Archivos de Zootecnia ISSN: Universidad de Córdoba España

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1 Archivos de Zootecnia ISSN: Universidad de Córdoba España Paiva, S. R.; Silvério, V. C.; de F. Paiva, D. A.; McManus, C.; Egito, A. A.; Mariante, A. da S.; Castro, S. R.; Albuquerque, M. S. M.; Dergam, J. A. Origin of the main locally adapted sheep breeds of Brazil: a RFLP-PCR molecular analysis Archivos de Zootecnia, vol. 54, núm , 2005, pp Universidad de Córdoba Córdoba, España Available in: How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative

2 ORIGIN OF THE MAIN LOCALLY ADAPTED SHEEP BREEDS OF BRAZIL: A RFLP-PCR MOLECULAR ANALYSIS ORIGEM DAS PRINCIPAIS RAÇAS DE OVINOS NATURALIZADOS DO BRASIL A PARTIR DE MARCADORES MOLECULARES RFLP-PCR Paiva, S.R. 1,3, V.C. Silvério 1, D.A. de F. Paiva 1, C. McManus 2, A.A. Egito 1, A. da S. Mariante 1, S.R. Castro 1, M.S.M. Albuquerque 1 and J.A. Dergam 3 1 Embrapa Genetics Resources and Biotechnology. CP 02372, CEP , Brasília, DF. Brazil. samuel@cenargen.embrapa.br 2 University of Brasília (UnB). FAV, CP 04508, CEP , Brasília, DF. Brazil. 3 University Federal of Viçosa (UFV). Department of Animal Biology. CEP , Viçosa, MG. Brazil. ADDITIONAL KEYWORDS Cytochrome oxidase I. Genetic of conservation. Ovis aries. PALAVRAS CHAVE ADICIONAIS Citocromo oxidase I. Genética da conservação. Ovis aries. SUMMARY Likewise other domestic animals, sheep were introduced in Brazil by the Portuguese settlers. Characterizing the geographic origins and the extent of variability of the genetic resources must also precede any conservation and management program. Recent work on variation of mtdna of Ovis aries indicates that this species may be subdivided in two haplogroups, a European and an Asian. These haplogroups may be readily distinguished by RFLP-PCR (Restriction Fragment Length Polymorphism- Polymerase Chain Reaction) analysis. This approach was employed to recognize the origins of the main Brazilian breeds. A 1052 Kb fragment of the mitochondrial cytochrome oxidase I gene region was amplified in 248 specimens of the Santa Inês, Crioula Lanada, Rabo Largo, Morada Nova, Somali and Bergamasca breeds. The patterns were assigned with HinfI recognition site to nucleotide positions in the COI gene. All animals were of European origin. However, these results also applied to the Brazilian breeds Somali and Morada Nova, considered as from African origin. The African breeds may share the same evolutionary history as the European breeds. A further analysis of mtdna sequences and microsatellites will include more Brazilian, European and African breeds. RESUMO Os ovinos foram introduzidos no Brasil pelos portugueses assim como a grande maioria dos animais domésticos. Para conservação dos recursos genéticos é desejável antes da implementação de qualquer programa de preservação ou manejo a caracterização dos estoques, bem como a avaliação de suas origens. Estudos independentes de pesquisadores europeus da região controle do mtdna demonstraram que o genoma mitocondrial de Ovis aries está dividido em dois grandes haplogrupos, um de origem européia e outro, provavelmente, de origem asiática. Estes haplótipos podem ser identificados a partir de Arch. Zootec. 54:

3 PAIVA ET AL. uma análise de RFLP-PCR (Restriction Fragment Length Polymorphism- Polymerase Chain Reaction) ao invés de sequenciamento. Dessa forma, o presente trabalho teve como objetivo realizar este teste diagnóstico para identificar as possíveis origens das principais raças de ovinos naturalizadas brasileiras. Foi amplificado um fragmento de 1052 pares de bases contendo parte do gene mitocondrial citocromo oxidase I (COI) de 248 animais pertencentes às raças Santa Inês, Crioula Lanada, Rabo Largo, Morada Nova, Somalis e Bergamácia. Depois de amplificados, os fragmentos foram submetidos à clivagem com a enzima de restrição HinfI. Todos os animais analisados foram classificados como de origem européia. Entretanto, algumas das raças naturalizadas brasileiras, como Somalis e Morada Nova tenham, provavelmente, origem Africana. Dessa maneira, os dados sugerem que as raças africanas possam apresentar uma história evolutiva semelhante das raças européias e compartilhar o mesmo haplótipo mitocondrial. Novos trabalhos serão desenvolvidos para testar essa hipótese a partir do sequenciamento da região controle do mtdna tanto das raças naturalizadas brasileiras como das raças européias e africanas. INTRODUCTION The process of adaptation and natural selection of European and African sheep breeds brought to Brazil at the time this country was settled by Portugal resulted in the formation of many locally adapted sheep breeds throughout the Brazilian territory. Generally, these animals are small and very efficient in terms of adaptation and survival in the most diverse Brazilian ecosystems. Until the present, these breeds had not undergone a process of selection and genetic improvement, being, therefore, little specialized in the intensive production of milk and/or meat. In addition, they possess high resistance to the illnesses and are used preferentially for the production of meat and skin. Since 1983, Embrapa has included in its Program of Research in Genetic Resources the domesticated and naturalized breeds threatened of extinction. Currently, the conservation of animal genetic resources is carried out by diverse Research Centers of Embrapa, Universities, State Research Companies, as well as private farmers by means of: (a) Nuclei of Conservation, kept in original habitats of the animals (in situ), and (b) the storage of semen, embryos and oocytes in Banks of Germoplasma (ex situ). Another strategy was to create banks of DNA of practically all the main domesticated and naturalized Brazilian species. Planning programs for conservation can be focused at many levels, from the ecosystems and communities to the individuals. In each one of these levels, molecular techniques have been considered powerful tools so as to identify the standards and processes responsible for the current situation of the flocks and to assist in the development of strategies for handling conservation units (Mace et al. 1996; Avise, 1994; Moritz, 1994). Over the last decades, there has been a general agreement that characterization of genetic variability is an essential aspect of management of natural resources (Hillis et al., 1996). Based on patterns of variation in the mitochondrial DNA control region, Wood and Phua (1996) and Hiendleder et al. (1998) determined, in Ovis aries, the existence of two large groups composed of related haplotypes Archivos de zootecnia vol. 54, núm , p. 396.

4 ORIGIN OF LOCALLY ADAPTED SHEEP BREEDS OF BRAZIL: A RFLP-PCR ANALYSIS (haplogroups), one European and the other of likely Asian origin. Their sequence identity (Hiendleder, 1999) may also be identified by Restriction Fragment Length Polymorphism- Polymerase Chain Reaction (RFLP- PCR). The aim of this work is to determine a test for diagnostic differences among Brazilian sheep breeds and their putative geographic origins. MATERIALS AND METHODS It was sampled 248 individuals of Santa Inês, Bergamasca, Rabo Largo, Morada Nova, Somali e Crioula Lanada breeds from the states of Goiás, Sergipe, Bahia, Distrito Federal, Ceará and Rio Grande do Sul (table I). DNA was extracted from leucocytes following Miller's (1988) salting-out protocols, and a fragment of mitochondrial cytochrome oxidase I (COI) was amplified with primers COIF (5 GCAGAGTTTGAAGCTGCT 3 ) and COIR (5 AGCTGACGTGAAGTAAGC 3 ) described for Hiendleder (1999). Reactions were performed in a MJ Research (PTC100) thermocycler with an initial denaturation step at 94 C for 5' followed by 25 cycles at 94 C 30'', 60ºC 1', and 72 C 1', and a final extension step of 72 C 5'. Amplified products were cleaved with restriction enzyme (RE) HinfI and analyzed 2 percent agarose gels with ethydium bromide. Individuals bearing a mutation at positions were considered as of European origin and as Asian if otherwise (Hiendleder, 1999). RFLP-PCR patterns were thus defined as European for specimens with two 477 e 359 base pair (bp) fragments, while the Asian pattern was charac- Table I. Samples used for RFLP-PCR analysis of Brazilian sheep breeds. N= sample sizes. (Raças de ovinos do Brasil utilizadas nas análises de RFLP- PCR. N= número de animais por localidade). Breed Locales Code N Santa Inês Central - Western farms OSI CO 15 Farms of Sergipe State OSI SE 17 Embrapa, Tabuleiros Costeiros, Ceará State OSI Embrapa 16 Bergamasca University of Brasília 1 EMPRAPA OB DF 33, Farms of Goiás State OB GO 13 Rabo Largo EBDA Bahia State ORL EB 28 Farms of Bahia State ORL JU 20 Morada Nova Red Line, EMBRAPA, Ceará State OMN VE 23 White Line, University Federal of Ceará OMN BR 25 Somali EMBRAPA, State of Ceará OS 48 Crioula Lanada EMBRAPA South, Rio Grande do Sul State OCL 10 TOTAL Individuals from Rio de Janeiro, São Paulo and Bahia states. Archivos de zootecnia vol. 54, núm , p. 397.

5 PAIVA ET AL. Figure 1. Patterns of COI fragments in 2 percent agarose gel. Left of 1Kb ladder, entire fragment of the gene. Right of 1kb ladder, two bands (477 and 359 bp) RFLP-PCR fragments diagnosing European origin in all sampled individuals. A-Santa Inês; B-Bergamasca; C- Rabo Largo; D- Morada Nova; E- Somali; F- Crioula Lanada. (Gel de agarose a 2 p.100 evidenciando os padrões de restrição enzimática do gene COI. A esquerda do padrão de tamanho molecular 1Kb se encontra o fragmento do gene COI (1053 pb) não clivado com enzima de restrição. A direita do padrão de 1kb se encontram duas bandas diagnósticas (477 e 359 pb) obtidas por RFLP- PCR que caracterizam o mtdna do tipo Europeu em todos os indivíduos analisados. A- Santa Inês; B- Bergamácia; C- Rabo Largo; D- Morada Nova; E- Somalis; F- Crioula Lanada). terized by a 836 pb fragment. RESULTS AND DISCUSSION All analyzed individuals presented the European haplotype (figure 1). This is not unexpected, because of the Portuguese colonization process in Brazil. However, some of the Brazilian breeds such as Somali and Morada Nova are currently considered as of African origins (Brazilian Breeds Encyclopedia - We concluded that these probable African races are recently derived from or share primitive haplotypes with European mitochondrial lineages. Other studies with sequencing of mtdna displacement region (D- Archivos de zootecnia vol. 54, núm , p. 398.

6 ORIGIN OF LOCALLY ADAPTED SHEEP BREEDS OF BRAZIL: A RFLP-PCR ANALYSIS LOOP) and microsatellites will be employed to increase the knowledge about the origins of Brazilian sheep breeds and the relationship with African breeds. REFERENCES Avise, J.C Molecular Markers, Natural History and Evolution. Chapman & Hall, Inc. 511 pp. Hiendleder, S., K. Mainz, Y. Plante and H. Lewalski Analysis of mitochondrial DNA indicates that domestic sheep are derived from two different ancestral maternal sources. No evidence for contributions from Urial and Argali sheep. Journal of Heredity, 89: Hiendleder, S., S.H. Phua and W. Hecht A diagnostic assay discriminating between two major Ovis aries mitochondrial DNA. Animal Genetics, 30: Hillis, D.M., C. Moritz and B.K. Mable Molecular Systematics. 2 nd edition. Sinauer Associates, Inc, Sunderland, 655 pp. Mace, G.M., T.B. Smith, M.W. Bruford and R.K. Wayne An overview of the Issues. In: Molecular Genetic Approaches in Conservation. (eds. Smith, T.B.; Wayne, R.K.), pp Oxford Press University, New York. Mariante, A. da S., M. do S.M. Albuquerque, A.A. Egito and C. McManus Advances in the Brazilian animal genetic resources conservation progamme. Animal Genetic Resources Information, 25: Miller, S.A., D.D. Dykes and H.F. Polesky A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16: Morais, O.R O melhoramento genético dos ovinos no Brasil: situação atual e perspectivas para o futuro. In: Simpósio Nacional de Melhoramento Animal, 3, Belo Horizonte. Anais. Belo Horizonte: FEPMVZ, Moritz, C Defining "Evolutionary Significant Units" for conservation. Trends in Ecology and Evolution, 9: Wood, N.J. and S.H. Phua Variation in the control region sequence of the sheep mitochondrial genome. Animal Genetics, 27: Archivos de zootecnia vol. 54, núm , p. 399.