Abstract: Objective: The objective of this study was to determine Growth Hormone (GH) gene polymorphisms and their association with productive traits, including body weight, at ages 1, 2, 3, 4, 5, 6, 7 and 8 weeks. Methodology: Polymorphisms in exon 1 of the GH gene were evaluated in two duck populations in West Sumatra Province Indonesia (Pitalah and Kumbang Janti ducks). For this purpose, blood samples were collected and DNA samples were extracted using the Promega Wizard® Genomic DNA Purification Kit. For this purpose, a total 225 ducks blood samples were collected from 145 male and 80 female ducks. Genetic polymorphisms were determined with the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method using the Eco721 restriction enzyme and agarose gel electrophoresis. Direct sequencing of some samples was used to confirm the results. Results: Two alleles (GHG and GHA) and three genotypes (GH/GG, GH/GA and GH/AA) were found in the studied duck samples at locus GH/Eco721. In both groups of ducks, the dominant allele was GHG. The most frequent genotype in the examined ducks was GH/GA. Three genotypes were observed in the Pitalah ducks, whereas two genotypes (GH/GA and GH/GG) were identified in the Kumbang Janti ducks and in the males. Pitalah ducks with the GH/GA genotype were characterized by a higher (p<0.01) body weight than the ducks with the GH/GG and GH/AA genotypes. This same trend was observed in the female Pitalah ducks; individuals with the GH/GA genotype had higher body weights (p<0.05 and p<0.01) than the birds with the two other detected genotypes. Kumbang Janti ducks with the GH/TT genotype were distinguished by higher values of all evaluated traits compared to the ducks with the GH/CT and GH/CC genotypes; however, most of the recorded differences were not significant. The only trait that was markedly impacted (p<0.05) by the polymorphism of GH gene intron 1 was the body weight at 5, 6, 7 and 8 weeks. Conclusion: This study found that the GH/TT genotype was associated with a higher body weight at 5, 6, 7 and 8 weeks of age in Pitalah and Kumbang Janti ducks.
INTRODUCTION
Advances in the molecular genetics of livestock animals have led to the identification of genes or markers associated with genes that influence growth, carcass and meat quality traits and reproductive features1,2. The molecular bases of these characteristics are being revealed by functional genomics methods and are providing opportunities for the enhancement of genetic improvement programs in farm animals through Marker-Assisted Selection (MAS)3.
Several genes have been used as candidate genes for marker-assisted selection for improved productive and reproductive performances of animals. One of these genes is the Growth Hormone gene (GH)4. The expressed product of the GH gene is the protein Growth Hormone (GH) (also called somatotropin), which is a member of the growth hormone/prolactin family produced in specific cells (somatotrophs) of the pituitary gland5. This gene has many physiological functions, such as promoting muscle growth6, bone growth and development7 and regulation of fat content8 and metabolism9. Additionally, GH plays important roles in the innate and acquired immune systems. The GH has been shown to affect the proliferation of lymphoid cells, the activity of phagocytic cells, thymulin excretion and the growth of the thymus10. Studies in animals have indicated that GH is involved in the sexual differentiation and pubertal maturation processes and participates in gonadal steroidogenesis, gametogenesis and ovulation11. In birds, GH has an important function in growth but is also involved in a variety of secondary functions, including egg production, aging and reproduction12.
Due to its functional importance, the GH gene has been studied in a wide range of species. The genomic structure of this gene has been examined in fish13, rats14, mice15, bovines16,17, sheep18, pigs19 and humans20. In birds, GH cDNA clones have been isolated and sequenced from chickens21, turkeys22 and ducks23. The genomic sequence of the avian GH gene was first reported in chickens24.
In all mammals, the GH gene extends over 2-3 kb and comprises five exons split by four introns25. The duck GH gene is 5.25 kb in size, consists of five exons and four introns and is structurally similar to the mammalian and chicken GH genes26. Furthermore, the GH gene is highly polymorphic in a variety of livestock animals. Many polymorphisms have been identified in the GH genes from pigs27, bovines17, goats28 and poultry29-31. In ducks, the effect of GH gene polymorphisms on important economic traits has been noted32,33. Moreover, a study conducted on ducks by Hiyama et al.34 suggested that variations in the GH promoter might influence the laying performance by changing the GH mRNA expression levels in the anterior pituitary gland.
The objectives of this study were to estimate the allele and genotype frequencies of GH/Eco721 polymorphisms in Pitalah and Kumbang Janti ducks. Additionally, we investigated the possible associations of duck growth hormone gene polymorphisms with body weight to identify a potential marker for use as a complementary parameter in the selection of ducks.
MATERIALS AND METHODS
Animals: The experiment was conducted in the Faculty of Animal Husbandry Andalas University, Indonesia. A total of 225 samples were used to genotype the GH gene. The ducks used for this study were two Indonesian native breeds (the Pitalah duck and the Kumbang Janti duck from Payakumbuh district, West Sumatra, Indonesia). The ducks used consisted of 145 Pitalah ducks (100 males and 45 females) and 80 Kumbang Janti ducks (45 males and 35 females). During the trial, the ducks were fed complete commercial diets ad libitum according to age as follows: A starter diet (from the 1st day to 3rd week of age) containing 20.0% CP and 11.7 MJ of Metabolizable Energy (ME) and a grower/finisher diet (from the 4th week of age to the end of the experiment) containing 18.5% CP and 12.2 MJ of ME. The ducks were weighed at ages 1, 2, 3, 4, 5, 6, 7 and 8 weeks.
Detection of polymorphisms in exon 1 of the GH gene: Blood samples were collected from the wing vein of each individual in tubes containing EDTA as an anticoagulant. The blood samples were stored at -20°C prior to DNA extraction. Genomic DNA was extracted from whole blood using the Wizard® Genomic DNA Purification Kit, Promega, Madison, USA. Genomic DNA from each duck was stored at -20°C prior to the allelic discrimination assays.
The GH genotypes were analyzed using the RFLP-PCR method. The 801 bp GH gene PCR product was amplified using the Master Mix from Thermo Scientific. The PCR conditions included 25 μL of the Master Mix, 2 μL (20 ng) of genomic DNA, 1.5 μL (15 nM) of each primer (forward primer 5’-CTG GAG CAG GCA GGA AAA TT-3’ and reverse primer 5’-TCC AGG GAC AGT GA AC-3’) and 20 μL of nuclease-free water. The following cycles were applied: Denaturation for 5 min at 94°C, followed by 40 cycles of 45 sec at 94°C, annealing for 45 sec at 60°C and extension for 60 sec at 72°C and a final extension for 5 min at 72°C. The PCR product (consisting of 801 bp) was digested with the Eco721 restriction enzyme for 4 h at 37°C. The digested fragments were separated on 2% agarose gels. The genotypes were identified against the molecular marker O’ Gene Ruler Low Range DNA Ladder (Thermo Scientific).
Statistical analysis: The genotype and allele frequencies were calculated in each group of ducks. The data used to compare the effects of GH gene polymorphisms on duck body weight were tested with a model that included the effect of each genotype at the GH/Eco721 locus. The genetic effects of the GH gene polymorphisms on body weight were analyzed using a General Linear Model (GLM) procedure35. The model used the Eq. 1:
| (1) |
where, Yijkl is the observed value of the dependent variable, μ is the overall mean, Gi is the fixed effect due to genotype GH/Eco721 (i = GH/CC, GH/CT or GH/TT), Dj is the fixed effect due to duck origin (j = Pitalah ducks or Kumbang Janti ducks), Sk is the fixed effect due to gender (k = males or females) and gijkl is the random residual error.
The Hardy-Weinberg equilibrium was assessed with the Chi-square test. The statistical significance of differences among the means was calculated in accordance with the SAS/STAT Software, Release 6.12 (SAS Institute Inc., Cary, NC, USA).
RESULTS
The allele frequencies of GH gene exon 1 in the two duck populations are listed in Table 1. The dominant allele of exon 1 of the duck GH gene was GHG for the two duck populations. No marked differences were observed between the frequencies of the GHG and GHA alleles in the Pitalah and Kumbang Janti ducks. The allelic distributions of the GH/Eco721 polymorphic sites in the Kumbang Janti duck populations followed a similar pattern. As a result of digestion of an 801 bp target region of the duck GH gene exon 1 by the Eco721 enzyme, the samples with an 801 bp fragment (uncut) were accepted as the GH/GG genotype, the samples with 801, 564 and 237 bp fragments were accepted as GH/GA and the samples with 564 and 237 bp fragments were accepted as the GH/AA genotype (Fig. 1). The genotype distributions of the GH gene in the two studied duck populations are presented in Table 2. The most frequent genotype in the examined duck groups was GH/GA. The highest degree of genetic polymorphism for the duck GH gene intron 1 was found in the Pitalah ducks. The genotype frequencies of the GH/Eco721 locus in this duck population were not in Hardy-Weinberg equilibrium (p>0.05).
Regarding the polymorphisms, 2 genotypes and 2 alleles were distinguished according to their restriction fragment lengths as follows: 801 bp (A allele) and 564 and 237 bp (G allele). The genotypes and alleles of the GH gene are shown in Fig. 1.
| Fig. 1: | GH/Eco721 genotype identification |
Lane 1, 2 and 3: Genotype GH/GG, Lane 4: Molecular weight marker 250, 500, 750, 1000 bp, Lane 5, 6 and 7: Genotype GH/GA-801, 564, 237 bp, Lanes 8, 9 and 10: Genotype GH/AA-801 bp) |
|
| Table 1: | Allele frequencies at the exon 1 locus of the GH gene |
| Table 2: | Association of GH gene polymorphisms in exon 1 with body weights at age 1-8 weeks in Pitalah ducks |
| NS: Non-significant | |
| Table 3: | Association of GH gene polymorphisms in exon 1 with body weights at age 1-8 weeks in Kumbang Janti ducks |
| NS: Non-significant | |
The results of the analysis of the associations between the GH/Eco721 polymorphisms and body weights in the Pitalah ducks are summarized in Table 2.
The association between different genotypes and body weights in the Pitalah ducks show no significant associations for 1, 2 and 3 weeks of age, significant associations for 4 weeks of age and highly significant association for 5, 6, 7 and 8 weeks of age (p<0.01). The ducks with the GH/GG genotype had the highest body weights, followed by the GG/AA and GH/GA genotypes.
The results of the analysis of associations between the GH/Eco721 polymorphisms and body weights in the Kumbang Janti ducks are summarized in Table 3.
The association between different genotypes and body weights showed similar results to those obtained for the Pitalah ducks. No significant association was found for 1, 2, 3 and 4 weeks of age, a significant association was found for 5 weeks of age and a highly significant association was found for 6, 7 and 8 weeks of age (p<0.01). The ducks with the GH/GG genotype had the highest body weights, followed by the GG/AA and GH/GA genotypes.
DISCUSSION
In the present study, polymorphisms of exon 1 of the duck GH gene were examined. Previous studies showed that polymorphisms of the avian GH gene could be identified not only at exon 1 but also in other regions. Polymorphisms in exonic regions of this gene were detected in ducks1 and geese31. Additionally, polymorphisms in the intronic regions of the avian GH gene were found in chickens at intron 130, in ducks at intron 232, in geese36 and chickens8 at intron 3 and in chickens at intron 437. There is no information in the literature concerning the identification of Eco721 polymorphisms in the first exon of the duck GH gene. However, we can compare the frequencies of the GH gene alleles and genotypes presented herein with the results described by Wu et al.32. The allelic frequencies reported in the above mentioned study, which was conducted in three duck populations, differed from the results of the present study. Wu et al.32 reported that usage of the BsmFI restriction enzyme in all duck populations enabled the detection of three genotypes. The observed differences may result from the origin of the experimental animals. Productive performances of poultry are affected by quantitative traits that can be influenced by many environmental factors and genes, such as the growth hormone gene. The GH gene polymorphisms have been studied in various poultry species, including chickens8, quail38, geese36 and ducks33. In these poultry species, a high degree of polymorphism has been detected in the DNA sequence of the GH gene. Chang et al.1found 19 SNPs in a 2087 base pair (bp) region in the duck GH gene. This study indicated that each SNP was associated with at least one duck fertility-related trait. However, available literature shows that some SNPs in the duck GH gene also affect growth and carcass traits. The effect of the avian growth hormone gene on the above mentioned characteristics was demonstrated by Wu et al.32, who first discovered the BsmFI polymorphism in the second intron of the duck GH gene. This Chinese research group tested three different breeds of duck (Cherry Valley, Muscovy and Jingjiang) slaughtered after 56 days of life. Considering the body weights of the ducks on the day of slaughter, the results of the present study partially confirm the observations of Wu et al.32. The authors noted that in one of the evaluated duck breeds (Jingjiang), individuals with genotype GH/TT were heavier than individuals with the GH/CT and GH/CC genotypes. However, findings of the present study regarding two other breeds were not in agreement with the findings described by Wu et al.32. In the Cherry Valley and Muscovy groups, the heavier birds had the GH/CT genotype32.
Due to a lack of any comparable results concerning the effect of the GH gene on body measurements in ducks, verification of our findings based on the results reported in previous studies is hampered. However, the results of present study showed that individuals with the GH/TT genotype displayed higher values of most of the assessed features compared to ducks of other genetic groups, which indicated that the GH gene might be a candidate marker for biometric traits in ducks. In conclusion, the highest degree of polymorphism in the first exon of the GH gene was observed in the Pitalah and Kumbang Janti ducks. The results of this study regarding the GH/Eco721 genotype showed a significant influence on BW.
SIGNIFICANCE STATEMENT
This study is the first attempt to explore the GH gene in Pitalah and Kumbang Janti ducks. This study discovered a new polymorphism (GH/Eco721) in intron 1 of the GH gene that was associated with body weight in Pitalah and Kumbang Janti ducks. These results can be beneficial for genetically assisted selection to improve these breeds. These findings are important for poultry farmers and policy makers when designing selection strategies for improving duck production and to ensure a protein supply for the general public.
ACKNOWLEDGMENTS
This study was funded and supported by the Directorate General of Higher Education of Ministry of Research, Technology and Higher Education through the Hibah Bersaing 2016 scheme with contract No. 01/H.16/HB/LPPM/2016. We also thank the Dean of the Faculty of Animal Science Andalas University, for housing support.