Polymorphism in the exon 7 to the 3’ flanking region of β-lactoglobulin (β-lg) gene in Turkish hair goat populations were investigated. The study was carried out including 233 hair goats using PCR-RFLP. Digestion of amplification product with SacII restriction enzyme revealed two alleles namely S1 and S2 (which was produced by a single nucleotide substitution) and three genotypes (S1S1, S1S2 and S2S2) in the studied population. The genotypic frequencies of S1S1 and S1S2 were almost equal. S2S2 genotype was found to be lower than other genotypes (S1S1 and S1S2) in the studied population. The allele frequencies of S1 and S2 at β-lg locus were 0.67 and 0.33 in hair goat population, respectively. Deviation from Hardy-Weinberg equilibrium was not detected.
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The first report of genetic polymorphism of bovine β-lactoglobulin (β-lg) was reported by Aschaffenburg and Drewry (1955). Since, then genetic polymorphism of milk proteins has been of considerable interest in animal breeding and in the dairy industry and possible relationships between milk protein polymorphism and production traits, milk composition and quality have been widely studied by Pagnacco and Caroli (1987), Ng-Kwai-Hang et al. (1990) and Baranyi et al. (1993). β-lg is a major whey protein in the milk of ruminants. It is also found in the milk of most mammals with exception of man, rodents and lagomorphs (Perez and Calvo, 1995). Until now, several variants of bovine and ovine β-lg have been described both at protein level and DNA level (Gaye et al., 1986; Erhardt, 1989; Elmaci et al., 2006). The two genetic variants (A and B) of β-lg were reported in goat milk (Moioli et al., 1998) at protein level. However, no variants resulting in amino acid substitution have been identified at DNA level in goat but Pena et al. (2000) and Yahyaoui et al. (2000) have described polymorphism in exon 7 and in the β-lg proximal promoter region of Spanish and French goats. β-lg gene polymorphism at the DNA level h as been a nalyzed by PCR-RFLP (Pena et al., 2000) and they also reported two novel genetic variants of the β-lg gene in goat breeds. β-lg polymorphism was reported in different goat populations at protein level in Turkey (Ozdil and Asal, 2002; Turkyilmaz, 2003; Gurcan, 2005) and two alleles, β-lgA and β-lgB, at β-lg locus in the studied goat populations were reported. However, no information has been reported in Turkish goats regarding β-lg polymorphism at the DNA level. Therefore, the aim of present research was to study the genetic polymorphism of the β-lg gene (exon 7) at DNA level in Turkish hair goat using PCR-RFLP method.
MATERIALS AND METHODS
Animal Resources and DNA Isolation
Hair goats are raised in all parts of Turkey, particularly in mountainous and bushy areas of Mediterranean. Hair goat is also called ordinary goat or black goat. It is major goat breed of Turkey. Blood samples were collected randomly from a total of 233 hair goats belonging to four different flocks around Bursa region in their natural habitats. An effort was made to collect samples from unrelated individuals based on information provided by farmers. Genomic DNA was isolated using Genomic DNA isolation kit following the manufacturer’s protocol. The quality of DNA was checked on 0.8% agarose gels and concentrations were measured by UV-spectrophotometer.
Amplification of β-lg Gene and Restriction Enzyme Digestion
PCR amplifications of the β-lg gene were performed in a 25 μL reaction volume containing 50-100 ng genomic DNA, 2X PCR Master Mix and 0.5 μM each of the primers, Forward 5’-CGGGAGCCTTGGCCCTCTGG-3’ and reverse 5’-CCTTTGTCGAGTTTGGGTGT -3’, (Pena et al., 2000). The amplification reactions were performed in an Techgene Thermal Cycler (Techne, Cambridge, UK) programmed for an initial denaturation at 95°C for 5 min, followed by 35 cycles each with denaturing at 95°C for 30 sec, annealing at 65°C for 1 min, extension at 72°C for 90 sec and a final extension at 72°C for 5 min. The products were visualized by staining with ethidium bromide after electrophoresis of 10 μL reaction mixture on a 2 % agarose gel.
PCR products (12 μL) were digested with 10 U of the SacII (Fermentas) restriction enzyme in a 20 μL total reaction volume overnight at 37°C. The restriction fragments were directly analyzed by electrophoresis in 3% agarose gels in 1X TAE buffer stained with ethidium bromide and visualized under UV light.
Direct counting was used to estimate genotypic and allelic frequencies of β-lg genetic variants. The chi-square test (χ2) was used to check whether the populations were in Hardy-Weinberg equilibrium. All calculations and the χ2 analysis were carried out using PopGene32 software (Yeh et al., 2000).
RESULTS AND DISCUSSIONS
β-lg polymorphism was reported in different goat populations at protein level in Turkey. Ozdil and Asal (2002), Turkyilmaz (2003) and Gurcan (2005) described two allele, β-lgA and β-lgB, at β-lg locus in different goat breed populations in Turkey. In Turkish hair goat population, β-lgB allele were found to be predominant at protein level (Ozdil and Asal, 2002; Gurcan, 2005). However, no information has been reported in Turkish goats regarding β-lg polymorphism at the DNA level.
The exon 7 to the 3’ flanking region of β-lg gene of Turkish hair goat were investigated. A fragment of 426 bp from exon 7 to the 3’ flanking region was successfully amplified by PCR and digested with SacII restriction enzymes to detect the presence of S1 or S2 variants. Digestion with SacII restriction enzyme reveals the polymorphic sites, which were produced by a single nucleotide substitution in position of +4601. Allele discrimination was based on size differentiation of digested fragments. Restriction digestion of 426 bp PCR products with SacII enzymes revealed three genotypes (Fig. 1) of S1S1 (349 and 77 bp), S1S2 (426, 349 and 77 bp ) and S2S2 (426 bp). Screening for allele frequency at β-lg locus at DNA level in goat population revealed the presence of two alleles, namely S1 and S2.
Table 1 shows the allelic and genotypic frequencies in investigated goat population. The genotypic frequencies of S1S1 and S1S2 were almost equal. S2S2 genotype was found to be lower than other genotypes (S1S1 and S1S2) in the studied population. The frequency of S2 allele at β-lg locus was found to be lower than that of S1 allele. It means that, the frequency of S1 allele was clearly predominant. The chi-square test results are shown in Table 1 and revealed that population was in Hardy-Weinberg equilibrium.
|Fig. 1:||Electrophoresis of exon 7 of goat β-lg gene amplified by PCR of animal with S1S1, S1S2 and S2S2 variants and digested with SacII. The 426 bp band was restricted into two bands 349 and 77 bp in the S1 polymorphism (Lane 1 and 4: S2S2: Lane 2: S1S1: Lane 3 and 5: S1S2: M: marker)|
|Table 1:||Gene and genotypic frequencies of β-lg locus in the studied Turkish hair goat population|
|NS: Non significant|
Polymorphism studies conducted in some Indian breeds (Kumar et al., 2006) showed higher frequencies of S2 allele than the present study. Kumar et al. (2000) showed that the frequency of S2S2 genotype was more frequent than other genotypes (percentage of S2S2 genotype varied from 41.7% to 100%) and S2 allele frequency was found to be the higher than S1 allele frequency. The frequencies of S1 and S2 allele in this study found to be similar to French and Spanish goat breeds studied previously by Pena et al. (2000). Consistent with the result of this study, an S1 allele frequency in French and Spanish goat breeds was higher than S2 allele frequency. On the other hand, some polymorphic sites were reported different region of goat β-lg gene. Graziano et al. (2003) and Ballester et al. (2005) described different polymorphism in proximal promoter region and in exons 1, 2, 3 and 6 of the goat β-lg gene in different goat breeds.
In the present study, we demonstrated that there was a genetic polymorphism in samples collected from Turkish hair goat breed. Milk protein polymorphism has been considered a potential tool for selection of goat breeds. The results of this study showed that Turkish hair goat breed have a genetic variability in the β-lg locus, which may lead to extensive study of genetic polymorphism of milk proteins in Turkish hair goat breed. This record may be useful for marker-assisted selection between different genotypes and the technological properties of milk. But, it is necessary to screen further goat breeds for determining the polymorphism at proximal promoter region of β-lg gene.
This study was supported by grants from the Scientific Research Projects Council of Uludag University (Project no: Z-2006/37), Bursa, Turkey.
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