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Origin and genetic diversity of Romanian Racka sheep using mitochondrial markers
Small Ruminant Research 144 (2016) 276–282
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Origin and genetic diversity of Romanian Racka sheep using mitochondrial markers Andreea Dudu a , Elena Ghit¸a˘ b , Marieta Costache a , Sergiu Emil Georgescu a,∗ a b
University of Bucharest, Faculty of Biology, Department of Biochemistry and Molecular Biology, Splaiul Independent¸ei 91-95, Bucharest, 050095, Romania National Research and Development Institute for Animal Biology and Nutrition, Calea Bucures¸ti 1, Balotes¸ti, 077015, Ilfov, Romania
a r t i c l e
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Article history: Received 16 November 2015 Received in revised form 9 August 2016 Accepted 13 October 2016 Available online 14 October 2016 Keywords: Racka Mitochondrial DNA Genetic diversity Phylogenetic network Breed origin
a b s t r a c t The current study investigates the genetic diversity of Racka sheep from Romania and its relationship with other breeds having different geographic origin. Mitochondrial markers (D-loop and cyt b gene) were sequenced in 40 unrelated individuals. A number of 12 distinct haplotypes, with haplotypes diversity (Hd) of 0.897 and nucleotide diversity (Pi) of 0.00437 was highlighted. The results were comparable with the ones from other sheep breeds and revealed that the Racka population has a good genetic variability. To infer the origin of Racka sheep, a number of 113 mtDNA sequences from different breeds were included into the data set. The network profiles showed that sheep breeds from different geographic regions intermixed. The sequences grouped within the network in clusters correspondent to the five haplogroups A, B, C, D, and E, with the Racka sequences distributed in A and B lineages. The genetic variation indicates that the Romanian Racka is an important reservoir of diversity and the presence of A and B haplotypes within the population is accordance with the findings for different breeds from Zackel group, in which Racka is considered to be included. In conclusion, our findings demonstrate that up to present the Romanian Racka breed was properly managed and has a good potential for conservation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The process of sheep domestication was very important for the development of human civilization, and the diversity of the breeds that appeared after domestication raises problems of origin and classification. The origin and name of the local Racka sheep are extremely controversial. Also, the relationship between the stocks of Racka sheep from Hungary, Romania and Serbia is still unclear. Thus, regarding the origin, Dr˘ag˘anescu (2005) proposed 2 hypotheses: Racka breed is either part of the old Egyptian sheep with corkscrew horns, or it is descended from the Mesopotamian sheep, where, however, only the rams had corkscrew horns, the sheep being hornless. The Balkan Peninsula is an important area for sheep breeds with primitive features, but unfortunately the majority of these are endangered. Thus in the former Yugoslav area (Kosovo, Montenegro and Serbia) there is at least one sheep breed resembling Racka sheep, and probably related to it, the Balusha sheep (Dr˘ag˘anescu, 2005). Presently, parts of the Zackel sheep are accepted as Racka
∗ Corresponding author. E-mail addresses: georgescu [email protected], [email protected] (S.E. Georgescu). http://dx.doi.org/10.1016/j.smallrumres.2016.10.016 0921-4488/© 2016 Elsevier B.V. All rights reserved.
sheep, including several breeds from the Balkan region, northern Greece, the former Yugoslav countries (Serbia, Montenegro, Bosnia and Herzegovina, Slovenia, Macedonia, Croatia) Albania, Bulgaria, Romania and Hungary. Generally, the sheep from Zackel group are dual purpose sheep and, although their productivity is moderate, they are very resilient to the harsh environmental conditions from the mountain areas where they live (Dr˘ag˘anescu, 2007; Savic et al., 2013). In terms of stocks, Racka breed is in a good state of conservation in Hungary, in critical state in Serbia (Savic et al., 2011), while in Romania it lost importance in favor of other breeds, being now bred in just a few locations in Banat area at the border with Serbia. Possible explanations for the low interest in this breed include that the breed is not economically competitive and that the productions of wool, milk and meat are lower than those of the sheep breeds that underwent breeding programs. According to the current estimations, in the five villages where it is still reared, there are some 4000 sheeps, of which just 500 are pure breed (Savic et al., 2013). In Romania there are two varieties of the breed: white (brown face and white wool) and black (black face and black wool), but it has been assimilated as variety of Tsurcana sheep up to present (Dr˘ag˘anescu, 2005). The rearing of Racka sheep is now subsidized through the program of animal genetic resources conservation.
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In Hungary, starting with 1960 significant imports of sheep from Romania was done and the breed was exposed to an intensive process of selection and conservation. However, the selection was, however, more efficient in Hungary since there only sheep with “V” shaped horns can be identified, while in Romania, there are still sheep with side-oriented horns. Additionally, the Romanian Racka sheep are larger and have longer, thicker wool (Dr˘ag˘anescu, 2005; Supplementary material Fig. S1 in the online version at DOI: 10.1016/j.smallrumres.2016.10.016). The name of the breed has many regional variants. Thus, in Serbia, this breed is also named “Vlachko-Vitoroga”, in Hungary “Raczka” or “Hungarian Zackel”, and in Romania “Rat¸ca”. Due to the multiple names given to the breed, and to the resemblance to other breeds from the Balkans, the Racka sheep breed has probably been mistaken or assimilated over time with other groups of sheep, in Romania being considered as a variety of Tsurcana sheep. Presently, the formal name of the breed is Vlashko-Vitoroga Zackel according to “FAO Domestic animal Diversity Information Service” and it is being considered a trans-border breed (Savic et al., 2013). Currently, three groups of wild sheep living in Eurasia, the mouflons (Ovis orientalis), urial (Ovis vignei) and argali (Ovis ammon), are considered to be the ancestors of the domesticated sheep, each of them having a contribution to the formation of breeds. The literature has several theories on the origin of the modern domesticated sheep. Thus, in 2002, Hiendleder et al. (Hiendleder et al., 2002) hypothesized that all the current breeds of domesticated sheep evolved from two different subspecies, each determining dam descendance (A and B). B haplotype is predominant in the sheep populations from Europe, being also the only haplotype observed in the European mouflon, but has a low frequency in the native breeds from Eastern Eurasia (Hiendleder et al., 1998a, 2002; Guo et al., 2005; Meadows et al., 2005). In 2013, Singh et al. (Singh et al., 2013) after surveying several studies on the origin and domestication of sheep, said that the process of sheep domestication is very complex and that it involves two main maternal lines (A and B) and three minor lines (C, D and E). The maternal haplotypes C, D and E appear to be more frequent in the sheep breeds form Near East. In this context, the purpose of our study was to evaluate the genetic diversity and to clarify the origin of the Romanian Racka sheep using the analysis of the mitochondrial markers D-loop and cytochrome b. By comparative analysis of specific mitochondrial markers and complex molecular phylogeny, this study aims to identify the clear descendance and interrelation of the Romanian Racka sheep with other sheep breeds. 2. Materials and methods 2.1. DNA extraction DNA samples were collected from 40 unrelated Racka sheep, of both sexes, from two private farms located in South-Western Romania (Caras¸-Severin County), at the border with Serbian Republic. Blood samples were collected on EDTA anticoagulant and DNA was extracted using the Wizard Genomic DNA Extraction Kit (Promega) according to manufacturer’s specifications.
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5 -gatctcccagctccatcaaa-3 ; R: 5 -tgagggggagtgttaagtgg-3 ; D-loop F: 5 -acccggagcatgaattgtag-3 ; R: 5 -gggggaagcgtgttaaaaat-3 ), 0.5 units of AmpliTaq Gold DNA Polymerase (5U/l), 50 ng of DNA template and nuclease-free water. The amplification reaction was done during 40 cycles, each one including denaturation at 95 ◦ C (30 s), hybridization at 58 ◦ C (30 s) and extension at 72 ◦ C (60 s). Furthermore, we also performed a step of initial denaturation at 95 ◦ C for 10 min and a stage of final extension at 72 ◦ C for 15 min. The primers were thus set as to amplify a fragment of D-loop region bordered by positions 16,345–16,520 within the sheep mitochondrial genome (NC001941, Hiendleder et al., 1998b), and a fragment from cytochrome b gene bordered by positions 14,216–14,958 (NC001941, Hiendleder et al., 1998b). The amplified fragments had a dimension of 175 bp for the control region and 743 bp for cyt b gene. The amplification products were thereafter purified with the Wizard PCR Preps DNA Purification System Kit (Promega). The purified fragments were further amplified in with the purpose of sequencing with the ABI Prism® BigDye Terminator Cycle Sequencing Reaction Kit (AppliedBiosytems) and then run on the automatic ABI Prism 3130 Genetic Analyzer. The sequences were processed using DNA Sequencing Analysis 5.1 Software (AppliedBiosytems), and the nucleotide sequence was aligned and edited using BioEdit software (Hall, 1999). The partial sequences obtained for Racka sheep breed were deposited into the GenBank, under the access numbers KP710097- KP710176. 2.3. Data analysis Both control region and cyt b sequences were aligned and compared with similar sequences from GenBank isolated in sheep breeds with different geographic origin from Europe, Asia and Middle and Near East (Supplementary material Table S1 in the online version at DOI: 10.1016/j.smallrumres.2016.10.016). The sequences were truncated to 175 bp for D-loop and respectively 743 bp for cyt b and for a higher accuracy of the analysis were concatenated to 918 bp dataset. The data set comprised 153 sequences isolated in different sheep breeds, including our 40 sequences from Romanian Racka. The sequences alignment was performed with ClustalW algorithm implemented in MEGA 5 (Tamura et al., 2011). The genetic diversity in terms of number of haplotypes, nucleotide diversity, haplotype diversity, average number of nucleotide differences and average number of nucleotide substitutions (Dxy) per site were calculated using DnaSP v5.1 (Librado and Rozas, 2009). Degree of genetic differentiation and gene flow among Racka breed and different haplogroups were determined by F-statistics using the same software. Mismatch distribution, as well as Fu’s FS (Fu, 1997) and Tajima’s D (Tajima, 1989) neutrality tests implemented in ARLEQUIN 3.5 (Excoffier et al., 2005) were used to assess departures from neutrality. DNA Alignment software v1.3.3.1 (http://www.fluxus-engineering.com) was used to convert the aligned sequences into RDF binary format. A haplotype network of mitochondrial sequences using a median-joining algorithm with default settings ( = 0) and the variable sites weighted equally (Weight = 10) was inferred with NETWORK v4.6.1.1 (Bandelt et al., 1999). The species Ovis orientalis anatolica was used as an outgroup species.
2.2. PCR amplification and sequencing 3. Results For the phylogenetic analysis we amplified fragments from the gene that encodes for cytochrome b and from the mitochondrial D-loop area. The reactions of PCR amplification were performed using a GeneAmp 9700 PCR System (AppliedBiosystems), in a final volume of 25 l which contained 10× PCR Buffer, 1.5 mM MgCl2 , 200 M dNTPs, 0.5 M of each primer (cytochrome b F:
3.1. mtDNA variation in Romanian Racka. Comparative analysis with other sheep breeds For the 153 mtDNA sequences, 66 haplotypes were identified, with an overall value of haplotypes diversity (Hd ) of 0.960 and a
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value of nucleotide diversity equal to 0.01719. These sequences were defined by 634 variable sites out of which 106 were parsimony informative and 528 were singleton. Among the 66 haplotypes in the set of analyzed sequences, 42 were observed once, while the most common haplotype occurred 22 times, including ten sequences of Romanian Racka (data not shown). By analyzing the concatenated data we identified a total number of 12 distinct haplotypes, with haplotype diversity (Hd ) of 0.897 and nucleotide diversity (Pi) of 0.00437 (Table 1). From the 12 haplotypes identified, haplotype 7 is the most common, characteristic for 10 individuals (with a frequency of 0.25), followed by haplotype 11, characteristic for a number of 6 individuals and presenting a frequency of 0.15. Most haplotypes (8 from the total 12) were found in each of two individuals from the total of 40 analyzed (Supplementary material Table S2 in the online version at DOI: 10.1016/j. smallrumres.2016.10.016). For Racka sequences 903 invariable and 15 variable sites were highlighted. The polymorphic sites were parsimony informative, no singleton mutations were identified. All the 15 polymorphisms were represented by transitions (Supplementary material Fig. S2 in the online version at DOI: 10.1016/j. smallrumres.2016.10.016). The Racka genetic diversity indices were compared to the ones obtained by analyzing 113 mtDNA sequences including all known haplogroups in sheep (Meadows et al., 2005, 2007, 2011). The haplotypes diversity (0.8974) in Racka breed revealed an intermediate value by comparison to the haplotypes diversity values characteristic for the haplogroups B (0.946) and A (0.772) and very similar with the haplotypes diversity of haplogroup C (0.895). The nucleotide diversity (Pi), calculated as the average number of nucleotide differences per site between two randomly selected sequences, varied from 0.00141 in the haplogroup A to 0.00490 in the haplogroup B, while the overall value of Pi for the all sheep sequences analyzed in the study is equal to 0.0073. With a nucleotide diversity index of 0.00437, the Racka sheep displays a value, higher than the value of Pi for haplogroups A and C and almost equal to that of haplogroup B. The analysis for the haplogroups D and E was performed based on a reduced number of mtDNA sequences due to the fact that these are less frequent in domestic sheep breeds. For each of these haplogroups a unique haplotype was observed thus leading to no haplotype diversity. Unfortunately, the data are inconclusive due to the reduced data set that we analyzed. 3.2. Genetic distance and gene flow analysis Nei’s DA genetic distance and the average number of pairwise differences (Dxy ) between Racka breed and haplogroups A, B and C were estimated and a distance matrix was generated (Table 2). The lowest distance between Racka and other haplogroup was observed in the case of Racka-haplogroup B (Dxy : 0.00451; DA : 0.00048), followed by the distance between Racka-haplogroup A (Dxy : 0.00512; DA : 0.00162). The degree of genetic differentiation between Racka breed and other breeds grouped in haplogroups A, B, and C was determined with Wright’s Fixation Index (Fst ), which can range from 0 (identical populations) to 1 (complete differentiation between populations). Gammast was also evaluated, as it represents an unbiased estimate of Fst that corrects for errors associated with incomplete sampling of populations and which is more suitable for mitochondrial haplotype data (Weir and Cockerham, 1984). Tajima’s D and Fu’s Fs neutrality tests were performed with the purpose of tracing the recent demographic events. Significant negative values are interpreted as a signal of purifying selection or alternatively as demographic expansion, while significant positive values indicate recent population bottleneck or population subdivision. For Racka population, Tajima’s D value was positive
(0.44193), while Fu F’s value was negative (−0.97675), but not significant from statistic point of view. Negative values were highlighted for both parameters in haplogroups A, B and C, indicating that these lineages had undergone population expansion event, but for haplogroup C the values were not statistically significant. Moreover, the mismatch distribution was fitted to the sudden expansion model (Rogers, 1995) and the analysis supported population expansion hypothesis in haplogroups A, B and C, since the Raggedness index for these groups was 0.137 (p = 0.55) for haplogroup A, 0.027 (p = 0.654) for haplogroup B and 0.0413 (p = 0.714) for haplogroup C, as expected in this case. Also, the sum of squared deviations (SSD) distribution presented low values (HPG A – 0.0151, HPG B – 0.0021 and HPG C – 0.0029), but none was significant (p = 0.12/0.43/0.76) supporting the sudden expansion model tested. 3.3. mtDNA haplotypes networks The phylogenetic network constructed based on mtDNA sequences for Romanian Racka revealed that the haplotypes were separated in two clusters connected by a median vector (Fig. 1). To assess the phylogenetic relationships between Racka and other sheep breeds we constructed MJ networks highlighting the maternal lineage/geographic distribution of the breeds (Fig. 2a, b). In relationship to previously defined sheep mtDNA haplogroups, all 40 sequences of Romanian Racka can be clearly grouped into A and B lineages, explaining thus the two clusters inferred by the Racka haplotypes network. The haplotypes from cluster I grouped together with haplotypes from A lineage, while the haplotypes from cluster II grouped together with haplotypes from B lineage. The sequences are grouped within the network in clusters correspondent to the five haplogroups A, B, C, D, and E, with the Racka sequences distributed in A and B lineages in proportion of 35 and 65% respectively (Fig. 2a). None of the Racka sheep haplotypes associated with C, D or E lineages were found in the sheep breeds of the Near East. Within the network profile there is one major haplotype in the center for A and B haplogroups, these parts of network displaying a “star like” shape. For the haplogroup A the central haplotype is shared by 16 sequences, among which two isolated in Romanian Racka breed, while in haplogroup B the central haplotypes is common to 22 sequences, including ten from Racka. The central haplotypes of the clades corresponding to A and B haplogroup are connected by six mutational steps. Haplogroups A and B were evidenced to have undergone population expansions. The star shaped phylogeny of lineages A and B are consistent with population expansion clearly confirmed by the neutrality tests results. The clade corresponding to the lineage C is characterized by a reduced number of sequences, while the D and E lineages are each represented by unique haplotypes. Haplogroup C appears to be derived from the haplogroup E, the central haplotypes of C lineage being connected with this one by only three mutational steps. Instead the D haplogroup is connected with the central haplotype of the haplogroup A by nine mutational steps. 4. Discussion An analysis of partial mitochondrial sequence of 40 individuals from Romanian Racka sheep has been presented in this study. Similar haplotypes diversity values with the one of Racka breed have been reported in other Asian and European sheep breeds (Chen et al., 2006; Pardeshi et al., 2007; Mariotti et al., 2013; Othman et al., 2015). Thus, in sheep breeds from Europe values of haplotype diversity ranging from 0.872 to 0.988 were highlighted (Mariotti et al., 2013; Othman et al., 2015). Moreover, comparable haplotype diver-
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Table 1 Diversity indices of Romanian Racka population. Breed
n
H
Hd ± SD
Pi ± SD
K
Variable sites
Singleton sites
Parsimony sites
Racka Haplogroup A Haplogroup B Haplogroup C Haplogroup D Haplogroup E Overall
40 33 57 18 2 3 153
12 12 36 8 1 1 66
0.89740 ± 0.02800 0.77200 ± 0.07500 0.94600 ± 0.02200 0.89500 ± 0.04100 0 0 0.95800 ± 0.00900
0.00437 ± 0.00032 0.00141 ± 0.00029 0.00490 ± 0.00121 0.00241 ± 0.00036 0 0 0.00700 ± 0.00060
4.01500 1.29700 4.49600 2.21600 0 0 6.70488
15 15 66 9 0 0 95
0 12 32 3 0 0 37
15 3 34 6 0 0 58
n = number of individuals; H = number of haplotypes; Hd = haplotype diversity; Pi-nucleotide diversity. K-average number of nucleotide differences. Table 2 Genetic distance and gene flow analysis between Racka breed and different haplogroups. Dxy – above; DA – below; Fst – above; Gammast – below. Dxy /DA
Racka HPG A HPG B HPG C
Fst /GammaSt Racka
HPG A
HPG B
HPG C
– 0.00162 0.00048 0.01055
0.00512 – 0.00358 0.00900
0.00451 0.00673 – 0.01271
0.01394 0.01091 0.01637 –
Racka HPG A HPG B HPG C
Racka
HPG A
HPG B
HPG C
– 0.21585 0.09395 0.32667
0.35898 – 0.31175 0.41179
0.05779 0.52846 – 0.35256
0.54183 0.56259 0.62578 –
Fig. 1. Median Joining network constructed based mtDNA haplotypes in Romanian Racka. Node size is proportional to haplotype frequency. The smallest node is representative of one haplotype. The red dots represent median vectors.
sity was obtained for Egyptian Ossimi breed (Hd – 0.8833) (Othman et al., 2015). In sheep breeds from China and India the values of haplotypes diversity in the analyzed sheep populations were ranging from 0.85 to 0.95 (Chen et al., 2006; Pardeshi et al., 2007). A study on Gymiesi Racka from Hungary revealed a higher number of haplotypes (23) and implicitly higher haplotypes diversity (0.950) in this population by comparison with Romanian Racka, but still both populations have significant haplotype diversity (Kusza et al., 2015). By comparing the genetic diversity indices for Racka with the ones of the analyzed haplogroups we observed that the values of haplotypes diversity are significant and similar. The overall haplotype variability was observed to be high (0.958 ± 0.009). The nucleotide diversity was similar with the one reported by other studies on sheep breeds from Europe and Asia that exhibited a composite genetic structure composed of A and B haplotypes (Arora et al., 2013; Demirci et al., 2013). Also, the analysis of nuclear markers in local sheep from Balkans region (Croatia and Bosnia and Herzegovina) also revealed considerable levels of genetic diversity (Salamon et al., 2014).
The gene flow analysis inferred by Fst and Gammast indices values that measure the short-term genetic differentiation revealed that the Racka breed is most diverged from haplogroups A and C, while between this breed and haplogroup B is a moderate genetic differentiation. Thus, the data confirm the distance data (DA and Dxy ) showing that Racka breed is more closely related to haplogroup B, predominant in the European sheep breeds. Both Tajima’s D and Fu’s neutrality test and the mismatch distribution analyzed under sudden expansion model were not significant and no conclusion can be formulated about the Racka population evolution. The haplotypes network highlighted the distribution of mtDNA sequences of Romanian Racka in two clusters corresponding to A and B lineages. The frequency of haplogroups A and B within Racka breed is not surprising since it was previously revealed that more than one maternal lineage is present in the majority of breeds. Previous reports (Tapio et al., 2006) described/identified individuals carrying type A haplotypes in European sheep breed from Austria, Ukraine, Poland, Russia and South-East Europe. Also, in different
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Fig. 2. Median Joining network of 40 Racka sequences and 113 sequences from GenBank. (A) Breeds distribution into haplogroups. Green – HPG A; Blue- HPG B; Yellow – HPG C; Black – HPG D; Orange – HPG E. (B) Breeds geographic origin. Blue – Europe; Green – Asia; Yellow – Turkey; Orange – Israel. For both networks the haplotypes of Romanian Racka are shown in purple. Node size is proportional to haplotype frequency. The smallest node is representative of one haplotype. The red dots represent median vectors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
sheep breeds from Turkey and Israel, Meadows et al. (2007) demonstrated that three or more lineages were identified within each breed. The network profile showed that sheep breeds from different geographic regions intermixed since the majority of the haplotypes were shared by individuals from different breeds across wide
geographic regions. The haplotypes distribution did not indicate a clear phylogeographic structure. Different breeds from Asia (India, Indonesia and Mongolia) presented exclusively type A haplotypes, while breeds sourced from Europe (eight breeds from Austria, Balkans Region, Russia and Romania) possessed only type B haplotypes.
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Three Romanian breeds (Tsurcana, Tsigai and Black Head Ruda) included in the study clustered in haplogroup B. The sheep breeds from Middle and Near East (Turkey and Israel) showed a higher heterogeneity in terms of lineages composition. No haplotypes pattern correlated with the geographic distribution was observed for the sheep breeds from this part of the world, supporting thus the hypothesis of a Near East domestication center for sheep. The presence of haplotypes A and B in Romanian Racka is in accordance with the finding of these haplotypes in other sheep breeds from Eastern Europe, including area of north and west, from the Black Sea and the Ural Mountains. Genetic studies performed on 12 local Zackel type breeds from East Adriatic region revealed the presence of both A and B haplotypes, with predominant B haplotype, but with a significant lower frequency of A haplotype comparative with the one found in Romanian Racka (Ferencakovic et al., 2013). Similar, haplogroup B was predominant (with a frequency of 95%) for sheep breeds from Mediterranean region (Lancioni et al., 2013). The differences regarding the prevalence of a certain haplotype are explainable if we take into account that domestication started in the Near East and continued towards Europe, where the domestication path run along the continent, along the Danube valley, or along the Mediterranean coast (Ryder, 1984). Sheep breeds from Europe, Caucasus and Central Asia are characterized by the presence of A, B and C haplotypes. Apart the three haplotypes, a new haplotype (D) was noticed in just one Caucasian breed. The frequency of B haplotype decreases towards the east, while that of haplotype A increases. The C haplotype has been noticed only in the breeds from Caucasus and Central Asia, and was not identified in any East-European breed (Tapio et al., 2006). The presence of both A and B haplotypes in populations from both Europe and Asia seems to be the consequence of introgression, as a result of a gene flow between breeds of domestic sheep from both continents (Meadows et al., 2005; Arora et al., 2013). Moreover, the proximity of Balkans with the Middle East, characterized by a heterogenic distribution of haplotypes within breeds, might be an explanation for the composite haplogroups of the breeds from Balkans.
5. Conclusions The Romanian Racka is characterized by an important genetic diversity which demonstrates that up to present the population was correctly managed and has a good potential for conservation. The presence of A and B haplotypes within the population is in accordance with the findings for different breeds from Zackel group and with their hypothesized origins. The distribution of Racka in the haplogroups A and B followed the pattern observed for other breeds with different geographic origins. Thus, the presence of both typically Asian and European haplotypes might be the results of gene flow and introgression events that have occurred in the history of this breed. The analysis of mtDNA sequences was informative to assess the diversity within Romanian Racka, but not useful for establishing the accurate origin of the breed and its detailed relationships with other breeds. In order to have a clear image about Racka biodiversity, further large scale studies are necessary. The research should be extended for Racka sheep populations from neighboring countries by using a larger panel of molecular markers in order to investigate the sheep in the regional context and to develop a gene pool for conservation purposes due the fragile status of the breed.
Conflict of interest The authors declare that there is no conflict of interest.
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Genetic diversity and differentiation of 12 eastern Adriatic and western Dinaric native sheep breeds using microsatellites. Animal 8 (2), 200–207. Savic, M., Jovanovic, S., Trailovic, R., Dimitrijevic, V., Dimitrijevic, B., 2011. Autochtonous breeds as livestock potential in sustainable farming in Serbia. In: Proceedings of the 19th Internationale Congress of Mediterranean Federation of Health and Production of Ruminants, Belgrade, Serbia, pp. 6–22. Savic, M., Trailovic, R., Petrujkic, B., Beckei, Z., Dimitrijevic, B., Dimitrijevic, V., 2013. Determining the value of Vlashko-Vitoroga Zackel sheep for the conservation process. Acta Vet. Beograd. 63 (5–6), 621–629.
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Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Origin and genetic diversity of Romanian Racka sheep using mitochondrial markers Andreea Dudu a , Elena Ghit¸a˘ b , Marieta Costache a , Sergiu Emil Georgescu a,∗ a b
University of Bucharest, Faculty of Biology, Department of Biochemistry and Molecular Biology, Splaiul Independent¸ei 91-95, Bucharest, 050095, Romania National Research and Development Institute for Animal Biology and Nutrition, Calea Bucures¸ti 1, Balotes¸ti, 077015, Ilfov, Romania
a r t i c l e
i n f o
Article history: Received 16 November 2015 Received in revised form 9 August 2016 Accepted 13 October 2016 Available online 14 October 2016 Keywords: Racka Mitochondrial DNA Genetic diversity Phylogenetic network Breed origin
a b s t r a c t The current study investigates the genetic diversity of Racka sheep from Romania and its relationship with other breeds having different geographic origin. Mitochondrial markers (D-loop and cyt b gene) were sequenced in 40 unrelated individuals. A number of 12 distinct haplotypes, with haplotypes diversity (Hd) of 0.897 and nucleotide diversity (Pi) of 0.00437 was highlighted. The results were comparable with the ones from other sheep breeds and revealed that the Racka population has a good genetic variability. To infer the origin of Racka sheep, a number of 113 mtDNA sequences from different breeds were included into the data set. The network profiles showed that sheep breeds from different geographic regions intermixed. The sequences grouped within the network in clusters correspondent to the five haplogroups A, B, C, D, and E, with the Racka sequences distributed in A and B lineages. The genetic variation indicates that the Romanian Racka is an important reservoir of diversity and the presence of A and B haplotypes within the population is accordance with the findings for different breeds from Zackel group, in which Racka is considered to be included. In conclusion, our findings demonstrate that up to present the Romanian Racka breed was properly managed and has a good potential for conservation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The process of sheep domestication was very important for the development of human civilization, and the diversity of the breeds that appeared after domestication raises problems of origin and classification. The origin and name of the local Racka sheep are extremely controversial. Also, the relationship between the stocks of Racka sheep from Hungary, Romania and Serbia is still unclear. Thus, regarding the origin, Dr˘ag˘anescu (2005) proposed 2 hypotheses: Racka breed is either part of the old Egyptian sheep with corkscrew horns, or it is descended from the Mesopotamian sheep, where, however, only the rams had corkscrew horns, the sheep being hornless. The Balkan Peninsula is an important area for sheep breeds with primitive features, but unfortunately the majority of these are endangered. Thus in the former Yugoslav area (Kosovo, Montenegro and Serbia) there is at least one sheep breed resembling Racka sheep, and probably related to it, the Balusha sheep (Dr˘ag˘anescu, 2005). Presently, parts of the Zackel sheep are accepted as Racka
∗ Corresponding author. E-mail addresses: georgescu [email protected], [email protected] (S.E. Georgescu). http://dx.doi.org/10.1016/j.smallrumres.2016.10.016 0921-4488/© 2016 Elsevier B.V. All rights reserved.
sheep, including several breeds from the Balkan region, northern Greece, the former Yugoslav countries (Serbia, Montenegro, Bosnia and Herzegovina, Slovenia, Macedonia, Croatia) Albania, Bulgaria, Romania and Hungary. Generally, the sheep from Zackel group are dual purpose sheep and, although their productivity is moderate, they are very resilient to the harsh environmental conditions from the mountain areas where they live (Dr˘ag˘anescu, 2007; Savic et al., 2013). In terms of stocks, Racka breed is in a good state of conservation in Hungary, in critical state in Serbia (Savic et al., 2011), while in Romania it lost importance in favor of other breeds, being now bred in just a few locations in Banat area at the border with Serbia. Possible explanations for the low interest in this breed include that the breed is not economically competitive and that the productions of wool, milk and meat are lower than those of the sheep breeds that underwent breeding programs. According to the current estimations, in the five villages where it is still reared, there are some 4000 sheeps, of which just 500 are pure breed (Savic et al., 2013). In Romania there are two varieties of the breed: white (brown face and white wool) and black (black face and black wool), but it has been assimilated as variety of Tsurcana sheep up to present (Dr˘ag˘anescu, 2005). The rearing of Racka sheep is now subsidized through the program of animal genetic resources conservation.
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In Hungary, starting with 1960 significant imports of sheep from Romania was done and the breed was exposed to an intensive process of selection and conservation. However, the selection was, however, more efficient in Hungary since there only sheep with “V” shaped horns can be identified, while in Romania, there are still sheep with side-oriented horns. Additionally, the Romanian Racka sheep are larger and have longer, thicker wool (Dr˘ag˘anescu, 2005; Supplementary material Fig. S1 in the online version at DOI: 10.1016/j.smallrumres.2016.10.016). The name of the breed has many regional variants. Thus, in Serbia, this breed is also named “Vlachko-Vitoroga”, in Hungary “Raczka” or “Hungarian Zackel”, and in Romania “Rat¸ca”. Due to the multiple names given to the breed, and to the resemblance to other breeds from the Balkans, the Racka sheep breed has probably been mistaken or assimilated over time with other groups of sheep, in Romania being considered as a variety of Tsurcana sheep. Presently, the formal name of the breed is Vlashko-Vitoroga Zackel according to “FAO Domestic animal Diversity Information Service” and it is being considered a trans-border breed (Savic et al., 2013). Currently, three groups of wild sheep living in Eurasia, the mouflons (Ovis orientalis), urial (Ovis vignei) and argali (Ovis ammon), are considered to be the ancestors of the domesticated sheep, each of them having a contribution to the formation of breeds. The literature has several theories on the origin of the modern domesticated sheep. Thus, in 2002, Hiendleder et al. (Hiendleder et al., 2002) hypothesized that all the current breeds of domesticated sheep evolved from two different subspecies, each determining dam descendance (A and B). B haplotype is predominant in the sheep populations from Europe, being also the only haplotype observed in the European mouflon, but has a low frequency in the native breeds from Eastern Eurasia (Hiendleder et al., 1998a, 2002; Guo et al., 2005; Meadows et al., 2005). In 2013, Singh et al. (Singh et al., 2013) after surveying several studies on the origin and domestication of sheep, said that the process of sheep domestication is very complex and that it involves two main maternal lines (A and B) and three minor lines (C, D and E). The maternal haplotypes C, D and E appear to be more frequent in the sheep breeds form Near East. In this context, the purpose of our study was to evaluate the genetic diversity and to clarify the origin of the Romanian Racka sheep using the analysis of the mitochondrial markers D-loop and cytochrome b. By comparative analysis of specific mitochondrial markers and complex molecular phylogeny, this study aims to identify the clear descendance and interrelation of the Romanian Racka sheep with other sheep breeds. 2. Materials and methods 2.1. DNA extraction DNA samples were collected from 40 unrelated Racka sheep, of both sexes, from two private farms located in South-Western Romania (Caras¸-Severin County), at the border with Serbian Republic. Blood samples were collected on EDTA anticoagulant and DNA was extracted using the Wizard Genomic DNA Extraction Kit (Promega) according to manufacturer’s specifications.
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5 -gatctcccagctccatcaaa-3 ; R: 5 -tgagggggagtgttaagtgg-3 ; D-loop F: 5 -acccggagcatgaattgtag-3 ; R: 5 -gggggaagcgtgttaaaaat-3 ), 0.5 units of AmpliTaq Gold DNA Polymerase (5U/l), 50 ng of DNA template and nuclease-free water. The amplification reaction was done during 40 cycles, each one including denaturation at 95 ◦ C (30 s), hybridization at 58 ◦ C (30 s) and extension at 72 ◦ C (60 s). Furthermore, we also performed a step of initial denaturation at 95 ◦ C for 10 min and a stage of final extension at 72 ◦ C for 15 min. The primers were thus set as to amplify a fragment of D-loop region bordered by positions 16,345–16,520 within the sheep mitochondrial genome (NC001941, Hiendleder et al., 1998b), and a fragment from cytochrome b gene bordered by positions 14,216–14,958 (NC001941, Hiendleder et al., 1998b). The amplified fragments had a dimension of 175 bp for the control region and 743 bp for cyt b gene. The amplification products were thereafter purified with the Wizard PCR Preps DNA Purification System Kit (Promega). The purified fragments were further amplified in with the purpose of sequencing with the ABI Prism® BigDye Terminator Cycle Sequencing Reaction Kit (AppliedBiosytems) and then run on the automatic ABI Prism 3130 Genetic Analyzer. The sequences were processed using DNA Sequencing Analysis 5.1 Software (AppliedBiosytems), and the nucleotide sequence was aligned and edited using BioEdit software (Hall, 1999). The partial sequences obtained for Racka sheep breed were deposited into the GenBank, under the access numbers KP710097- KP710176. 2.3. Data analysis Both control region and cyt b sequences were aligned and compared with similar sequences from GenBank isolated in sheep breeds with different geographic origin from Europe, Asia and Middle and Near East (Supplementary material Table S1 in the online version at DOI: 10.1016/j.smallrumres.2016.10.016). The sequences were truncated to 175 bp for D-loop and respectively 743 bp for cyt b and for a higher accuracy of the analysis were concatenated to 918 bp dataset. The data set comprised 153 sequences isolated in different sheep breeds, including our 40 sequences from Romanian Racka. The sequences alignment was performed with ClustalW algorithm implemented in MEGA 5 (Tamura et al., 2011). The genetic diversity in terms of number of haplotypes, nucleotide diversity, haplotype diversity, average number of nucleotide differences and average number of nucleotide substitutions (Dxy) per site were calculated using DnaSP v5.1 (Librado and Rozas, 2009). Degree of genetic differentiation and gene flow among Racka breed and different haplogroups were determined by F-statistics using the same software. Mismatch distribution, as well as Fu’s FS (Fu, 1997) and Tajima’s D (Tajima, 1989) neutrality tests implemented in ARLEQUIN 3.5 (Excoffier et al., 2005) were used to assess departures from neutrality. DNA Alignment software v1.3.3.1 (http://www.fluxus-engineering.com) was used to convert the aligned sequences into RDF binary format. A haplotype network of mitochondrial sequences using a median-joining algorithm with default settings ( = 0) and the variable sites weighted equally (Weight = 10) was inferred with NETWORK v4.6.1.1 (Bandelt et al., 1999). The species Ovis orientalis anatolica was used as an outgroup species.
2.2. PCR amplification and sequencing 3. Results For the phylogenetic analysis we amplified fragments from the gene that encodes for cytochrome b and from the mitochondrial D-loop area. The reactions of PCR amplification were performed using a GeneAmp 9700 PCR System (AppliedBiosystems), in a final volume of 25 l which contained 10× PCR Buffer, 1.5 mM MgCl2 , 200 M dNTPs, 0.5 M of each primer (cytochrome b F:
3.1. mtDNA variation in Romanian Racka. Comparative analysis with other sheep breeds For the 153 mtDNA sequences, 66 haplotypes were identified, with an overall value of haplotypes diversity (Hd ) of 0.960 and a
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value of nucleotide diversity equal to 0.01719. These sequences were defined by 634 variable sites out of which 106 were parsimony informative and 528 were singleton. Among the 66 haplotypes in the set of analyzed sequences, 42 were observed once, while the most common haplotype occurred 22 times, including ten sequences of Romanian Racka (data not shown). By analyzing the concatenated data we identified a total number of 12 distinct haplotypes, with haplotype diversity (Hd ) of 0.897 and nucleotide diversity (Pi) of 0.00437 (Table 1). From the 12 haplotypes identified, haplotype 7 is the most common, characteristic for 10 individuals (with a frequency of 0.25), followed by haplotype 11, characteristic for a number of 6 individuals and presenting a frequency of 0.15. Most haplotypes (8 from the total 12) were found in each of two individuals from the total of 40 analyzed (Supplementary material Table S2 in the online version at DOI: 10.1016/j. smallrumres.2016.10.016). For Racka sequences 903 invariable and 15 variable sites were highlighted. The polymorphic sites were parsimony informative, no singleton mutations were identified. All the 15 polymorphisms were represented by transitions (Supplementary material Fig. S2 in the online version at DOI: 10.1016/j. smallrumres.2016.10.016). The Racka genetic diversity indices were compared to the ones obtained by analyzing 113 mtDNA sequences including all known haplogroups in sheep (Meadows et al., 2005, 2007, 2011). The haplotypes diversity (0.8974) in Racka breed revealed an intermediate value by comparison to the haplotypes diversity values characteristic for the haplogroups B (0.946) and A (0.772) and very similar with the haplotypes diversity of haplogroup C (0.895). The nucleotide diversity (Pi), calculated as the average number of nucleotide differences per site between two randomly selected sequences, varied from 0.00141 in the haplogroup A to 0.00490 in the haplogroup B, while the overall value of Pi for the all sheep sequences analyzed in the study is equal to 0.0073. With a nucleotide diversity index of 0.00437, the Racka sheep displays a value, higher than the value of Pi for haplogroups A and C and almost equal to that of haplogroup B. The analysis for the haplogroups D and E was performed based on a reduced number of mtDNA sequences due to the fact that these are less frequent in domestic sheep breeds. For each of these haplogroups a unique haplotype was observed thus leading to no haplotype diversity. Unfortunately, the data are inconclusive due to the reduced data set that we analyzed. 3.2. Genetic distance and gene flow analysis Nei’s DA genetic distance and the average number of pairwise differences (Dxy ) between Racka breed and haplogroups A, B and C were estimated and a distance matrix was generated (Table 2). The lowest distance between Racka and other haplogroup was observed in the case of Racka-haplogroup B (Dxy : 0.00451; DA : 0.00048), followed by the distance between Racka-haplogroup A (Dxy : 0.00512; DA : 0.00162). The degree of genetic differentiation between Racka breed and other breeds grouped in haplogroups A, B, and C was determined with Wright’s Fixation Index (Fst ), which can range from 0 (identical populations) to 1 (complete differentiation between populations). Gammast was also evaluated, as it represents an unbiased estimate of Fst that corrects for errors associated with incomplete sampling of populations and which is more suitable for mitochondrial haplotype data (Weir and Cockerham, 1984). Tajima’s D and Fu’s Fs neutrality tests were performed with the purpose of tracing the recent demographic events. Significant negative values are interpreted as a signal of purifying selection or alternatively as demographic expansion, while significant positive values indicate recent population bottleneck or population subdivision. For Racka population, Tajima’s D value was positive
(0.44193), while Fu F’s value was negative (−0.97675), but not significant from statistic point of view. Negative values were highlighted for both parameters in haplogroups A, B and C, indicating that these lineages had undergone population expansion event, but for haplogroup C the values were not statistically significant. Moreover, the mismatch distribution was fitted to the sudden expansion model (Rogers, 1995) and the analysis supported population expansion hypothesis in haplogroups A, B and C, since the Raggedness index for these groups was 0.137 (p = 0.55) for haplogroup A, 0.027 (p = 0.654) for haplogroup B and 0.0413 (p = 0.714) for haplogroup C, as expected in this case. Also, the sum of squared deviations (SSD) distribution presented low values (HPG A – 0.0151, HPG B – 0.0021 and HPG C – 0.0029), but none was significant (p = 0.12/0.43/0.76) supporting the sudden expansion model tested. 3.3. mtDNA haplotypes networks The phylogenetic network constructed based on mtDNA sequences for Romanian Racka revealed that the haplotypes were separated in two clusters connected by a median vector (Fig. 1). To assess the phylogenetic relationships between Racka and other sheep breeds we constructed MJ networks highlighting the maternal lineage/geographic distribution of the breeds (Fig. 2a, b). In relationship to previously defined sheep mtDNA haplogroups, all 40 sequences of Romanian Racka can be clearly grouped into A and B lineages, explaining thus the two clusters inferred by the Racka haplotypes network. The haplotypes from cluster I grouped together with haplotypes from A lineage, while the haplotypes from cluster II grouped together with haplotypes from B lineage. The sequences are grouped within the network in clusters correspondent to the five haplogroups A, B, C, D, and E, with the Racka sequences distributed in A and B lineages in proportion of 35 and 65% respectively (Fig. 2a). None of the Racka sheep haplotypes associated with C, D or E lineages were found in the sheep breeds of the Near East. Within the network profile there is one major haplotype in the center for A and B haplogroups, these parts of network displaying a “star like” shape. For the haplogroup A the central haplotype is shared by 16 sequences, among which two isolated in Romanian Racka breed, while in haplogroup B the central haplotypes is common to 22 sequences, including ten from Racka. The central haplotypes of the clades corresponding to A and B haplogroup are connected by six mutational steps. Haplogroups A and B were evidenced to have undergone population expansions. The star shaped phylogeny of lineages A and B are consistent with population expansion clearly confirmed by the neutrality tests results. The clade corresponding to the lineage C is characterized by a reduced number of sequences, while the D and E lineages are each represented by unique haplotypes. Haplogroup C appears to be derived from the haplogroup E, the central haplotypes of C lineage being connected with this one by only three mutational steps. Instead the D haplogroup is connected with the central haplotype of the haplogroup A by nine mutational steps. 4. Discussion An analysis of partial mitochondrial sequence of 40 individuals from Romanian Racka sheep has been presented in this study. Similar haplotypes diversity values with the one of Racka breed have been reported in other Asian and European sheep breeds (Chen et al., 2006; Pardeshi et al., 2007; Mariotti et al., 2013; Othman et al., 2015). Thus, in sheep breeds from Europe values of haplotype diversity ranging from 0.872 to 0.988 were highlighted (Mariotti et al., 2013; Othman et al., 2015). Moreover, comparable haplotype diver-
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Table 1 Diversity indices of Romanian Racka population. Breed
n
H
Hd ± SD
Pi ± SD
K
Variable sites
Singleton sites
Parsimony sites
Racka Haplogroup A Haplogroup B Haplogroup C Haplogroup D Haplogroup E Overall
40 33 57 18 2 3 153
12 12 36 8 1 1 66
0.89740 ± 0.02800 0.77200 ± 0.07500 0.94600 ± 0.02200 0.89500 ± 0.04100 0 0 0.95800 ± 0.00900
0.00437 ± 0.00032 0.00141 ± 0.00029 0.00490 ± 0.00121 0.00241 ± 0.00036 0 0 0.00700 ± 0.00060
4.01500 1.29700 4.49600 2.21600 0 0 6.70488
15 15 66 9 0 0 95
0 12 32 3 0 0 37
15 3 34 6 0 0 58
n = number of individuals; H = number of haplotypes; Hd = haplotype diversity; Pi-nucleotide diversity. K-average number of nucleotide differences. Table 2 Genetic distance and gene flow analysis between Racka breed and different haplogroups. Dxy – above; DA – below; Fst – above; Gammast – below. Dxy /DA
Racka HPG A HPG B HPG C
Fst /GammaSt Racka
HPG A
HPG B
HPG C
– 0.00162 0.00048 0.01055
0.00512 – 0.00358 0.00900
0.00451 0.00673 – 0.01271
0.01394 0.01091 0.01637 –
Racka HPG A HPG B HPG C
Racka
HPG A
HPG B
HPG C
– 0.21585 0.09395 0.32667
0.35898 – 0.31175 0.41179
0.05779 0.52846 – 0.35256
0.54183 0.56259 0.62578 –
Fig. 1. Median Joining network constructed based mtDNA haplotypes in Romanian Racka. Node size is proportional to haplotype frequency. The smallest node is representative of one haplotype. The red dots represent median vectors.
sity was obtained for Egyptian Ossimi breed (Hd – 0.8833) (Othman et al., 2015). In sheep breeds from China and India the values of haplotypes diversity in the analyzed sheep populations were ranging from 0.85 to 0.95 (Chen et al., 2006; Pardeshi et al., 2007). A study on Gymiesi Racka from Hungary revealed a higher number of haplotypes (23) and implicitly higher haplotypes diversity (0.950) in this population by comparison with Romanian Racka, but still both populations have significant haplotype diversity (Kusza et al., 2015). By comparing the genetic diversity indices for Racka with the ones of the analyzed haplogroups we observed that the values of haplotypes diversity are significant and similar. The overall haplotype variability was observed to be high (0.958 ± 0.009). The nucleotide diversity was similar with the one reported by other studies on sheep breeds from Europe and Asia that exhibited a composite genetic structure composed of A and B haplotypes (Arora et al., 2013; Demirci et al., 2013). Also, the analysis of nuclear markers in local sheep from Balkans region (Croatia and Bosnia and Herzegovina) also revealed considerable levels of genetic diversity (Salamon et al., 2014).
The gene flow analysis inferred by Fst and Gammast indices values that measure the short-term genetic differentiation revealed that the Racka breed is most diverged from haplogroups A and C, while between this breed and haplogroup B is a moderate genetic differentiation. Thus, the data confirm the distance data (DA and Dxy ) showing that Racka breed is more closely related to haplogroup B, predominant in the European sheep breeds. Both Tajima’s D and Fu’s neutrality test and the mismatch distribution analyzed under sudden expansion model were not significant and no conclusion can be formulated about the Racka population evolution. The haplotypes network highlighted the distribution of mtDNA sequences of Romanian Racka in two clusters corresponding to A and B lineages. The frequency of haplogroups A and B within Racka breed is not surprising since it was previously revealed that more than one maternal lineage is present in the majority of breeds. Previous reports (Tapio et al., 2006) described/identified individuals carrying type A haplotypes in European sheep breed from Austria, Ukraine, Poland, Russia and South-East Europe. Also, in different
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Fig. 2. Median Joining network of 40 Racka sequences and 113 sequences from GenBank. (A) Breeds distribution into haplogroups. Green – HPG A; Blue- HPG B; Yellow – HPG C; Black – HPG D; Orange – HPG E. (B) Breeds geographic origin. Blue – Europe; Green – Asia; Yellow – Turkey; Orange – Israel. For both networks the haplotypes of Romanian Racka are shown in purple. Node size is proportional to haplotype frequency. The smallest node is representative of one haplotype. The red dots represent median vectors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
sheep breeds from Turkey and Israel, Meadows et al. (2007) demonstrated that three or more lineages were identified within each breed. The network profile showed that sheep breeds from different geographic regions intermixed since the majority of the haplotypes were shared by individuals from different breeds across wide
geographic regions. The haplotypes distribution did not indicate a clear phylogeographic structure. Different breeds from Asia (India, Indonesia and Mongolia) presented exclusively type A haplotypes, while breeds sourced from Europe (eight breeds from Austria, Balkans Region, Russia and Romania) possessed only type B haplotypes.
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Three Romanian breeds (Tsurcana, Tsigai and Black Head Ruda) included in the study clustered in haplogroup B. The sheep breeds from Middle and Near East (Turkey and Israel) showed a higher heterogeneity in terms of lineages composition. No haplotypes pattern correlated with the geographic distribution was observed for the sheep breeds from this part of the world, supporting thus the hypothesis of a Near East domestication center for sheep. The presence of haplotypes A and B in Romanian Racka is in accordance with the finding of these haplotypes in other sheep breeds from Eastern Europe, including area of north and west, from the Black Sea and the Ural Mountains. Genetic studies performed on 12 local Zackel type breeds from East Adriatic region revealed the presence of both A and B haplotypes, with predominant B haplotype, but with a significant lower frequency of A haplotype comparative with the one found in Romanian Racka (Ferencakovic et al., 2013). Similar, haplogroup B was predominant (with a frequency of 95%) for sheep breeds from Mediterranean region (Lancioni et al., 2013). The differences regarding the prevalence of a certain haplotype are explainable if we take into account that domestication started in the Near East and continued towards Europe, where the domestication path run along the continent, along the Danube valley, or along the Mediterranean coast (Ryder, 1984). Sheep breeds from Europe, Caucasus and Central Asia are characterized by the presence of A, B and C haplotypes. Apart the three haplotypes, a new haplotype (D) was noticed in just one Caucasian breed. The frequency of B haplotype decreases towards the east, while that of haplotype A increases. The C haplotype has been noticed only in the breeds from Caucasus and Central Asia, and was not identified in any East-European breed (Tapio et al., 2006). The presence of both A and B haplotypes in populations from both Europe and Asia seems to be the consequence of introgression, as a result of a gene flow between breeds of domestic sheep from both continents (Meadows et al., 2005; Arora et al., 2013). Moreover, the proximity of Balkans with the Middle East, characterized by a heterogenic distribution of haplotypes within breeds, might be an explanation for the composite haplogroups of the breeds from Balkans.
5. Conclusions The Romanian Racka is characterized by an important genetic diversity which demonstrates that up to present the population was correctly managed and has a good potential for conservation. The presence of A and B haplotypes within the population is in accordance with the findings for different breeds from Zackel group and with their hypothesized origins. The distribution of Racka in the haplogroups A and B followed the pattern observed for other breeds with different geographic origins. Thus, the presence of both typically Asian and European haplotypes might be the results of gene flow and introgression events that have occurred in the history of this breed. The analysis of mtDNA sequences was informative to assess the diversity within Romanian Racka, but not useful for establishing the accurate origin of the breed and its detailed relationships with other breeds. In order to have a clear image about Racka biodiversity, further large scale studies are necessary. The research should be extended for Racka sheep populations from neighboring countries by using a larger panel of molecular markers in order to investigate the sheep in the regional context and to develop a gene pool for conservation purposes due the fragile status of the breed.
Conflict of interest The authors declare that there is no conflict of interest.
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