In this study chicken growth hormone (cGH) gene polymorphism has been investigated in broiler by PCR-RFLP. The genomic DNA was extracted from 150 samples by using modified spin column technique. The DNA fragment of the growth hormone gene with 776 bp was amplified by PCR using specific primers. Then the PCR products were digested with MspI restriction enzyme and analyzed on 8% polyacrylamide gel. The allelic frequency of exon-1 and partial intron-1 locus for A and B allele were 0.39 and 0.61, respectively. The genotypic frequency for AA, AB and BB in Broilers were 0.22, 0.28 and 0.50, respectively. The result showed that the exon-1 and partial intron-1 of cGH is polymorphic.
Poultry production is an important and diverse component of agriculture all over the world. Today, more attention has been given to poultry due to their quality of meat and sustainable production (Kaya and Yıldız, 2008). Growth hormone (GH) is secreted from the anterior pituitary gland and affects a wide variety of physiological functions in birds (Kansaku et al., 2008). The chicken growth hormone (cGH) gene is one of the effective gene that influenced the chicken performance traits because it plays a crucial role in growth and metabolism (Vasilatos-Younken et al., 2000; Nie et al., 2005). Polymorphisms in the cGH gene were widely studied by restriction fragment length polymorphisms (RFLPs) or sequencing (Mou et al., 1995; Tanaka et al., 1992). Molecular technology has provided new opportunities to evaluate genetic variability at the DNA level (Kaya and Yildiz, 2008). Many candidate genes associated with poultry productivity have been identified (Abdi et al., 2014; Fatemi et al., 2012; Xu et al., 2011a; Xu et al., 2011b). A significant correlation was reported between GH gene and meat quality in Anka and Rugao hens (Sheng et al., 2008). Polymorphism in the chicken growth hormone gene has positive correlation with resistance and egg production (Kuhnlein et al., 1997). The purpose of the present research was to estimate genetic variability of cGH gene and to investigate their associations with the growth traits of the broiler in India.
Material and Methods
This experiment was conducted by Animal Genetics and Breeding department, College of Veterinary and Animal Science, Bikaner with all essential procedures of sample collection were performed strictly as specified by Institutional Ethical Committee with minimal stress to Birds.
For present investigation blood samples were collected from the 150 unrelated broilers were selected from poultry farm situated in the campus of College of Veterinary and Animal Science, Rajasthan University of Veterinary and Animal Science, Bikaner and approximately 0.5-1.0 ml of venous blood samples were collected in sterile vacutainer tubes containing 0.5 M EDTA as anticoagulant. The blood samples were gently mixed with EDTA and kept in ice pack to prevent cell lysis and stored at 4˚C until the isolation of genomic DNA.
DNA Extraction and PCR
Genomic DNA was extracted using the spin column method (Sambrook and Russell, 2001) under manufacture protocols with slight modification through genomic DNA Isolation kit (Himedia Pvt. Ltd). The quality of the extracted DNA was checked by Agarose gel electrophoreses. Based on cGH gene sequence (GenBank accession number: EF452679) primers were designed through online primer-3 software. The specificity of primer pairs was confirmed on the basis of available sequence of cGH gene at the National Center of Biotechnology Information (NCBI). The forward and reverse primers were 5’ATCCCCAGGCAACATCCTC3′ and5’CCTCGAC ATCCAGGTCACAT3′, respectively. The PCR reactions were performed with a total volume of 25 ml. The cycling conditions were as follow: 5 min at 95°C for initiation of denaturation, then 45 s at 94°C (denaturation), 1 min at 55°C (annealing), 60 s at 72°C for extension (35 cycles), followed by 10 min at 72°C for final extension.
The polymorphism of cGH was detected with PCR-RFLP. The 30 µl reaction mixture was prepared to adding 17 µl nuclease free water, 2 µl 10X Buffer and 10 µl PCR product were digested with 1 µl MspI restriction enzyme. This enzyme acts on 5’…C↓CGG…3’ and 3’…GGC↑C…5’. Then it kept for digestion in incubator at 37˚C overnight. After the digestion, heat inactivation of the enzyme was done at 80˚C for 20 min. The restriction fragments were resolved by polyacrylamide gel electrophoresis on 8% gel in 1X TBE buffer at 120V for 1.5 hrs.
Traits and Statistical Analysis
The genotypes were detected by seeing the RFLP patterns of each sample in the RFLP gels. The frequency of exon-1 and partial intron-1 of growth hormone genotypic patterns were estimated by standard procedure (Falconer and Mackey, 1996).
Genotyping patterns frequencies =
Gene frequencies =
Where, D = number of birds homozygous for a particular allele, H = number of heterozygote birds and N = total number of birds
Analysis of Association between PCR-RFLP Genotypes Variants and Body Weight Traits
The association between genotypic patterns of chicken growth hormone gene (cGH) with body weight traits in broiler was carried out through General Linear Model (GLM) procedure of SPSS by following statistical mode –
Yij = µ + Gi + eij
Where, Yij = body weight of the ith Genotype, µ = Overall mean, Gi = Fixed effect of jth genotype, eij = Random error NID (0,σ2).
Result and Discussion
PCR Amplification of cGH gene
The PCR products with 776 bp length which run on 1.5% agarose gel were presented in Fig. 1.
Fig. 1: Amplification of cGH (exon-1 and partial intron-1) gene using the specific primer of polymerase chain reaction in broiler visualize under UV Illuminator stained with EtBr. A commercial DNA marker was used for size analysis.
RFLP Analysis of cGH Gene
Three genotypic patterns viz. AA (235 and 541 bp fragments), AB (126, 148, 235, 267 bp fragments) and BB (148, 235, and 393 bp fragments) were detected by digesting the 776 bp chicken growth hormone gene region by MspI restriction endonuclease which has C^ CGG as its restriction site. These genotypes were found in all the strains. These restriction patterns were in concurrence with the observations made earlier by Kaur et al. (2008) in Panjabi broiler and Singh et al. (2008) in Rhode Island red and White Leghorn layer (Fig. 2).
Fig. 2: 8% polyacrylamide gel electrophoresis and restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified product of exon-1 and partial intron-1 in Chicken Growth Hormone (cGH) gene digested with MspI in broiler. Lane 1: DNA marker, Lane 1-2: genotype AA, Lane 3-4: genotype AB, Lane 5-6: genotype BB.
Gene and Genotypic Frequency of Chicken Growth Hormone (cGH) Gene
The gene and genotypic frequency were observed for RFLP restricted genotypic patterns. The three genotypes AA, AB and BB were distributed in the frequency of 0.22, 0.28 and 0.5, respectively. The genotypic pattern ‘BB’ was observed in most (n=75) of the birds. The results indicated that genotypic patterns BB were dominant in the chicken population. The genotypic patterns ‘AA’ and ‘AB’ were present in low number. The estimated allele frequency of A and B allele as observed is 0.39 and 0.61, respectively. The allele A is found to be less frequent in comparison to allele B.
Table 1: Association study of genotypic patterns of cGH gene with body weights in broiler
|Genotypic Patterns||Body Weight (gms)|
|Mean ± SE|
NS: Non significant
The statistical analysis involving association of genotypic patterns of chicken growth hormone gene with body weight parameters in broiler was carried out using General Linear Model (GLM) procedure of SPSS. The three genotypic patterns ‘AA’, ‘AB’ and ‘BB’ of cGH exon-1 and partial intron-1 gene in broiler did not reveal any significant association with body weight at any stage of life. However, non-significantly higher body weight was observed for ‘BB’ genotypic pattern at day-14, day-21, day-28 and day-35 of age.
The results of the current study indicated that exon-1 and partial intron-1 of cGH is polymorphic in broiler. Therefore, the cGH gene could be exploited as a candidate gene for marker-assisted selection (MAS) in broiler. Further studies are required to evaluate the association of production traits with cGH gene polymorphism broiler.
The authors are thankful to dean and Faculty Chairman of the Institute, for providing all facilities and financial support to carry out the work. The helps rendered by the staffs of Poultry Farm are also duly acknowledged.