PCR-RFLP Polymorphism of Growth Hormone Gene and Its Association with Growth and Morphometric Traits in Ganjam and Baigani Goats of Odisha

Animal breeders use the genetic variation reflected in the phenotypic variation in the economic traits to select superior animals to improve the population. As now a days polymorphism in the genes could be identified with molecular genetic tools, which can serve as marker for selection for better animals and increase efficiency of selection. Growth hormone gene is such a candidate gene that has got bearing on the economic traits of the livestock. Growth hormone (GH) is an anabolic hormone synthesized and secreted by the somatotroph cells of the anterior lobe of the pituitary in a circadian and pulsatile manner (Ayuk and Sheppard, 2006). Growth hormone (GH) influences animal processes such as growth (Breier, 1999), lactation (Baldi, 1999), reproduction (Scaramuzzi et al., 1999) and metabolism (Bauman, 1999).
 
Growth hormone is a peptide hormone of 217 amino acid in goats (Kioka et al.,1989) and  is encoded by 2500 base pairs (bp), consisting of five exons, separated by four intervening intron sequences (Gordon et al.,1983). PCR-RFLP polymorphism in exon 2 and 3 of GH gene was studied by Singh et al., (2015) in Sirohi and Barbari goats which revealed   two alleles with non-significant association of them with body length and withers height from birth to 180 days using PCR-RFLP. Hua et al., (2009) studied GH gene of Boer bucks by PCR-SSCP and found two SNPs-A781G (Ser/Sly 35) and A1575G (Leu147). AA genotype resulted significant decrease in chest girth at birth and weaning weight comparing to AB genotype, while CC genotype contributed to weaning height greater than CD genotype. The present investigation was undertaken to explore the polymorphism in the growth hormone gene, estimate the gene and genotype frequencies and association of the alleles with the morphometric traits in Ganjam, a recognized breed and Baigani, a lesser-known goat type of Odisha.

Location and sampling of animals
 
The investigation was conducted on unrelated 100 adult animals of each breed. Ganjam goats were sampled from farmers of three field units Chhatrapur, Rambha and Khallikote of All India Coordinated Research Project on Goat Improvement in the 4^th Agroclimatic zone of Odisha and ‘Baigani goats’  from local farmers of coastal area during 2015-16. Blood samples (10 ml) were collected in 15ml tube with 10 mg EDTA as anticoagulant. The data on morphometry and morphological traits were recorded. A total of twelve morphometric traits were recorded viz. Body weight (kg), Body length (cm), Wither height (cm), Chest girth (cm), Brisket height (cm), Head length (cm), Horn length (cm), Ear length (cm), Tail length (cm), Rump height (cm), Rump width (cm) and neck Circumference (cm).
 
DNA isolation and PCR amplification
 
Genomic DNA isolation from blood was carried out by using Hipura Blood DNA isolation kit from Himedia. The quality was determined by 1% agarose gel. The concentration of DNA samples were measured by Nanodrop technique. The purity was evaluated by UV spectrophotometry by OD at 260 nm and 280 nm. The yield of DNA ranged from 10 to 400 µg/ml with the OD 260/280 ratio ranging from 1.7 to 2.0.
       
The oligonucleotide primers forward 5'CTCTGCCTGC CCTGGACT3' and reverse 5'GGAGAAGCAGAAGGCAAC C3' described by Hua et al., (2009) were used to amplify 422 bp of the exon 2 and 3 of GH gene.
       
The PCR reactions were carried out in a thermal cycler with a final volume of 25µl containing 2.5 µl of 10x PCR buffer, approximately 1.0 µl of (10 pmol) of each primers,  0.5µl of each of dNTPs (10.0 mM), 0.2 µl (1 unit) of Taq DNA Polymerase, approximately 1.0 µl (100ng) of genomic DNA and 17.3 µl of nuclease free water. A manual hot-start PCR was performed under the following thermal cycling profile. Initial denaturation was performed at 94°C for 10 min, there after Taq polymerase was added and a total of 35 cycles of amplifications were employed. Each cycle of amplification comprised of denaturation at 94°C for 1 min, annealing at 58°C for 1min and extension at 72°C for 1 min. A final extension step was carried out at 72°C for 10 min. PCR amplified products were verified on 1.5% agarose horizontal gel electrophoresis.
 
PCR-RFLP assay
 
Restriction digestion of amplicon was conducted in a total volume of 20µl reaction mixture having 10X Buffer (2µl), PCR product (10µl), restriction enzyme 1 µl (10 units/µl) and nuclease free water (17µl). The digestion with restriction enzyme Hae III was carried out at 37°C for a duration of 2 hr 30 minutes. The digested products were separated on 2% agarose gel stained with 1µg/ml Ethidium Bromide and visualized under UV transilluminator (Life Science). The gene and genotype frequencies were calculated by scoring the band pattern.

Data analysis
 
The data on morphometric traits were analysed  using SAS software (SAS Institute Inc. 2009) using analysis of variance taking genotype and breed as fixed effects. The model used was indicated as follows: Where
Y_ijk was phenotypic value of traits; µ was the population mean; Gi was fixed effect of i^th genotype; B_j was fixed effect of j^th breed and e_ijk were random residual error.

Gene and genotype frequencies
 
The PCR-RFLP pattern for 422 bp product of exon 2 and 3 of GH gene using the restriction enzyme Hae III yielded two alleles resulting in three genotypes. The variant with no restriction site was scored as B type and the one having one restriction site with 366bp and 56bp was scored A-type. The band pattern of the three genotypes are presented in Fig 1 and Fig 2 respectively. The details of the gene and genotype frequencies are presented in Table 1. The test of homogeneity of both the population with respect to genotypes revealed that both the population had significantly (P<0.05) different genotypic frequencies. In other word, it can be suggested that Ganjam goat population had higher percentages of heterozygotes as compared to the Baigani goat population.
 

 

 

 
Out of the 100 Ganjam goats, 17 were of AA, 75 of AB and 8 were of BB genotype with corresponding genotype frequency of 0.17, 0.75 and 0.08 respectively. The allele frequency for ‘A’ and ‘B’ were 0.55 and 0.45 respectively.  In Baigani goats the frequency for A and B alleles were 0.46 and 0.54 respectively and the genotype frequency was 0.15, 0.63 and 0.22 for AA, AB and BB respectively. The allele frequencies of A and B alleles reported in this study were in agreement with the studies of Singh et al., (2015) where frequency of A and B alleles were 0.41 and 0.59 in Sirohi goats and 0.45 and 0.55 in Barbari goats.  In Ganjam goats the frequency of ‘A’ allele was more than ‘B’ allele where as in Baigani goats frequency of B allele is high. Similarly the allele frequency of intermediate range (A allele: 0.5812 and B allele: 0.4188) was also reported by Hua et al., (2009) in a Boer goat population. Similar allelic frequencies of 0.620 for A allele was reported by Alakilli et al., (2012) in Barki goats. Othman et al., (2015) also obtained gene frequencies of similar range in Egyptian goat breeds. The polymorphic variants of GH gene have also been reported in Malabari goats by Chitra et al., (2004) using PCR-RFLP. Polymorphism in different exons of GH gene has been reported in Black Bengal goats by Gupta et al., (2007), in Sirohi goats by Kumar et al., (2008), in Jakhrana goats by Gupta et al., (2009) and in a crossbred goat population by An et al., (2010) using PCR-SSCP.
 

 

 
Association of genotypes with morphometric traits
 
The means for  growth and  morphometric trait such as  Body weight (kg), Body length (cm), Wither height (cm), Chest girth (cm), Brisket height (cm), Head length (cm), Horn length (cm), Ear length (cm), Tail length (cm), Rump height (cm), Rump width (cm) and neck Circumference (cm) are presented in Table 2 for the three genotypes and in Table 3 for both the breeds. In all the twelve morphometric traits Ganjam goats had significantly (P<0.01) higher values than the Baigani goats, whereas head length was only significantly (P<0.03) greater than Baigani goats. Rump width was the only exception where no significant difference was observed between the two goat populations. The comparison of trend suggested that Baigani is smaller as compared to Ganjam goats.
 
AB genotypes had slightly higher values in most of the morphometric traits studied as compared to AA and BB genotypes, though the superiority was statistically not significant. Similar observations were reported by Singh et al., (2015) in Sirohi and Barbari goats for body length at all ages. Similar findings were reported by Wickramaratne et al., (2010) in Sangamneri goats for different regions of GH gene. Hua et al., (2009) reported non-significant effect of genotypes on birth weight, body length, birth height, chest girth at birth, body weight at eleven month. However, he reported significant effect of genotype on chest girth at birth and weaning weight. Similarly Zhang et al., (2008) in Nanjiang Huang goats, Deng et al., (2010) in Xinong Saanen goats reported significant association of GH gene with body length at different ages. The differences in the performance of different genotypes might be due to the differential expression of the genotypes in different environmental situations and complex genotype-environment interactions.

The associations of the genotypes studied with types traits such as head profile, body coat colour pattern, ear type and horn type were presented in Table 4. It revealed that genotypes were significantly associated with head profile and horn types of the animals whereas there was no significant association of genotypes with coat colour profile and ear types of the animals.
 

The exploratory study revealed that the target genetic locus of growth hormone gene had two genetic variants in both the population of Ganjam and Baigani goats as detected by the PCR-RFLP method with Hae III and three genotypes namely AA, AB and BB. There were more number of heterozygotes in both the population. The effect of genotypes was found to be statistically non-significant on the morphometric traits though the AB genotype had slightly higher measurements than both the genotypes for most of the morphometric traits.

The study was supported by Indian Council of Agricultural Research, New Delhi under All India Coordinated Research Project on goat improvement.