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Vol. 4, N.3: pp. 276-282, August, 2013 ISSN: 2179-4804

Journal of Biotechnology and Biodiversity

Genetic Variability of Guzerat and Senepol Bovine Breeds

by Microsatellite Markers

Ednaldo da Silva Filho1,*, Yasmin Martins dos Santos1, Yvana Melyssa Mandú Margarido1, Telmo José Mendes2, Luiz Marcelo de Lima Pinheiro3, Paulo Henrique de Souza1, José Elivalto Guimarães Campelo 4

ABSTRACT

Bovine production plays economic importance in Brazil and Guzerat and Senepol breeds are producer of meat. It was aimed to analyze the genetic variability of Guzerat and Senepol breeds by microsatellite markers. The breeds were collected and genotyped for ten microsatellite loci by automatic sequencer and statistically analysed. A total of 53 alleles were observed being the average number was 5.3 in both breeds. The effective numbers of alleles were 3.36 for Guzerat and 3.11 for Senepol cattle. The Shannon indexes were 1.36 for Guzerat and 1.26 for Senepol cattle. The expected heterozigosity were 0.71 and PIC values were 0.64 in both breeds. The FIS were 0.01 and 0.11 for Guzerat and Senepol breeds, respectively and Hardy-Weinberg equilibrium were P>0.05 for Guzerat and P<0.05 for Senepol cattle. The combined discrimination powers were 0.99 in both breeds and combined exclusion powers (PE1) were 0.99 in both breeds and combined exclusion powers (PE2) were 0.96 and 0.95 for Guzerat and Senepol breeds respectively. There is genetic variability in both breed, but there are evidences of inbreeding enabling genetic drift and should be necessary to use major number of microsatellite loci to analyze with high efficiency the exclusion power (PE2).

KEYWORDS:Cattle, STRs, Diversity, Livestock conservation.

Variabilidade Genética de Bovinos das Raças Guzerá e

Senepol por Marcadores Microssatélites

RESUMO

A bovinocultura desempenha função importante na economia Brasileira e as raças Guzerá e Senepol são produtoras de carne. Objetivou-se analisar as variabilidades genéticas das raças Guzerá e Senepol através dos marcadores microssatélites. Os animais foram coletados e genotipados para dez loci microssatélites em um sequenciador automático de DNA e analisado estatisticamente. Um total de 53 alelos foi observado com número médio de 3,36

para Guzerá e 3,11 para a raça Senepol. Os índices de Shannon foram 1,36 para Guzerá e 1,26 para raça Senepol. As

heteroziosidades esperadas foram 0,71 e os valores de PIC foram 0,64 em ambas as raças. Os valores de FIS foram

0,01 e 0,11 para Guzerá e Senepol, respectivamente e as proporções de Hardy-Weinberg foram não significativas em Guzerá (P>0,05) e significativas em Senepol (P<0,05). Os poderes de discriminação combinados foram 0,99 em ambas as raças e os poderes de exclusões combinados (PE1) foram 0,99 em ambas as raças e os poderes de exclusão combinados (PE2) foram 0,96 e 0,95 em Guzerá e Senepol, respectivamente. Existe variabilidade genética em ambas as raças, mas existem evidências de endocruzamentos por consequência da deriva genética e seria necessário

avaliar um maior número de loci microssatélites para aumentar a eficiência no poder de exclusão (PE2 ).

PALAVRAS-CHAVE: Gado, STRs, Diversidade, Conservação animal.

*Author for correspondence.

1Federal Rural University of Amazon, Zip Code 66.077-901,Belém-PA, Brazil, silva.filho@ufra.edu.br .

2 Federal University of Maranhão, Zip Code 65.500-000, Chapadinha-MA, Brazil.

3 Federal University of Pará, Zip Code 68.800-000, Breves-PA, Brazil.

4 Federal University of Piauí, Zip Code 64.049-550, Teresina-PI, Brazil.

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https://doi.org/10.20873/jbb.uft.cemaf.v4n3.silvafilho

Silva-Filho, E.et al. 277

INTRODUCTION

Bovine livestock in Brazil is a factor of the great economy importance, principally the

beef cattle. Thus, Brazil is the country that has a biggest production of bovine livestock

in the world. The official number of bovines that has been registered in Brazil by “Pesquisa

da Pecuária Municipal” of “InstitutoBrasileiro deGeografia e Estatística” (IBGE, 2011) is

about 212 million of animals. This number is considering animals used for both meat and dairy producti on.

Genetic variability of resources it is, actually, a world preoccupation to future for next generations, principally for genetic resources which serve as a food, both animal and vegetable origin. Moreover, the importance to maintain the genetic background as genetic variability source may to contribute with preservations of breeds and in improvement animal programs (Silva Filho et al., 2007). With advancement of molecular biology has

been very importance to study the genetic

variability, through many tools. Thus, several studies about genetic variability in Brazilian

bovines were made using differences tools, from RAPD (Serrano et al., 2004) to

microsatellite markers (Egito et al., 2007). Actually, many papers are being published in

differences sciences journals disseminating the current genetic structure of some Brazilian bovine breeds using Microsatellites markers (Carneiro et al., 2007).

Microsatellite DNA is a class within of the repetitive sequences of DNA in eukaryote

applied a lot in conservations studies of

various organisms, from vegetable to animals. There are several advantage to use the

microsatellite markers in detriment others molecular markers. Initially, microsatellites

were applied in mapping genomic projec t (Weissenbach et al., 1992). It has been

extensively used in association analyses of genes to disease susceptive (Rubinsztein

1999) and demonstrated extreme efficiency in paternity analyses of individuals (Edwards et al., 1992) and populations studies (Paetkau et al., 1995). Variation in microsatellite is used in hybridizations studies between correlated species (Roy et al., 1994). Therefore, it was aimed to analyze the genetic variability of two bovine breeds produced in Brazil, Senepol and Guzerat breeds, being a taurine and a zebuine respectively, utilizing microsatellite markers used in paternity exams routine recommend by International Society of Animal Genetics (ISAG).

MATERIALS AND METHODS

A total of 58 animals were casually selected from two differences Brazilian herd, being 29

animals of Senepol breed (Taurine) from a farm in Uberlândia town of Minas Gerais

State and 29 animals of Guzerat breed (Zebuine) from a farm in Imperatriz town of Maranhão State. There were collected 5 mL of blood tissue by vacutainer blood collection and after they were conserved on freezer at - 20°C to extraction of DNA. Extraction of

genome and it is better known among DNA was realized by

markers. They are sequences of genome that Phenol:Chloroform:Isoamyl Alcohol

consist of repeats from one to nine nucleotides in many copies in tanden found in vertebrates, insects, fungi and vegetables (Charlesworth et al., 1994). Historically, the microsatellite term is applied in rep etitions

with two nucleotides (CA) and (GT) and

various others names are used to describe these repetitive sequences in tanden as Simple

Sequence Repeat (SSRs) or Short Tanden Repeat (STRs) (Edwards et al., 1991).

Recently, microsatellites markers are bein g

(25:24:1) method Sambrook et al., (1989). A set of ten loci of microsatellite markers were selected from loci used to paternity exam routine in bovine recommended by International Society of Animal Genetics

(ISAG): BM1818, BM1824, BM2113,

ETH10, ETH225, ETH3, INRA023, SPS115, TGLA122 and TGLA126 loci .

PCR reactions were realized in Multiplex System for a final volume of 15 µL under the

following conditions: Promega’s STR 10X

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Silva-Filho, E.et al. 278

Buffer (1X); MgCl2 (1.5 mmol); every forward primer were marked using fluorescent dyes (FAM, HEX or TAMRA) (1 -

4 pmol) and reverse primer unlabeled (1- 4 pmol); Platinum Taq DNA polymerase®

(Invitrogen, USA) (0.5 unit) and genomic DNA (100 ng/µL). PCR reactions were in a

Mastercycler PCR Thermocycler (Eppendorf, Germany) under the following conditions: hot

start at 94°C for 4 min; 30 cycles of 94°C for 1 min, 58°C for 1 min and 72°C for 1 min; final extension temperature at 72°C for 4 min. Products of PCR were separated and detected in the ABI Prism® 3100 Genetic Analyzer (Applied Biosystems) following the manufacturer’s standard protocol. Data was analyzed with GeneScan® v3.7 and Genotyper® v3.7 software (Applied Biosystems).

POPGENE 1.32 software (Yeh et al., 1999) was used to determine the allelic frequencies per locus as well as some diversity indexes as:

Number of observed alleles (Na) per locus ;

number of effective alleles (Ne) per locus and Shannon index (SI). Expected heterozygosity

(He) per locus was estimated according to

1984) within of each population using

GENEPOP 3.1d software (Raymond and Rousset, 1995) .

Power of discrimination (PD) per locus was estimated according to Kloosterman et al.

(1993). Probability of exclusion (PE) was determined in two situations: (PE1 ),

estimated by 1a equation, when the genotypes of matrix, offspring and bull are compared,

this equation is appropriated when the matrix is confirmed as genitor at time of birth and (PE2), estimated by 2a equation, when at least one parental genotype not available (Jamieson and Taylor, 1997). Values per locus of PD , PE1 and PE2 were combined (PDc and PEc ) to all loci evaluated (k loci) according to Fisher (1951).

RESULTS AND DISCUSSION

A total of 53 alleles were observed for ten loci

analyzed in both breeds. However, the less polymorphism loci were BM1824 and

INRA23, in the Guzerat and Senepol breeds, respectively, while the most polymorphism

loci were BM1818 in Guzerat breed and

Ney and Roychoudhury (1974). BM2113 and TGLA126 in Senepol breed.

Polymorphism Informative Contend (PIC ) was estimated according to Botstein et al.

(1980).

A exact test was used to determine possible deviations of Hardy-Weinberg equilibrium (HWE) and existence of random associations of genotypes applying Markovian Chain method as well as it was determined the inbreeding coefficient (FIS) (Weir and Cocker,

The average proportion of effective allele in relation to observed allele was about 0.60 in

the two breeds. Therefore, TGLA126 locus was the great proportion of effective allele in relation to observed allele (0.87) in Guzerat breed, while BM1818 (0.77) and BM1824 (0.77) loci were in Senepol breed (Tab. 1 and 2).

Table 1. Parameters of diversity in Guzerat breed. Loci Naa Neb SI c

He d

PIC e

Fis f

PHWE g

BM1818 7 3.68 1.57 0.75 0.70 0.25 0.07

BM1824 3 2.16 0.91 0.56 0.48 -0.05 1.00

BM2113 6 4.75 1.65 0.82 0.76 0.02 0.88

ETH10 5 2.59 1.17 0.64 0.56 -0.05 0.15

ETH225 6 4.00 1.56 0.78 0.72 -0.01 0.48

ETH3 5 3.40 1.36 0.73 0.65 0.09 0.92

INRA23 6 2.07 1.08 0.53 0.49 -0.01 0.11

SPS115 5 3.86 1.41 0.77 0.69 -0.13 0.81

TGLA122 6 3.64 1.54 0.75 0.70 0.06 0.51

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Silva-Filho, E.et al. 279

TGLA126

Average Sd h

4

5.30 1.16

3.48

3.36 0.85

1.30

1.36 0.24

0.74

0.71 0.10

0.66

0.64 0.10

- 0.10

0.01 0.11

0.44 0.25 i

aNumber of observed alleles, bnumber of effective alleles, cShannon index, dexpectedheterozigosity, e polymorphism informative contend, fdeficit of heteroziosity, gProbability for Hardy-Weinberg equilibrium, hstandard deviation and

iprobability for all loci .

Table 2. Parameters of diversity in Senepol breed.

Loci Naa Neb SIc Hed PICe Fisf PHWE g

BM1818 5 3.84 1.45 0.76 0.69 0.27 0.04

BM1824 4 2.18 0.96 0.56 0.49 0.62 039

BM2113 7 5.22 1.73 0.84 0.78 -0.13 0.26

ETH10 5 3.83 1.47 0.77 0.70 -0.01 0.26

ETH225 5 2.16 1.10 0.55 0.50 0.31 0.00

ETH3 5 3.37 1.33 0.73 0.66 -0.26 0.04

INRA23 3 1.42 0.57 0.32 0.29 0.33 0.02

SPS115 6 2.57 1.21 0.64 0.58 0.00 0.55

TGLA122 6 3.05 1.31 0.69 0.61 -0.16 0.70

TGLA126 7 3.48 1.46 0.74 0.67 0.10 0.37 Average 5.30 3.11 1.26 0.71 0,64 0.11 0.00 i

Sdh 1.25 1.08 0.32 0.15 0.14 0.27

aNumber of observed alleles, bnumber of effective alleles, cShannon index, dexpectedheterozigosity, e polymorphism informative contend, fdeficit of heteroziosity, gProbability for Hardy-Weinberg equilibrium, hstandard deviation and

iprobability for all loci .

Average Shannon index (SI) were over 1 in the two breeds. The expected heterozygosity

varied from 0.53 to 0.82 in Guzerat breed, while Senepol breed varied from 0.32 to 0.84

and the average heterozygosity were over 0.70 in two breeds. The values of PIC var ied

from 0.48 to 0.76 in Guzerat breed, while Senepol varied from 0.29 to 0.78 and the average heterozygosis were over 0.60 in both breeds (Tab. 1 and 2).

The inbreeding coefficients (FIS) varied from - 0.10 to 0.25 in Guzerat breed, while Senepol breed varied from -0.26 to 0.62. All breeds had positive average heterozygosis deficit indicating presence of consanguine breeding. Nothing locus had significant (P>0.05)

deviation from HW equilibrium in Guzerat breed, while Senepol breed had some loci

with significantdeviations from HWE ( P <0.05) (Tab. 1 and 2).

The values of PD parameter varied from 0.70 to 0.90 and combined of 0.99 in Guzerat

breed, while Senepol breed varied from 0.43 to 0.89 and combined of 0.99. The PE1 values varied from 0.28 to 0.58 and combined of 0.99 in Guzerat breed, while Senepol breed varied from 0.16 to 0.62 and with combined of 0.99. The PE2 values varied from 0.14 to 0.35 and with combined of 0.96, while Senepol breed varied from 0.05 to 0.45 and combined of 0.95 (Tab. 3).

Table 3. Analyses of discrimination and exclusion powers.

Loci

PD

Guzerat breed PE1

PE2

PD

Senepol breed PE1

PE2

BM1818 0.81 0.52 0.33 0.87 0.50 0.33

BM1824 0.72 0.28 0.14 0.72 0.30 0.15

BM2113 0.90 0.58 0.41 0.89 0.62 0.45

ETH10 0.74 0.36 0.20 0.87 0.52 0.34

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Silva-Filho, E.et al. 280

ETH225 0.80 0.53 0.35 0.66 0.33 0.16

ETH3 0.87 0.45 0.28 0.78 0.46 0.29

INRA23 0.70 0.32 0.15 0.43 0.16 0.05

SPS115 0.86 0.49 0.32 0.79 0.39 0.22

TGLA122 0.87 0.52 0.33 0.80 0.42 0.26

TGLA126 0.83 0.45 0.28 0.85 0.48 0.31

Combined 0.99 0.99 0.96 0.99 0.99 0.95

Comparing the polymorphism with those

observed in some European breeds (Kantanem et al., 2000), they were very

similar and compared with Brazilian breeds as: Nelore that was more polymorphic than this work (Cevini et al., 2006; Carneiro et al., 2007). O SI indicated high genetic variability (over 1.0) as affirmed by Fatima et al. (2008) evaluating goats breeds and by Arif et al. (2010) evaluating antelope breeds using microsatellite markers. Average expected heterozigosities and average PIC indicated high genetic variability in both breeds as compared with the taurine and zebuine breeds created in Brazil, including the Guzerat breed (Egito et al., 2007). Same conditions were

observed by Carneiro et al. (2007) analyzing

Nelore breed on Amazon conditions. The deficits of heterozigosity were literally same

those observed in some breeds as Hariana and Hissar (Rehman and Khan, 2009), Brown

Swiss breed (Dalvit et al., 2008), Hallikar cattle (Shekar et al., 2011) and taurine and

zebuine breeds from Brazil (Egito et al., 2007). Therefore, some breeds were not observed deficits of heterozigosity as Kangayam cattle (Karthickeyan et al., 2009) and Burlina and Hostein Frisian breeds (Davit et al., 2008). Although all loci have showed considered variability, they were viable for analyses of discriminations as those observed in the Nelore breed (Carneiro et al., 2007) and Yugoslav Pied cattle (Stevanovic et al., 2010), but should be necessary to extend the number of loci to have more efficiency in paternity analyses (Silva Filho et al., 2007).

CONCLUSION

All breeds analyzed indicated considerable genetic variability using 11 microsatellite

loci, but the deficit of heterozigosity may be

due to consanguine breeding within each breed being necessary the use of molecular

markers assisted selection (MAS) by animal improvement program and to use more microsatellite loci in these breeds for better efficiency in paternity analyses.

ACKNOWLEDGMENTS

We thank all the owners of the farms that kindly gave samples of the two breeds.

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