Association of single nucleotide polymorphisms in the insulin and growth hormone gene with growth traits of Mia Chicken

Local chicken breeds play an important role in contributing to the total income of farm households in the developing countries. They make up from 50% to 70% of the country’s total chicken population (Berthouly et al., 2010; Desvaux et al., 2008; Hanh et al., 2007). The native chicken meat, with a traditional breeding method, is preferred by local consumers due to better taste, good meat texture, low fat and cholesterol and a high protein content (Promwatee et al., 2013). However, the native chickens generally show a slower growth, a higher FCR and a higher production cost than commercial broilers (Minh et al., 2006; FAO, 2008). 
        
According to Moula et al., (2011), indigenous breeds were estimated to represent over 80% of the total chicken flock of Vietnam. The native chickens, with more than 30 breeds, are a flagship of the Vietnamese domestic animal breeds. (Minh et al., 2006; Moula et al., 2011; Pham et al., 2013). The Mia breed, considered as one of the ancient Vietnamese breeds, is classified as threatened with extinction, partly due to modest production performance. Body weight of Mia chicken at 7 weeks of age is only 494 g and and FCR is higher than (4.29) other exotic breeds (Ly et al., 2001). Consequently, conservation of this Vietnamese heritage necessitates a selection for the improvement of growth performance.
        
Recently, application of molecular genetics in livestock breed selection for improvement of a number of important production traits has allowed obtaining better selection performances than phenotypes-only based methods. The selection based on candidate genes not only improves the selection of the favorable alleles, but also significantly shortens the selection time (Dekkers, 2004; Nadaf et al., 2007; Qiu et al., 2006; R. Jiang et al., 2004). The chicken insulin (INS) and growth hormone (GH) genes are among the candidate genes for growth performance in chickens (Lei et al., 2007; Nie et al., 2005; Qiu et al., 2006; Zhang et al., 2007). Insulin is a peptide hormone secreted by the β cells of the pancreatic islets of Langerhans. The effect of insulin is to enhance the uptake of glucose into the cells, where it is metabolized and stored as glycogen or used as an energy substrate in the synthesis of proteins or fats (Hadley, 2000; Wilcox, 2005). Therefore, the INS gene is usually considered as a candidate gene in the genetic analysis of animal complex traits like growth rate, body composition and fat deposition (Amills et al., 2003; Qiu et al., 2006). The GH gene encodes a mature growth hormone protein with 191 amino acids and a signal peptide with 25 amino acids (Tanaka et al., 1992). It is a polypeptide hormone that is secreted from the anterior pituitary gland and plays a crucial role in growth and metabolism rates of the chickens (Vasilatos-Younken et al., 2000). The present study was to determine the relationship between the four SNP of INS and GH genes and growth traits of Mia chicken.

Animals and sample collection
 
A total of 128 Mia chickens at 1 week of age belonging to the Breeding Center of Vietnam National University of Agriculture were used in this study. All birds were raised in floor pens and given commercial feed (commercial corn-soybean diets meeting National Research Council requirements) and water ad libitum. One and half milliliter blood samples were collected from the wing veins in tubes containing EDTA. All samples were stored at 4°C for DNA extraction.
 
DNA extraction, amplification and genotyping
 
DNA extraction from the blood sample of 128 chickens was performed using the method of Albariño et al., (1994) with slight modifications. The concentration and purity of DNA was checked on 1% agarose gel and measured using ODA260/A280. Then, DNA was diluted at a concentration of 50 ng/µl.
        
Two pairs of primers for the A3971G and T3737C SNP in the INS gene (Qiu et al., 2006) and two other for G662A and C423T in the GH gene (Nie et al., 2005) were used to amplify the gene fragments. However, systems and conditions of reaction slightly differed from what were previously reported (Qiu et al., 2006; Nie et al., 2005). Information on primer sequences and restriction enzyme are shown in Table 1.
 

        
The PCR amplification was performed using 50ng of genomic DNA, 1.5mM MgCl₂, 0.2 mM dNTPs, 0.5 µM primers and 2U of Taq DNA polymerase and PCR buffer in a 25 µl final volume. Amplification conditions of the genes were 3 min at 94°C, followed by 35 cycles of 30s at 94°C, 45s at certain annealing temperatures (ranged from 58°C to 60°C for each primer), 1 min at 72°C and a final extension of 5 min at 72°C. The PCR products were analyzed on a 1% agarose gel to assess the correct size and quality of the fragments. The lengths of the PCR product are 280bp, 370bp, 466bp and 518bp for A3971G, T3737C of the INS gene and G662A, C423T in the GH gene, respectively.
        
Then, the products were digested by MspI for A3971G, T3737C of the INS gene and G662A of the GH gene and PagI for C423T in the GH gene in a total volume of 20µL of solution, containing 8µL of PCR product, 3.0 units of restriction enzymes, buffer and H₂O volume of 20µL of solution. The sample was then incubated at 37°C for 8 hours. The SNP genotype of each individual was determined by 3% agarose gel electrophoresis after restriction digestion.
 
Measurement of growth traits
 
Each animal was identified individually. Chickens were weighed individually at week 2, 4, 6, 8, 10, 12, 14 and 16. The average daily gains (ADG) were calculated according to the ratio of total growth to the total number of days of feeding.
 
Statistical analysis
 
Genotype and allele frequencies were calculated. Hardy-Weinberg equilibrium was tested by comparing expected and observed genotype frequencies using a Chi-squared test (c²). P-values < 0.05 were considered as significant. The analysis of the association of the INS and GH genes with growth traits was performed using a general linear model (GLM) with the SAS software, version 9.1 to identify significant sources of variation. The following statistical model was used:

 
                                y_ij = μ + G_i + ε_ij,

 
where:
y_ij is the observed trait value for the j^th animal;
µ is the population mean value;
G_i is the individual genotype effect for genotype i of either A3971G INS gene, T3737C INS gene, G662A GH gene or C423T GH gene;
ε_ij is the random residual for this observation.

Polymorphisms of INS and GH genes
 
For SNP A3971G INS gene, two specific alleles were found to be a 281 bp fragment for allele A and fragment of 233 for allele G. The three different genotype patterns of AA, GA and GG among Mia chicken are shown in Fig 1. The T3737C SNP was digested with the restriction enzyme MspI and two specific alleles were identified, a 372 bp fragment for allele T and two fragments of 234 bp and 138 bp for allele C. In our sample of the Mia chicken breed, only two genotypes (TT and TC) were observed.
 

        
For the GH gene, two polymorphisms were segregating in the Mia breed. For the G662A SNP, the three genotypes, namely GG (226, 125 and 115 bp), AG (240, 226, 125 and 115 bp) and AA (240 and 226 bp) were identified. The C423T SNP was digested with the restriction enzyme PagI and three genotypes were identified, a 518 bp uncut fragment for genotype TT, two fragments of 308 and 210 bp for genotype CC and three fragments of 518, 308 and 210 bp for the TC genotype.
 
Genotype and allele frequencies
 
All genotype and allele frequencies of the chicken INS and GH genes of Mia breed are shown in Table 2. The three possible genotypes of the SNP A3971G of INS gene, G662A and C423T of GH were observed while only 2 genotypes were obtained for the SNP T3737C of INS gene. In Mia breed population, allele A of A3971G INS gene, T of T3737C INS gene, A of G662A GH gene and C of C423T GH gene represent the major allele. Several previous studies indicated that there was association between A3971G polymorphism of INS gene and weight gain and carcass yield traits of chicken, with allele G contributing to improve weight gain, and presented with low frequency (Amills et al., 2003; Lei et al., 2007; Qiu et al., 2006). The result of polymorphism A3971G INS gene of low growth rate as Mia chicken showed that low frequencies of GG genotype were also found. For the T3737C SNP of INS, no CC genotype was observed. In previous studies, the CC genotype was observed with quite low frequency: 1% in the Coob lines and 3% in the Noi chicken breed (Khoa et al., 2013). The reason of non-occurrence of the CC genotype in this study is probably due to the low frequency of the C allele in this population and the small sample size (expected number of CC under HWE conditions is only 1.72). At G662A of GH gene, the AA genotype frequency in Mia breed was higher (0.50) as compared to the GG frequency (0.03). This result is consistent with a previous study, which showed that the AA genotype was major and GG was minor in local chicken breeds in Vietnam (Khoa et al., 2013). At the C423T SNP of GH gene, CC genotype had a higher frequency than TT. The result of Chi-square (c²) testing indicated that all genotype frequencies for each locus of INS and GH genes in Mia breed were found to be in Hardy-Weinberg equilibrium (P>0.05).
 

 
Associations of INS and GH polymorphisms with growth traits of Mia chicken
 
The association of the INS and GH genes with growth traits is shown in Table 3 and 4. There was no significant association of the INS A3971G SNP with any growth traits in the Mia chicken. The T3737C genotypes were significantly associated with BW at 10, 11 and 12 weeks and ADG at 6-8 and 12-weeks of age (P<0.05). Mia chicken with TT genotype showed higher BW and ADG compared to the CT genotype.
 

 

        
According to Qiu et al., (2006) for the crossing of Chinese native Xinghua chickens and White Recessive Rock chickens, the A3971G and T3737C genotypes were associated with BW at 28 and 56 days of age (4 and 8 weeks of age). In the present study, no significant different among three genotypes of the polymorphism A3971G of INS gene but a significant association was found in the polymorphism T3737C of INS gene. Mia chicken with TT genotype has higher BW compared to the CT genotype at 10, 11 and 12 weeks of age (P<0.05).
        
As shown in Table 4, GH G662A genotypes were significantly associated with later BW (from 7 to 16 weeks of age) and ADG (4-6; 6-8; 8-10; 10-12 and total 2-16 weeks of age) of Mia chicken (P < 0.05). Mia chicken with GG genotype had significantly higher BW and ADG values than with the AA and AG genotypes. The presence of the G allele seems to be associated with a positive effect on both BW and ADG. For GH C423T, there was no significant association of the SNP genotypes with any growth traits in the Mia chicken (P > 0.05).
        
The chicken GH gene has numerous SNPs, some of which have been associated with body weight and skeletal growth (Nie et al., 2005). Mehdi and Reza (2012) observed that SNP at G662A had a significant effect on body growth. Our result are consistent with previous studies of Mehdi and Reza (2012). The GH gene is one of the most important genes affecting the chicken growth performance traits and plays a critical role in both growth and metabolism rates (Feng et al., 1997; Vasilatos-Younken et al., 2000). In the present study, the significant association identified between the G662A SNP of GH gene and growth traits seems to indicate that this gene could play an important role in the growth traits of Mia chicken.
        
In conclusion, it was found that polymorphisms of the A3971G INS gene and the C423T GH gene show no association with growth traits of the Mia chicken. The SNP T3737C INS gene was associated with BW at 10 to 12 and ADG at 6-8 weeks of age. The SNP G662A GH gene was significantly associated with later BW (from 7 to 16 weeks of age) and ADG (4-6; 6-8; 8-10; 10-12 and total 2-16 weeks of age) of Mia chicken (P < 0.05). Although currently infrequent in the population, the GG genotype corresponds to higher BW and ADG than the other genotypes. However, before the G662A SNP of the GH gene can be used as a candidate marker gene for genetic improvement of growth traits in Mia chicken breed, further investigation with large sample size is necessary.