Livestock Science 136 (2011) 277–280
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Livestock Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i
Y chromosomal characterization of Turkish native sheep breeds Y. Oner a,⁎, J.H. Calvo b,c, C. Elmaci a a b c
Department of Animal Science Faculty of Agriculture University of Uludag, Turkey Unidad de Tecnología en Producción Animal, CITA, Zaragoza 50059, Spain ARAID researcher, Spain
a r t i c l e
i n f o
Article history: Received 22 March 2010 Received in revised form 23 August 2010 Accepted 24 August 2010 Keywords: Sheep Y chromosome SRY SRYM18
a b s t r a c t In this study, ten native Turkish sheep breeds were sampled to evaluate Y chromosomal genetic variation. Two regions of the SRY gene, one region each of the DBY and AMEL genes and SRYM18 Y-speciﬁc microsatellite locus were sequenced. While no base substitutions were found in the sequenced regions of SRY, DBY and AMELY, most of the Turkish sheep breeds have some variations of SRYM18. In total, we found three different SRYM18 alleles that were 141, 143 and 145 bp in length. The distribution of these alleles was different among fat-tailed sheep breeds and the thin-tailed sheep breeds. While the most common allele was 143 bp among the breeds, the 141-bp allele was also observed in both fat-tailed and thin-tailed breeds. The 145-bp allele was only found in the fat-tailed sheep at low frequency. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Today, it is well known that the domestication of sheep occurred approximately 10,000 to 11,000 years ago in the region that spans from northern Zagros to southeastern Anatolia (Uerpmann, 1996; Vigne et al., 1999). The genetic structure of native Turkish breeds could be very important because of the probability that they gave rise to most of the modern European sheep breeds (Koban, 2004). Asia Minor provides an important geographic link between the Middle East, Asia and Europe, and for this reason, this region manifests a genetic constitution that reﬂects the consequences of numerous gene ﬂow, admixture and local differentiation processes spanning from the beginning of the domestication to the present day (Cavalli-Sforza et al., 1994). It is very important to reveal the genetic structure of native Turkish breeds to understand their contribution to modern European sheep breeds' gene pools and the domestication process. Determining the genetic structure of sheep breeds is also necessary for an effective genetic conservation program, which is needed in Turkey as uncontrolled breeding systems have reduced the number and population of native ⁎ Corresponding author. Tel.: + 90 224 2941561; fax: + 90 224 442 81 52. E-mail address: [email protected]
(Y. Oner). 1871-1413/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2010.08.015
sheep breeds. Disorganization in sheep breeding systems can cause both genetic pollution and genetic erosion. The uni-paternal and haploid inheritance of Y-speciﬁc microsatellites and SNPs make these genetic markers extremely sensitive for detecting genetic history, the domestication process of breeds, population relationships and male bias gene ﬂow. With respect to genders, asymmetric introgression between geographic regions might have occurred during the domestication process. Therefore, by using Y-speciﬁc markers, it may be possible to gain insight into male-speciﬁc gene ﬂow. Several studies have been carried out using microsatellite markers (Uzun et al., 2006; Koban, 2004), mitochondrial DNA (Pedrosa et al., 2005; Meadows et al., 2007) and Ychromosomal markers (Meadows and Kijas, 2009) to reveal the genetic structure of some Turkish sheep breeds. However, the number of studies carried out on sheep using the Ychromosome markers has been limited when compared to other livestock species such as cattle (Hanotte et al., 2000; Pérez-Pardal et al., 2009) and goats (Pidancier et al., 2006; Sechi et al., 2009). In sheep, only one microsatellite locus (SRYM18) and eight SNPs, located in the 5′ promoter region of the sex-determining gene (SRY), have been identiﬁed (Meadows et al., 2006; Meadows and Kijas, 2009). In the present study, the distribution of Y-chromosomal haplotypes
Y. Oner et al. / Livestock Science 136 (2011) 277–280
was studied using variations in the 5′ promoter region of the SRY gene and the SRYM18 microsatellite locus in ten Turkish sheep breeds. In addition, polymorphisms in two fragments of the 5′ upstream region of the SRY gene (AY604734 and AY604735), the 3′ untranslated region of the DBY gene (171 bp) and a 182-bp region from AMELY (DQ469593) were examined in ten native Turkish sheep breeds.
2. Material and methods 2.1. Animal resources and DNA isolation Blood samples used for DNA isolation were collected from 147 animals belonging to ten native Turkish sheep breeds, which were reared on 42 distinct farms in different regions of Turkey. Hemsin (HMS, n = 15), Morkaraman (MRK, n = 10) and Ivesi (Awassi) (AWS, n = 15) sheep were sampled from ﬁve, one, and thirteen farms found in eastern Turkey, respectively. Karayaka animals (KRY, n = 9) were collected from three farms. Daglic (DGL, n = 13) and Akkaraman (AKK, n = 15) sheep were sampled from four and seven farms, respectively. Animals from SKZ (SKZ, n = 17) and CCB (CCB, n = 12) were selected from only one and three ﬂocks, respectively. Kivircik (KVR, n = 22) sheep were sampled from three farms. Finally, Gokceada (GDA, n = 19) animals were collected from two distinct farms (Fig. 1). Total DNA was extracted using a genomic DNA puriﬁcation kit (K0512, Fermentas, Lithuania) according to the instructions provided in the manual. All animal samples were sequenced for the two 5′upstream regions of the SRY gene and genotyped for SRYM18 microsatellite region. To search for novel sequence variants located in the region from AMELY and DBY 171 bp and 182 bp of lengths were sequenced in two or three animals per breed carrying different SRYM18 length variants.
2.2. PCR ampliﬁcations and sequence analysis PCR ampliﬁcations for two regions (549 and 598 bp in length) of the SRY gene and a fragment of 3′ untranslated region of the DBY gene (171 bp) were performed as described by Meadows et al. (2004). Ampliﬁcation of the SRYM18 microsatellite locus was performed according to Meadows et al. (2006) using a ﬂuorescently labeled forward primer. PCR products were visualized on an ABI 310 (Applied Biosystems, USA). To determine the allele size of SRYM18, a sample of Spanish Mouﬂon previously validated as 143 bp by Meadows et al. (2006) was also included in each run. The AMELY fragment (182 bp) was selected because Dervishi et al. (2008) observed ampliﬁcation failures in some Spanish breeds, possibly due to DNA variations. The AMELY fragment (182 bp) was puriﬁed from gel using the Macherey–Nagel-Extract II puriﬁcation kit according to the manufacturer's instructions. The puriﬁed PCR products were then sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) on an automated Genetic Analyser ABI 3100 (Applied Biosystem, USA). Sequences were aligned using CLUSTALW (http://www.ebi.ac.uk/clustalw/) software and were compared against the GenBank and EMBL databases. 3. Results and discussion No polymorphisms were found in the DBY region, the AMELY region or in the two regions of the SRY gene analyzed. All individuals carried the A allele at the oY1 SNP in the SRY gene. The AMELY gene demonstrated 100% identity with the Ovis Aries chromosome Y amelogenin variant 2 gene (GenBank acc. DQ469593) described by Weikard et al. (2006). The classiﬁcation of haplotypes was performed according to Meadows et al. (2006) using the oY1 SNP in the SRY gene and the genotype of the SRYM18 microsatellite locus. Three
Fig. 1. Distribution of SRYM18 alleles among Turkish native breeds according to distinct regions.
Y. Oner et al. / Livestock Science 136 (2011) 277–280
Table 1 Distribution of frequency of SRYM18 alleles among Turkish sheep breeds according to their tail type.
H8 H6 H4
16 121 10
– 0.867 0.133
0.500 0.200 0.300
– 0.923 0.077
– 0.933 0.067
0.500 0.417 0.083
– 0.877 0.133
– 1 –
– 1 –
0.053 0.947 –
0.444 0.556 –
distinct paternal haplotypes were revealed among Turkish native sheep breeds. The haplotypes identiﬁed were H4, H6 and H8, characterized by allele lengths of 145, 143 and 141 bp, respectively, at the SRYM18 locus. These results are concordant with those described by Meadows et al. (2006) and Meadows and Kijas (2009), which revealed four SRYM18 alleles (139 bp, 141 bp, 143 bp, and 145 bp) for domestic sheep breeds. Meadows and Kijas (2009) investigated seven native Turkish sheep breeds (Cine Capari, Karakas, Karayaka, Morkaraman, Norduz, Sakiz and Tuj) and a crossbreed (Karya) and found ﬁve haplotypes. Two animals from the Karakas breed, which is accepted as a variety of the Akkaraman breed, were grouped in the H7 haplotype characterized by a G allele for oY1. The authors also found six individuals from the Sakiz breed that were grouped in the H12 characterized by the 139 bp allele of SRYM18; however, in the present study, all of our Sakiz animals carried only the H6 haplotype (Fig. 1). This situation can be explained by our sampling strategy concerning the Sakiz individuals, which were obtained from only one ﬂock as mentioned in the materials and method section. Fig. 1 shows the haplotype distribution among the Turkish sheep breeds. Although the H6 haplotype predominated among the Turkish sheep breeds (with a frequency of 0.826), as reported by Meadows and Kijas (2009) as well, the H8 haplotype was predominant in the MRK and CCB breeds. These breeds showed the highest haplotype diversity with three haplotypes. CCB animals were sampled from three ﬂocks, whereas the MRK animals were sampled from a single ﬂock without genealogic information. The variation observed in the MRK population may reﬂect the highly heterogeneous nature of this breed, and this variability was greater than previously reported (Meadows and Kijas, 2009). These inconsistencies can be due to the small sample sizes used in both of these studies. Table 1 shows the haplotype frequencies for the different sheep breeds. As shown on Fig. 1, although there were no differences between the geographic regions of Turkey, there was a signiﬁcant difference between thin- and fat-tailed sheep breeds in regard to allele distribution. Whereas the 145 bp allele of SRYM18 has not been detected among the thin-tailed sheep breeds, the allele was found in the fat-tailed sheep at low frequency. This ﬁnding is in concordance with the results from African sheep breeds (Ouna et al., 2006). In 491 male individuals from 35 distinct breeds in Africa, no 145 bp allele of SRYM18 was found in thin-tailed breeds. Interestingly, Meadows and Kijas (2009) did not ﬁnd the H4 haplotype as deﬁned by a combination of the 145 bp allele and oY1-G among 254 individuals belonging to 34 European sheep breeds. In contrast, these results are in agreement with the study reported by Uzun et al. (2006) using microsatellite
markers, in which a clear separation was seen between breeds with fat-tails and breeds with thin-tails (Table 1). In our study, the H4 haplotype was not found among the thintailed breeds. Furthermore, these breeds showed a lower haplotype diversity overall. Analysis of mtDNA of Turkish native breeds revealed ﬁve maternal haplogroups and a high genetic diversity of maternal lineages (Pedrosa et al., 2005; Meadows et al., 2007). Although a majority of the investigated Turkish native breeds had haplogroup B, the distribution of frequencies of the haplogroups varied considerably (Pedrosa et al., 2005; Meadows et al., 2007). There are few studies which have been carried out using nuclear markers with limited number of breeds (Uzun et al., 2006; Devrim et al., 2007), and the ﬁndings from these studies are not in agreement with mtDNA data (Pedrosa et al. 2005; Meadows et al., 2007). In addition, Uzun et al. (2006) and Devrim et al. (2007) found a similarity between the AKK and MRK breeds using microsatellite and RAPD markers. Uzun et al. (2006) suggested that this similarity could be explained by a probable common paternal founder for these two breeds. Although these two breeds (AKK and MRK) share two haplotypes (H4 and H6), it is difﬁcult to explain this similarity using results from only one paternal marker. In the present study, ten Turkish native sheep breeds were characterized using known Y-speciﬁc markers for sheep. To better understand the paternal lines of sheep breeds, additional MSY markers are needed. Acknowledgements This study was ﬁnancially supported by the Scientiﬁc Research Project Council of Uludag University (Project number: Z-2009/29). This manuscript was edited by American Journal Experts (AJE). References Cavalli-Sforza, L.L., Menozzi, P., Piazza, A., 1994. The History and Geography of Human Genes. Princeton University Press, Princeton. Dervishi, E., Martinez-Royo, A., Sánchez, P., Alabart, J.L., Cocero, M.J., Folch, J., Calvo, J.H., 2008. Reliability of sex determination in ovine embryos using amelogenin gene (AMEL). Theriogenology 70, 241–247. Devrim, A.K., Kaya, N., Guven, A., Kocamış, H., 2007. A study of genomic polymorphism and diversity in sheep breeds in northeastern Anatolia. Small Rum. Res. 73, 291–295. Hanotte, O., Tawah, C.L., Bradley, D.G., Okomo, M., Verjee, Y., Ochieng, J., Rege, J.E., 2000. Geographic distribution and frequency of a taurine Bos taurus and an indicine Bos indicus Y speciﬁc allele among sub-Saharan African cattle breeds. Mol. Ecol. 9, 387–396. Koban, E., 2004. Genetic Diversity of Native and Crossbreed Sheep Breeds in Anatolia. PhD thesis. Middle East Technical University. Ankara. 138 pp. Meadows, J.R.S., Kijas, J.W., 2009. Re-sequencing regions of the ovine Y chromosome and wild sheep reveals novel paternal haplotypes. Anim. Genet. 40, 119–123.
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