STUDY OF POLYMORPHISM IN POPULATIONS OF TWO BRAZILIAN’S
CAIPIRAS CHICKENS LINEAGES USING THREE SSR MARKERS
ESTUDO DO POLIMORFISMO EM DUAS POPULAÇÕES DE LINHAGENS DE
GALINHAS CAIPIRAS BRASILEIRAS UTILIZANDO-SE TRÊS MARCADORES
SSR
Mari Helen Pagani Possamai1, Marcos Edgar Herkenhoff2, Fábio Pértile3, Ediane Paludo4,
Jaqueline Battilana5, Carlos André da Veiga Lima-Rosa6
Abstract:
This study aimed to investigate whether there was the maintenance of genetic variability
characteristic of birds in two groups of Brazilian’s caipiras lineage analyzing three
microsatellite loci. We collected blood samples from 92 birds of Paraíso Pedrês and Rubro
Negra. We used the technique of polymerase chain reaction (PCR) to amplify samples and
amplified products were subjected to electrophoresis on an automated sequencer. The average
alleles per locus were 22 and 21 for the line Paraíso Pedrês (PP) and Rubro Negra (RN),
respectively. The average observed and expected heterozygosity was 0,643 and 0,889 for PP
and 0,647 and 0,881 for RN. All analyzed loci were informative (PIC>0,5). We can conclude
that there was the maintenance of genetic variability of caipira’s birds in these lineages.
Keywords: native chickens, strains, microsatellites, genetic variability.
Resumo:
Este trabalho teve como objetivo investigar se houve a manutenção da variabilidade genética,
característica de aves caipiras, em duas populações de linhagens de galinhas caipiras
brasileiras através da análise de três lócus de microssatélites. Foram coletadas amostras de
sangue de 92 aves das linhagens Paraíso Pedrês e Rubro Negra. Foi utilizada a técnica de
reação em cadeia pela polimerase (PCR) para a amplificação das amostras e os produtos
amplificados foram submetidos à eletroforese em um sequenciador automático. A média de
alelos por loco foi de 22 e 21 para a linhagem Paraíso Pedrês (PP) e Rubro Negra (RN),
respectivamente. A heterozigosidade média observada e esperada foi de 0,643 e 0,889 para PP
e 0,647 e 0,881 para RN. Todos os lócus analisados foram informativos (PIC>0,5). Podemos
concluir que houve a manutenção da variabilidade genética das aves caipiras nestas linhagens.
Palavras-chave: galinhas nativas, linhagens, microssatélites, variabilidade genética.
Brazilian poultry industry began with the arrival of Portuguese settlers who brought to
Brazil the first examples of pure-bred birds. These birds have been released, providing a
random mating, emerging, so-called Brazilian’s caipiras chickens (Albino et al., 2005).
The Brazilian poultry development has evolved with the use of technologies in genetics and
health, leading to more productive birds, causing the free-range chickens, poor
productivity, stay forgotten in the yards of the interior (Albino & Moreira, 2006).
In early1980, there was a change in consumption habits of a portion of the population,
_______________________
1
Médica Veterinária, estudante do curso de pós-graduação em Ciência Animal da Universidade do Estado de
Santa Catarina, UDESC/CAV, Lages, SC.
2
Médico Veterinário, estudante do curso de pós-graduação em Ciência Animal da UDESC/CAV.
3
Médico Veterinário, estudante do curso de pós-graduação em Medicina Veterinária, UNESP.
4
Bióloga, estudante do curso de pós-graduação em Ciência Animal da UDESC/CAV.
5
Bióloga, Dra. Genética e Biologia Molecular, UDESC/CAV.
6
Médico Veterinário, Dr. Genética e Biologia Molecular, UDESC/CAV.
Correspondência: Av. Luiz de Camões, 2090, bairro Conta Dinheiro, cep 88.520-000, Lages, SC.
E-mail: [email protected]
enhancing the natural products, making the range chickens alternative high commercial
value (Gessulli, 1999). With the increased demand for these products was the creation of
strains hicks, birds that blend the hardiness and resilience of birds with the original hillbillies
industrial productivity (Albino et al., 2001). In Brazil, there are some strains produced for this
purpose, and have a greater genetic variability when compared to typical industrial poultry.
However, there are few studies of a scientific nature to support this feature. Microsatellites are
sequences of nuclear DNA composed of tandem repeat units. Most of them are repetitions of
1-6 nucleotides, are co-dominant inheritance, are easy to identify and present high
polymorphism (Kaya & Yildiz, 2008). The aim of this study was to investigate whether there
was the maintenance of genetic variability in populations of two strains of Brazilian freerange chickens, using three microsatellite loci.
Were collected blood samples from 92 birds of Paraíso Pedrês lineage (42) and Rubro
Negra (50), from Birds of Paradise Farm, the Itatiba, São Paulo. Were used primers labeled
with fluorophores for the three microsatellite loci, LEI0192, LEI0212, LEI0214. For the
amplification of desired fragments was performed PCR (Polymerase Chain Reaction) from
DNA extracted. These fragments were amplified after subjected to electrophoresis on an
automated sequencer, and then viewed and analyzed from the Gene Mapper software version
3.2.1. The calculation for the allele frequencies, observed and expected heterozygosity, and
fitness to the Hardy-Weinberg equilibrium were performed using the program Arlequin
version 3.5 (Excofier & Lischer, 2010). The Polymorphic Information Content (PIC) was
estimated by the program Cervus version 3.0 (Kalinowski et al., 2007).
The values of the polymorphisms analyzed are described in Table 1. In line Paraíso
Pedrês (PP) were found 16 alleles for locus LEI0192, 32 alleles for the locus LEI0212 and 18
for LEI0214 site. In line Rubro Negra (RN) 15 alleles were found for the locus LEI0192, 31
for LEI0212 and 17 to the locus LEI0214. The average alleles per locus was 22 and 21 for the
line PP and RN, respectively. The average observed heterozygosity (Ho) and expected (He)
was 0,643 and 0,889 for PP and 0,647 and 0,881 for RN. In the two populations were
observed deviations from Hardy-Weinberg equilibrium (HWE). The average Polymorphic
Information Content (PIC) for the PP line was 0,780 and 0,740 of strain RN.
Table 1 – Values of various parameters of polymorphism of three microsatellite loci
analyzed in two groups of Brazilian’s caipiras chickens lineages.
Paraíso Pedrês
Loco
LEI0192
LEI0212
LEI0214
Mean
Na1
16
32
18
22
T2(pb)
246-342
338-470
131-309
135,33
Ho3
He4
Valor de p5
s.d.6
PIC7
0,738
0,852
0,001
0,000
0,601
0,857
0,948
0,202
0,000
0,836
0,333
0,867
0,000
0,000
0,904
0,643
0,889
0,780
Rubro Negra
LEI0192
15
186-342
0,800
0,853
0,286
0,000
0,508
LEI0212
31
350-309
0,880
0,956
0,001
0,000
0,835
LEI0214
17
123-309
0,260
0,834
0,000
0,000
0,876
Mean
21
127,67
0,647
0,881
0,740
1
Number of alleles per locus, 2allele size base pairs; 3Observed heterozygosity,
4
Expected heterozygosity, 5Hardy-Weinberg equilibrium in situ (p> 0,05), 6Standard
deviation, 7Content information polymorphic.
The number of alleles per locus is obtained in accordance with the minimum number
of alleles suggested by Barker (1994), which must be greater than four. The average number
2
of alleles of the strains PP and newborn compared with values found by Berthouly et
al. (2008) in commercial birds, indicates that the development of these lineages must have
occurred to maintain genetic variability found in their ancestral hillbillies. The average values
of Ho and He were similar to that found in other breeds native to Ethiopia (Hassen et al.,
2009), and obtained higher values when compared to commercial chickens (Granevitze et al.,
2007). The selection and mating led to the formation of these strains were important reasons
for the occurrence of deviation from HWE. All markers had PIC values greater than
0,5. According to Botstein et al. (1980), markers with values above 0,5 indicate that a PIC
greater genetic information can be provided by locus.
From the results presented for these three markers, we conclude that there was the
maintenance of genetic variability of caipira’s birds in these lineages.
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