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Short communication
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Characterization of 37 breed-specific single nucleotide polymorphisms in sheep.
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L. Pariset1, I. Cappuccio1, P. Ajmone-Marsan2, M. Bruford3, S. Dunner4, O. Cortes4, G.
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Erhardt5, G. Lühken5, C. Peter5, S. Joost7, G. Pinto-Juma3, I.J. Nijman6, J.A. Lenstra6, T.
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Perez3, A. Valentini1 and Econogene Consortium8
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Dipartimento di Produzioni Animali, Università della Tuscia, Viterbo, Italy
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Istituto di Zootecnica, Università Cattolica del Sacro Cuore, Piacenza, Italy
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School of Biosciences, University of Wales, Cardiff, United Kingdom
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Departimento de Producción Animal, Universidad Complutense, Madrid, Spain
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Department of Animal Breeding and Genetics, Justus-Liebig-Universität, Giessen, Germany
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Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
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http://lasig.epfl.ch/projets/econogene
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Correspondence: L. Pariset, tel. +39 761 357447, fax +39 761 357434, e-mail: pariset@unitus.it
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Author to whom reprint requests should be addressed: corresponding author
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Summary
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We identified 37 single nucleotide polymorphisms (SNPs) in sheep and screened 16 individuals
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from 8 different sheep breeds selected throughout Europe. Population-genetic measures based on
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the genotyping about 30 sheep from the same 8 breeds are reported. To date, there are not sheep
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SNPs documented in the NCBI dbSNP database. Therefore, the markers presented here contribute
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significantly to those currently available.
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Keywords
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Sheep, single nucleotide polymorphisms, allele frequency.
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The several complete-genome projects have led to the emergence of single nucleotide
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polymorphisms (SNPs) as most modern genetic markers. SNPs occur frequently in the mammalian
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genome (Brouillette et al. 2000; Shubitowski et al. 2001) and are useful for rapid, large-scale and
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cost effective genotyping (Schlotterer, 2004; Syvanen et al., 2001; Vitalis et al., 2001; Vignal et al.,
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2002), for ecological and conservation studies (Morin et al., 2004; Seddon et al., 2005; Vignal et al.
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2002), and for population and evolutionary studies (Kuhner et al., 2000; Sunnucks 2000; Glaubitz et
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al., 2003). However, SNPs are still scarce in species where little DNA sequence data are available
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for non-model organisms, primarly due to the effort needed to find SNPs in species where little
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DNA sequencedata are available, (Aitken et al 2004) and their full potential is not yet exploited.
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The EU-sponsored Econogene project led to a collection of samples from sheep breeds from
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different European and Near-Eastern regions. This allowed an SNP discovery by across-breed
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comparison of 16 unrelated individuals belonging to eight breeds representingwide phenotypic and
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geographic variation in Western Eurasia: Akkaraman (Turkey), Bergamasca (Italy), Karagouniko
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(Greece), Rhönsheep (Germany), Rubia del Molar (Spain), Turcana (Romania), Welsh Mountain
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(Great Britain), Zelazna (Poland). DNA was isolated after collection of whole blood using standard
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techniques. Primers designed using sheep sequences (where available) or the consensus sequences
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of the closest species in Genebank were used both for PCR amplification and sequencing of the
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corresponding genomic fragment. Sequences were BLAST aligned to establish homology. Thirty-
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seven SNPs were identified in 29 genes (Table 1). Most of these were transitions, while we found
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five transversions and one deletion.
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Subsequently, these SNPs were genotyped in usually 30 individuals from each of the same eight
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breeds, sampling no more than 3 individuals per farm. Standard population statistics (Weir, 1996,
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Botstein et al., 1980) for each locus and over all populations were computed using the programs
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Powermarker (Liu and Muse, 2001) and Genepop 3.3 (Raymond and Rousset, 1995) and shown in
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Table 1. Thirty-two SNPs show an overall frequency of the rare allele higher than 5% and are thus
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generally applicable to population-genetic studies. FST values are variable, but several values above
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0.2 suggest that SNPs are indicative for breed-specific selection. The values of 0.82 for both SNPs
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in the MC1R coat colour gene are caused by a high frequency of the minor alleles only in the Welsh
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Mountain sheep.
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Diversity parameters for individual breeds were calculated using the program FSTAT (Goudet,
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2000) and listed in table 2. From the 5 SNPs in 3 genes with overall frequencies below 5%, four
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have appreciable frequencies (0.1 to 0.266) in specific breeds: CAPN_1 in Karagouniko and
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Rhönsheep, CAPN_2 in Turcana and in Welsh Mountain, LEP_1 in Welsh Mountain; and LEP_2 in
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Turcana and and Welsh Mountain. The minor allele of DES_1 was found only at a low frequency
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(0.052) in Rhönsheep Such SNPs may be informative to a reconstruction of the breed history or
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may be under breed-specific selection.
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Our SNP data contribute to the collection of about 100 SNPs identified so far and to the eventual
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use of these markers for the genetic analysis of breed history a variety of phenotypes.
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Acknowledgements:
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This work has been partially supported by the EU Econogene contract QLK5-CT-2001-02461.
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Major objectives of the project and a list of participants can be found on the website
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http://lasig.epfl.ch/projets/Econogene. We would like to thank all the farmers who kindly provided
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the material and CBS Technologies for help during sampling.
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The content of the publication does not represent necessarily the views of the Commission or its
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services.
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References
25
Aitken, N., Smith, S., Schwarz, C., Morin, P.A. (2004) Single nucleotide polymorphism (SNP)
26
discovery in mammals: a targeted-gene approach. Molecular Ecology, 13, 1423-31.
27
Alvarez-Busto, J., Ruiz-Nunez, A., Jugo, B.M. (2004) Detection of polymorphisms in the tumour
28
necrosis factor alpha candidate gene in sheep. Eur. J. Immunogenet., 31, 155-158
1
Botstein, D., White, R.L., Skolnick, M., Davis, R.W. (1980) Construction of a genetic linkage map
2
in man using restriction fragment length polymorphisms. American Journal of Human Genetics, 32,
3
314-331.
4
Brouillette, J.A., Andrew J.R., Venta P.J. (2000) Estimate of nucleotide diversity in dogs with a
5
pool-and-sequence method. Mammalian Genome, 11, 1079–86.
6
Glaubitz, JC, Rhodes, O E Jr, Dewoody, J A (2003) Prospects for inferring pairwise relationships
7
with single nucleotide polymorphisms. Molecular Ecology, 12, 1039–1047
8
Goudet, J (2000) FSTAT, a program to estimate and test gene diversities and fixation indices
9
(version 291) Available from http://wwwunilch/izea/softwares/fstathtml. Updated from Goudet
10
(1995).
11
Kuhne,r M.K., Beerli, P., Yamato, J., Felsenstein, J. (2000) Usefulness of single nucleotide
12
polymorphism data for estimating population parameters. Genetics 156, 439–447.
13
Liu, K, Muse S (2001) PowerMarker: new genetic data analysis software Version 3.0. Free program
14
distributed by the author over the internet from http://www.powermarker.net.
15
Lühken, G., Hiendleder, S., Prinzenberg, E. M., Erhardt, G. (2000): Rapid communication: a single-
16
strand conformation polymorphism in the ovine interleukin-2 (IL-2) gene. J. Anim. Sci. 78, 2754-
17
2755
18
Lühken, G., Weimann, C., Kraus, M., Goldammer, T., Womack, J. E., Erhardt, G. (2002): Genetic
19
and physical mapping of the ovine interleukin-2 gene (IL2). Anim. Genet. 33, 245-247
20
Lühken, G., Buschmann, A., Groschup, M. H., Erhardt, G. (2004): Prion protein allele A136
21
H154Q171 is associated with high susceptibility to scrapie in purebred and crossbred German
22
Merino land sheep. Arch. Virol. 149, 1571-1580
23
Morin, P A, Luikart, G, Wayne, R K (2004) SNP workshop Group SNPs in ecology, evolution and
24
conservation. Trends in Ecology and Evolution, 19, 208–216.
25
Pariset, L, Cappuccio, I, Joost, S, D’Andrea, MS, Marletta, D, Ajmone-Marsan, P, Valentini, A,
26
ECONOGENE Consortium (2006) Characterization of single nucleotide polymorphisms in sheep
27
and their variation as an evidence of selection. Animal Genetics, in press.
1
Raymond, M., Rousset, F. (1995) GENEPOP (version 12): a population genetics software for exact
2
tests and ecumenicism. J. Heredity, 86, 248-249.
3
Schlötterer, C. (2004) The evolution of molecular markers — just a matter of fashion? Nature
4
Reviews Genetics, 5, 63–69.
5
Seddon, J M, Parker, H G, Ostrander, E A, Ellegren H (2005) SNPs in ecological and conservation
6
studies, a test in the Scandinavian wolf population. Molecular Ecology, 14, 503–511.
7
Shubitowski, D.M., Venta, P.J., Douglass, C.L., Zhou, R.X., Ewart, S.L. (2001) Polymorphism
8
identification within 50 equine gene-specific sequence tagged sites. Animal Genetics, 32, 78–88.
9
Sunnucks, P. (2000) Efficient genetic markers for population biology. Trends in Ecology and
10
Evolution, 15, 199–206.
11
Syvanen, A.C. (2001) Accessing genetic variation: genotyping single nucleotide polymorphisms.
12
Nature Genetics Reviews, 2, 930-942
13
Våge, D.I., Klungland, H., Lu, D., Cone, R.D. (1999) Molecular and pharmacological
14
characterization of dominant black coat color in sheep. Mammalian Genome, 10, 39-43.
15
Vignal, A., Milan, D., San Cristobal, M., Eggen, A. (2002) A review on SNP and other types of
16
molecular markers and their use in animal genetics. Genetics Selection Evolution, 34, 275–305.
17
Vitalis, R., Dawson, K., Boursot, P. (2001) Interpretation of Variation Across Marker Loci as
18
Evidence of Selection. Genetics, 158, 1811–1823.
19
20
Weir, B.S. (1996) Genetic data analysis II, Sunderland, MA: Sinauer Associates, Inc.
21
Weir, B.S., Cockerham, C.C. (1984) Estimating F-statistics for the analysis of population structure.
22
Evolution, 38, 1358-1370.
23