The Associations of GH and GHR Genes with Carcass Components in Indonesian Kampung and Broiler Chicken Cross

The chicken growth hormone (GH) and its receptor (growth hormone receptor, GHR) play important roles in chicken performances due to their crucial functions in growth. The variations of GH and GHR genes were then thought to be associated with the variations of the performances. This experiment was designed to identify the g.2248G>A GH and the g.565G>A GHR loci polymorphisms and to evaluate their associations with carcass components in Kampung and broiler chicken cross. A total of 215 chickens including 4 chicken populations (Kampung, Cobb broiler, F1, and F2 Kampung x broiler chicken cross) were screened to identify polymorphism using PCR-RFLP technique with EcoRV and Eco72I restriction enzyme for GH and GHR loci, respectively. The carcass components were recorded at 26 weeks of age on F2 Kampung and broiler chicken cross (42 chickens) for association study. Both the g.2248G>A GH and the g.565G>A GHR loci were polymorphic with two alleles (G and A) and three genotypes (GG, AG, and AA). The GG genotype and the G allele of GH locus were predominant in all chicken populations. While in GHR locus, the AA genotype and the A allele were found to be higher in all chicken populations. The association study showed that the g.565G>A GHR locus polymorphism had significant effect on carcass components, including live weight, carcass weight, breast weight, thighs weight, breast muscle weight, and thighs muscle weight. There was no significant association was found between the g.2248G>A GH genotype and carcass components. It could be concluded that the g.2248G>A GH and the g.565G>A GHR loci were polymorphic in Kampung and broiler chicken cross and the g.565G>A GHR locus was accosiated with carcass components. This g.565G>A GHR SNP might be an important candidate marker for chicken growth and muscle mass improvement.


INTRODUCTION
Native chickens do not only contribute to the conserva tion of poultry genetic resources, but also play an important role in rural economies in most of the developing and undeveloped countries (Moharrery & Mirzaei, 2014;Padhi, 2016). Kampung chicken is Indonesian native chicken, which can be found easily in rural area with strong economic and social relations to community (Nataamijaya, 2010). Zein & Sulandari (2012) reported that Kampung chicken's population is getting expanded with high genetic diversity. However, growth is the main challenge for Indonesian native chicken production. By intensive rearing system, Indonesian Kampung chicken reaches slaughter weight in 4.5 months or more (FAO, 2008).
Improvement of the performance of Kampung chicken could be done by selection and crossbreeding or by utilization of both selection and crossbreeding (Sheng et al., 2013;Padhi, 2016). In order to improve the growth and carcass performance of Kampung chicken, an F2 intercross between Kampung and commercial broiler chicken was designed and then followed by selection based on genes controlling growth. Abdurrahman et al. (2016) reported that the local chicken crossbreds had lower fat and cholesterol contents due to a higher muscular contraction compared to modern breeds. Meanwhile, at the gene level, molecular genetics provides rapid and accurate identification and selection tools for individuals to improve their performances permanently (Fulton, 2008;Padhi, 2016). Furthermore, in poultry breeding program, selection in molecular approach could be done before the traits were expressed or shortly after hatching (Fulton, 2008).
Kata kunci: ayam silangan Kampung, growth hormone, growth hormone receptor, keragaman genetik, karkas binding, activates a variety of signaling molecules that contributes to the GH-induced changes in enzymatic activity, transport function, and gene expression (Ciftci et al., 2013). Study of Xu et al. (2013) proposed that lacking of the GHR gene might be involved in dwarfism formation in chicken. Studies in broiler chicken shows that the GH gene is located in chromosome 27, has 4.1 kb overall length and consists of five exons and four introns (Stephen et al., 2001;Nie et al., 2005). The GHR gene, which is located in chromosom Z, has 4.0 kb length and consists of ten exons and nine introns (Nie et al., 2005). Nie et al. (2005) reported that 46 SNPs in GH gene and the g.2248G>A GH (G+1705A) polymorphism in intron 3 had strong associations with growth traits in F2 White Recessive Rock and Xinghua chicken. A number of 33 SNPs in GHR gene was reported by Nie et al. (2005), and the g.565G>A GHR polymorphism was reported had significant associations with fatness and muscle fiber traits in Chinese local chickens (Lei et al., 2007).
Studies in other poultries reported the associations of the GH gene with growth and carcass traits in Pitalah and Kumbang Janti Indonesian ducks (Yurnalis et al., 2017), Huoyan Chinese native goose , and Japanese quail (Johari et al., 2013). In other farm animals, the GH gene was reported in cattle breeds (Dolmatova & Ilyasov, 2011), pig (Ashok et al., 2014), salmonids fish (Kamenskaya et al., 2015), sheep (Jia et al., 2014), goat , camel (Shawki et al., 2015), and passerine bird (Arai & Iigo, 2010). The effect of GH and GHR gene polymorphisms on carcass component in Kampung and broiler chicken cross has not been examined yet. The objectives of this study were to identify the g.2248G>A GH and the g.565G>A GHR loci polymorphisms using PCR-RFLP and to evaluate their effect on carcass components in Kampung and broiler chicken cross. This polymorphism information might serve as a platform for development of molecular marker in carcass and meat selections in chicken.

Animal and Phenotypic Data Collection
Animal care procedures were approved by the Animal Care and Use Committee of Bogor Agricultural University (No. 22-2016 IPB). A number of 215 individuals consisting of 4 chicken populations: Kampung (49), commercial Cobb broiler (30), F1 Kampung x Cobb broiler cross (43), and F2 Kampung x Cobb broiler cross (93) were obtained from Animal Breeding and Genetics Division, Faculty of Animal Science, Bogor Agricultural University (IPB), Indonesia, were used in this experiment. The F2 cross was set by crossing individuals from the Kampung and commercial Cobb broiler lines as parental (P). Kampung chicken was selected as roosters, collected from traditional farmers in Ciawi, District of Bogor, West Java. Commercial Cobb broiler parent stock was used as hen. The F1 was generated by crossing Kampung and broiler lines. The F1s were then bred to generate F2. Blood sample from each individual (P, F1, and F2) was obtained from the wing vein and collected in tube containing EDTA. The experimental chickens were raised in the same environmental condition and had free access to feed and water.
F2 Kampung x Cobb broiler cross (42 chickens: 20 males and 22 females) were slaughtered at 26 weeks of age to collect carcass component data. Carcass component measurement included live weight, carcass weight, commercial cuts (breast, thighs, drum sticks, and wings) weights, muscle (breast muscle, thigh muscle, and drum sticks muscle) weights, and percentage of carcass, commercial cuts and muscles. The phenotypic of carcass measurement is provided in the Table 1.

Genomic DNA Extraction
Genomic DNA was isolated from blood. The DNA extraction protocol was performed according to Sambrook & Russel (2001) with minor modification. Briefly, each 20 μL of blood sample was added with 800 μL RBC lysis buffer, homogenized, and centrifuged (800 rpm) for 5 min. Then, the supernatant part was removed. The precipitation part was added with 40 μL 10% SDS, 10 μL Proteinase K 5 mg/mL, and 300 μL 1 x STE, and slowly shaken at 55 o C for 2 h. Then, each sample was added with 400 μL phenol solution, 400 μL CIAA, and 40 μL NaCl 5M, and slowly shaken at room temperature for an hour, and centrifuged (12000 rpm) for 5 min. About 400 μL liquid from top layer was removed into a new tube and added with 800 μL 96% EtOH and 40 μL NaCl 5M. Then, the sample was frozen overnight. DNA molecule was centrifuged (12000 rpm) for 5 min, and the supernatant part was discarded. For the DNA precipitation, the pellet was air dried and 100 μL 80% TE was added. DNA sample was frozen stored for long term usage.

Amplification and Genotyping
Specific fragments of GH and GHR genes were produced with the polymerase chain reaction (PCR) method using thermocycler machine (GeneAmp® PCR System 9700, Applied Bio SystemsTM, Foster City, USA). Primers (Table 2) were designed using Primer Designing Tool (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/). The PCR process was run with 35 cycles consisting of denaturation at 95 o C for 10 s, annealing at 60-61 o C for 20 s, and extension at 72 o C for 30 s. Amplification was performed with a total volume of 25 μL containing 50 ng/mL DNA sample, 0.5 pmol primer, 0.5 unit GoTaq Green Master Mix (Promega, Madison, USA), and water.
Genotyping was done using a restriction fragment length polymorphism (RFLP). PCR product and restriction enzyme (Thermo Fisher Scientific, EU, Lithuania) were incubated at 37 o C for 12 h. Genotype was visualized through 2% agarose gel electrophoresis (v/w), which was stained with FluoroSafe DNA Staining (1 st Base, Singapore) above UV Transilluminator (Alpha Imager, Alpha Innotech, Santa Clara, USA). Primers annealing position and restriction site of GH and GHR were shown in Figures 1 and 2

Data Analysis
Polymorphism information and sequencing. Polymorphism parameters (genotype frequency, allele frequency, and the Hardy-Weinberg Equilibrium) were analyzed according to Nei & Kumar (2000). All sequence results (ABI trace files) were analyzed in Molecular Evolutionary Genetic Analysis (MEGA) 6.0 according to Tamura et al. (2013) and BioEdit (Hall, 2011). The Basic Local Alignment Search Tool (BLAST) was used to identify similarity (homology) with genes data in GenBank (www.ncbi.nhl.nih.gov./BLAST).

Statistical analysis.
The association of genotype with carcass components was analyzed using SAS GLM procedure (SAS Institute, 2008) and Duncan multiple range test. The genetic effects were analyzed using this model: where y ijk was the observed phenotypic trait (carcass components) for k th individual with i th sex (i= male and female) and j th genotype (j= AA, AG, and GG), μ was the overall mean, S i was the genetic effect of i th sex, G j was the genetic effect of j th genotype, and ε ijk was the normally distributed residual error.

Polymorphisms of GH|EcoRV and GHR|Eco72I Loci
The partial fragment of GH and GHR genes in all individuals were successfully amplified and showed a 339 and 326 bp bands, respectively (Figure 3). The lengths of PCR products were in good agreements with the reference sequences (GenBank accession number: AY461843.1 and AJ506750.1 for GH and GHR, respectively). Genotyping analysis of GH locus was performed

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using EcoRV restriction enzyme. This genotyping identified two alleles (A and G) and three genotypes (AA, AG, and GG). The A allele was indicated by 191 and 148 bp bands (restricted), while the G allele was indicated by 339 bp (unrestricted, Figure 4). Mutation was found in 190 bp from G to A nucleotide, ( Figure 5). According to GenBank (accession number: AY461843.1), this SNP is located in g.2248G>A GH. Moreover, Eco72I enzyme was used for genotyping of GHR locus. The genotyping of GHR|Eco72I locus generated the same allele and genotype with GH|EcoRV locus. The A allele was indicated by a 326 bp (unrestricted), while the G allele was indicated by 212 and 114 bp (restricted, Figure 4). Mutation from G to A nucleotide was occurred in the position of 115 bp ( Figure 6). According to GenBank (accession number: AJ506750.1), this SNP is located in g.565G>A GHR. All chicken populations were polymorphic for both GH and GHR loci ( Table 3). The GG genotype and the G allele of GH locus were predominant in all chicken populations. The AA genotype was barely found (0.005) in overall population. The AA genotype was only found in Cobb population (0.033), not in Kampung, F1, and F2 chicken populations. While in GHR locus, the AA genotype and the A allele were found to be higher in all chicken populations. The x 2 analysis of GH locus showed that all chicken populations were in the Hardy-Weinberg equilibrium. In the GHR locus, the Cobb broiler and F2 cross chicken populations were in the Hardy-Weinberg disequilibrium. This result indicated that the allele and genotype frequencies were not con-stant from generation to generation in these two populations (Allendorf et al., 2013).

Effect of Gene Polymorphism on Carcass Components
Association analyses showed that the g.565G>A GHR locus polymorphism had significant effect on chicken carcass components, but no significant associations between g.2248G>A GH genotype and carcass components were observed (Table 4). The GG genotype of g.565G>A GHR locus had higher live weight, carcass weight, breast weight, thigh weight, breast muscle weight, and thigh muscle weight than the AA genotype (P<0.05) in F2 Kampung x Cobb broiler chicken cross. No significant differences were found in carcass percentage, commercial cuts percentages, and muscle weight percentages.

DISCUSSION
The essential roles of GH and GHR genes in chicken have been explored since decades for improving meat productivity of chicken. The GH molecule has two binding sites which each interacts with the extracellular region of the performed GHR dimer. This binding leads a functionally dimerized complex that induces intracellular signaling (Kopchick, 2016;List et al., 2013). The GH-GHR complexes are then resulted in the endoplasmic reticulum (van den Eijnden & Strous, 2007). This complexes travel to the cell surface and activate the JAK2 via the STAT5/MAPK pathway (Sedek et al., 2014).  bold shows EcoRV restriction sites; box shows g.2248G>A GH SNP target (GenBank accession number: AY461843.1) . Kim et al. (2010) explain that the GH-GHR complexes activate hepatic IGF-I secretion, which stimulates the differentiation and proliferation of bone and muscle cell.
Our study in the GH|EcoRV genotyping generated the GG genotype predominantly (0.749), the AG genotype as a second majority (0.247), and almost no AA genotype was found (0.005) in overall population. The AA genotype was detected only in Cobb broiler population in a very low frequency. Similarly, in the F2 population generated from White Recessive Rock and Xinghua chicken, 18 chickens with the AA genotype were found from total 451 chickens (AA genotype freq= 0.04 ;Nie et al., 2005). Higher GG genotype frequency was also reported by Lei et al. (2007) in Xinghua (0.67) and White Plymouth Rock (1.00) chickens, Zhang et al. (2007) in  497 maturity in semi-intensive rearing system (Yuwanta & Fujihara, 2000). At the age of 26 weeks, chicken growth is in the stationary phase. Thus, in this phase, chicken has reached the maximum growth (Knízetová et al., 1995). Results of our study showed no significant effect between the g.2248G>A GH polymorphism and chicken carcass components. This result was in line with Zhang et al. (2007) in the Chinese indigenous chicken populations. Different to our findings, Nei et al. (2005) andAnh et al. (2015) found any associations of this SNP with chicken growth and carcass traits. Nie et al. (2005) reported that the AA genotype had positive effects (higher body weight, shank length, and ADG) in a F2 population derived from a cross of a fast-growing line, White Recessive Rock, and a slow-growing line, Xinghua. However, Anh et al. (2015) found that the AA genotype negatively affected carcass traits (lower body weight and ADG) in Thai broiler chicken. From those various studies, effects of this g.2248G>A GH polymorphism in different chicken breeds generated varied results. Kansaku et al. (2008) presumed that this indicated the various involvements of GH in production parameters. Moreover, Harvey (2013) concludes from large literature that pituitary GH has no effect on avian growth.   Khoa et al., 2013), Noi (1.00, Khoa et al., 2013), Xinghua (1.00, Lei et al., 2007), and White Plymouth Rock (1.00, Lei et al., 2007) chickens. Ouyang et al. (2008) could not detect the g.565G>A GHR polymorphism using DHPLC detection in Leghorn layer, White Recessive Rock broiler, Taihe Silkies, and Xinghua chickens.
This study evidenced the association of the g.565G>A GHR genotype with live weight, carcass weight, breast weight, thigh weight, breast muscle weight, and thigh muscle weight, for the first time. Our finding proposed that the GG genotype of the g.565G>A GHR had positive effect on chicken carcass and yielded proportional body composition. The inhibition of normal human and animal skeletal muscle growths and fat depositions was reported to be caused by mutation in GHR gene by causing the inhibition of GH signal transduction (Lin et al., 2012). However, the molecular mechanism of GHR introns and its effect on muscle growth in chicken is unclear. It is believed that the genetic potential of Kampung chicken can be improved through crossbreeding and selection. In this study, the G allele of the g.565G>A GHR locus tends to have a positive effect on body and carcass weight in F2 Kampung and broiler chicken cross.
This locus can be recommended as a good candidate to select chickens with better growth and heavier carcass weights. However, further analysis in larger populations and study on the gene expression and protein level is necessary to be proven to reinforce this hypothesis.

CONCLUSION
This study investigated the polymorphisms of GH and GHR genes and emphasized that the g.565G>A GHR locus polymorphisms had significant association with carcass components in F2 Kampung and broiler chicken cross. Here, GHR is a potential marker for carcass traits in chicken.

ACKNOWLEDGEMENT
Authors thank to PMDSU Research Grant 2016 (Batch I) from The Ministry of Research, Technology, and Higher Education, Republic of Indonesia, which financially supported this work.