eastern region of Bosnia Herzegovina; samples collected from Stolac area from 6 breeders Bosnia; samples collected in Vranica area from 5 breeders Bosnia; samples collected from Kupres area from 5 breeders Bosnia; samples collected from Vlasic area from 8 breeders Breed originates from Bosnia (Vlasic) but sampled in Udbine area (Croatia) Croatia; samples collected from Unesic, Planjanje Gornje, Planjanje Donje, Medvida, Benkovacko selo and Jesenice Croatia; samples collected from one island from the archipelago of Kornati Croatia; samples collected from Vodice, Orle, Punta Kriz area from 4 breeders Croatia; samples collected from Baska Draga area from 4 breeders German Brown Mountain Sheep sampled in Germany; originates from Alpine area of south Germany, Austria, Switzerland, and north Italy €rttemberger, sampled in Germany; originates from Known as Wu crosses of Spanish merino with local sheep strains from south and middle Germany Known as Coburger Fox Sheep sampled and originates from middle Germany. Known as Grey Horned Heath Sheep, originates from northern Germany
CFS GGH
50 34 48
15 16 17 18
Known as German Blackheaded Mutton, sampled in Germany; originates from crosses of English sheep breeds in German landraces of County Westfalen East Friesian Milk Sheep, sampled in Germany; originates from North-West Germany
SFS
Meat-wool Meat-landscape management Meat-wool
OFS
Milk
48
Sampled in Germany; originates from Netherland Texel Island Sampled in Germany; originates from England County Suffolk
TXL SUF
Meat Meat
51 22
For comparison purposes, these breeds are grouped into seven Pramenka strains from Croatia and Bosnia-Herzegovina (1–7), three Croatian insular sheep breeds (8–10) and eight central and north-western European breeds (11–18). All samples were collected taking into account minimal relationship within the flocks.
Genetic diversity within breeds was estimated using observed (HO) and expected heterozygosity (HE) according to (Nei 1987). The total and mean number of alleles, Wright’s fixation indices (FST, FIT) and the significance level were estimated using FSTAT v2.9.3.2 (Goudet 2002). Alleles were classified into three groups according to their frequency: common alleles (observed in all 18 sheep subpopulations), private (alleles observed in a single breed or strain) and rare alleles (alleles with frequency
