- Email: [email protected]
An historical and biogeographical assessment of European Merino sheep breeds by microsatellite markers
Small Ruminant Research 177 (2019) 76–81
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
An historical and biogeographical assessment of European Merino sheep breeds by microsatellite markers
T
⁎
Vincenzo Landia, Emiliano Lasagnab, , Simone Ceccobellib, Amparo Martineza, Fatima Santos-Silvac, Jose Luis Vega-Plad, Francesco Panellaa, Daniel Allaine, Isabelle Palhieree, Maciej Murawskif, Susana Dunnerg, Luìs Telo Da Gamah, Cecilio Barbai, Juan Vicente Delgadoa, Francesca Maria Sartib a
Departamento de Genética, Universidad de Córdoba, Edificio Méndel C5, Campus Rabanales, 14071, Cordoba, Spain Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università degli Studi di Perugia, Borgo XX giugno 74, 06121, Perugia, Italy INIAV, Quinta da Fonte Boa, 2005-048, Vale de Santarém, Portugal d Laboratorio de Investigación Aplicada, Crıa Caballar de las Fuerzas Armadas, Cordoba, Spain e INRA, UMR1388 Génétique, Physiologie et Systèmes d’Elevage, Castanet-Tolosan, France f Department of Animal Biotechnology, Agricultural University of Krakow, ul. Redzina 1B, 30-248 Krakow, Poland g Departamento de Producción Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain h CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal i Department of Animal Production, Faculty of Veterinary Sciences, University of Córdoba, 14071 Córdoba, Spain b c
A R T I C LE I N FO
A B S T R A C T
Keywords: STR Genetic diversity Population structure Merino breed
Although the Merino breed originated in the Iberian Peninsula, the Merino and Merino-derived sheep breeds have nowadays been widely distributed across the world both as purebreds and as admixed populations. In Europe, the Merino trunk has both high phenotypic and genetic diversity which originated from several genetic types adapted to different economic and farming environments. For these reasons, Merino and Merino-derived breeds must be presently considered cosmopolitan breeds that have an important role worldwide in the production of high-quality wool. This paper presents the results of the genetic characterization by 35 STR markers of 539 animals which are the most important 16 Merino and Merino-derived sheep breeds in Europe. The results highlighted a remarkable level of genetic variability in all breeds. Overall, microsatellite polymorphism analysis revealed the Merino Pozo Blanco as the most homogeneous breed. The subdivision between the studied breeds was shown by the STRUCTURE analysis even though a genetic admixture is particularly evident in the three Italian Merino derived breeds. In this study, the historical background of the Merino breed has been assessed from the genetic point of view, and the Spanish origin of the Merinos branch seems to be confirmed. In general, despite their common ancestral origin, nowadays the investigated Merino and Merino-derived breeds present a genetic identity in accordance with their geographical location probably due to different selection strategies.
1. Introduction
For a long time, selling Merino outside Spain had been banned; subsequently, the first sporadic export of Merino animals was under a strict royal control. The Merino was firstly introduced to the south of Italy during the Aragon king dominion in 1435, which originated the breed known as the Gentile di Puglia (Tortorelli, 1984). Subsequently, many other introductions were reported in the north as well as the south of the Italian Peninsula. In addition, Merino rams were introduced to the centre of Italy (Church State) during the pontificate of Pio VI (end of XVIII) to crossbreed the local Vissana sheep population,
Although the ethnological origin of Merino strain is still unclear according to several authors (Piper and Ruvinsky, 1997; Esteban Muñoz, 2004a; Ciani et al., 2015), Merino would derive from Ovis aries tudertanus which the ancient Spanish prehistoric populations reared around 1000 years BC. In addition, according to several Roman authors (Ciani et al., 2014), the original area of the Merino breed was the Iberian Peninsula (Pedrosa et al., 2007).
⁎ Corresponding author at: Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università degli Studi di Perugia, Borgo XX giugno, 74, 06121 Perugia PG, Italy. E-mail address: [email protected] (E. Lasagna).
https://doi.org/10.1016/j.smallrumres.2019.06.018 Received 31 January 2019; Received in revised form 6 May 2019; Accepted 19 June 2019 Available online 20 June 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
Roja Mallorquina (RMA) the unique fat-tail Spanish breed, were included as non-Merino-derived breeds. Table S1 and table S2 shows additional information of the studied breeds. The blood samples collection were conducted as part of a routine health screen by qualified veterinarians. Since collecting blood samples from the animals was part of the veterinarians’ routine work, no Ethics Committee Approval was required. To ensure a representative sample, the animals were randomly chosen from several flocks (Figure S1). In addition, to minimize the chance of sampling closely related individuals, herdbook of each breed was consulted and Merino-related animals that were unrelated for at least two generations were selected, and the shepherds were asked about the animal’s genealogical relationships belonging to the control breeds.
resulting in the Merino-derived breed, known as the Sopravissana (Bonadonna, 1976). France is certainly the country where the productive performances of the breed were genetically improved. During the second half of 1700, the presence of the Merino breed officially began in France although historical traces in this country had been reported at least 100 years before (Sarti, 1996). Between 1700 and 1800, the Spanish government gave permission to export the breed to several other European countries. In 1765, the king of Spain exported approximately 200 animals to the prince of Saxony (actual Germany), and other exportation of sheep were also registered in the 1788, 1789, and 1811. Improved strains of Merino were finally exported from Germany and France to Poland, Romania, and Russia (Semyonov and Selkin, 1989; Martyniuk and Rzepecki, 1995). Although the origin of Merino in Portugal is still not clear, it is suggested that the sheep came from Spain due to both the geographic proximity of the two countries and the transhumance routes of Spanish Merino herds (Laguna Sanz, 1986); in addition, both countries were integrated in the same kingdom between 1580 and 1640. After the nineteenth century, new breeds, which originated from the Iberian Merino (Merino Precoce Merino Rambouillet, Ile de France), were imported to improve the herds in Portugal, (Matos, 1986). Presently, there are three Merino breeds in Portugal: Merina Branca, Merina Preta, and Merina da Beira Baixa. Regardless of their ancestral origin, these breeds were certainly influenced by Spanish and French Merino. After spreading in all Europe, the Merino acquired high phenotypic and genetic diversity generating several genetic types adapted to different economic and farm environments. Thus, today Merino and Merino-derived breeds must be considered cosmopolitan breeds that have an important role in the production of high-quality wool (Ciani et al., 2015). Several authors studied the genetic characterization of Merino and Merino-derived breeds in their own countries. For example, Diez Tascon et al. (2000) and Arranz et al. (2001) studied the principal Merino breeds (Spanish Merino, Merino Précoce, Merino Fleischschaf, Merino Preto, Merino Branco, and New Zeland Merino) with STR markers proving a considerable genetic differentiation; however, several important Merino breeds (i.e. Italian Merino-derived breeds, Polish Merino, Hungarian Merino, etc.) were not considered in these studies. In addition, further studies were carried out on Italian Merino-derived breeds (Lasagna et al., 2011), on Chinese Merino breeds (Zhong et al., 2016), and on Australian Merino (Al-Atiyat et al., 2016). Moreover, several studies considered an extensive number of Merino breeds from different countries (Tapio et al., 2010), while other studies considered the breeds from a specific territory or nation were taken into consideration (Tolone et al., 2012; Al-Atiyat et al., 2014; Pons et al., 2015; Sassi-Zaidy et al., 2016; Gaouar et al., 2016; Othman et al., 2016; Yilmaz et al., 2016). The aim of this study was to achieve a better knowledge on the molecular characterization and population structure by means of microsatellite markers of European Merino branch considering the historical events that influenced their genetic makeup. The molecular information in this study can serve as a guideline for management and breeding strategies (reducing inbreeding and crossbreeding) in order to obtain better utilization and conservation of Merino and Merino-derived sheep breeds.
2.2. Microsatellite genotyping Genomic DNA was extracted from whole blood by the GenElute Blood Genomic DNA kit (Sigma-Aldrich, St. Louis, MO). A panel of 35 microsatellite loci were chosen according to the recommendations of FAO and International Society for Animal Genetics (ISAG) (Table S3). The markers were subjected to multiplex PCR amplification using a Biometra TGradient 96 with the following conditions: an initial denaturation step of 5 min at 94 °C, 35 cycles of 30 s at 95 °C, 45 s at the annealing T° of each multiplex PCR, 30 s at 72 °C and a final extension of 15 min at 72 °C. The reaction volume of 10 μl contained 25 ng of genomic DNA, 2.5 mM MgCl2, 1 μl of 10X PCR buffer, 0.5 U Hot start Taq (Sigma–Aldrich, St. Louis, MO, USA), 200 μM dNTPs and 0.2 pmol of each primer. PCR products were separated by electrophoresis with an automatic sequencer (ABI PRISM 3130xl, Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Allele sizes were estimated by using the internal size standard GeneScan-400 HD ROX (Applied Biosystems, Foster City, CA). Genotypes were read and interpreted with a computer software GeneMapper version 4.0 (Applied Biosystems, Foster City, CA). Reference samples were used in each run to ensure the consistency of allele assignments. 2.3. Statistical analyses The total number of alleles per locus (A), observed (HO) and expected (HE) heterozygosities, and mean number of alleles (MNA) per breed were estimated using MICROSATELLITES TOOLKIT software (Park, 2001). To calculate the average allelic richness (Ar) for each population, the rarefaction method implemented in HP-RARE version 1.0 software was used, using a sample of 5 individuals, as suggested by Kalinowski (2005). Fisher’s exact test for establishing Hardy-Weinberg equilibrium (dHWE) across loci and breeds, and linkage disequilibrium (LD) among loci were estimated with GENEPOP package version 4.0.10 (Rousset, 2008). Levels of significance were adjusted using the false discovery rate procedure (Benjamini and Hochberg, 1995). FIS for each population was calculated via bootstrapping using 1000 replicates with GENETIX software version 4.05 (Belkhir et al., 2001). The index of pairwise FST of Weir and Cockerham (Weir and Cockerham, 1984) between populations and their associated 95% confidence intervals were estimated using GDA software (Lewis and Zaykin, 1999). Reynolds’ genetic distances (Reynolds et al., 1983) were calculated with POPULATIONS software (Langella, 1999), and a Neighbour-net dendrogram was constructed with the SPLITSTREE4 package (Huson and Bryant, 2006). Population genetic structure across all the studied breeds was investigated using a Bayesian approach implemented in STRUCTURE software version 2.3.4 (Pritchard et al., 2000) to estimate ancestral clusters (K). The assignment of individuals to populations, considered
2. Materials and methods 2.1. Sheep sampling Individual blood samples were collected from 539 animals of both sexes, which represented the most important Merino and Merino-derived sheep breeds in the European countries. In addition, 149 samples from Canaria de Pelo (CAN), the only wool-les European breed, and 77
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
(CAN and RMA). The highest FIS (0.116) was detected in MBX, while the lowest was detected in MEL (0.01). The MBX value is probably due to the very low number of heads (6.859 heads enrolled in the herdbook in 2013, http:// www.ovinosecaprinos.com/mbb.html). A negative FIS (-0.014) was estimated in RAM, which was an unexpected value because this breed is reared in closed flock. The number of locus deviated from Hardy-Weinberg equilibrium per breed was quite low and ranged between 0 and 3 in all the sampled populations; for this reason, all the markers were included in the statistical analysis. In some breeds, other researchers (Pons et al., 2015) identified deviations from Hardy-Weinberg equilibrium in specific loci which could be caused by the presence of null alleles, the inbreeding, or geographic isolation. Moreover, no significant linkage disequilibrium was detected between pairs of loci in each studied breed after False Discovery Rate correction. Overall, microsatellite polymorphism analysis highlighted MPB as the most homogeneous breed (lowest allele richness and gene diversity value). It has to be considered that the origin and evolution of Merino-derived breeds outside Spain is rather different. Indeed during the 15th century, Merino rams were exported from Aragon (Spain) to Southern Italy, and mated to native ewes, giving rise to the Gentile di Puglia breed. In the 18th century, purebred Merino rams were transferred to Germany and France where they originated the Merino Landschaf and Rambouillet breeds, respectively. The Rambouillet breed was also exported to Central Italy where it gave rise first to the Sopravissana breed and, only more recently, to the Merinizzata Italiana breed (Sarti et al., 2006; Lasagna et al., 2011). All of those breeds originated from small migrant flocks having genetic drift effects in their foundation (Pedrosa et al., 2007). Some of these breeds were thereafter bred in purity in other different countries, while others had a genetic introgression from local breeds showing some migration effect (Esteban Muñoz, 2004b). The pairwise FST distance among the breeds is reported in Table 2 that shows significant comparison after 1000 permutations. All the FST values were significantly different from zero. For example, MPP and MBX presented the lowest differentiation (0.014), which could be explained by their geographical proximity and reciprocal uncontrolled crossbreeding; moreover, these two breeds are reared for both dairy and meat production (Tibério and Diniz, 2014). This finding is in agreement with the Gaouar et al. (2016) hypothesis regarding the effective role of gene flow in declining the genetic differentiation among the breeds, particularly among those reared within the same or close geographical location. MPB is the breed that differs the most from the others showing FST values ranging from 0.276 (same as MPZ and FLC) to 0.106 (same as HUG), which indicate that this breed is genetically well isolated. The Neighbor-net dendrogram constructed from the Reynold’s genetic distances (Fig. 1) clearly confirms the genetic differentiation of MPB from the other studied breeds; whereas, RAM, FLC, and MPZ appear to be less genetically differentiated compared to MPB. On the contrary, some breeds clustered together such as MDA and MEL; MBA; MPP and MBX; HUG and ROM: this topology was in agreement with the geographical origin. Neighbor-net networks showed the presence of reticulations which could be due to a recent divergence from a common ancestor or to population admixture after divergence, as also reported by Ciani et al. (2013) in the Italian sheep breeds. To confirm the considerable differentiation observed, the population’s structure was modelled on the Bayesian clustering approach implemented in STRUCTURE software with values of K ranging between 1 and 18. Following Evanno et al. (2005), the modelling revealed that the highest delta-K values were obtained at K = 3, K = 5, K = 7, and K = 9 (Figure S2). Meirmans (2015) hypothesized that besides the large uncertainty in the estimation of K, there are often few biological reasons to assume only a single value of K. Different values may simply reflect different demographic processes and therefore warrant interpretation. In a similar approach analysis on dog breeds, Leroy et al. (2009) hypothesized that the highest values obtained for small K are biased with
an ancestry model with admixture, correlated allele frequencies and defined sampling location for each individual. Ten independent runs with 500,000 MCMC (Markov Chain Monte Carlo) iterations and a burn-in of 200,000 steps were performed for 2 ≤ K ≤ 18 to estimate the most likely number of clusters present in the dataset. The algorithm of Evanno et al. (2005) was adopted in order to evaluate the most probable values of K. Genetic differentiation on a geographical basis was investigated using R software. The R-script available in POPS1 algorithm was used to display spatial interpolations of the Q matrices obtained with STRUCTURE 2.3.4 based on kriging methods (Jay et al., 2012). Geographical coordinates were recorded from Google Earth software 7.3.1.4507 according to municipality data (Table S1). Because spatial interpolation software is normally designed for wildlife animals in which each sampling point (individual) is a coordinate, it does not consider that farm animal individuals have the same or almost the same coordinate. In this scenario Kriging algorithm is not able to generate a spatial correlation due to few variabilities between point. To overcome this problem, coordinates were randomly dispersed using the rnorm command setting a standard deviation of 0.01 starting from the central geographical point of the sampling area of each breed (François et al., 2006). 3. Results and discussion In the 16 studied breeds, the total number of described alleles in the thirty-five studied loci reached 580 (Table S4). Polymorphism was high for all analyzed loci. According to the FAO/ISAG (1998) recommendation for the minimum number of alleles in genetic characterization studies, the OarFCB304 locus showed the highest number of alleles (34), while ETH10 showed the lowest number of alleles (5). In this study, the detected level of genetic variability was higher than the variability detected in other previous studies (Arranz et al., 2001; DiezTascon et al., 2000; Al-Atiyat et al., 2016). This could be due to the use of a different number of loci as well as to the noticeable genetic variability of these breeds which is still present today. Moreover, it is important to consider that some of the studied breeds had very good genetic management practices to avoid inbreeding. All the markers used in this study showed high PIC values per locus, with exception of ETH10 (0.16) and OarFCB193 (0.40). The mean expected and observed heterozygosities were 0.70 ± 0.02 and 0.66 ± 0.01, respectively, with a within-breed significant deficit of heterozygosities over loci and breeds of 0.07. These values reflect a good level of genetic variability and are similar to other studies on Merino (Arranz et al., 2001; Lasagna et al., 2011, and Al-Atiyat et al., 2016), European (Lawson Handley et al., 2007), and Chinese sheep breeds (Zhong et al., 2016). The mean number of alleles detected in the studied breeds was high (Table 1). RAM presented the lowest value (3.00 ± 0.97); whereas, MPP showed the highest value (8.59 ± 2.80). It is important to highlight that RAM presented the lowest value of allelic richness (2.73); whereas, MPP showed the highest value (5.68) once again. It is also important to note that since its importation to France, the RAM breed has been maintained as a small and closed flock (Nguyen et al., 1992) where matings between related animals occurred. The values of HE and HO were high and quite similar in all the studied breeds suggesting these breeds are characterized by a remarkable level of genetic variability. The MPB had lower values (0.49 and 0.48 respectively) than the other studied breeds. MPB is a variety of Spanish breeds, recovered in the last years with a scarce available information on the breed and traditionally reared together with the Iberian pig in extensive production grazing systems named “The dehesa” (Benito et al., 1998). The reduced number of heads can justify the low values of HE and HO in this breed despite the good genetic management to maintain a low inbreeding rate. In the 16 studied breeds, all the genetic variability parameters are comparable to the parameters observed in the two non-Merino breeds 78
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
Table 1 Studied sheep breeds, Acronym (ID), sample size of each breed, mean number of observed alleles (MNA), mean allelic richness per locus corrected for sample size and breed based on a minimum sample size of five diploid individuals (Ar), mean observed (HO) and expected (HE) heterozygosity, inbreeding coefficient (FIS) per breed, and number of locus deviated from Hardy-Weinberg equilibrium per breed (dHWE). Breed
ID
MNA ± DS
Ar
HE ± DS
HO ± DS
FIS [IC 95%]
dHWE
Merino D’Arles Merino Est a Laine Merino Precoce Merino Rambouillet Merino Fleischschaf Hungarian Merinos Gentile di Puglia Sopravissana Merinizzata Italiana Polish Merinos Merino Branco Merino da Beira Baixa Merino Preto Romanian Merinos Merino Pozo Blanco Spanish Merinos Canaria de Pelo Roja Mallorquina
MDA MEL MPZ RAM FLC HUG GEP SOP MEI POL MBA MBX MPP ROM MPB MES CAN RMA
7.94 6.72 5.34 3.00 5.74 7.25 7.68 8.43 8.29 7.86 7.94 8.31 8.59 7.63 3.33 7.69 8.64 8.35
5.59 5.20 4.21 2.73 4.39 5.08 5.36 5.46 5.65 5.51 5.62 5.61 5.68 5.39 2.86 5.13 5.11 5.08
0.76 0.74 0.66 0.53 0.67 0.74 0.72 0.73 0.75 0.75 0.75 0.73 0.74 0.74 0.49 0.71 0.73 0.71
0.71 0.73 0.61 0.54 0.65 0.70 0.68 0.69 0.70 0.70 0.70 0.65 0.69 0.70 0.48 0.66 0.67 0.61
0.068 [-0.006 - 0.010] 0.010 [-0.052 - 0.031] 0.073 [0.004 - 0.086] −0.014 [-0.114 - 0.036] 0.018 [-0.049 - 0.048] 0.061 [-0.004 - 0.089] 0.067 [-0.008 - 0.09] 0.057 [0.013 - 0.076] 0.067 [0.013 - 0.087] 0.063 [0.002 - 0.086] 0.062 [0.009 - 0.079] 0.116 [0.060 - 0.135] 0.068 [0.012 - 0.087] 0.050 [-0.026 - 0.090] 0.021 [-0.049 - 0.051] 0.082 [0.035 - 0.101] 0.088 [0.050 - 0.111] 0.139 [0.102 - 0.163]
1 0 1 0 2 1 2 2 1 2 2 2 0 2 2 3 4 5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.62 1.90 2.17 0.97 1.85 1.95 2.66 2.88 2.62 2.88 2.98 2.87 2.80 2.75 1.09 2.35 2.97 2.83
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.03 0.03 0.04 0.03 0.02 0.02 0.03 0.02 0.02 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.03
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01
MNA, mean number of observed alleles; HO, mean observed heterozygosity; HE, expected heterozygosity; Ar, mean allelic richness per locus corrected for sample size and breed; FIS, inbreeding coefficient per breed and number of locus deviated from Hardy-Weinberg equilibrium per breed (dHWE). *Significantly different from zero (P < 0.05).
Evanno’s method when the number of breeds was high. At K = 3 (Fig. 2), the 16 studied breeds were separated in three different clusters in which the major cluster grouped the breeds originated in France, Portugal and Poland (RAM, MDA, MEL, MBX, MBA, MPP, and POL). At K = 5, the three French breeds of this cluster appeared separated from the Portuguese and Poland breeds and splitted in two subclusters (RAM, MDA, and MEL). At K = 7, these three French breeds were now grouped in one cluster. Similarly, the three Portuguese Merino were clustered together. The POL clustered together with ROM and HUG breeds even if all the POL samples showed a remarkable genetic admixture. The history of Merino sheep in Eastern Europe is in fact quite similar: the dispersal of the Merino trunk in this geographic area started in the 18th century. In Hungary, Merino sheep breeding began in 1774 when Empress Maria Theresa bought 300 Merino individuals from Spain (Ciani et al., 2015). In Romania, it is a supposition that ROM breed was produced in 19th century by some cross with Merino during the transhumance in Crimea and North Caucasus in order to increase the wool quality, and more recent, to improve the fattening and carcass quality, some specialized breeds (such as Merino Fleischschaf) for meat production were used (Ilişiu et al., 2013). Besides, the first Spanish Merino
Fig. 1. Neighbor-net dendrogram constructed from the Reynold’s genetic distances among 18 sheep breeds.
Table 2 Pairwise FST distance among 16 Merino and Merino-derived sheep breeds*.
MEL MPZ RAM FLC HUG GEP MEI SOP POL MBA MBX MPP ROM MPB MES CAN RMA
MDA
MEL
MPZ
RAM
FLC
HUG
GEP
MEI
SOP
POL
MBA
MBX
MPP
ROM
MPB
MES
CAN
0.025 0.063 0.182 0.058 0.081 0.026 0.030 0.040 0.032 0.045 0.042 0.044 0.086 0.166 0.035 0.034 0.038
– 0.089 0.192 0.067 0.090 0.033 0.041 0.049 0.046 0.050 0.048 0.048 0.089 0.170 0.045 0.039 0.033
– 0.160 0.094 0.066 0.087 0.072 0.113 0.121 0.107 0.109 0.110 0.083 0.276 0.096 0.105 0.080
– 0.158 0.238 0.120 0.133 0.145 0.135 0.124 0.138 0.140 0.242 0.239 0.140 0.093 0.095
– 0.059 0.068 0.056 0.078 0.101 0.093 0.099 0.092 0.065 0.276 0.074 0.088 0.067
– 0.038 0.043 0.045 0.037 0.042 0.047 0.048 0.025 0.106 0.043 0.021 0.034
– 0.020 0.038 0.044 0.040 0.036 0.040 0.042 0.239 0.050 0.049 0.040
– 0.031 0.061 0.054 0.052 0.049 0.045 0.226 0.033 0.042 0.032
– 0.074 0.057 0.059 0.053 0.047 0.239 0.061 0.058 0.058
– 0.070 0.067 0.061 0.039 0.238 0.086 0.077 0.074
– 0.021 0.015 0.053 0.244 0.072 0.055 0.065
– 0.014 0.053 0.248 0.075 0.059 0.057
– 0.053 0.243 0.068 0.050 0.057
– 0.118 0.044 0.018 0.026
– 0.247 0.264 0.254
– 0.056 0.041
– 0.064
* all values are significantly different from zero (P < 0.05). 79
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
Fig. 2. Population structure of 16 Merino and Merino-derived sheep breeds obtained from 10 runs for each number of assumed populations (K) value ranging from 2 to 18 using the model-based STRUCTURE software.
sheep were introduced to Poland from Spain in 1786. After 1860, Rambouillet and Merino Precoce were brought to Poland, and work towards a dual-purpose type sheep was begun. After 1945, Merino Fleischschaf and Merino Landschaf were introduced into some of the Merino flocks which survived the war (Porter et al., 2016). At K = 7, a genetic admixture is detectable also in the three Italian Merino derived breeds (SOP, GEP, and MEI). At K = 9, a similar situation was observed with exception of MPZ, FLC, and POL that now clustered separately. When K = 18, most of the studied breeds clustered separately, showing some grouping on a geographical basis (France, Portugal, Romania, and Hungary). Genetic admixture is particularly evident in the three Italian Merino derived breeds (SOP, GEP, and MEI). As a matter of fact, the two ancient breeds (GEP, SOP) were reared in neighboring territories and commercial exchanges of mating heads between them were presumably frequent. Moreover, it has to be taken into consideration that both of these two breeds were the genetic base to constitute the MEI breed (Sarti et al., 2006). It is important to highlight that MPB breed was clearly separated from all the other breeds (from K = 3 to K = 18). From K7 level, the two non-Merino breeds (CAN and RMA) clustered isolated from the 16 studied breeds as expected. The population substructures highlighted in the STRUCTURE analysis at the most probable K value showed that the investigated Merino and Merino-derived breeds, presented a genetic identity in accordance with their geographical location despite their common ancestral origin. Probably, these breeds historically underwent different selection strategies in the different countries. The ancestry coefficient map displays the correlation between the Q matrix obtained from STRUCTURE software and the geographical coordinates (Fig. 3, Figure S1). This method permits to search for hidden relationship between genetics structure and territory conformation. At first glance, we can see a clear gradient separating northern Europe from Mediterranean regions. This partition clustered Portuguese, French, and Polish Merinos. Similar findings were already reported by Peter et al. (2007) in a study on 57 European and Middle-Eastern sheep breeds. This situation could be in fact explained by historical information that reported the importation of French animals both to Poland and to Portugal in order to improve local populations. Diez‐Tascón et al. (2000) reported that, since 1930, French Mutton Merino was introduced in order to direct the production towards early maturity in Portuguese Branco Merino. The other clusters put together MES, MPZ, and FLC with Italian Merino-derived SOP and MEI. MPZ, and FLC have been reared in Spain since 1913, when all importation from France were interrupted and were widely admixed with MES, and this could be the reason for the separation from the French breeds. Italian breeds have been linked to MES since their origin, and a strong genetic relationship was confirmed in a previous study (Lasagna et al., 2011). GEP shows a small gradient (blue in the map) probably due to genetic introgression with MPZ and other Merino-derived breeds. HUG and ROM are jointly clustered in the green gradient with MPB.
Fig. 3. Geographic map representing Q matrix from Structure software at K = 3.
This information should be considered with caution because this group of animals, which namely belong to Merino breeds, showed a strong divergence that can lead to STRUCTURE software overestimation of genetics partition. 4. Conclusions In this paper, the above-mentioned historical information has been investigated from the genetic point of view. According to our results, the Spanish origin of the Merinos branch seems to be confirmed, although most of the French Merinos showed a high genetic identity compared with the Spanish ancestors. Probably because of the efficient selection, the French Merino breeds showed recent introgression in the Iberian Merinos, as in some Eastern countries such as Poland. Although some of the studied breeds were affected by the admixture phenomenon there is still genetic identity among breeds in accordance with their geographical location, as result of different selection strategies in the different countries. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.smallrumres.2019.06. 018. 80
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
References
Hewitt, G.M., 2007. Genetic structure of European sheep breeds. Heredity 99, 620–631. https://doi.org/10.1038/sj.hdy.6801039. Leroy, G., Verrier, E., Meriaux, J.C., Rognon, X., 2009. Genetic diversity of dog breeds: between-breed diversity, breed assignation and conservation approaches. Anim. Genet. 40, 333–343. https://doi.org/10.1111/j.1365-2052.2008.01843.x. Lewis, P.O., Zaykin, D., 1999. Genetic Data Analysis. Computer Program for the Analysis of Allelic Data. Version 1.0. Lewis Labs. Univ of Connecticut, Storrs, CT. Martyniuk, E., Rzepecki, R., 1995. Sheep husbandry in Poland-an outline. Cahiers Options Mediterraneennes 11, 121–131. Matos, C.A.P., 1986. Avaliacao das capacidades produtivas e reprodutivas das racas ovinas Merino Branco e Campanica, Relatorio de estagio. Universidad de Tras-osMontes e Alto Douro, Vila Real. Meirmans, P.G., 2015. Seven common mistakes in population genetics and how to avoid them. Mol. Ecol. 24, 3223–3231. https://doi.org/10.1111/mec.13243. Nguyen, T.C., Morera, L., Llanes, D., Leger, P., 1992. Sheep blood polymorphism and genetic divergence between French Rambouillet and Spanish Merino: role of genetic drift. Anim. Genet. 23, 325–332. https://doi.org/10.1111/j.1365-2052.1992. tb00154.x. Othman, O.E.M., Payet-Duprat, N., Harkat, S., Laoun, A., Maftah, A., Lafri, M., Da Silva, A., 2016. Sheep diversity of five Egyptian breeds: genetic proximity revealed between desert breeds: local sheep breeds diversity in Egypt. Small Rumin. Res. 144, 346–352. https://doi.org/10.1016/j.smallrumres.2016.10.020. Park, S.D.E., 2001. Trypanotolerance in West African Cattle and the Population Genetic Effects of Selection. PhD Dissertation. University of Dublin. Pedrosa, S., Arranz, J.J., Brito, N., Molina, A., San Primitivo, F., Bayón, Y., 2007. Mitochondrial diversity and the origin of Iberian sheep. Genet. Sel. Evol. 39, 91–103. https://doi.org/10.1186/1297-9686-39-1-91. Peter, C., Bruford, M., Perez, T., Dalamitra, S., Hewitt, G., Erhardt, G., Econogene Consortium, 2007. Genetic diversity and subdivision of 57 European and Middle‐Eastern sheep breeds. Anim. Genet. 38, 37–44. https://doi.org/10.1111/j. 1365-2052.2007.01561.x. Piper, L., Ruvinsky, A., 1997. The Genetics of Sheep. CAB INTERNATIONAL (Eds.), New York, USA. Pons, A.L., Landi, V., Martinez, A., Delgado, J.V., 2015. The biodiversity and genetic structure of Balearic sheep breeds. J. Anim. Breed. Genet. 132, 268–276. https://doi. org/10.1111/jbg.12129. Porter, V., Alderson, L., Hall, S.J., Sponenberg, D.P., 2016. Mason’s World Encyclopedia of Livestock Breeds and Breeding Vol. 2. Cabi (Eds.), pp. 885. Pritchard, J.K., Stephens, M., Donnelly, P., 2000. Inference of population structure using multilocus genotype data. Genetics. 155, 945–959. Reynolds, J., Weir, B.S., Cockerham, C.C., 1983. Estimation of the coancestry coefficient: basis for a short-term genetic distance. Genetics. 105, 767–779. Rousset, F., 2008. Genepop’007: a complete reimplementation of the Genepop software for Windows and Linux. Mol. Ecol. Resour. 8, 103–106. https://doi.org/10.1111/j. 1471-8286.2007.01931.x. Sarti, D.M., 1996. Le razze derivate Merinos ad attitudine carne nel centro sud europeo. L’Allevatore di Ovini e Caprini, pp. 1–2 7/8. Sarti, F.M., Lasagna, E., Panella, F., Lebboroni, G., Renieri, C., 2006. Wool quality in Gentile di Puglia sheep breed as measure of genetic integrity. Ital. J. Anim. Sci. 5, 371–376. https://doi.org/10.4081/ijas.2006.371. Sassi-Zaidy, Y.B., Maretto, F., Charfi-Cheikhrouha, F., Mohamed-Brahmi, A., Cassandro, M., 2016. Contribution of microsatellites markers in the clarification of the origin, genetic risk factors, and implications for conservation of Tunisian native sheep breeds. Genet. Mol. Res. 15, gmr.15017059. https://doi.org/10.4238/gmr. 15017059. Semyonov, S.I., Selkin, I.I., 1989. Sheep. In: Dimitriev, N.G., Ernst, L.K. (Eds.), Animal Genetic Resources of the USSR. FAO-UNEP, Rome, pp. 154–271. Tapio, M., Ozerov, M., Tapio, I., Toro, M.A., Marzanov, N., Ćinkulov, M., Goncharenko, G., Kiselyova, T., Murawski, M., Kantanen, J., 2010. Microsatellite-based genetic diversity and population structure of domestic sheep in northern Eurasia. BMC Genet. 11, 76. https://doi.org/10.1186/1471-2156-11-76. Tibério, M.L., Diniz, F., 2014. Sheep and goat production in Portugal: a dynamic view. Mod. Econ. 5, 703. https://doi.org/10.4236/me.2014.56066. Tolone, M., Mastrangelo, S., Rosa, A.J.M., Portolano, B., 2012. Genetic diversity and population structure of Sicilian sheep breeds using microsatellite markers. Small Rumin. Res. 102, 18–25. https://doi.org/10.1016/j.smallrumres.2011.09.010. Tortorelli, N., 1984. Allevamento della pecora. Edagricole (Eds.). Weir, B.S., Cockerham, C.C., 1984. Estimating F‐statistics for the analysis of population structure. Evolution 38, 1358–1370. https://doi.org/10.1111/j.1558-5646.1984. tb05657.x. Yilmaz, O., Cemal, İ., Karaca, O., Ata, N., 2016. Molecular genetic characterization of Kivircik sheep breed raised in Western Anatolia. Development 28, 3. Zhong, T., Ma, Y.H., Gao, H.J., He, J.N., Liu, N., Zhao, Y.J., Zhang, J.H., Huang, Y.F., 2016. Conservation genetics in Chinese sheep: diversity of fourteen indigenous sheep (Ovis aries) using microsatellite markers. Ecol. Evol. 6, 810–817. https://doi.org/10. 1002/ece3.1891.
Al-Atiyat, R.M., Salameh, N.M., Tabbaa, M.J., 2014. Analysis of genetic diversity and differentiation of sheep populations in Jordan. Electron. J. Biotechnol. 17, 168–173. https://doi.org/10.1016/j.ejbt.2014.04.002. Al-Atiyat, R., Flood, W., Franklin, I., Kinghorn, B., Ruvinsky, A., 2016. Microsatellitebased genetic variation and differentiation of selected Australian Merino sheep flocks. Small Rumin. Res. 136, 137–144. https://doi.org/10.1016/j.smallrumres. 2016.01.018. Arranz, J.J., Bayón, Y., San Primitivo, F., 2001. Differentiation among Spanish sheep breeds using microsatellites. Genet. Sel. Evol. 33, 529–542. https://doi.org/10.1186/ 1297-9686-33-5-529. Belkhir, K., Borsa, P., Chikhi, L., Raufaste, N., Bonhomme, F., 2001. GENETIX 4.02, Logiciel Sous Windows TM Pour La Génétique Des Populations. Laboratoire Génome, Populations, Interactions, CNRS UPR 9060. Université de Montpellier II, Montpellier. France. Benito, J., Ferrera, J.L., Vazquez, C., Menaya, C., Garcia, J.M., 1998. El cerdo ibérico: poblador de la dehesa. In: Benítez, W. (Ed.), Los cerdos locales en los sistemas tradicionales de producción. FAO, Roma, pp. 71–96. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300. Bonadonna, T., 1976. Etnologia Zootecnica. UTET (Eds.). Ciani, E., Ciampolini, R., D’Andrea, M., Castellana, E., Cecchi, F., Incoronato, C., D’Angelo, F., Albenzio, M., Pilla, F., Matassino, D., Cianci, D., 2013. Analysis of genetic variability within and among Italian sheep breeds reveals population stratification and suggests the presence of a phylogeographic gradient. Small Rumin. Res. 112, 21–27. https://doi.org/10.1016/j.smallrumres.2012.12.013. Ciani, E., Crepaldi, P., Nicoloso, L., Lasagna, E., Sarti, F.M., Moioli, B., Napolitano, F., Carta, A., Usai, G., D’Andrea, M., Marletta, D., Ciampolini, R., Riggio, V., Occidente, M., Matassino, D., Kompan, D., Modesto, P., Macciotta, N., Ajmone-Marsan, P., Pilla, F., 2014. Genome-wide analysis of Italian sheep diversity reveals a strong geographic pattern and cryptic relationships between breeds. Anim. Genet. 45, 256–266. https:// doi.org/10.1111/age.12106. Ciani, E., Lasagna, E., D’andrea, M., Alloggio, I., Marroni, F., Ceccobelli, S., Delgado Bermejo, J.V., Sarti, F.M., Kijas, J., Lenstra, J.A., Pilla, F., the International Sheep Genomics Consortium, 2015. Merino and Merino-derived sheep breeds: a genomewide intercontinental study. Genet. Sel. Evol. 47, 64. https://doi.org/10.1186/ s12711-015-0139-z. Diez‐Tascón, C., Littlejohn, R.P., Almeida, P.A.R., Crawford, A.M., 2000. Genetic variation within the Merino sheep breed: analysis of closely related populations using microsatellites. Anim. Genet. 31, 243–251. https://doi.org/10.1046/j.1365-2052. 2000.00636.x. Esteban Muñoz, C., 2004a. The Sheeps in America. Role of Merino Sheep Breed in the Formation of the American Sheep Stock. Esteban Muñoz, C., 2004b. Razas Ganadera Espanola Ovinas. Ministerio de Agricoltura, Madrid. Evanno, G., Regnaut, S., Goudet, J., 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–2620. https://doi.org/10.1111/j.1365-294X.2005.02553.x. François, O., Ancelet, S., Guillot, G., 2006. Bayesian clustering using hidden markov random fields in spatial population genetics. Genetics 174, 805–816. https://doi.org/ 10.1534/genetics.106.059923. Gaouar, S.B.S., Kdidi, S., Ouragh, L., 2016. Estimating population structure and genetic diversity of five Moroccan sheep breeds by microsatellite markers. Small Rumin. Res. 144, 23–27. https://doi.org/10.1016/j.smallrumres.2016.07.021. Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23, 254–267. https://doi.org/10.1093/molbev/msj030. Ilişiu, E., Dărăban, S., Radu, R., Pădeanu, I., Ilişiu, V.C., Pascal, C., Rahmann, G., 2013. The Romanian Tsigai sheep breed, their potential and the challenges for research. Appl. Agric. For. Res. 2, 161–170. https://doi.org/10.3220/LBF_2013_161-170. Jay, F., Manel, S., Alvarez, N., Durand, E.Y., Thuiller, W., Holderegger, R., Taberlet, P., François, O., 2012. Forecasting changes in population genetic structure of alpine plants in response to global warming. Mol. Ecol. 21, 2354–2368. https://doi.org/10. 1111/j.1365-294X.2012.05541.x. Kalinowski, S.T., 2005. HP–RARE 1.0: a computer program for performing rarefaction on measures of allelic richness. Mol. Ecol. Notes 5, 187–189. https://doi.org/10.1111/j. 1471-8286.2004.00845.x. Laguna Sanz, E., 1986. Historia del Merino, Madrid (Eds.), Ministerio de Agricultura Pesca y Alimentacion y direccion general de la produccion agraria. Madrid. . Langella, O., 1999. POPULATIONS 1.2.30: Population genetic software. Gif-sur-Yvette, France: Laboratoire Evolution, Génomes et Spéciation. Lasagna, E., Bianchi, M., Ceccobelli, S., Landi, V., Martínez, A.M., Vega Pla, J.L., Panella, F., Delgado Bermejo, J.V., Sarti, F.M., 2011. Genetic relationships and population structure in three Italian Merino-derived sheep breeds. Small Rumin. Res. 96, 111–119. https://doi.org/10.1016/j.smallrumres.2010.11.014. Lawson Handley, L.J., Byrne, K., Santucci, F., Townsend, S., Taylor, M., Bruford, M.W.,
81
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
An historical and biogeographical assessment of European Merino sheep breeds by microsatellite markers
T
⁎
Vincenzo Landia, Emiliano Lasagnab, , Simone Ceccobellib, Amparo Martineza, Fatima Santos-Silvac, Jose Luis Vega-Plad, Francesco Panellaa, Daniel Allaine, Isabelle Palhieree, Maciej Murawskif, Susana Dunnerg, Luìs Telo Da Gamah, Cecilio Barbai, Juan Vicente Delgadoa, Francesca Maria Sartib a
Departamento de Genética, Universidad de Córdoba, Edificio Méndel C5, Campus Rabanales, 14071, Cordoba, Spain Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università degli Studi di Perugia, Borgo XX giugno 74, 06121, Perugia, Italy INIAV, Quinta da Fonte Boa, 2005-048, Vale de Santarém, Portugal d Laboratorio de Investigación Aplicada, Crıa Caballar de las Fuerzas Armadas, Cordoba, Spain e INRA, UMR1388 Génétique, Physiologie et Systèmes d’Elevage, Castanet-Tolosan, France f Department of Animal Biotechnology, Agricultural University of Krakow, ul. Redzina 1B, 30-248 Krakow, Poland g Departamento de Producción Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain h CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal i Department of Animal Production, Faculty of Veterinary Sciences, University of Córdoba, 14071 Córdoba, Spain b c
A R T I C LE I N FO
A B S T R A C T
Keywords: STR Genetic diversity Population structure Merino breed
Although the Merino breed originated in the Iberian Peninsula, the Merino and Merino-derived sheep breeds have nowadays been widely distributed across the world both as purebreds and as admixed populations. In Europe, the Merino trunk has both high phenotypic and genetic diversity which originated from several genetic types adapted to different economic and farming environments. For these reasons, Merino and Merino-derived breeds must be presently considered cosmopolitan breeds that have an important role worldwide in the production of high-quality wool. This paper presents the results of the genetic characterization by 35 STR markers of 539 animals which are the most important 16 Merino and Merino-derived sheep breeds in Europe. The results highlighted a remarkable level of genetic variability in all breeds. Overall, microsatellite polymorphism analysis revealed the Merino Pozo Blanco as the most homogeneous breed. The subdivision between the studied breeds was shown by the STRUCTURE analysis even though a genetic admixture is particularly evident in the three Italian Merino derived breeds. In this study, the historical background of the Merino breed has been assessed from the genetic point of view, and the Spanish origin of the Merinos branch seems to be confirmed. In general, despite their common ancestral origin, nowadays the investigated Merino and Merino-derived breeds present a genetic identity in accordance with their geographical location probably due to different selection strategies.
1. Introduction
For a long time, selling Merino outside Spain had been banned; subsequently, the first sporadic export of Merino animals was under a strict royal control. The Merino was firstly introduced to the south of Italy during the Aragon king dominion in 1435, which originated the breed known as the Gentile di Puglia (Tortorelli, 1984). Subsequently, many other introductions were reported in the north as well as the south of the Italian Peninsula. In addition, Merino rams were introduced to the centre of Italy (Church State) during the pontificate of Pio VI (end of XVIII) to crossbreed the local Vissana sheep population,
Although the ethnological origin of Merino strain is still unclear according to several authors (Piper and Ruvinsky, 1997; Esteban Muñoz, 2004a; Ciani et al., 2015), Merino would derive from Ovis aries tudertanus which the ancient Spanish prehistoric populations reared around 1000 years BC. In addition, according to several Roman authors (Ciani et al., 2014), the original area of the Merino breed was the Iberian Peninsula (Pedrosa et al., 2007).
⁎ Corresponding author at: Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università degli Studi di Perugia, Borgo XX giugno, 74, 06121 Perugia PG, Italy. E-mail address: [email protected] (E. Lasagna).
https://doi.org/10.1016/j.smallrumres.2019.06.018 Received 31 January 2019; Received in revised form 6 May 2019; Accepted 19 June 2019 Available online 20 June 2019 0921-4488/ © 2019 Elsevier B.V. All rights reserved.
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
Roja Mallorquina (RMA) the unique fat-tail Spanish breed, were included as non-Merino-derived breeds. Table S1 and table S2 shows additional information of the studied breeds. The blood samples collection were conducted as part of a routine health screen by qualified veterinarians. Since collecting blood samples from the animals was part of the veterinarians’ routine work, no Ethics Committee Approval was required. To ensure a representative sample, the animals were randomly chosen from several flocks (Figure S1). In addition, to minimize the chance of sampling closely related individuals, herdbook of each breed was consulted and Merino-related animals that were unrelated for at least two generations were selected, and the shepherds were asked about the animal’s genealogical relationships belonging to the control breeds.
resulting in the Merino-derived breed, known as the Sopravissana (Bonadonna, 1976). France is certainly the country where the productive performances of the breed were genetically improved. During the second half of 1700, the presence of the Merino breed officially began in France although historical traces in this country had been reported at least 100 years before (Sarti, 1996). Between 1700 and 1800, the Spanish government gave permission to export the breed to several other European countries. In 1765, the king of Spain exported approximately 200 animals to the prince of Saxony (actual Germany), and other exportation of sheep were also registered in the 1788, 1789, and 1811. Improved strains of Merino were finally exported from Germany and France to Poland, Romania, and Russia (Semyonov and Selkin, 1989; Martyniuk and Rzepecki, 1995). Although the origin of Merino in Portugal is still not clear, it is suggested that the sheep came from Spain due to both the geographic proximity of the two countries and the transhumance routes of Spanish Merino herds (Laguna Sanz, 1986); in addition, both countries were integrated in the same kingdom between 1580 and 1640. After the nineteenth century, new breeds, which originated from the Iberian Merino (Merino Precoce Merino Rambouillet, Ile de France), were imported to improve the herds in Portugal, (Matos, 1986). Presently, there are three Merino breeds in Portugal: Merina Branca, Merina Preta, and Merina da Beira Baixa. Regardless of their ancestral origin, these breeds were certainly influenced by Spanish and French Merino. After spreading in all Europe, the Merino acquired high phenotypic and genetic diversity generating several genetic types adapted to different economic and farm environments. Thus, today Merino and Merino-derived breeds must be considered cosmopolitan breeds that have an important role in the production of high-quality wool (Ciani et al., 2015). Several authors studied the genetic characterization of Merino and Merino-derived breeds in their own countries. For example, Diez Tascon et al. (2000) and Arranz et al. (2001) studied the principal Merino breeds (Spanish Merino, Merino Précoce, Merino Fleischschaf, Merino Preto, Merino Branco, and New Zeland Merino) with STR markers proving a considerable genetic differentiation; however, several important Merino breeds (i.e. Italian Merino-derived breeds, Polish Merino, Hungarian Merino, etc.) were not considered in these studies. In addition, further studies were carried out on Italian Merino-derived breeds (Lasagna et al., 2011), on Chinese Merino breeds (Zhong et al., 2016), and on Australian Merino (Al-Atiyat et al., 2016). Moreover, several studies considered an extensive number of Merino breeds from different countries (Tapio et al., 2010), while other studies considered the breeds from a specific territory or nation were taken into consideration (Tolone et al., 2012; Al-Atiyat et al., 2014; Pons et al., 2015; Sassi-Zaidy et al., 2016; Gaouar et al., 2016; Othman et al., 2016; Yilmaz et al., 2016). The aim of this study was to achieve a better knowledge on the molecular characterization and population structure by means of microsatellite markers of European Merino branch considering the historical events that influenced their genetic makeup. The molecular information in this study can serve as a guideline for management and breeding strategies (reducing inbreeding and crossbreeding) in order to obtain better utilization and conservation of Merino and Merino-derived sheep breeds.
2.2. Microsatellite genotyping Genomic DNA was extracted from whole blood by the GenElute Blood Genomic DNA kit (Sigma-Aldrich, St. Louis, MO). A panel of 35 microsatellite loci were chosen according to the recommendations of FAO and International Society for Animal Genetics (ISAG) (Table S3). The markers were subjected to multiplex PCR amplification using a Biometra TGradient 96 with the following conditions: an initial denaturation step of 5 min at 94 °C, 35 cycles of 30 s at 95 °C, 45 s at the annealing T° of each multiplex PCR, 30 s at 72 °C and a final extension of 15 min at 72 °C. The reaction volume of 10 μl contained 25 ng of genomic DNA, 2.5 mM MgCl2, 1 μl of 10X PCR buffer, 0.5 U Hot start Taq (Sigma–Aldrich, St. Louis, MO, USA), 200 μM dNTPs and 0.2 pmol of each primer. PCR products were separated by electrophoresis with an automatic sequencer (ABI PRISM 3130xl, Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. Allele sizes were estimated by using the internal size standard GeneScan-400 HD ROX (Applied Biosystems, Foster City, CA). Genotypes were read and interpreted with a computer software GeneMapper version 4.0 (Applied Biosystems, Foster City, CA). Reference samples were used in each run to ensure the consistency of allele assignments. 2.3. Statistical analyses The total number of alleles per locus (A), observed (HO) and expected (HE) heterozygosities, and mean number of alleles (MNA) per breed were estimated using MICROSATELLITES TOOLKIT software (Park, 2001). To calculate the average allelic richness (Ar) for each population, the rarefaction method implemented in HP-RARE version 1.0 software was used, using a sample of 5 individuals, as suggested by Kalinowski (2005). Fisher’s exact test for establishing Hardy-Weinberg equilibrium (dHWE) across loci and breeds, and linkage disequilibrium (LD) among loci were estimated with GENEPOP package version 4.0.10 (Rousset, 2008). Levels of significance were adjusted using the false discovery rate procedure (Benjamini and Hochberg, 1995). FIS for each population was calculated via bootstrapping using 1000 replicates with GENETIX software version 4.05 (Belkhir et al., 2001). The index of pairwise FST of Weir and Cockerham (Weir and Cockerham, 1984) between populations and their associated 95% confidence intervals were estimated using GDA software (Lewis and Zaykin, 1999). Reynolds’ genetic distances (Reynolds et al., 1983) were calculated with POPULATIONS software (Langella, 1999), and a Neighbour-net dendrogram was constructed with the SPLITSTREE4 package (Huson and Bryant, 2006). Population genetic structure across all the studied breeds was investigated using a Bayesian approach implemented in STRUCTURE software version 2.3.4 (Pritchard et al., 2000) to estimate ancestral clusters (K). The assignment of individuals to populations, considered
2. Materials and methods 2.1. Sheep sampling Individual blood samples were collected from 539 animals of both sexes, which represented the most important Merino and Merino-derived sheep breeds in the European countries. In addition, 149 samples from Canaria de Pelo (CAN), the only wool-les European breed, and 77
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
(CAN and RMA). The highest FIS (0.116) was detected in MBX, while the lowest was detected in MEL (0.01). The MBX value is probably due to the very low number of heads (6.859 heads enrolled in the herdbook in 2013, http:// www.ovinosecaprinos.com/mbb.html). A negative FIS (-0.014) was estimated in RAM, which was an unexpected value because this breed is reared in closed flock. The number of locus deviated from Hardy-Weinberg equilibrium per breed was quite low and ranged between 0 and 3 in all the sampled populations; for this reason, all the markers were included in the statistical analysis. In some breeds, other researchers (Pons et al., 2015) identified deviations from Hardy-Weinberg equilibrium in specific loci which could be caused by the presence of null alleles, the inbreeding, or geographic isolation. Moreover, no significant linkage disequilibrium was detected between pairs of loci in each studied breed after False Discovery Rate correction. Overall, microsatellite polymorphism analysis highlighted MPB as the most homogeneous breed (lowest allele richness and gene diversity value). It has to be considered that the origin and evolution of Merino-derived breeds outside Spain is rather different. Indeed during the 15th century, Merino rams were exported from Aragon (Spain) to Southern Italy, and mated to native ewes, giving rise to the Gentile di Puglia breed. In the 18th century, purebred Merino rams were transferred to Germany and France where they originated the Merino Landschaf and Rambouillet breeds, respectively. The Rambouillet breed was also exported to Central Italy where it gave rise first to the Sopravissana breed and, only more recently, to the Merinizzata Italiana breed (Sarti et al., 2006; Lasagna et al., 2011). All of those breeds originated from small migrant flocks having genetic drift effects in their foundation (Pedrosa et al., 2007). Some of these breeds were thereafter bred in purity in other different countries, while others had a genetic introgression from local breeds showing some migration effect (Esteban Muñoz, 2004b). The pairwise FST distance among the breeds is reported in Table 2 that shows significant comparison after 1000 permutations. All the FST values were significantly different from zero. For example, MPP and MBX presented the lowest differentiation (0.014), which could be explained by their geographical proximity and reciprocal uncontrolled crossbreeding; moreover, these two breeds are reared for both dairy and meat production (Tibério and Diniz, 2014). This finding is in agreement with the Gaouar et al. (2016) hypothesis regarding the effective role of gene flow in declining the genetic differentiation among the breeds, particularly among those reared within the same or close geographical location. MPB is the breed that differs the most from the others showing FST values ranging from 0.276 (same as MPZ and FLC) to 0.106 (same as HUG), which indicate that this breed is genetically well isolated. The Neighbor-net dendrogram constructed from the Reynold’s genetic distances (Fig. 1) clearly confirms the genetic differentiation of MPB from the other studied breeds; whereas, RAM, FLC, and MPZ appear to be less genetically differentiated compared to MPB. On the contrary, some breeds clustered together such as MDA and MEL; MBA; MPP and MBX; HUG and ROM: this topology was in agreement with the geographical origin. Neighbor-net networks showed the presence of reticulations which could be due to a recent divergence from a common ancestor or to population admixture after divergence, as also reported by Ciani et al. (2013) in the Italian sheep breeds. To confirm the considerable differentiation observed, the population’s structure was modelled on the Bayesian clustering approach implemented in STRUCTURE software with values of K ranging between 1 and 18. Following Evanno et al. (2005), the modelling revealed that the highest delta-K values were obtained at K = 3, K = 5, K = 7, and K = 9 (Figure S2). Meirmans (2015) hypothesized that besides the large uncertainty in the estimation of K, there are often few biological reasons to assume only a single value of K. Different values may simply reflect different demographic processes and therefore warrant interpretation. In a similar approach analysis on dog breeds, Leroy et al. (2009) hypothesized that the highest values obtained for small K are biased with
an ancestry model with admixture, correlated allele frequencies and defined sampling location for each individual. Ten independent runs with 500,000 MCMC (Markov Chain Monte Carlo) iterations and a burn-in of 200,000 steps were performed for 2 ≤ K ≤ 18 to estimate the most likely number of clusters present in the dataset. The algorithm of Evanno et al. (2005) was adopted in order to evaluate the most probable values of K. Genetic differentiation on a geographical basis was investigated using R software. The R-script available in POPS1 algorithm was used to display spatial interpolations of the Q matrices obtained with STRUCTURE 2.3.4 based on kriging methods (Jay et al., 2012). Geographical coordinates were recorded from Google Earth software 7.3.1.4507 according to municipality data (Table S1). Because spatial interpolation software is normally designed for wildlife animals in which each sampling point (individual) is a coordinate, it does not consider that farm animal individuals have the same or almost the same coordinate. In this scenario Kriging algorithm is not able to generate a spatial correlation due to few variabilities between point. To overcome this problem, coordinates were randomly dispersed using the rnorm command setting a standard deviation of 0.01 starting from the central geographical point of the sampling area of each breed (François et al., 2006). 3. Results and discussion In the 16 studied breeds, the total number of described alleles in the thirty-five studied loci reached 580 (Table S4). Polymorphism was high for all analyzed loci. According to the FAO/ISAG (1998) recommendation for the minimum number of alleles in genetic characterization studies, the OarFCB304 locus showed the highest number of alleles (34), while ETH10 showed the lowest number of alleles (5). In this study, the detected level of genetic variability was higher than the variability detected in other previous studies (Arranz et al., 2001; DiezTascon et al., 2000; Al-Atiyat et al., 2016). This could be due to the use of a different number of loci as well as to the noticeable genetic variability of these breeds which is still present today. Moreover, it is important to consider that some of the studied breeds had very good genetic management practices to avoid inbreeding. All the markers used in this study showed high PIC values per locus, with exception of ETH10 (0.16) and OarFCB193 (0.40). The mean expected and observed heterozygosities were 0.70 ± 0.02 and 0.66 ± 0.01, respectively, with a within-breed significant deficit of heterozygosities over loci and breeds of 0.07. These values reflect a good level of genetic variability and are similar to other studies on Merino (Arranz et al., 2001; Lasagna et al., 2011, and Al-Atiyat et al., 2016), European (Lawson Handley et al., 2007), and Chinese sheep breeds (Zhong et al., 2016). The mean number of alleles detected in the studied breeds was high (Table 1). RAM presented the lowest value (3.00 ± 0.97); whereas, MPP showed the highest value (8.59 ± 2.80). It is important to highlight that RAM presented the lowest value of allelic richness (2.73); whereas, MPP showed the highest value (5.68) once again. It is also important to note that since its importation to France, the RAM breed has been maintained as a small and closed flock (Nguyen et al., 1992) where matings between related animals occurred. The values of HE and HO were high and quite similar in all the studied breeds suggesting these breeds are characterized by a remarkable level of genetic variability. The MPB had lower values (0.49 and 0.48 respectively) than the other studied breeds. MPB is a variety of Spanish breeds, recovered in the last years with a scarce available information on the breed and traditionally reared together with the Iberian pig in extensive production grazing systems named “The dehesa” (Benito et al., 1998). The reduced number of heads can justify the low values of HE and HO in this breed despite the good genetic management to maintain a low inbreeding rate. In the 16 studied breeds, all the genetic variability parameters are comparable to the parameters observed in the two non-Merino breeds 78
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
Table 1 Studied sheep breeds, Acronym (ID), sample size of each breed, mean number of observed alleles (MNA), mean allelic richness per locus corrected for sample size and breed based on a minimum sample size of five diploid individuals (Ar), mean observed (HO) and expected (HE) heterozygosity, inbreeding coefficient (FIS) per breed, and number of locus deviated from Hardy-Weinberg equilibrium per breed (dHWE). Breed
ID
MNA ± DS
Ar
HE ± DS
HO ± DS
FIS [IC 95%]
dHWE
Merino D’Arles Merino Est a Laine Merino Precoce Merino Rambouillet Merino Fleischschaf Hungarian Merinos Gentile di Puglia Sopravissana Merinizzata Italiana Polish Merinos Merino Branco Merino da Beira Baixa Merino Preto Romanian Merinos Merino Pozo Blanco Spanish Merinos Canaria de Pelo Roja Mallorquina
MDA MEL MPZ RAM FLC HUG GEP SOP MEI POL MBA MBX MPP ROM MPB MES CAN RMA
7.94 6.72 5.34 3.00 5.74 7.25 7.68 8.43 8.29 7.86 7.94 8.31 8.59 7.63 3.33 7.69 8.64 8.35
5.59 5.20 4.21 2.73 4.39 5.08 5.36 5.46 5.65 5.51 5.62 5.61 5.68 5.39 2.86 5.13 5.11 5.08
0.76 0.74 0.66 0.53 0.67 0.74 0.72 0.73 0.75 0.75 0.75 0.73 0.74 0.74 0.49 0.71 0.73 0.71
0.71 0.73 0.61 0.54 0.65 0.70 0.68 0.69 0.70 0.70 0.70 0.65 0.69 0.70 0.48 0.66 0.67 0.61
0.068 [-0.006 - 0.010] 0.010 [-0.052 - 0.031] 0.073 [0.004 - 0.086] −0.014 [-0.114 - 0.036] 0.018 [-0.049 - 0.048] 0.061 [-0.004 - 0.089] 0.067 [-0.008 - 0.09] 0.057 [0.013 - 0.076] 0.067 [0.013 - 0.087] 0.063 [0.002 - 0.086] 0.062 [0.009 - 0.079] 0.116 [0.060 - 0.135] 0.068 [0.012 - 0.087] 0.050 [-0.026 - 0.090] 0.021 [-0.049 - 0.051] 0.082 [0.035 - 0.101] 0.088 [0.050 - 0.111] 0.139 [0.102 - 0.163]
1 0 1 0 2 1 2 2 1 2 2 2 0 2 2 3 4 5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.62 1.90 2.17 0.97 1.85 1.95 2.66 2.88 2.62 2.88 2.98 2.87 2.80 2.75 1.09 2.35 2.97 2.83
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.03 0.03 0.04 0.03 0.02 0.02 0.03 0.02 0.02 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.03
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01
MNA, mean number of observed alleles; HO, mean observed heterozygosity; HE, expected heterozygosity; Ar, mean allelic richness per locus corrected for sample size and breed; FIS, inbreeding coefficient per breed and number of locus deviated from Hardy-Weinberg equilibrium per breed (dHWE). *Significantly different from zero (P < 0.05).
Evanno’s method when the number of breeds was high. At K = 3 (Fig. 2), the 16 studied breeds were separated in three different clusters in which the major cluster grouped the breeds originated in France, Portugal and Poland (RAM, MDA, MEL, MBX, MBA, MPP, and POL). At K = 5, the three French breeds of this cluster appeared separated from the Portuguese and Poland breeds and splitted in two subclusters (RAM, MDA, and MEL). At K = 7, these three French breeds were now grouped in one cluster. Similarly, the three Portuguese Merino were clustered together. The POL clustered together with ROM and HUG breeds even if all the POL samples showed a remarkable genetic admixture. The history of Merino sheep in Eastern Europe is in fact quite similar: the dispersal of the Merino trunk in this geographic area started in the 18th century. In Hungary, Merino sheep breeding began in 1774 when Empress Maria Theresa bought 300 Merino individuals from Spain (Ciani et al., 2015). In Romania, it is a supposition that ROM breed was produced in 19th century by some cross with Merino during the transhumance in Crimea and North Caucasus in order to increase the wool quality, and more recent, to improve the fattening and carcass quality, some specialized breeds (such as Merino Fleischschaf) for meat production were used (Ilişiu et al., 2013). Besides, the first Spanish Merino
Fig. 1. Neighbor-net dendrogram constructed from the Reynold’s genetic distances among 18 sheep breeds.
Table 2 Pairwise FST distance among 16 Merino and Merino-derived sheep breeds*.
MEL MPZ RAM FLC HUG GEP MEI SOP POL MBA MBX MPP ROM MPB MES CAN RMA
MDA
MEL
MPZ
RAM
FLC
HUG
GEP
MEI
SOP
POL
MBA
MBX
MPP
ROM
MPB
MES
CAN
0.025 0.063 0.182 0.058 0.081 0.026 0.030 0.040 0.032 0.045 0.042 0.044 0.086 0.166 0.035 0.034 0.038
– 0.089 0.192 0.067 0.090 0.033 0.041 0.049 0.046 0.050 0.048 0.048 0.089 0.170 0.045 0.039 0.033
– 0.160 0.094 0.066 0.087 0.072 0.113 0.121 0.107 0.109 0.110 0.083 0.276 0.096 0.105 0.080
– 0.158 0.238 0.120 0.133 0.145 0.135 0.124 0.138 0.140 0.242 0.239 0.140 0.093 0.095
– 0.059 0.068 0.056 0.078 0.101 0.093 0.099 0.092 0.065 0.276 0.074 0.088 0.067
– 0.038 0.043 0.045 0.037 0.042 0.047 0.048 0.025 0.106 0.043 0.021 0.034
– 0.020 0.038 0.044 0.040 0.036 0.040 0.042 0.239 0.050 0.049 0.040
– 0.031 0.061 0.054 0.052 0.049 0.045 0.226 0.033 0.042 0.032
– 0.074 0.057 0.059 0.053 0.047 0.239 0.061 0.058 0.058
– 0.070 0.067 0.061 0.039 0.238 0.086 0.077 0.074
– 0.021 0.015 0.053 0.244 0.072 0.055 0.065
– 0.014 0.053 0.248 0.075 0.059 0.057
– 0.053 0.243 0.068 0.050 0.057
– 0.118 0.044 0.018 0.026
– 0.247 0.264 0.254
– 0.056 0.041
– 0.064
* all values are significantly different from zero (P < 0.05). 79
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
Fig. 2. Population structure of 16 Merino and Merino-derived sheep breeds obtained from 10 runs for each number of assumed populations (K) value ranging from 2 to 18 using the model-based STRUCTURE software.
sheep were introduced to Poland from Spain in 1786. After 1860, Rambouillet and Merino Precoce were brought to Poland, and work towards a dual-purpose type sheep was begun. After 1945, Merino Fleischschaf and Merino Landschaf were introduced into some of the Merino flocks which survived the war (Porter et al., 2016). At K = 7, a genetic admixture is detectable also in the three Italian Merino derived breeds (SOP, GEP, and MEI). At K = 9, a similar situation was observed with exception of MPZ, FLC, and POL that now clustered separately. When K = 18, most of the studied breeds clustered separately, showing some grouping on a geographical basis (France, Portugal, Romania, and Hungary). Genetic admixture is particularly evident in the three Italian Merino derived breeds (SOP, GEP, and MEI). As a matter of fact, the two ancient breeds (GEP, SOP) were reared in neighboring territories and commercial exchanges of mating heads between them were presumably frequent. Moreover, it has to be taken into consideration that both of these two breeds were the genetic base to constitute the MEI breed (Sarti et al., 2006). It is important to highlight that MPB breed was clearly separated from all the other breeds (from K = 3 to K = 18). From K7 level, the two non-Merino breeds (CAN and RMA) clustered isolated from the 16 studied breeds as expected. The population substructures highlighted in the STRUCTURE analysis at the most probable K value showed that the investigated Merino and Merino-derived breeds, presented a genetic identity in accordance with their geographical location despite their common ancestral origin. Probably, these breeds historically underwent different selection strategies in the different countries. The ancestry coefficient map displays the correlation between the Q matrix obtained from STRUCTURE software and the geographical coordinates (Fig. 3, Figure S1). This method permits to search for hidden relationship between genetics structure and territory conformation. At first glance, we can see a clear gradient separating northern Europe from Mediterranean regions. This partition clustered Portuguese, French, and Polish Merinos. Similar findings were already reported by Peter et al. (2007) in a study on 57 European and Middle-Eastern sheep breeds. This situation could be in fact explained by historical information that reported the importation of French animals both to Poland and to Portugal in order to improve local populations. Diez‐Tascón et al. (2000) reported that, since 1930, French Mutton Merino was introduced in order to direct the production towards early maturity in Portuguese Branco Merino. The other clusters put together MES, MPZ, and FLC with Italian Merino-derived SOP and MEI. MPZ, and FLC have been reared in Spain since 1913, when all importation from France were interrupted and were widely admixed with MES, and this could be the reason for the separation from the French breeds. Italian breeds have been linked to MES since their origin, and a strong genetic relationship was confirmed in a previous study (Lasagna et al., 2011). GEP shows a small gradient (blue in the map) probably due to genetic introgression with MPZ and other Merino-derived breeds. HUG and ROM are jointly clustered in the green gradient with MPB.
Fig. 3. Geographic map representing Q matrix from Structure software at K = 3.
This information should be considered with caution because this group of animals, which namely belong to Merino breeds, showed a strong divergence that can lead to STRUCTURE software overestimation of genetics partition. 4. Conclusions In this paper, the above-mentioned historical information has been investigated from the genetic point of view. According to our results, the Spanish origin of the Merinos branch seems to be confirmed, although most of the French Merinos showed a high genetic identity compared with the Spanish ancestors. Probably because of the efficient selection, the French Merino breeds showed recent introgression in the Iberian Merinos, as in some Eastern countries such as Poland. Although some of the studied breeds were affected by the admixture phenomenon there is still genetic identity among breeds in accordance with their geographical location, as result of different selection strategies in the different countries. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.smallrumres.2019.06. 018. 80
Small Ruminant Research 177 (2019) 76–81
V. Landi, et al.
References
Hewitt, G.M., 2007. Genetic structure of European sheep breeds. Heredity 99, 620–631. https://doi.org/10.1038/sj.hdy.6801039. Leroy, G., Verrier, E., Meriaux, J.C., Rognon, X., 2009. Genetic diversity of dog breeds: between-breed diversity, breed assignation and conservation approaches. Anim. Genet. 40, 333–343. https://doi.org/10.1111/j.1365-2052.2008.01843.x. Lewis, P.O., Zaykin, D., 1999. Genetic Data Analysis. Computer Program for the Analysis of Allelic Data. Version 1.0. Lewis Labs. Univ of Connecticut, Storrs, CT. Martyniuk, E., Rzepecki, R., 1995. Sheep husbandry in Poland-an outline. Cahiers Options Mediterraneennes 11, 121–131. Matos, C.A.P., 1986. Avaliacao das capacidades produtivas e reprodutivas das racas ovinas Merino Branco e Campanica, Relatorio de estagio. Universidad de Tras-osMontes e Alto Douro, Vila Real. Meirmans, P.G., 2015. Seven common mistakes in population genetics and how to avoid them. Mol. Ecol. 24, 3223–3231. https://doi.org/10.1111/mec.13243. Nguyen, T.C., Morera, L., Llanes, D., Leger, P., 1992. Sheep blood polymorphism and genetic divergence between French Rambouillet and Spanish Merino: role of genetic drift. Anim. Genet. 23, 325–332. https://doi.org/10.1111/j.1365-2052.1992. tb00154.x. Othman, O.E.M., Payet-Duprat, N., Harkat, S., Laoun, A., Maftah, A., Lafri, M., Da Silva, A., 2016. Sheep diversity of five Egyptian breeds: genetic proximity revealed between desert breeds: local sheep breeds diversity in Egypt. Small Rumin. Res. 144, 346–352. https://doi.org/10.1016/j.smallrumres.2016.10.020. Park, S.D.E., 2001. Trypanotolerance in West African Cattle and the Population Genetic Effects of Selection. PhD Dissertation. University of Dublin. Pedrosa, S., Arranz, J.J., Brito, N., Molina, A., San Primitivo, F., Bayón, Y., 2007. Mitochondrial diversity and the origin of Iberian sheep. Genet. Sel. Evol. 39, 91–103. https://doi.org/10.1186/1297-9686-39-1-91. Peter, C., Bruford, M., Perez, T., Dalamitra, S., Hewitt, G., Erhardt, G., Econogene Consortium, 2007. Genetic diversity and subdivision of 57 European and Middle‐Eastern sheep breeds. Anim. Genet. 38, 37–44. https://doi.org/10.1111/j. 1365-2052.2007.01561.x. Piper, L., Ruvinsky, A., 1997. The Genetics of Sheep. CAB INTERNATIONAL (Eds.), New York, USA. Pons, A.L., Landi, V., Martinez, A., Delgado, J.V., 2015. The biodiversity and genetic structure of Balearic sheep breeds. J. Anim. Breed. Genet. 132, 268–276. https://doi. org/10.1111/jbg.12129. Porter, V., Alderson, L., Hall, S.J., Sponenberg, D.P., 2016. Mason’s World Encyclopedia of Livestock Breeds and Breeding Vol. 2. Cabi (Eds.), pp. 885. Pritchard, J.K., Stephens, M., Donnelly, P., 2000. Inference of population structure using multilocus genotype data. Genetics. 155, 945–959. Reynolds, J., Weir, B.S., Cockerham, C.C., 1983. Estimation of the coancestry coefficient: basis for a short-term genetic distance. Genetics. 105, 767–779. Rousset, F., 2008. Genepop’007: a complete reimplementation of the Genepop software for Windows and Linux. Mol. Ecol. Resour. 8, 103–106. https://doi.org/10.1111/j. 1471-8286.2007.01931.x. Sarti, D.M., 1996. Le razze derivate Merinos ad attitudine carne nel centro sud europeo. L’Allevatore di Ovini e Caprini, pp. 1–2 7/8. Sarti, F.M., Lasagna, E., Panella, F., Lebboroni, G., Renieri, C., 2006. Wool quality in Gentile di Puglia sheep breed as measure of genetic integrity. Ital. J. Anim. Sci. 5, 371–376. https://doi.org/10.4081/ijas.2006.371. Sassi-Zaidy, Y.B., Maretto, F., Charfi-Cheikhrouha, F., Mohamed-Brahmi, A., Cassandro, M., 2016. Contribution of microsatellites markers in the clarification of the origin, genetic risk factors, and implications for conservation of Tunisian native sheep breeds. Genet. Mol. Res. 15, gmr.15017059. https://doi.org/10.4238/gmr. 15017059. Semyonov, S.I., Selkin, I.I., 1989. Sheep. In: Dimitriev, N.G., Ernst, L.K. (Eds.), Animal Genetic Resources of the USSR. FAO-UNEP, Rome, pp. 154–271. Tapio, M., Ozerov, M., Tapio, I., Toro, M.A., Marzanov, N., Ćinkulov, M., Goncharenko, G., Kiselyova, T., Murawski, M., Kantanen, J., 2010. Microsatellite-based genetic diversity and population structure of domestic sheep in northern Eurasia. BMC Genet. 11, 76. https://doi.org/10.1186/1471-2156-11-76. Tibério, M.L., Diniz, F., 2014. Sheep and goat production in Portugal: a dynamic view. Mod. Econ. 5, 703. https://doi.org/10.4236/me.2014.56066. Tolone, M., Mastrangelo, S., Rosa, A.J.M., Portolano, B., 2012. Genetic diversity and population structure of Sicilian sheep breeds using microsatellite markers. Small Rumin. Res. 102, 18–25. https://doi.org/10.1016/j.smallrumres.2011.09.010. Tortorelli, N., 1984. Allevamento della pecora. Edagricole (Eds.). Weir, B.S., Cockerham, C.C., 1984. Estimating F‐statistics for the analysis of population structure. Evolution 38, 1358–1370. https://doi.org/10.1111/j.1558-5646.1984. tb05657.x. Yilmaz, O., Cemal, İ., Karaca, O., Ata, N., 2016. Molecular genetic characterization of Kivircik sheep breed raised in Western Anatolia. Development 28, 3. Zhong, T., Ma, Y.H., Gao, H.J., He, J.N., Liu, N., Zhao, Y.J., Zhang, J.H., Huang, Y.F., 2016. Conservation genetics in Chinese sheep: diversity of fourteen indigenous sheep (Ovis aries) using microsatellite markers. Ecol. Evol. 6, 810–817. https://doi.org/10. 1002/ece3.1891.
Al-Atiyat, R.M., Salameh, N.M., Tabbaa, M.J., 2014. Analysis of genetic diversity and differentiation of sheep populations in Jordan. Electron. J. Biotechnol. 17, 168–173. https://doi.org/10.1016/j.ejbt.2014.04.002. Al-Atiyat, R., Flood, W., Franklin, I., Kinghorn, B., Ruvinsky, A., 2016. Microsatellitebased genetic variation and differentiation of selected Australian Merino sheep flocks. Small Rumin. Res. 136, 137–144. https://doi.org/10.1016/j.smallrumres. 2016.01.018. Arranz, J.J., Bayón, Y., San Primitivo, F., 2001. Differentiation among Spanish sheep breeds using microsatellites. Genet. Sel. Evol. 33, 529–542. https://doi.org/10.1186/ 1297-9686-33-5-529. Belkhir, K., Borsa, P., Chikhi, L., Raufaste, N., Bonhomme, F., 2001. GENETIX 4.02, Logiciel Sous Windows TM Pour La Génétique Des Populations. Laboratoire Génome, Populations, Interactions, CNRS UPR 9060. Université de Montpellier II, Montpellier. France. Benito, J., Ferrera, J.L., Vazquez, C., Menaya, C., Garcia, J.M., 1998. El cerdo ibérico: poblador de la dehesa. In: Benítez, W. (Ed.), Los cerdos locales en los sistemas tradicionales de producción. FAO, Roma, pp. 71–96. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300. Bonadonna, T., 1976. Etnologia Zootecnica. UTET (Eds.). Ciani, E., Ciampolini, R., D’Andrea, M., Castellana, E., Cecchi, F., Incoronato, C., D’Angelo, F., Albenzio, M., Pilla, F., Matassino, D., Cianci, D., 2013. Analysis of genetic variability within and among Italian sheep breeds reveals population stratification and suggests the presence of a phylogeographic gradient. Small Rumin. Res. 112, 21–27. https://doi.org/10.1016/j.smallrumres.2012.12.013. Ciani, E., Crepaldi, P., Nicoloso, L., Lasagna, E., Sarti, F.M., Moioli, B., Napolitano, F., Carta, A., Usai, G., D’Andrea, M., Marletta, D., Ciampolini, R., Riggio, V., Occidente, M., Matassino, D., Kompan, D., Modesto, P., Macciotta, N., Ajmone-Marsan, P., Pilla, F., 2014. Genome-wide analysis of Italian sheep diversity reveals a strong geographic pattern and cryptic relationships between breeds. Anim. Genet. 45, 256–266. https:// doi.org/10.1111/age.12106. Ciani, E., Lasagna, E., D’andrea, M., Alloggio, I., Marroni, F., Ceccobelli, S., Delgado Bermejo, J.V., Sarti, F.M., Kijas, J., Lenstra, J.A., Pilla, F., the International Sheep Genomics Consortium, 2015. Merino and Merino-derived sheep breeds: a genomewide intercontinental study. Genet. Sel. Evol. 47, 64. https://doi.org/10.1186/ s12711-015-0139-z. Diez‐Tascón, C., Littlejohn, R.P., Almeida, P.A.R., Crawford, A.M., 2000. Genetic variation within the Merino sheep breed: analysis of closely related populations using microsatellites. Anim. Genet. 31, 243–251. https://doi.org/10.1046/j.1365-2052. 2000.00636.x. Esteban Muñoz, C., 2004a. The Sheeps in America. Role of Merino Sheep Breed in the Formation of the American Sheep Stock. Esteban Muñoz, C., 2004b. Razas Ganadera Espanola Ovinas. Ministerio de Agricoltura, Madrid. Evanno, G., Regnaut, S., Goudet, J., 2005. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611–2620. https://doi.org/10.1111/j.1365-294X.2005.02553.x. François, O., Ancelet, S., Guillot, G., 2006. Bayesian clustering using hidden markov random fields in spatial population genetics. Genetics 174, 805–816. https://doi.org/ 10.1534/genetics.106.059923. Gaouar, S.B.S., Kdidi, S., Ouragh, L., 2016. Estimating population structure and genetic diversity of five Moroccan sheep breeds by microsatellite markers. Small Rumin. Res. 144, 23–27. https://doi.org/10.1016/j.smallrumres.2016.07.021. Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23, 254–267. https://doi.org/10.1093/molbev/msj030. Ilişiu, E., Dărăban, S., Radu, R., Pădeanu, I., Ilişiu, V.C., Pascal, C., Rahmann, G., 2013. The Romanian Tsigai sheep breed, their potential and the challenges for research. Appl. Agric. For. Res. 2, 161–170. https://doi.org/10.3220/LBF_2013_161-170. Jay, F., Manel, S., Alvarez, N., Durand, E.Y., Thuiller, W., Holderegger, R., Taberlet, P., François, O., 2012. Forecasting changes in population genetic structure of alpine plants in response to global warming. Mol. Ecol. 21, 2354–2368. https://doi.org/10. 1111/j.1365-294X.2012.05541.x. Kalinowski, S.T., 2005. HP–RARE 1.0: a computer program for performing rarefaction on measures of allelic richness. Mol. Ecol. Notes 5, 187–189. https://doi.org/10.1111/j. 1471-8286.2004.00845.x. Laguna Sanz, E., 1986. Historia del Merino, Madrid (Eds.), Ministerio de Agricultura Pesca y Alimentacion y direccion general de la produccion agraria. Madrid. . Langella, O., 1999. POPULATIONS 1.2.30: Population genetic software. Gif-sur-Yvette, France: Laboratoire Evolution, Génomes et Spéciation. Lasagna, E., Bianchi, M., Ceccobelli, S., Landi, V., Martínez, A.M., Vega Pla, J.L., Panella, F., Delgado Bermejo, J.V., Sarti, F.M., 2011. Genetic relationships and population structure in three Italian Merino-derived sheep breeds. Small Rumin. Res. 96, 111–119. https://doi.org/10.1016/j.smallrumres.2010.11.014. Lawson Handley, L.J., Byrne, K., Santucci, F., Townsend, S., Taylor, M., Bruford, M.W.,
81