Genetic Polymorphisms in Exon-3 region of Growth Hormone Gene in the Egyptian Goat Breeds

Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidate important biological, chemical, or physical mechanisms of broad physiological significance.

Background: In Egypt, the importance of goats comes from its potential source of meat and milk production.Thus, goat productivity improvement acts as a global strategy to provide Egyptians by important source of protein feeding.Molecular techniques have been employed to analyze growth hormone (GH) gene that promotes goat muscles, bone formation, regulating fat content and other important traits in goat.Our objective was to document and find out an accurate genetic marker sites, single nucleotide polymorphisms (SNPs) in exon-3 region of GH gene in some Egyptian goat breeds.Methods: Fifteen blood samples were collected from three Egyptian goat breeds (Baladi, Barki and Zaraibi), DNA was isolated and primer was used to amplify GH gene region corresponding to exon-3 region.Finally, sequencing methodology was applied for PCR products regarding the three goat breeds.Results: The results showed four nucleotide substitutions; The first SNP (CT) was located at nucleotide position 106, the second SNP( GA) was located at nucleotide position118 and the third SNP (GA) was located at nucleotide position 128 .Finally, the fourth SNP (TC) was located at nucleotide position 136.It was found that the SNP (GA) at nucleotide position 128 caused an amino acid change from Glycine to Serine in the protein sequence of GH gene.Conclusion: Our findings demonstrated several genetic variations across exon-3 region of GH gene which can be used as marker in selection of goats with high valued traits.
Goat breeds are largely classified according to their geographical location, morphological features and prolific nature.
In Egypt, the most common goat breeds distributed around different regions are Baladi, Barki and Zaraibi breeds (Galal et al., 2005).The native Egyptian goat breed located in the Nile Delta and along the Nile is known as Baladi breed.This breed shows highly phenotypic variation among different subpopulations.They are polyestrous animals; kidding all the year.Additionally, they are characterized by good fertility rate with high prolific and high milk yield (Haider and Abdelsalam, 1993).Barki desert goat is the only goat breed raised in the coastal zone of the western desert in Egypt.They are estimated to be 10% of total goat population in Egypt (Haider et al., 1994).Zaraibi breed is one of the most important Egyptian goat breeds distributed across the North East of the Nile Delta.They have a great economic value in Egypt due to their highly production of meat and milk as well as their great litter size rate (Marai et al., 2002).
In Egypt, most of livestock breeds lack molecular characterization which is required for conserving important genetic resources besides improving animal productivity.This can be achieved by identification and utilization of different genetic variations within and among different animal breeds (Agha et al., 2008).Genetic improvement schemes in goat were depended on the selective breeding where the estimated breeding value was derived only from phenotypes.These breeding protocols do not allow for optimal control over precise phenotypic traits resulting in loosing or displacing of many important breeds without knowing their genetic significance (Rout et al., 2008).Nowadays, the evaluation of genetic variability in domestic goats is depending on the detection of different genetic markers that are associated with an important economic trait via Marker Assisted Selection (MAS).
Marker Assisted Selection (MAS) involved in the identification of genetic markers (DNA markers) for specific trait that are linked to several Quantitative Trait Loci (QTL) in different genes (Williams, 2005).The genes attributed with different economic traits including growth, reproduction, meat and milk production traits as well as disease resistance trait is known as candidate genes (Supakorn, 2009).These genes were identified by advanced molecular techniques such as Restriction Fragment Length Polymorphism (RFLP), Single Strand Conformation Polymorphism (SSCP) and sequencing techniques (Yang et al., 2013).Growth hormone gene is one of the most essential candidate genes which is required for animal's cells activity.It is secreted by somato-tropic cells of the anterior lobe of the pituitary gland.GH gene influences different animal processes such as growth, lactation, reproduction and metabolism (Burton et al., 1994).Goat growth hormone gene is encoded by 2500 base pairs (bp).It consists of 5 exons and separated by 4 intervening introns (Kioka et al., 1989).
Many publications have reported that the polymorphisms of this gene have been identified in the promoter regions, UTRs, coding and non-coding regions.Certainly, a few of these polymorphic sites have been precisely characterized for nucleotide and amino acid changes along the GH gene sequence in different goat breeds (Yu et al., 2004).The current study concerned with the identification of SNP abundance in exon-3 region of GH gene to identify the nucleotide and amino acid changes across the three Egyptian goat breeds (Baladi, Barki and Zaraibi) using polymerase chain reaction and sequencing technique.

Animal
population and blood collection: a total of 15 blood samples were collected from three Egyptian goat breeds (Baladi, Barki and Zaraibi) by 5 samples per breed.The three goat breeds were reared in the research farms of the department of Animal Production, Faculty of Agriculture, Cairo University.Genomic DNA extraction: Genomic DNA was isolated using phenolchloroform extraction technique (Sambrook and Russell, 2006).The quantity was determined using spectrophotometer and quality of DNA samples were checked on 1.5 % agarose gel electrophoresis.The samples of good quality were used for PCR amplification.

Polymerase Chain Reaction (PCR):
PCR reactions were performed in 15 μl reaction mixture containing 1 μl of DNA (50 ng), 7.5 μl Go-Taq green mater mix which consisted of (1X PCR buffer, 1.5 mM MgCl2, 200 μM of each dNTPs and 1 U/μl of Taq DNA polymerase).Then, 2μl of a primer mix (10 pmol of each primer) was added where the primer used for exon-3 amplification was previously described by Wickramaratne et al., (2010) in studying GH gene in Osmanabadi and Sangamneri goat breeds.
The volume was completed with ddH2O to 15 μl.The thermal cycling conditions (Bio-Rad MJ-mini, USA) included an initial denaturation at 95°C for 3 min followed by 30 cycles of denaturation at 95°C for 15s; annealing at 65°C for 30s and extension at 72°C for 45s then a final extension at 72°C for 5 min.The PCR products were detected using 1.5% agarose gel electrophoresis to predict the presence of band which representing the amplified fragment.

DNA sequencing:
The amplified fragments of exon-3 region from the three goat breeds were sequenced in ABI 310 automated DNA sequencer (Applied Bio System).Sequence analysis and single nucleotide polymorphism (SNP) detection: Sequence analysis and alignment of GH gene were carried out using NCBI/BLAST (blastn) https://blast.ncbi.nlm.nih.gov/Blast.cgiagainst database sequence with the accession number (D00476.1) to identify single nucleotide substitution in the three goat breeds.Additionally, the protein sequence was predicted using EXPASY software https://www.expasy.org/.

RESULTS AND DISCUSSION
Recently, applications of molecular genetics tools allow genotyping of specific genetic loci of multiple genes in different living individuals (Dekkers, 2004).Growth Hormone gene is one of the most important candidate genes which affect a wide variety of physiological parameters.There is evidence of an association between plasma levels of GH gene and its genetic variants in goat (Mousavizadeh et al., 2009).This work was designed for screening of SNPs in exon-3 region of GH gene in three different Egyptian goat breeds.

Target amplification:
The primer used in this study flanked a 449 bp fragment corresponding to exon-3.The amplified fragments obtained from all tested goat animals appeared at 449 bp in 1.5% agarose gel electrophoresis as represented in (Fig. 1).

Sequence analysis:
Sequence data resulted from sequencing of exon-3 fragments was aligned against database sequence with the accession number (D00476.1).The results revealed four nucleotide substitutions in exon-3 region among the three goat breeds as indicated in (Table 1).Where; bp = base pair

Nucleotide substitutions:
The first nucleotide substitution (CT) was detected at nucleotide position 106 in exon-3 of GH gene in the three goat breeds as represented in (Fig. 2).The second nucleotide substitution (GA) was detected at nucleotide position 118 as represented in (Fig. 3).
The third nucleotide substitution (GA) was detected at nucleotide position 128 as represented in (Fig. 4) while the last nucleotide substitution (TC) was detected at nucleotide position 136 as represented in (Fig. 5).
The translation of the sequenced segment of exon-3 region of GH gene using EXPASY software refers to a change in the amino acid sequence as a result of the nucleotide substitution (GA) which is located at nucleotide position 128.This nucleotide substitution caused an amino acid change from Glycine to Serine between G and A alleles as represented in (Fig. 6).

Fig. 1 :
Fig.1: Amplified PCR products of exon-3 region appeared at 449 bp in the three goat breeds

Fig. 2 :
Fig. 2: The first nucleotide substitution (CT) at nucleotide position 106: (A) Reference sequence with nucleotide C (B) GH sequence with SNP T