Identification of nucleotide variation of growth hormone gene in rabbit populations reared in Bulgaria

Five rabbit populations of New Zealand White (NZW), Californian (CAL), crossbred NZW×GW and two generations of the synthetic population – SPF1 and SPF2 reared in Bulgaria were included in the present study with the aim of detecting the genetic variability of the growth hormone encoding gene (GH) via polymerase chain reaction with the restriction fragment length polymorphism analysis and direct sequencing. The targeted region of the rabbit GH gene was amplified and a fragment of a total of 231 bp was obtained in all studied populations. Allele identification was determined after enzymatic digestion, where two fragments of 62 and 169 bp correspond to allele C and an undigested fragment of 231 bp corresponds to allele T. Two additional bands of 107 and 124 bp evidenced A/G genetic polymorphism in the rabbit GH gene. Thirtyeight percent of the studied rabbits were carriers of the double mutation (C/T+A/G) in the same locus as the studied GH gene. The sequence analysis revealed two nucleotide substitutions – g.111C>T and g.156A>G in the non-coding region between the regulatory TATA box and 5’ UTR region, and a novel g.255G>A genetic variant in intron 1 of GH gene. The A>G transition was most frequent (40.57%), compared to the other ones, G>A (28.57%) and C>T (10.80%), respectively. The most frequent genotype in the NZW population was homozygous TT (0.93), with a prevalence of the T allele (0.97) over allele C (0.03) for g.111C>T SNP site. The distribution of the allele and genotype frequencies at the sites g.156A>G and g.255G>A in this rabbit group was identical, with the highest value of 0.93 for alleles A and G, respectively. The rabbit populations CAL and NZW×GW showed equal frequencies of the prevalent T allele (0.83) and for homozygous TT genotype (0.67) according to g.111C>T SNP. The highest values were obtained for the allele А (0.83) and for homozygous AA genotype (0.67) at c.33A>G SNP in these rabbit groups. The highest values (0.67, 0.60 and 0.80) for the heterozygous genotypes at g.111C>T, g.156A>G and g.255G>A SNPs, respectively, were detected among the SPF2 rabbit population, compared to the both homozygous genotypes. The results obtained in the present research indicates a significant degree of genetic variability of the studied polymorphic GH locus in the SPF2 rabbit group.