Considered the rabbit Growth Hormone Receptor (GHR) gene as a candidate gene for growth efficiency, understanding the genetic variation in this locus is of particular relevance. The aim of the present research was to investigate populations from the rabbits (Oryctolagus cuniculus) and to identify single nucleotide polymorphism (SNP) with respect to the GHR gene through PCR-RFLP assay. Genotype profiles were established in a total of 100 rabbits from two populations (New Zealand White, NZW, n=51 and Californian, n=49), reared at The Institute of Animal Science, Kostinbrod. As expected, a 479bp amplicon of the polymorphic site (exon 3) of GHR gene was amplified using PCR and digested with endonuclease enzymes HinfI. The restriction pattern obtained in agarose gel electrophoresis was constituted by three bands (210bp, 162bp and 107 bp) for allele C and by two bands for allele G (317bp and 162 bp). The obtained restriction fragments revealed three genotypes: CC, CG and GG, observed in 7.8%, 53% and 39.2% of the NZW rabbit population and in 28.6%, 51% and 20.4% of the Californian rabbit population, respectively, without departure from the Hardy-Weinberg equilibrium (P>0.20) in the investigated groups. The allele frequencies determined a prevalence of the G allele (0.657) over the C allele (0. 343) in NZW rabbit population, while in the Californian rabbits, the frequency of the C allele (0.541) was higher than allele G (0.459). Observed heterozygosity was higher than expected, resulting in a negative inbreeding coefficient (Fis= -0.174 for NZW rabbits and Fis= -0.027 for Californian rabbits), indicating a sufficient number of heterozygous forms in both studied groups of rabbits. The obtained results from the present investigation confirmed the presence of the SNP in rabbit GHR gene. Therefore, the genetic variability established in this polymorphic locus could be applied in further association studies with growth traits in domestic rabbits

Deyana Gencheva

Krasimir Velikov

Svetlana Georgieva

Svetlin Tanchev

Tsvetoslav Koynarski

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ISSN 1314-6246 Gencheva et al. J. BioSci. Biotechnol. 2017, 6(3): 197-201RESEARCH ARTICLE  197 http://www.jbb.uni-plovdiv.bg Introduction The European rabbit (Oryctolagus cuniculus) is one of the domesticated animal species with the broadest ranges of economic and scientific applications and fields of investigation (Bertolini et al., 2014). On the other hand, domestic rabbits have been recently considered as a good alternative source of animal protein for the increasing human population in the developing countries (Lukefahr & Cheeke, 1991; El-Sabrout & Aggag, 2017). In this connection, revelation the single nucleotide polymorphism in the Growth Hormone Receptor encoding (GHR) gene is of particular relevance, taking into consideration that this locus is a candidate gene for growth efficiency in rabbits due to its biological roles.  As a member of the cytokine receptor superfamily, GHR is constituted by three functional domains – an extracellular (ligand-binding) domain, one transmembrane domain and a signal-transducing, cytoplasmic domain (VanderKuur et al., 1994; Postel-Vinay & Finidori, 1995; Rosenboom, 2000). The binding of Growth Hormone (GH) to GHR causes receptor-dimerization and initiates signaling cascades via the cytoplasmic domain that leads to cell growth and differentiation, increased blood glucose levels, increased free fatty acid levels, decreased fat mass, increased lean body mass, as well as increased linear growth and organ size (Frank, 2001).  Deyana Gencheva 1 Svetlana Georgieva 1 Krasimir Velikov 2 Tsvetoslav Koynarski 3 Svetlin Tanchev 1 Single nucleotide polymorphism of the Growth Hormone Receptor (GHR) encoding gene in Oryctolagus cuniculus Authors’ addresses: 1 Department of Genetics, Breeding and Reproduction, Faculty of Agriculture, Trakia University, Students town, Stara Zagora 6000,  Bulgaria . 2 Department of Special branches, Institute of Animal Science – Kostinbrod, spirka Pochivka,  Sofia 2232,  Bulgaria. 3 Department of General Livestock Breeding, Faculty of Veterinary Medicine, Trakia University,  Students town, Stara Zagora 6000,  Bulgaria.  Correspondence: Deyana G. Gencheva Department of Genetics, Breeding and Reproduction, Faculty of Agriculture,  Trakia University, Students town, Stara Zagora 6000,  Bulgaria. Tel.: +359 889 90 87 84 е-mail: dhristova@uni-sz.bg   Article info: Received: 27 November 2017 Accepted: 23 December 2017  ABSTRACT Considered the rabbit Growth Hormone Receptor (GHR) gene as a candidate gene for growth efficiency, understanding the genetic variation in this locus is of particular relevance. The aim of the present research was to investigate populations from the rabbits (Oryctolagus cuniculus) and to identify single nucleotide polymorphism (SNP) with respect to the GHR gene through PCR-RFLP assay. Genotype profiles were established in a total of 100 rabbits from two populations (New Zealand White, NZW, n=51 and Californian, n=49), reared at The Institute of Animal Science, Kostinbrod. As expected, a 479bp amplicon of the polymorphic site (exon 3) of GHR gene was amplified using PCR and digested with endonuclease enzymes HinfI. The restriction pattern obtained in agarose gel electrophoresis was constituted by three bands (210bp, 162bp and 107 bp) for allele C and by two bands for allele G (317bp and 162 bp). The obtained restriction fragments revealed three genotypes: CC, CG and GG, observed in 7.8%, 53% and 39.2% of the NZW rabbit population and in 28.6%, 51% and 20.4% of the Californian rabbit population, respectively, without departure from the Hardy-Weinberg equilibrium (P>0.20) in the investigated groups. The allele frequencies determined a prevalence of the G allele (0.657) over the C allele (0. 343) in NZW rabbit population, while in the Californian rabbits, the frequency of the C allele (0.541) was higher than allele G (0.459). Observed heterozygosity was higher than expected, resulting in a negative inbreeding coefficient (Fis= -0.174 for NZW rabbits and Fis= -0.027 for Californian rabbits), indicating a sufficient number of heterozygous forms in both studied groups of rabbits. The obtained results from the present investigation confirmed the presence of the SNP in rabbit GHR gene. Therefore, the genetic variability established in this polymorphic locus could be applied in further association studies with growth traits in domestic rabbits.  Key words:  Oryctolagus cuniculus, Growth Hormone Receptor (GHR) gene, single nucleotide polymorphism (SNP), PCR-RFLP ISSN 1314-6246 Gencheva et al. J. BioSci. Biotechnol. 2017, 6(3): 197-201RESEARCH ARTICLE  198 http://www.jbb.uni-plovdiv.bg The rabbit GHR genes have been already sequenced and Leung et al. (1987) established ten exons encoding 638 amino acids. In this gene, SNP polymorphisms and their association with body weight and growth performance traits in different domestic rabbit breeds and strains were successfully determined. The genetic variability in the rabbit GHR gene have been reported by Deng et al. (2008), who sequenced a part of exon 10 and identified two polymorphisms (g.63537066 C>T and g.63537228 A>G) that were used in an association study with carcass traits and feed efficiency. Also, by Zhang et al. (2012), who sequenced three exons of this gene and identified a missense polymorphism that was associated with carcass traits and body weight in three rabbit breeds. All coding exons and portions of flanking and intronic regions of the GHR gene have been re-sequenced in a panel of several rabbits by Fontanesi et al. (2016) in order to identify polymorphisms and performed an association study between a SNP (g.63453192C>G or c.106C>G if based on the predicted cDNA), located in exon 3 and finishing weight in a commercial meat rabbit line, genotyped by PCR-RFLP. The single nucleotide polymorphism examined by authors is a missense mutation that changes the amino acid at position 36 of the GHR protein (p.L36V). The 1st exon of the growth hormone receptor gene with expected amplicon size of 263 bp had been carried out by Sahwan et al. (2014) for revealing GHR gene polymorphism in three rabbit breeds reared under Egyptian conditions and to investigate the association between growth performance and detecting of SNP in this locus.  The present investigation was carried out for identification C/G nucleotide polymorphism in exon 3 of the GHR gene in New Zealand White and Californian rabbit populations reared in Bulgaria and to establish the genetic structure, through PCR-RFLP assay. Materials and Methods Sample source and DNA isolation  The present investigation was carried out with a total of 100 rabbits from 2 populations, reared in The Institute of Animal Science, Kostinbrod: New Zealand White (22♀ and 29♂; Figure 1) and Californian (25♀ and 24♂; Figure 2). Blood samples (3 ml) were obtained from the auricular vein of rabbits in sterile EDTA tubes, mixed thoroughly and stored at -20°C until the genetic assay. Genomic DNA was extracted from the whole rabbit blood using Illustra Blood Genomic Prep DNA Purification Kit (GE Healthcare, UK). The quality and quantity (about 30-70 ng) of the obtained DNA was determined using NanoVue Plus Spectrophotometer (GE Healthcare) and through agarose gel  electrophoresis (Figure 3).  PCR amplification and genotyping  The genotypes of the studied rabbits with respect to the exon 3 of the GHR gene were established through PCR-RLFP analysis. PCR amplifications were carried out in total volume of 20 μl, containing 100 ng DNA template, 2×Red Taq DNA Polymerase Master mix (VWR, Belgium) and 10 pM of each primer with sequence described by Fontanesi et al. (2016). PCR reactions were performed in Gradient thermocycler (VWR) under the following conditions: an initial denaturation at 94ºС/5 min, followed by 30 cycles at Figure 1. New Zealand White rabbit reared in The Institute of Animal Science, Kostinbrod. Figure 2. Californian rabbit reared in The Institute of Animal Science, Kostinbrod. Figure 3. Changes in seed coat colours in the M2 generation and the control variant (upper left corner). ISSN 1314-6246 Gencheva et al. J. BioSci. Biotechnol. 2017, 6(3): 197-201RESEARCH ARTICLE  199 http://www.jbb.uni-plovdiv.bg 94ºС/30 sec, primer annealing at 56.4ºС/45 sec, extension at 72ºС/1 min and final extension at 72ºС/7 min. The digestion reactions were carried out in 25 μl final volume, containing 10 μl PCR product, incubated at 37ºC/15h using 10 U/μl enzyme HinfI (Bioneer). The obtained PCR products and restriction fragments were separated on 2.5% agarose gel and visualized using Electrophoresis Gel Imaging Analysis System (Bio-Imaging Systems, Israel). Statistical analysis The data were processed using PopGene 32, v.1.31 software (Yeh & Yong, 1999; Labate, 2000). In both studied  rabbit populations, the following parameters were calculated: allele and genotype frequencies, observed (Ho) and Nei-expected (He) heterozygosity, coefficient of inbreeding (Fis) and chi-square (χ2) for testing deviation from Hardy-Weinberg Equilibrium (HWE). Results and Discussion As expected, a 479bp amplicon of the target polymorphic site (exon 3) of the GHR gene was successfully amplified in both rabbit populations, through gradient PCR (Figure 4). The restriction pattern with endonuclease enzyme HinfI, obtained in 2.5 % agarose gel electrophoresis was constituted by three bands (210bp, 162bp and 107 bp) for allele C and by two bands for allele G (317bp and 162 bp). The obtained restriction fragments revealed three genotypes: two homozygous CC and GG and one heterozygous CG, observed in 7.8%, 53% and 39.2% of the New Zealand White rabbit population and in 28.6%, 51% and 20.4% of the Californian rabbit population, respectively (Figure 5). The presented on Figure 6 allele frequencies determined a prevalence of the G allele (0.657) over the C allele (0.343) in NZW rabbit population. On the contrary, in the Californian rabbits, the frequency of the C allele (0.541) was higher than allele G (0.459).   Our results for the NZW breed agreed with data reported by Zhang et al. (2012) for higher prevalence of allele G (frequency 0.676) over allele C (frequency 0.323) in Tianfu rabbit population, also in Iraq rabbits (0.610 for allele G and 0.384 for allele C). The genotype frequencies of the GHR gene in each of rabbit populations are presented in Figure 7. The heterozygous CT genotype was detected in 27 NZW rabbits, with frequency 0.529, while the homozygous CC genotype was found out only in 4 rabbits (0.078). In this population, homozygous GG genotype was presented in 20 rabbits, with frequency 0.392. Figure 4. PCR gradient-obtained amplicons of exon 3 at rabbit GHR gene on 2.5% agarose gel. Figure 5. HinfI - restriction fragments represented different GHR genotypes of rabbit populations. Figure 6. Allele frequencies of the polymorphic site in GHR gene in the studied rabbit populations. ISSN 1314-6246 Gencheva et al. J. BioSci. Biotechnol. 2017, 6(3): 197-201RESEARCH ARTICLE  200 http://www.jbb.uni-plovdiv.bg In the Californian rabbit population, the frequency of the heterozygous CT genotype was 0.510 (25 rabbits), whereas the homozygous CC and TT genotypes had frequencies of 0.286 (14 rabbits) and 0.204 (10 rabbits), respectively. In a study by Zhang et al. (2012) comprising 111 rabbits from Champagne breed, the frequency of homozygous genotype GG was higher (0.657) compared to another homozygous CC genotype (0.018). This implied a prevalence of the G allele over allele C (0.819 vs 0.180) in this group of rabbits. The values of theoretically expected heterozygosity in the studied rabbit populations with respect to GHR gene are presented on Figure 7. The values of theoretically expected heterozygosity ranging from 0.451 for NZW rabbit population tо 0.497 for Californian rabbits were lower than observed ones (0.529 and 0.510, respectively, see Figure 7). Therefore, this tendency resulting in a negative inbreeding coefficient (Fis= -0.174 for NZW rabbits and Fis= -0.027 for Californian rabbits), indicating a sufficient number of heterozygous forms in both studied groups of rabbits. The values of the inbreeding coefficient as a measure of heterozygosity deficiency or excess in a given population are presented in Figure 8. The chi-square test for Hardy-Weinberg equilibrium (Table 1) showed a value of χ2 with level of probability P>0.2 and at degree of freedom df=1, confirming the validity of the HWE for both rabbit populations in the present research. Conclusions  The obtained experimental results based on PCR-RFLP analysis confirmed the presence of C/G SNP at exon 3 of the GHR gene in studied rabbit populations. The distribution of allele frequencies with respect to GHR gene suggested that the observed genetic polymorphism could be a useful marker in future research on association analysis for growth performance traits in rabbits. Therefore, additional investigations are planned to estimate the favourable GHR genotypes that would allow accurate rabbit selection. Acknowledgement The present research was realized in the DNA laboratory of the Faculty of Agriculture in Trakia University, Stara Zagora, within the frame of the scientific project number 1AF/16. We would like to thank to the staff of the Institute of Animal Science – Кostinbrod who collected the blood rabbit samples. References Bertolini F, Schiavo, G, Scotti, E, Ribani, A, Martelli, PL, Casadio, R and Fontanesi, L. 2014. High‐throughput SNP discovery in the rabbit (Oryctolagus cuniculus) genome by next‐generation semiconductor‐based sequencing. Animal genetics, 45(2): 304-307. Deng XS, Wan J, Chen SY, Wang Y, Lai SJ, Jiang MS, Xu M. 2008. The correlations between polymorphism of growth hormone receptor gene and butcher traits in rabbit. Hereditas, 30(11): 1427-1432. Figure 7. Genotype frequencies of the polymorphic site in GHR gene in the studied rabbit populations. Figure 8. Coefficient of inbreeding (Fis) for the polymorphic locus in GHR gene in the studied rabbit populations. Table 1. Nei-expected heterozygosity, chi-square test of HWE (χ2) and P-value for the polymorphic locus in GHR gene in the investigated rabbit populations. Rabbit  populations n Nei* χ2  P-value New  Zealand White 51 0.451 3.707 0.230 Californian 49 0.497 4.437 0.906 *Expected heterozygosity calculated as per Nei (1973).  ISSN 1314-6246 Gencheva et al. J. BioSci. Biotechnol. 2017, 6(3): 197-201RESEARCH ARTICLE  201 http://www.jbb.uni-plovdiv.bg El-Sabrout K, Aggag SA. 2017. Associations between single nucleotide polymorphisms in multiple candidate genes and body weight in rabbits. Veterinary World, 10(1): 136-139. Fontanesi L,  Sparacino G,  Utzeria VJ, Scottia E,  Fornasini D,  Dall'Olioa S,  Frabetti A. 2016. Identification of polymorphisms in the Rabbit Growth Hormone Receptor (GHR) gene and association with finishing weight in a commercial meat rabbit line. Animal Biotechnology, 27(2): 77-83. Frank SJ. 2001. Growth hormone signalling and its regulation: preventing too much of a good thing. Growth Horm IGF Res., 11: 201-212. Labate J. 2000. Software for population genetic analyses of molecular marker data. Crop Sci., 40: 1521-1528. Leung DW, Spencer SA, Cachianes G, Hammonds RG, Collins C, Henzel WJ, Barnard R, Waters MJ, Wood WI. 1987. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature, 330 (6148): 537-543. Lukefahr SD, Cheeke PR. 1991 Rabbit project development strategies in subsistence farming systems. World Animal Review, 68: 60-70 Nei M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. U.S.A. 70: 3321-3323. Postel-Vinay MC, Finidori J. 1995. Growth hormone receptor: structure and signal transduction. Eur. J. Endocrinol., 133: 654-659. Postel-Vinay MC, Finidori J. 1995. Growth hormone receptor: structure and signal transduction. Eur. J. Endocrinol., 133: 654-659. Sahwan FM, El-Sheik, AI, Sharaf, MM, El-Nahas, AF. 2014. Genetic Polymorphism in growth hormone receptor gene (GHR) and its relationship with growth trait in pure and hybrid Rabbit breeds. Alexandria Journal for Veterinary Sciences, 43 (1). VanderKuur JA, Wang X, Zhang L, Campbell GS, Allevato G, Billestrup N, Norstedt G, Carter-Su C. 1994. Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase. J. Biol. Chem., 269: 21709-21717. Yeh F, Yong R. 1999. POPGENE version 1.31 (02.04.2011). Microsoft based Freeware for Population Genetic Analysis. University of Alberta, Edmonton, Canada, http://www.ualberta.ca/~fyeh/fyeh. Zhang WX, Zhang GW, Peng J, Lai SJ. The polymorphism of GHR gene associated with the growth and carcass traits in three rabbit breeds. Proc 10th World Rabbit Congr – September 3–6, 2012 Sharm El- Sheikh, Egypt, 75-78. Zhang WX, Zhang GW, Peng J, Lai SJ. 2012. The polymorphism of GHR associated with the growth and carcass traits in three Rabbit breeds. 10th World Rabbit Congress, Sharm El-Sheikh, Egypt, 75-78.  

Single nucleotide polymorphism of the Growth Hormone Receptor (GHR) encoding gene in Oryctolagus cuniculus

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Single nucleotide polymorphism of the Growth Hormone Receptor (GHR) encoding gene in Oryctolagus cuniculus

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