INTER AND INTRASPECIFIC COMPARATIVE ANALYSIS OF GROWTH HORMONE GENE FOR SOME FARM RUMINANT SPECIES

olecular genetics techniques help in discovery of the genes or candidate genes which affect the important economic traits, the use of this techniques for genetic improvement depend on the ability to genotype individuals with better potentialities for specific economic traits. The information from candidate genes helps in planning breeding programs through increasing the accuracy of selection, selection differential and consequently the response to selection.


INTER AND INTRASPECIFIC COMPARATIVE ANALYSIS OF GROWTH HORMONE GENE FOR SOME FARM
olecular genetics techniques help in discovery of the genes or candidate genes which affect the important economic traits, the use of this techniques for genetic improvement depend on the ability to genotype individuals with better potentialities for specific economic traits. The information from candidate genes helps in planning breeding programs through increasing the accuracy of selection, selection differential and consequently the response to selection.
Growth hormone gene (GH) plays a fundamental role in the regulation of growth and metabolism in vertebrates (Davidson, 1987;Sami, 2007). This gene is a about 1800 bp in length with four intervening sequences and consists of five exons of about 648 nucleotides (Gordon et al., 1983) with chromosome region 19q26 in cattle, 11q25 in sheep (Hediger et al., 1990), 19q22 in goat (Schibler et al., 1998;Pinton et al., 2000). The gene produces growth hormone from the anterior pituitary, is necessary for prenatal and postnatal growth and metabolism in animals (Yamano et al., 1988;Supakorn, 2009). Furthermore, Malveiro et al. (2001), Hattori, (2009 and Hua et al. (2009) reported that this hormone is influencing animal growth, milk yield traits and immune stimulant properties.
Polymorphism at DNA level might be responsible for the alteration of gene functions leading to changes in a disease situation or impair/enhance a production trait. Furthermore, GH gene polymorphism was known to give possible selection criterion for fertility, weight and milk production traits (Mullen et al., 2011;Ishida et al., 2010;Ibeagha-Awemu et al., 2008).

DNA samples
Blood samples were collected from three species (cattle, sheep and goat). The samples were randomly taken from three breeds of cattle viz. Simmental, Brown Swiss and Baladi, and three breeds of sheep viz. Abodlik, Awasi and Rahmani and two breeds of goat viz. Damascus and Baladi. Blood samples of Damascus and Awassi were obtained from farms of National Research Centre (NRC), while Baladi cattle samples were obtained from M Animal Production Research Institute (APRI) and Brown Swiss and Simmental samples were obtained from Sanad farm ‫‬ , Abodlik and Baladi goat samples were obtained from local herds in the south of Egypt (Halaib and Shalateen) and Rahmani samples were obtained from those collected by Kayali (2013) from Animal Production Research Institute (APRI), Agricultural Research Center.
Approximately 5 ml blood per animal were obtained by jugular venipuncture in a K3-EDTA tube containing anticoagulant (Tripotassium-ethylene diamine tetracetic acid (K3-EDTA). The genomic DNA was extracted from whole blood using a commercial kit (ISOLATE II Genomic DNA Kit, Bioline, Cat No. BIO-52066).

PCR conditions
According to the conserved regions of available sequences of bovine, ovine and caprine GH genes that obtained from GenBank, NCBI, one sets of universal primers were designed to amplify intron 2, exons 3 and intron 3 of the GH gene. The primer sequence was: Forward F: 5´-TGCTG ACACC TTCAA AGAGT-3´; Reverse R: 5´-CAGTG AGATG TGAAG CAGCT-3´.
PCR products were electrophoretically separated on 1.5% agarose gel, stained with ethidium bromide to test the amplification success. PCR reaction was performed in a final volume of 25 μl, containing 1 μl genomic DNA, 1 μl each primer, 12.5 μl GoTaq® Green Master

DNA sequencing and analysis
Purified fragments were sequenced by sequencing service (Macrogen, Netherlands). Analysis of the sequencing data was performed using the Geneious program v8.1.

RESULTS AND DISCUSSION
The sequence lengths for GH gene of cattle breeds were 599, 573 and 403 bp for Simmental, Brown Swiss and Baladi, respectively. While sequence lengths of sheep breeds were 490, 486 and 484 bp for Abodlik, Awassi and Rahmani, respectively. Whereas sequence lengths of goat breeds were 535 and 598 bp for Damascus and Baladi, respectively. The variations in sequence lengths were due to cutting out of unsure bases.

Variations among species
Variation between the consensus sequences of the three species was detected and then compared with the available sequences of bovine, ovine and caprine GH genes that obtained from GenBank, NCBI to exclude the polymorphic variation within species. There are 20 interspecific genetic variations (monomorphic within every species, polymorphic between species). All of these variations located in intronic region, whereas exon 3 didn't have any species-specific genetic variations. It is observed that intron 2 has a large number of this genetic variation, thirteen, while intron 3 have only seven. This means that the variations between species in this region centralize at the regulatory level.

Some of interspecific genetic variations caused variations in transcription factors-
binding site prediction (Fig. 1).
Although intronic variation cannot change the amino acids sequence of the protein, there is augment evidence that variants in intronic region also play significant roles in amendment gene expression patterns (Le Hir et al., 2003;Pagani and Baralle, 2004). The influence of noncoding regulatory variants on complex traits may be more than the influence of coding-region variants (Clop et al., 2006;Pagani and Baralle, 2004). Intronic mutations may affect splice sites and consequently mRNA stability and may lead to truncated protein products or to the lack of them (Ibeagha-Awemu et al., 2008). Generally, Introns function in a number of different ways, such as sources of noncoding RNA, carriers of transcriptional regulatory elements, contributors to alternative splicing, enhancers of meiotic crossing over within coding sequences and signals for mRNA export from the nucleus as reported by Fedorova and Fedorov (2003) and Dario et al. (2008).

Variations among cattle breeds
There is no single nucleotide polymorphisms (SNPs) identified between the three breeds of cattle.

Variations among goat breeds
Five SNPs were identified between the two breeds; all of them are transition (Table 1). Two SNPs were detected in exon 3, one SNPs led to an amino acid change, G353A which alters the amino acid codon GGC to AGC. This substitution led to an amino acid mutation serine to glycine (39ser>gly).
Variations in the exons may lead to changes in amino acids sequence of the expressed protein and may affect positively or negatively its function and consequently traits that are influenced by this protein. The effects of this substitution may lead to eliminating, inactivating, unstable product or amendment protein functions to suit different environments (King et al., 2006;Ibeagha-Awemu et al., 2008).
The other variations in exons did not change in amino acid sequences. Some researcher stated association between synonymous variation and production trait (Yao et al., 1996;Silveira et al., 2008;Chung et al., 1996;Dybus, 2002;Fan et al., 2010). Synonymous mutations can alter mRNA folding, led to a decrease or an increase in the mRNA stability and translation, and also can alter the methylation pattern which leads to alterations in chromatin structure and either to a decrease or an increase in the rate of transcription. (Duan et al., 2003;Capon et al., 2004;Ramser et al., 2008). Table (2), the T>C Transition changed a common codon AAC to a less used codon AAT (both coding asparagine). This may be having important consequences for cellular processes but cannot be deduced from simple analysis of DNA sequence, it needs further analysis before drawn any conclusion.

Variations among sheep breeds
There are two SNPs identified between the three breeds, all of them are Transition (Table 3). G353A alter the amino acid codon GGC to AGC. This substitution led to an amino acid mutation Serine to Glycine (39Ser>Gly).

CONCLUSION
Inter-specific genetic variations can be considered as species differentiating marker, and requires further studies to know their effects on the gene expression.
Based on the newly discovered finding with regard to the synonymous mutation, we need to look at them thoroughly and requires further studies to know their effects on the production traits to be used for genetic improvement of livestock animals. Baladi goat has a large variation and could be used in genetic improvement.

SUMMARY
Growth hormone affects a lot of physiological processes and traits, such as metabolism, milk and meat production. Polymorphism at DNA level might affect gene function and consequently the trait. The aim of this study was to identify the variation in the growth hormone gene between and within species (cattle, sheep and goat). The results showed that all variations between species located at intronic region, whereas exon 3 didn't have any species-specific genetic variations. There is no SNPs identified between the breeds of cattle, whereas the variation within breeds of sheep and goat located at an intronic and exonic region. 1. is a Purine (Adenine or Guanine), 2 is a Pyrimidine (Cytosine or Thymine), Nt. no is nucleotide number