POLYMORPHISM OF INSULIN-LIKE GROWTH FACTOR 1 GENE IN NAJDI CATTLE POPULATIONS

The Insulin-like Growth Factor 1 (IGF-1) system pla ys a critical endocrine role controlling nutrient metabolism in dairy cattle and it is a group of str uc urally-related polypeptides that regulate the gr owth of many types of mammalian cells. In liver, IGF-1 is d ynamically regulated by lactation and energy balanc e. Less is known about the regulation of IGF-1 and IGF -binding protein mRNA in reproductive tissues and plays an important role in bovine follicular growth , acquisition of oocyte competence and embryo viabi lity. The present study describes polymorphism of IGF-1 g ene in Najdi cattle populations. In order to characterization polymorphism, blood sample were co lle ted from 84 Najdi cattles from Shadegan, Mahshar cities and Shushtar station. DNA extraction was based on Boom method and exons 1 of the IGF-1 gene were amplified to produce a 249 bp fragment. T he amplified fragment were digested with Eco105I (SnaBI) restriction endonuclease and then subjected to e lectrophoretic separation in ethidium bromidestained 2% agarose gel. The results were revealed t wo alleles, A and B. Three genotypes were observed, Frequencies were 2.38, 14.28 and 83.33% for AA, AB and BB, respectively. This gives frequencies of 0/0952 and 0/9048 for A and B alleles. The populati ons were in hardy-weinberg equilibrium. It is possi ble that this gene has not exposed to selection. The he terozygosity is low and inbreeding is estimated hig h.


INTRODUCTION
Quantitative traits are often controlled by a great number of genes. Localization of Quantitative Trait Loci (QTLs) can be done by linkage disequilibrium analysis or by candidate gene approach. Candidate genes have known biological functions related to the development or physiology of an important trait (Rothschild et al., 1997). Such genes can encode structural proteins or a member in a regulatory or biochemical pathway affecting the expression of the trait (Bryne and McMullen, 1996) and can be tested as putative QTLs (Yao et al., 1996). The Growth Hormone (GH) and Insulin-like Growth Factor 1 (IGF-1) genes are candidates for growth in bovine, since they play a key role in growth regulation and development (Breier, 1999). Genetic polymorphism in native breeds is a major concern considering the necessity of preserving genetic resources. It is very important to characterize genetically indigenous breeds (Bastos et al., 2001). Insulin-like Growth Factors 1 and 2 (somatomedins-IGF-1 and IGF-2) are structurally related proteins, playing a key role in cell differentiation, embryogenesis, growth and regulation of metabolism. IGF1 and IGF2 were first identified by Salmon and Daughaday (1956;1957) and designated 'sulphationfactor' due to their ability to incorporate AJBB Science Publications sulphate into ratcartilage in vitro. They were also known As Non-Suppressible Insulin-Like Activity (NSILA) I and II (Froesch et al., 1963). A decade later, the terms sulphation factor and NSILA were replaced by the term 'somatomedin' (Daughaday et al., 1972) and subsequently they were renamed 'IGFs 1 and 2' due to their structural similarity with insulin and their growthpromoting activities (Rinderknecht and Humbel, 1976a;1976b). IGF1 is one of two ligands of the IGF family (Hwa et al., 1999;Spicer, 2004). The established components of the IGF system also include two receptors, six high-affinity IGF-Binding Proteins (IGFBPs) and IGFBP proteases (Giudice, 1995;Hwa et al., 1999;Spicer, 2004). Furthermore, another group of lowaffinity binding proteins, known as IGFBP-related Proteins (IGFBP-rPs), belongs to the IGF family. However, no final nomenclature has been agreed for these proteins, as several research groups have identified the same protein and each group has used adifferent nomenclature (Hwa et al., 1999;Rosenfeld et al., 2001). Potential receptors for IGFBP(s) and IGFBP-rP(s) have also been reported to be part of the IGF family (Hwa et al., 1999) (Table 1). Due to similarities in structure and sharing intracellular signalling cascades with other members of the IGF system, insulin, its receptor and a hybrid insulin/IGF receptor are also considered to be part of the IGF superfamily (Jones and Clemmons, 1995;Monget and Martin, 1997;McCusker, 1998;Poretsky et al., 1999;Butler and Roith, 2001;Juul, 2003). Insulin-like Growth Factor I (IGF1) is known to play an important role in various aspects of muscle growth and development (Davis and Simmen, 2006). Due to the effect of IGF1 on the hypertrophy of muscle cells, muscle fiber diameter can be affected by IGF1. Since increasing muscle fiber diameter may decrease tenderness (Herring et al., 2009), it can be postulated that IGF1 may also affect tenderness by increasing the size of the muscle fibers (Koohmaraie et al., 1995). The aim of this study was to investigate relationship between DNA polymorphisms in the IGF1 gene and tenderness and muscle development. IGF-1 is a polypeptide of the molecular weight 7.5 kDa built of 70 amino acids (Daughaday and Rotwein, 1989). The amino acid sequence of IGF-1 is identical in humans, cattle, dogs and pigs (Nixon et al., 1999). In humans the IGF-1 gene contains 6 exons and is about 90 kbp-longs (Rotwein et al., 1986;Steenbergh et al., 1991). Due to an alternative splicing of exons 1 and 2, two different transcripts are formed: the one with exon 1 containing 1155 nucleotides (nt), while the other one, with exon 2, is shorter and contains 750 nt. Production of these transcripts is controlled by two different promoters both containing canonical regulatory sequences-TATA-box and CCAAT-box (Jansen et al., 1991). It was shown that transcripts of both classes are differentially expressed in various tissues, being, however, most abundant in liver (Wang et al., 2006). In vertebrates, the Insulin-Like Growth Factor 1 (IGF1) or somatomedin gene plays a key role in various physiological and metabolic processes, where IGF1 and growth hormone or somatotrophin is involved in the somatotropic axis. IGF1 is a mediator of many biological effects; for example, it increases the absorption of glucose, stimulates myogenesis, inhibits apoptosis, participates in the activation of cell cycle genes, increases the synthesis of lipids, stimulates the production of progesterone in granular cells and intervenes in the synthesis of DNA, protein, RNA and in cell proliferation (Etherton, 2004). The bovine IGF1 gene was mapped on chromosome 5, in the centimorgan 73.5 (Grosse et al., 1999). The provisional nucleotide sequence is approximately 72 kb (ID number 281239). In humans, pigs, goats, rats and chickens, the IGF1 nucleotide sequence is about 70-90 kb (Rose, 2002). Exon number is different between species; for example, goats, pigs and sheep have 1-6 exons (Mikawa et al., 1995) and humans and rats 1-5 (Rotwein et al., 1986). The IGF-1 gene is extremely conserved among species and few polymorphisms are described. The presence of a microsatellite at the promoter region of this gene in bovine, human and horse allows to analyze genetic variations related to this locus (Kirkpatrick, 1992). Evidence of selection effects on allele frequencies at these two loci was found in the traditional lineage of Canchim (Regitano et al., 1999). Nucleotide sequence polymorphisms were identified in the bovine IGF-1 gene and their correlations with animals' growth rate and meat performance traits were found. The Short Tandem Repeat (STR) polymorphism in the 5'-flanking region and the Single Strand Conformation Polymorphism (SSCP) in intron 3 of the IGF-1 were reported by Kirkpatrick (1992). In Hereford cattle the STR polymorphism was shown to be associated with body weight at birth and at weaning and with the growth rate (Moody et al., 1996). Such associations were not found in other beef breeds (Curi et al., 2005). The SSCP in the 5'-flanking region of IGF-1 was found by Ge et al. (1997) in Angus cattle. This polymorphism was then identified as T/C transition, also recognizable as RFLP-SnaBI (Ge et al., 2001). Two alleles and three genotypes were found. Allele A (with nt T at position -472) appeared significantly more frequent than allele B (with nt C) in a group of animals selected for the high IGF-1 content of blood. However, the BB genotype (with nucleotides CC) was found to be associated with higher body weight at weaning (Li et al., 2004). No association was found between IGF-1 RFLP-SnaBI and dairy production traits in Holstein cattle (Hines et al., 1998). Two polymorphisms in the IGF-1 were reported by Lien et al. (2000) in Norwegian cattle: the TTTG Insertion/Deletion (InDel) in intron 4 and the RFLP-DpnI in intron 5. However, the effects of these polymorphisms on beef or milk production traits were not investigated. The objective of the present study was to Investigate of IGF-1 gene polymorphism in najdi cattle breed populations in khouzestan province of IRAN using PBR (PCR Based RFLP) technique.

MATERIALS AND METHODS
In current study, Random blood samples were collected from 84 Najdi Cattle from three populations involve: Shushtar station, Shadegan and Mahshar cities in Khuzestan province of Iran (Fig. 1). Approximately, 5 ml blood sample was gathered from venom in EDTA tube and was transferred to -20°C freezer. Genomic DNA was isolated by using DNA Extraction Kit (Diatom) and was based on Boom et al. (1989) method. Quantity was determined by measuring the absorbance at 260 nm and the concentration, purity and quality were determined by measuring the absorbance at 260/280 nm and 230/260 ratios using a NanoDropTM 1000 spectrophotometer (Thermo Scientific). DNA extractions were appropriately labeled and stored at -20°C for analysis. The study was concentrated on a 249 bp fragment of IGF-1 gene spanning over exon 1. Primer sequence for PCR were established by Ge et al. (2001); Li et al. (2004) and Laureano et al. (2009) (Table 2). The PCR reaction volume of 25 µL contained approximately 100 ng of genomic DNA, 1unit Taq DNA Polymerase, 1x PCR Buffer, 1.5 mM MgCl2, 200 µM dNTPs and 0.3 pMol of each primer. Amplification conditions included an initial denaturation at 95°C for 5 min, followed by 35 cycles at 94°C for 30 s, 63°C for 30 s and 72°C for 40 s, followed by a final extension at 72°C for 5 min. The amplified fragment was digested with Eco105I (SnaB I). The digestion reaction contained by 5 µL of PCR product, 2 µL Buffer 10X, 5 U of Eco105I (SnaB I) and H 2 O up to a total volume of 20 µ L then were incubated at 37°C for 12-16 h. The digestion products were electrophoresed on 2% agarose gel in 0.5X TBE and visualized by ethidium bromide staining for 40 min at 100 V. The frequencies of genotype, alleles, mean expected, observed and Nei's heterozygosities and Hardy-Wienberg equilibrium test were calculated using Popgene32 software (version 1.31).

RESULTS
The amplified Insulin-like Growth Factor 1 (IGF-1) resulted in a DNA fragment with 249 bp including the sequences of spanning over exon 1 regions with PCR technique (Fig. 2). From the analysis, two alleles (A and B) were observed, resulting in three genotypes. The Eco105I (SnaB I) digestion of the PCR products produced digestion fragments of 226 and 23 bp. The animals with both alleles were assigned with AB genotype, whereas those possessing only A or B alleles were assigned with AA or BB genotypes, respectively. Genotype AA showed the two band pattern (bands of approximately 226 and 23 bp). Genotype BB showed one band pattern (approximately 249 bp), while AB animals displayed a pattern with all three bands (249, 226 and 23) (Fig. 3). The genotypes of all animals were used to determine the allele frequencies. A and B allele frequencies were 0.0952 and 0.9048, respectively and were similar to the 0.98 and 0.02 (Laureano et al., 2009), to the 0.7 and 0.3 (Kim et al., 2004) and to the 0.97 and 0.03 (Reyna et al., 2010) previously reported. The high frequency of the B allele suggests that this allele Variant might have been favored by selection for production traits. The observed genotype frequencies were 0.023 for AA, 0.142 for BB and 0.833 for AB (Table 3). najdi population was in Hardy-Weinberg equilibrium (p<0.05). The observed and expected heterozygosity were 0.1429 and 0.1734, respectively. Effective allele and true allele estimates were 1.20 and 2.00, respectively. This difference between effective all and true allele number and low diversity is due to more frequency of allele B compare to allele A, that reduced frequency in any locus. This number is larger, if there are more loci with same combination of alleles. In Shadegan and Mahshar cities, A and B allele frequencies were 0.1538 and 0.8462, respectively but in shushtar station, A and B allele frequencies were 0.0000 and 1.0000, respectively. In Shadegan city, the observed genotype frequencies were 0.051 for AA, 0.743 for BB and 0.205 for AB. In Mahshar city, the observed genotype frequencies were 0.000 for AA, 0.692 for BB and 0.307 for AB and In Shushtar station, the observed genotype frequencies were 0.000 for AA, 1.000 for BB and 0.000 for AB (Table 4). Two Populations of Najdi Cattle (Shadegan and Mahshahr cities) were in Hardy-Weinberg equilibrium but Shushtar station was in hardy-weinberg disequilibrium (p<0.05). In Shadegan city, the observed and expected heterozygosity were 0.2000 and 0.2600, respectively. In Mahshar city, the observed and expected heterozygosity were 0.3000 and 0.2700 and In Shushtar station, the observed and expected heterozygosity were 0.0000 and 0.0000, respectively ( Table 5).

DISCUSSION
Candidate genes have known biological functions related to the development or physiology of an important trait. Such genes can encode structural proteins or a member in a regulatory or biochemical pathway affecting the expression of the trait (Bryne and McMullen, 1996) and can be tested as putative QTLs (Yao et al., 1996). The study by Ge et al. (2001) characterizes a G→A transition polymorphism within an Eco130I site of intron 3 of the IGF1 gene. Polymorphisms in the bovine IGF-I gene are associated with circulating IGF-I concentrations and growth traits. Growth in animals is controlled by a complex system, in which the somatotropic axis plays a key role. Genes that operate in the somatotropic axis are responsible for the postnatal growth, mainly GH that acts on the growth of bones and muscles mediated by IGF-1.

CONCLUSION
Najdi cattle breed showed a low degree of genetic diversity for the IGF-1 locus. Although we observed the low variability for this locus, in the other hand, this data provide evidence that Iranian najdi cattle populations have a good polymorphism for IGF-1 gene, which opens interesting prospects for future selection programs, especially marker assistant selection between different genotypes of different locus and milk, gain and meat traits. Present results showed that PCR-RFLP (PBR) is appropriate tools for evaluating genetic variability.

ACKNOWLEDGMENT
The current study was supported by Faculty of Animal Sciences and Food Industries, Ramin Agricultural and Natural Resources universities and Department of Genetic and Biotechnology in the Varamin Pishva branch of Islamic Azad University in the Iran.