Archivos de Zootecnia ISSN: Universidad de Córdoba España

Archivos de Zootecnia

ISSN:

0004-0592

pa1gocag@lucano.uco.es

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. 206-207, 2005, pp. 395-399

Universidad de Córdoba

Córdoba, España

Available in: http://www.redalyc.org/articulo.oa?id=49520743

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

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. Castro1_{, M.S.M. Albuquerque}1 _{and J.A. Dergam}3

1_{Embrapa Genetics Resources and Biotechnology. CP 02372, CEP 70770-900, Brasília, DF. Brazil.}

E-mail: samuel@cenargen.embrapa.br

2_{University of Brasília (UnB). FAV, CP 04508, CEP 70910-900, Brasília, DF. Brazil.}

3_{University Federal of Viçosa (UFV). Department of Animal Biology. CEP 36571-000, Viçosa, MG. Brazil.}

A

DDITIONALKEYWORDS

Cytochrome oxidase I. Genetic of conservation. Ovis aries.

P

ALAVRASCHAVEADICIONAIS

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 5562-5566 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 re-cursos 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

Archivos de zootecnia vol. 54, núm. 206-207, p. 396.

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 frag-mento 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 natura-lizadas brasileiras, como Somalis e Morada Nova tenham, provavelmente, origem Africana. Dessa maneira, os dados sugerem que as raças africa-nas 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

na-tural selection of European and African

sheep breeds brought to Brazil at the

time this country was settled by

Portu-gal 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

prefe-rentially 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

Conser-vation, 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

(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

mitochon-drial 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 5562-5566 were

consi-dered 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 Brasília1

, 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 248

Archivos de zootecnia vol. 54, núm. 206-207, p. 398.

PAIVA ET AL.

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

Portu-guese 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

-www.boletimpecuário.com.br). 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-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).

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. 1994. Molecular Markers, Natural History and Evolution. Chapman & Hall, Inc. 511 pp.

Hiendleder, S., K. Mainz, Y. Plante and H. Lewalski. 1998. Analysis of mitochondrial DNA indicates that domestic sheep are derived from two different ancestral maternal sources. No evidence for contributions from Urial and Ar-gali sheep. Journal of Heredity, 89: 113-120. Hiendleder, S., S.H. Phua and W. Hecht. 1999. A diagnostic assay discriminating between two major Ovis aries mitochondrial DNA. Animal Genetics, 30: 211-213.

Hillis, D.M., C. Moritz and B.K. Mable. 1996. Molecular Systematics. 2nd _{edition. Sinauer}

Associates, Inc, Sunderland, 655 pp. Mace, G.M., T.B. Smith, M.W. Bruford and R.K.

Wayne. 1996. An overview of the Issues. In: Molecular Genetic Approaches in Conser-vation. (eds. Smith, T.B.; Wayne, R.K.), pp. 3-25. Oxford Press University, New York.

Mariante, A. da S., M. do S.M. Albuquerque, A.A. Egito and C. McManus. 1999. Advances in the Brazilian animal genetic resources conservation progamme. Animal Genetic Resources Information, 25: 109-123. Miller, S.A., D.D. Dykes and H.F. Polesky. 1988.

A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research, 16: 1215.

Morais, O.R. 2000. O melhoramento genético dos ovinos no Brasil: situação atual e perspecti-vas para o futuro. In: Simpósio Nacional de Melhoramento Animal, 3°, Belo Horizonte. Anais. Belo Horizonte: FEPMVZ, 266-272. Moritz, C. 1994. Defining "Evolutionary Significant

Units" for conservation. Trends in Ecology and Evolution, 9: 373-375.

Wood, N.J. and S.H. Phua. 1996. Variation in the control region sequence of the sheep mito-chondrial genome. Animal Genetics, 27: 25-33.