Polymorphism of Insulin-like Growth Factor I Gene in Six Chicken Breeds and Its Relationship with Growth Traits

The polymorphism of insulin-like growth factor I (IGF I) in 6 chicken breeds (total n=515) was detected by PCR-Pst I- RFLP, and allele A (621 bp) or allele B (364 and 257 bp) were observed. In these chicken breeds, it was found that exotic chicken carried high frequencies of allele B, while Chinese native chicken breeds carried high frequencies of allele A. Meanwhile the role of IGF I was investigated in 133 Ningdu Yellow chicken and 162 Wanzhai Yellow chicken. Five growth traits were recorded for analyzing the association between IGFI gene polymorphism and performance. In both the Ningdu and Wanzhai Yellow breeds, body weight at 4 months was significantly higher with BB genotype than with AA genotype (p<0.05). Furthermore, body weight at 2 months in the Wanzhai Yellow breeds was also higher with BB genotype than with AA genotype (p<0.05). There were no differences among the genotypes for the other traits studied. Based on these results, it is necessary to do more studies on IGFI before making the IGFI locus into the application of maker-assisted selection programms. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 3 : 301-304)


INTRODUCTION
As growth in Chicken is highly regulated by growth axis, recent work revealed the mechanism of signal pathway from the release of growth hormone (GH) from pituitary to the expression of animal growth.The activity of GH is brought about by its binding to a specific site of GH receptor followed by the alteration of conformation, so that the intracellular domains of GH receptor associate with a tyrosine kinase of the Janus family (JAK2).The signal transduction commenced from the binding of GH to its receptor can active a specific gene coding insulin-like growth factor I (IGF I).The IGF I released into the circulation binds to its specific receptor called by IGF type-1 receptor and finally stimulates cell proliferation (Okumura and Kita, 1999).
Insulin-like growth factor I (IGF I) is a 70 residue, single chain peptide that has multiple effects on cellular growth and metabolism.IGF I plays a major role in mammals such as mediating the growth-promoting actions of growth hormone and also functions as a locally regulated autocrine or paracrine growth stimulator.The chicken IGF I has been characterized and shown to be 70 amino acid polypeptides (Ballard et al., 1990).Some literatures pointed out an important role for IGF I in the control of growth and metabolism in chicken, as in mammals.After hatching, plasma concentration and hepatic gene as expression of IGF I increase rapidly with aging, reach a peak before sexual maturity, and then decline (Kikuchi et al., 1991).In most circumstances, IGF I makes a complex with specific high affinity IGF-binding proteins (IGFBPs) (Nagao et al., 2001).
Chicken IGF I (cICF I) is structurally similar to mammalian IGF I, but there are some fundamental differences with respect to IGF I physiology between mammals and poultry.Yoshitaka Kajimoto and Peter Rotwein (1991) cloned and sequenced the chicken IGF I, and found that the cIGF I gene contains four exons and three introns, and distributes over more than 50 kb of chromosomal DNA which is thus approximately two-thirds the length of rat IGF I and within the gene are seven regions that contain repetitive elements.By fluorescence in situ hybridization and genetic linkage analysis, the cIGF I was located to chicken chromosome 1p1.4-p1.3 and shown highly conserved syntenic group to other vertebrates (Klein et al., 1996).
The effects of IGF I gene variants on performance traits had been described for mouse (Collins et al., 1993;Horvat et al., 1995) and pig (Casas-Carrillo et al., 1997;Wang et al., 2002).In chicken, associations between variants at the IGF I locus and performance traits are required further investigation.
In this study we investigated the polymorphism of IGF I in six chicken breeds and the effects of one IGF I restriction fragment length polymorphism (RFLP) described by Nagaraja et al. (2000) on growth traits in Ningdu Yellow chickens and Wanzai Yellow chickens.

IGF I genotypes defined by PCR-RFLP
Genomic DNA was extracted from blood, using a phenol/chloroform extraction method followed by ethanol precipitation.Working dilutions of extracted DNA were prepared for each individual at a concentration of 50 ng/µg.Primers 5′-GAC TAT ACA GAA AGA ACC CAC-3′ and 5′-TAT CAC TCA AGT GGC TCA AGT-3′ were used for polymerase chain reaction (PCR) amplification of insulinlike growth factor I gene (Nagaraja et al., 2000).The PCR mixture containing 50 ng genomic DNA, 25 pmol of each primer, 25 µM of each dNTP, of unit Taq DNA polymerase (TAKARA) and 1×reaction buffer in a 25 µL reaction volume.PCR were processed on PE9600 (PERKIN ELMER) according the procedure: first 94°C for 300 s then 35 cycles: 94°C for 60 s, 55°C for 120 s, 72°C for 90 s, at last 72°C for 480 s.The 621 bp PCR products were subsequently digested by Pst I and revealed allele A (fragment of 621 bp) or allele B (fragments of 364 and 257 bp).The restriction digests were separated using 2.0% agrose gel in 1×TAE at a constant current of 50 mA.The gels were stained with ethidium bromide and the fragments were visualized using a UV transilluminator.

Statistical analysis
Least square means (LSM) method was used for statistical analysis for data collected.Associations between IGF I genotypes and growth traits were analyzed using SAS system (1989), significance between genotypes was assessed by Student's t-tests.The model include breed, environment and genotypes of IGF I gene effects.

RESULTS
The genotype frequencies and allele frequencies of IGF I in 6 breeds was list in Table 1.
From Table 1, the BB genotype in Chinese native chicken breeds was lower than exotic chicken breeds, especially in Chongren Chicken, it frequency was 0.71%.While in exotic breeds, the BB genotype was high and Lohmann (AB ♂) was the highest (66.7%).Meanwhile the B frequency in exotic breeds was high, especially in Lohmann (AB ♂), with its frequency of 79.1%.In Chinese native breeds, the A frequency was high (above 50%) and in Dongxiang Luke the A frequency was 67.5%.
Table 2 listed the observed genotypes and their frequencies.The AA genotype in two chicken breeds was higher than BB genotype and the frequency in two breeds was 39.85 and 34.57% respectively.
In Ningdu Yellow chickens, body weight (BW) at 4 months of age was significantly associated with IGF I genotypes.The chicken with BB genotype had higher than the ones with AA genotype for body weight at 4 months of  age (p<0.05).But no significant difference was observed among three genotypes for birth weight, 1 month BW, 2 months BW and 3 months BW.
In Wanzhai Yellow breed, the chickens with BB genotype had higher than the ones with AA or AB genotypes for BW at 2 months of age (p<0.05), and the chickens with BB genotype had higher than the ones with AA genotype for BW weight at 4 months of age (p<0.05).

DISCUSSION
The chicken IGF I gene has been shown to be in the short arm of chromosome 1 near the centromere and to be conserved in several vertebrates species (Klein et al., 1996).Also, the RFLP analysis of the chicken IGF I gene revealed a single Pst I polymorphism in the 5′ region (Nagaraja et al., 2000;Seo et al., 2001).In this study, the PCR-Pst I-FRLP for the IGF I gene revealed polymorphic fragments of 621, 364 and 257 bp, which is consistent with the results reported by Nagaraja et al. (2000) and Seo et al. (2001).The comparison for two allele sequences indicated the loss of the Pst I restriction enzyme site (CTGCA↓G) by the point of mutation.
In Table 1, the AA genotype frequencies and A allele frequencies of Chinese native chicken breeds were higher than those of exotic chicken breeds.In Table 2, the two chicken breeds, AA genotypes were also high, this was similar to the results by Nagaraja et al. (2000) and Seo et al. (2001).Nagaraja et al. (2000) found that AA genotype frequencies were low (3.5%) in white Leghorn, and Seo et al. (2001) found that AA genotype frequencies were high (16.9%) in Korea Native Ogol Chicken (KNOC).These results implied that exotic chickens had higher AA genotype and A allele frequencies.chi-square test found that the genotype frequencies were independently distributed among breeds (data not given) (χ 2 =104.03,p<0.001), that meant the distribution at this locus among breeds had breed specify.After using Hardy-Weinberg equilibrium testing, only Dongxiang Luke, Chongren Chicken and Ningdu Yellow chicken were out of equilibrium.Seo et al. (2001) found that the distribution of genotypes was not different from the expected distribution under the assumption of the Hardy-Weinberg equilibrium for the IGF I alleles in KNOC, while the results of Nagaraja et al. (2000) was different.Seo et al. (2001) thought such differences may reflect differences in hereditary characters among chicken breeds.
It has been reported that IGF I have significant effects on the overall body and muscle growth chicken (Duclos et al., 1999).A study for repeated divergent selection for high or circulator IGF I concentration concluded that the IGF I is a heritable character in chicken (Beccavin et al., 2001).In the two different pure chicken breeds, effects of different genotypes on body weight were differed.In Ningdu Yellow breed, only chickens with BB genotype had higher 4 months BW than those with AA genotype (p<0.05),but in Wanzhai Yellow breed, chickens with BB genotype had higher 2 months BW than the ones with AA genotype (p<0.05) and had higher 4 months BW than the ones with AA and AB genotypes (p<0.05).Seo et al. (2001) found that different genotypes had no significant differences for female body weight, but the male chickens with BB genotype had higher for 30 weeks body weight than those with AA genotype.And Nagaraja et al. (2000) found that different genotypes had no significant differences for 140, 265 and 365 days weight, but the influential trends are same, such as BB genotype chickens have more body weight than AA genotype chickens.In our study, Ningdu Yellow chicken and Wanzhai Yellow chickens were all hens, so we could not analyzed the genotypes effects on growth rates in both sexes.Seo et al. (2001) found that the male chickens at the same days had higher serum IGF I concentration than females, which meant that serum IGF I was important for body growth in male, while the IGF I was important for egg production in the female.Now many studies of IGF I are only focused on its biochemical functions and nutrient modulation.But few studies were conducted to review whether polymorphism of IGF I gene could be used as a genetic marker in chicken and breeding system, thus the results could provide a theoretical and an experimental reference for chicken genetic improvement.

Table 1 .
The genotype frequencies and allele frequencies of chicken IGF I gene in 6 chicken breeds * Digits in the blanket is the number of chicken.

Table 2 .
Effects of different genotypes on the body weight in two pure chicken breeds