The contribution of goats to the national meat supply in Indonesia is only 2.9% while goats population increase from 2003-2007 was just around 2-3% per annum. The goats population in Indonesia is approximately 13.491 million and involves 2.7 million farmers (Anonymous, 2015). Among local goat in Indonesia, Ettawa grade goat is one of potential asset that can be developed due to their dual purpose in milk and meat production. The Ettawa grade goats originally descended from cross-breeding between Kacang and Ettawa goats. This breed consists of three groups: Black head hair with white body, brown head hair with white body and mix color. To increase the income, the farmers prefer keep the goat with black head and neck color for competition purpose rather than for milk purpose due to the high price compare to brown color. Even though, farmers believed that color differences will not have impact on the productivity of goats. Initially, these perception need to be confirmed based on genetic molecular basis, which can describe the distribution of allele of gene, which responsible to the hair color differences. In order to detect the genotype variability of the type of both goats, an analysis of gene polymorphism need to be conducted. Furthermore, this molecular approach is of major significance for many fundamental knowledge and applied areas of animal genetics. This advent of gene technology leads to give basic evidence to prove the group of farmer perceptions. This study also gives an implication of socio-economic policy to gain the benefit of farmers.
The melanocortin-1-receptor (MC1R) gene plays a central role in regulation of animal coat color formation. The gene has been widely used to identify the coat color in various ruminants such as in sheep, cattle, goat and rabbit. The haplotype AATGT in MC1R was uniquely associated with black coat color in minxian black-fur breed (Yang et al., 2013). The coat color extension gene (E), which encoded the transmembrane domain MC1R have been indicated affecting the coat color in French cattle breed (Rouzaud et al., 2000). In goat, the p.267W missence mutation located in coding region of MC1R was present in all Murciano-Granadina black goats, whereas it was never identified in the brown ones (Fontanesi et al., 2009). The c.[124A, 125_130del6] was suggested responsible for a MC1R variant determining eumelanin production in the black area of the rabbit (Fontanesi et al., 2010). Moreover, the mutation at the position 676 bp of MC1R gene had been detected in Boer goat with red head and neck color (Wu et al., 2006). Therefore, the objective of this study was to identify the genetic profile of Ettawa grade goat with different head color based on MC1R gene. Due to the cross bred nature of Ettawa grade goats, the research hypothesis is that the genotype is heterozygote.
MATERIALS AND METHODS
Animal and sampling: Thirty Ettawa grade goats, which divided in three groups, CP: Brown head and neck color with white body color, RP: Brown or black head and neck color with various body color and HP: Black head and neck color with white body color were reared in the field laboratory in Faculty of Animal Science, Universitas Gadjah Mada (FAS UGM) with the same environments condition. The blood samples were collected for genomic DNA isolation using gSYNC DNA Extraction Kit (Genetika Science, Indonesia).
DNA amplification and genotyping: The primer sequences according to Wu et al. (2006), annealing temperature for PCR amplification and the restriction enzyme for PCR-RFLP are shown in Table 1. Amplifications were performed in 10 min at 94̊C for pre-denaturation, 30 sec at 94̊C for denaturation, annealing at 64̊C for 30 sec, elongation (extension) at 72̊C for 30 sec and a final extension at 72̊C for 10 min. The PCR products were visualized in 1.5% standard agarose gels stained with ethidium bromide.
The PCR products were sequenced using the same primers for PCR by PT Genetika Science, Indonesia. The DNA sequences were analyzed with the BioEdit program ver. 7.00 (Tom Hall, Ibis Therapeutics, California, USA) and the SNPs was confirmed based on the electrophoregram results. The SNP G.676A>G was used for genotyping using PCR-restriction fragment length polymorphism (PCR-RFLP) method. The restriction enzyme digestion was performed in 20 μL reaction volumes with approximately 15 μL of PCR products and 2 units of each restriction enzyme. The digested products were run on 3% agarose gels.
Statistical analysis: A chi-square test was performed to test the allele and genotype frequencies for Hardy Weinberg equilibrium.
||Primers, annealing temperature and restriction enzyme for genotype of Ettawa grade goat
The following mathematical model was:
where, χ2 is chi-square value, Oi is observed frequency, Ei is expected frequency and n is the number of possible outcomes of each event.
RESULTS AND DISCUSSION
The SNPs in MC1R gene was initially identified by direct sequencing using PCR product pool. The SNP was confirmed by BioEdit program. As the results, three SNPs of MC1R gene were identified: SNP 676A>G, SNP 748T>G and SNP 801C>G (Fig. 1a, b). Among three SNPs, the SNP 676A>G and SNP 748T>G were found to have amino acid variants. The amino acid variant K-E (lysine to glutamic) was detected in SNP 676A>G and variant F-V (phenylalanine to valine) in SNP 748T>G. However, no amino acid variant have been detected in SNP 801C>T. Similar result have been observed in Boer goat that the amino acid variant K-E detected in SNP 676A>G (Wu et al., 2006). In sheep, two nonsynnonymous mutations previously associated with coat color (c.218 T>A, p.73 Met>Lys c.361 G>A, p.121 Asp>Asn) and three synonymous mutations (c.429 C>T, p.143 Tyr>Tyr; c.600 T>G, p.200 Leu>Leu, c.735 C>T, p.245 Ile>Ile) were identified in the CDS of MC1R gene (Yang et al., 2013).
The SNP 676A>G was used for genotyping using PCR-RFLP. Homozygote AA and GG were defined when the fragments size being recognized at 163, 253 and 416 bp, respectively. The heterozygote AG existed by PCR-RFLP method at the same position of the homologous chromosome with 163, 253 and 416 bp of fragments size (Fig. 2). The results showed most of animals in three groups have heterozygote (AG) genotype. Only one animal have GG genotype in HP group. Based on Table 2, the results indicated the spread of both alleles A and G were equal in all groups. The A and G allele frequencies were similar 0.5 in both CP and RP group. However, the A allele frequency (0.55) was slightly higher than G allele (0.50) in HP group.
|| Identification of three SNPs based on (a) Electrophoregram and (b) Alignment sequence of PCR products
PCR-RFLP genotyping results for SNP 676A>G of MC1R in Ettawa grade goats, heterozygote animal with AG genotype was defined when the fragment size being recognized with 163, 253 and 416 bp and GG genotype with 416 bp
|Table 2:||Frequencies allele and genotype of three groups Ettawa grade goats based on PCR-RFLP results using SNP g.676A>G
|CP: Brown head and neck color with white body color, RP: Brown or black head and neck color with various body color, HP: Black head and neck color with white body color|
The AG genotype frequencies were similar 1.00 in both CP and RP group. This suggested that both allele A and G of the MC1R gene may control the black and red color in head and neck of Ettawa grade goat since, they are descended originally from crossing between the Kacang and Ettawa goats. Most of Kacang goats have red (brown) color and Ettawa have mix black and white color. Thus, most of goats in CP and RP groups are heterozygote genotypes. However, no AA genotype was identified in the study. Compare to Wu et al. (2006) using similar SNP, three genotypes of Boer goats have been detected. The AA genotype indicated has association with red head and neck color in Boar goats. Breed differences may due to pattern genotype variances.
The results of Pearsons chi-square (χ2) test indicated that the genotypes of the goats were deviated from the Hardy-Weinberg equilibrium (HWE) since χ2>3.84 (5% significance level for 1 degree of freedom). The deviation may due to variation of causes. Mutation, gene flow, non-random mating (Assortative mating), genetic drift and selection may lead to deviate from HWE (Falconer and Mackay, 1996). In case of this study, non-random mating and small sample size may due to the deviation.
In conclusion, all the groups of Ettawa grade goats with different head and neck color have equal allele distribution and same heterozygote genotypes based on MC1R gene. These results may give evidence to group of farmer perceptions in order to select their animal. Furthermore, these results also suggest that the SNP 676A<G of MC1R may control the genotype profile of Ettawa grade goats.
This molecular approach is of major significance for many fundamental knowledge and applied areas of animal genetics. This advent of gene technology leads to give basic evidence to prove the group of farmer perceptions. This study also gives an implication of socio-economic policy to gain the benefit of farmers.
This study was supported by a grant namely "Competency Research Grant 2015" from The Indonesian Directorate General of Higher Education (DIKTI) with contract No. 203/LPPM/2015. Furthermore, all the authors report no financial interests or potential conflicts of interest in this study.