GHR GENE POLYMORPHISM IN BREEDING STOCKS OF CHINCHILLAS

Daniel Polasik
Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland

ABSTRACT

The aim of this study was to establish genetic characteristics of Chinchilla herds and compare them in relation to the polymorphism in the GHR gene. For the analysis sample of 545 standard Chinchillas was used, obtained from two farms with different breeding cycle. A screening test based on ACRS, AS-PCR, PCR-RFLP methods was designed for polymorphism detection. Analyzing polymorphisms statistically significant differences were found between herds in allele and genotype frequency. In the case of polymorphism 135G>C in the herd with closed breeding cycle, a presence of one allele was observed. It was also confirmed that herd with open breeding cycle was characterized by a higher coefficient of average heterozygosity.

Key words: breeding cycle, Chinchilla, fur animals, GHR gene, polymorphism.

INTRODUCTION

Rapid growth of Chinchilla breeding, which occurred in Poland at the beginning of the nineties of XX century has contributed to greater interest in this species in our country as well as in the whole world [1]. There are a lot of investigations on Chinchillas concerning mainly anatomical traits [4, 22], semen traits and its cryopreservation [3, 26], changeability of phenotypic traits [27], estimation of hematological parameters [20] and ethology [7]. However it is hard to find information about DNA polymorphism of this species. Only Hidas et al. [9] reported RAPD polymorphism in Chinchilla breeding stocks.

Growth hormone (GH) and growth hormone receptor (GHR) genes are important candidate genes for identification of genetic markers for growth, carcass and milk traits in livestock [8]. Exon 10 of the Chinchilla GHR gene was sequenced by [10] to establish phylegenetic relationships among South American hystricognath rodents of the Octodontoidea superfamily. Exon 10 encodes intracellular domain of the receptor, which is responsible for signal transduction. Polymorphism in this exon was studied in lots of aspects. In human most of investigations concern Laron type dwarfism [12, 13, 29], association [6, 28, 31] and population [30] studies. In animals association between GHR gene polymorphism in exon 10 of and performance traits was studied mainly. Most of them were carried out on cows [5, 8, 11, 14, 19] chickens [15, 18, 21] and pigs [16, 24]. Results of  these researches proved an influence of GHR variants on economically important traits. Usefulness of GHR gene polymorphism in investigation of  population structure in goats was also confirmed by [23].

Because exon 10 of GHR gene in lots of species is characterized by high variability, associations between phenotypic traits and there are no information about genes diversity in Chinchilla, we decided to determine genetic structure of polish Chinchillas herds with different breeding cycle and compare them basing on the previously detected polymorphism [25].

MATERIALS AND METHODS

Investigations were carried out on a randomly chosen sample consisting of 545 standard Chinchillas individuals, derived from the following breeding farms:

• farm M located in Lesser Poland (n=277);
• farm N located in West Pomerania (n=268).

Conditions of feeding and rearing were equalized for both the farms. On farm M open breeding cycle is applied, in which outside individuals are acceptable for heard replacement. However, on farm N closed breeding cycle is applied, in which only own individuals are possible for herd replacement.

Tissues for DNA isolation (ears) were collected after slaughter and were frozen in -20°C. DNA was isolated using High Pure PCR Template Preparation Kit (Roche). Based on the earlier detected polymorphism in exon 10 of Chinchilla GHR gene (acc. no AY701337) [25], the following screening tests were designed:

• ACRS (Amplification-Created Restriction Site) – polymorphism in position 135bp (135G>C);
• PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) – polymorphism in positions 352 and 354bp (352CAG>AAA); 
• AS-PCR (Allele-specific Polymerase Chain Reaction) – polymorphism in position 641bp (641C>A).

Position of polymorphisms are given according to sequence AF520660. Primers for PCR were designed manually or by use Primer3 software (Table 1). In the case of AS-PCR method TrueSNPTM Allele-Specific PCR Primers with LNA (Locked Nucleic Acids) were used (Proligo). In the case of ACRS method reverse primer introduced artificial restriction site by mismatch (T-A) at the position 3. from 3’ end. 

Thermal profiles were selected experimentally however in the case of AS-PCR recommendations of primers manufacturer were applied. Composition of PCR mix and thermal profiles was different, depending on method that was used:

• ACRS: 50–80 ng DNA, 0.12 μM of each primer, 100 μM of dNTP mix, 2.5 mM MgCl2, 0.75 U Taq polymerase (Eurx) and 1 x PCR buffer (filled up to 15 μl by ddH2O);  95°C/5min, 35 x (95°C/1 min, 62°C/1 min, 72°C/2 min), 72°C/10 min;
• PCR-RFLP: 50–80 ng DNA, 0.12 μM of each primer, 100 μM of dNTP mix, 1.2 mM MgCl2, 0.5 U Taq polymerase (Eurx) and 1 x PCR buffer (filled up to 15 μl by ddH2O); 95°C/5 min, 35 x (95°C/45 s, 60°C/1 min, 72°C/50 s), 72°C/5 min;
• AS-PCR: 20 ng DNA, 0.4 μM of each primer, 200 μM of dNTPs mix, 4 mM MgCl2, 1 U Taq polymerase (Eurx), 1 x PCR buffer (filled up to 25 μl by ddH2O); 95°C/7 min, 30 x (95°C/30 s, 57°C/30 s, 72°C/30 s), 72°C/7 min.

Obtained amplicons were digested overnight by 3 U of EcoRI and Bpu10I restriction enzymes (Fermentas) for 135 G>C and 352 CAG>AAA polymorphism respectively. Restriction fragments or amplicons (641 C>A) were separated in agarose gels stained with Ethidinium Bromide. Results of separation were visualized in UV light and recorded using Vilber Lourmat system.

Statistical analysis concerning genetic characteristics of Chinchilla herds and their comparison were carried out by using STAT-GEN (ProSoft) and PowerMarker [17] software. For each herd frequency of individual genotypes and alleles were calculated. The number of observed and expected genotypes were also compared to determine Hardy-Weinberg equilibrium. In order to compare the herds following parameters were taken into account: frequency of individual alleles and genotypes, heterozygosity and gene diversity (expected heterozygosity). For each marker polymorphic information content (PIC) was also calculated [2].

RESULTS AND DISCUSSION

In the case of each polymorphism in exon 10 of Chinchilla GHR gene 2 alleles determining presence of 3 genotypes was observed. Figs. 1–3. represent electropherograms with individual genotypes determined by use of ACRS-PCR, PCR-RFLP, AS-PCR methods.

The frequency of individual genotypes and alleles with its comparison between herds is shown in Table 2.

In the case of the polymorphism 135 G>C the presence of an allele G in the herd with closed breeding (N) cycle was observed. In the second herd both the alleles determining 3 genotypes were present, however allele C was characterized by low frequency – 0.110. It is most probable that allele C was introduced into the herd M by its replacement from different breeding farms. Allele C could be also characteristic for this farm.

Analyzing the polymorphism 352 CAG>AAA difference (P≤0.01) was found in the frequency of alleles CAG and AAA between the herds M and N.

There were also observed differences (P≤0.05) in the frequency of homozygous genotypes between herds. Genotype CAG/CAG appeared more often in the herd M, but genotype AAA/AAA in the herd M. By examination of the last polymorphism – 641 C>A differences (P≤0.01) was affirmed in the frequency of alleles between investigated herds. In both of the herds allele C was present more often, however in the herd N it was mentioned with higher frequency. Analyzing frequency of genotypes difference (P≤0.01) was also observed between the herds in the case of genotype CC. In the herd M it was present more rarely (0.408) than in the herd N (0.522). It is difficult to clarify what caused differences in frequency of genotypes and alleles between the herds. It seems that most affecting factor is different breeding cycle.

Genetic equilibrium could be determine by the comparison of observed and expected genotypes, which are counted according to Hardy-Weinberg law. Based on calculations for the polymorphisms 352 CAG>AAA and 641 C>A we confirmed that the herds M and N follow the Hardy-Weinberg law. However we observed loss of genetic equilibrium in the herd M by analyzing the polymorphism 135 G>C. It indicates that the Hardy-Weinberg law cannot be applied to all loci because different factors have an influence on herds e.g. selection, which favors some genotypes whereas others do not.

A parameter which describes variability in population is heterozygosity. As we expected the herd M was characterized by its higher value (0.367) than the herd N (0.290). Higher variability in the herd M is probably caused by introduction different alleles from the other breeding herds. In the herd M lower heterozygosity in relation to gene diversity (0.383) was observed. However, in the herd N heterozygosity was slightly higher than expected (0.288).

Polymorphic information content (PIC) also shows variability in population and determine usefulness of the marker for different application e.g. gene mapping or paternity testing. PIC was low for polymorphism 135 G>C (0.100), however for 352 CAG>AAA and 641 C>A was medium and amounted 0.367 and 0.339 respectively. These differences result from the monomorphism of the SNP 135 G>C in the herd N.

It is not easily to discuss with obtained results because there is only one case of polymorphism studied in Chinchilla. Hidas et al. [9] tested 20 RAPD markers and their combination to compare and analyze Chinchilla herds. They chose 7 out of them, which generated 20 variable fragments and affirmed that some markers were also monomorphic, what was similar to our results. Frequency of other markers showed also high variations between the herds. These researchers did not find fragments characteristic for herd. In our study we found allele which was specific for one of the herd (C), but it appeared with low frequency. As opposed to RAPD markers which are codominant, wide dispersed in genome and very sensitive to changes in PCR conditions we proposed screening tests based on ACRS-PCR, PCR-RFLP, AS-PCR methods. They characterize repeatability as well as possibility to distinguish homozygotes from heterozygotes (dominant character of markers). Polymorphism which we studied include known coding region which influence on pheneotypic traits whereas RAPD markers may be localized also in noncoding parts of genome.

SUMMARY

The results clearly show that the polymorphism in Chinchilla GHR gene can be applied in studies on the population genetics. However, investigations should be carried out on a larger sample, larger number of herds and using more markers. Because polymorphism in exon 10 of Chinchilla GHR causes aminoacid substitution in the intracellular domain of the receptor, our results can be a starting point to association study and expression analysis in relation to performance traits or dwarfism appearing in Chinchilla farms

REFERENCES

1. Barabasz B., 2003. Review of main trends in investigations concerning performance traits of chinchillas [Przegląd głównych kierunków badań nad doskonaleniem cech użytkowych szynszyli]. Zeszyty Naukowe Przeglądu Hodowlanego, 68 (6), 9–17 [in Polish].
2. Botstein D., White R.L., Skolnick M., Davis R.W., 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. The American Journal of Human Genetics, 32 (3), 314–331.
3. Busso J.M., Ponzio M.F., De Cuneo M.F., Ruiz R.D., 2005. Year-round testicular volume and semen quality evaluations in captive Chinchilla lanigera. Animal Reproduction Science, 90 (1–2), 127–134.
4. Cevik-Demirkan A., Ozdemir V., Demirkan I., Turkmenoglu I., 2007. Gross morphological features of plexus brachialis in the chinchilla (Chinchilla lanigera). Journal of the South African Veterinary Association, 78 (1), 21–24.
5. Di Stasio L., Destefanis G., Brugiapaglia A., Albera A., Rolando A., 2005. Polymorphism of the GHR gene in cattle and relationships with meat production and quality. Animal Genetics, 36 (2), 138–140.
6. Du Y., Hirose N.,  Ping J.,  Ishida Y., Kojima T., Arai Y., Inagaki H., Gondo Y., Sakaki Y., Haneda M., Ito S.,  Isobe K., 2006. Analysis of growth hormone receptor polymorphism in Japanese semisuper centenarians. Geriatrics & Gerontolpgy International, 6 (2), 82–86.
7. Dzierżanowska-Góryń D., Grzeszczak A., 2006. The preliminary investigations of exploratory behaviours in small chinchilla (Chinchilla lanigera) [Wstępne badania zachowań eksploracyjnych szynszyli małej (Chinchilla lanigera)]. Konferencja „Zachowanie-Fizjologia-Środowisko” Warszawa, 22–24 września. Materiały Zjazdowe I Kongresu Polskiego Towarzystwa Etologicznego [in Polish].
8. Ge W., Davis M.E., Hines H.C., Irvin K.M., Simmen R.C.M., 2003. Association of single nucleotide polymorphisms in the growth hormone and growth hormone receptor gene with blood serum insulin-like growth factor I concentration and growth traits in Angus cattle. Journal of Animal Science, 81 (3), 641–648.
9. Hidas A., Edvi M.E., Pothaczky I., Toth Á., 2002. RAPD polymorphism in chinchilla breeding stocks. Scientifur, 26 (1), 22.
10. Honeycutt R.L., Rowe D.L., Gallardo M.H., 2003. Molecular systematics of the South American caviomorph rodents: relationships among species and genera in the family: Octodontidae. MolecularPhylogenetics and Evolution, 26 (3), 476–489.
11. Hradecka E., Rehout V., Cítek J., 2006. The polymorphism of growth hormone receptor gene in Holstein and Czech Pied bulls. Acta fytotechnica et zootechnica, 9, 224–227.
12. Iida K., Takahashi Y., Kaji H., Onodera N., Takahashi M.O., Okimura Y., Abe H., Chihara K., 1999. The C422F mutation of the growth hormone receptor gene is not responsible for short stature. The Journal of ClinicalEndocrinology &Metabolism, 84 (11), 4214–4219.
13. Johnson L.B., Pashankar F., Camacho-Hubner C., Savage M.O., Clark A.J.L., 2000. Analysis of the intracellular signalling domain of the human growth hormone receptor in children with idiopathic short stature. Clinical Endocrinology, 52, (4), 463–469.
14. Kaminski S., Brym P., Rusc A., Wojcik E., Ahman A., Magi R., 2006. Associations between milk performance traits in Holstein cows and 16 candidate SNPs identified by arrayed primer extension (APEX) microarray. Animal Biotechnology, 17 (1), 1–11.
15. Lau J. & Leung F., 2001. Detection of a single nucleotide polymorphism in exon 10 of the chicken growth hormone receptor. Proceedings of 7th World Congress on Genetics Applied to Livestock Production. Montpellier, France, 19–23 August.
16. Li J.T.,  Mu Y.L.,  Zhang L., Yang S.L.,  Li K.,  Feng S.T., 2007. New mutations in growth hormone and receptor genes from Chinese Wuzhishan miniature pig. Acta Agriculturae Scandinavica, Section A - Animal Science, 57 (2), 97–100.
17. Liu K. & Muse S.V., 2005. Powermarker: an integrated analysis environment for genetic marker analysis. Bioinformatics, 1 (9), 2128–2129.
18. Nie Q., Lei M., Ouyang J., Zeng H., Yang G., Zhang X., 2005. Identification and characterization of single nucleotide polymorphisms in 12 chicken growth-correlated genes by denaturing high performance liquid chromatography. Genetics Selection Evolution, 37 (3), 339–360.
19. Oleński K., Suchocki T., Kaminski S., 2010. Inconsistency of associations between growth hormone receptor gene polymorphism and milk performance traits in Polish Holstein-Friesian cows and bulls. Animal Science Papers and Reports, 28 (3), 229–234.
20. Ostojic H., Cifuentes V., Monge C., 2002. Hemoglobin affinity in Andean rodents. Biological Research, 35 (1), 27–30.
21. Ouyang J.H., Xie L., Nie Q., Luo C., Liang Y., Zeng H., Zhang X., 2008. Single nucleotide polymorphism (SNP) at the GHR gene and its associations with chicken growth and fat deposition traits. British Poultry Science, 49 (2), 87–95.
22. Ozdemir V., Cevik-Demirkan A., Turkmenoglu I., 2008. The right coronary artery is absent in the chinchilla (Chinchilla lanigera). Anatomia, Histologia, Embryologia, 37 (2): 114–117.
23. Pariset L., Cappuccio I., Ajmone-Marsan P., Bruford M., Dunner S., Cortes O., Erhardt G., Prinzenberg E.M., Gutscher K., Joost S., Pinto-Juma G., Nijman I.J., Lenstra J.A., Perez T., Valentini A., Econogene Consortium., 2006. Characterization of 37 breed-specific single-nucleotide polymorphisms in sheep. Journal of Heredity, 97 (5), 531–534.
24. Pierzchala M., Wyszynska-Koko J., Urbanski P., Blicharski T., 2006. Association between allele frequency of genes from MyoD family and chosen genes of somatotropic axis with meatiness in Large White and Landrace boars [Zależność między frekwencją alleli genów z rodziny mody oraz wybranych genów osi somatotropowej a mięsnością knurów hodowlanych ras wbp i pbz]. III Międzynarodowa Konferencja: Zastosowanie osiągnięć naukowych z zakresu genetyki, rozrodu, żywienia oraz jakości tusz i mięsa w nowoczesnej produkcji świń. Ciechocinek, 29–30 czerwca. Wydawnictwo Uczelniane Akademii Techniczno-Rolniczej [in Polish].
25. Polasik D., Kmiec M., Liefers S., Terman A., 2005. Single nucleotide polymorphism in exon 10 of the chinchilla growth hormone receptor (GHR) gene. Journal of Applied Genetics, 46 (4), 403–406.
26. Ponzio M.F., Busso J.M., Fiol De Cuneo M., Ruiz R.D., Ponce A.A., 2008.  Functional activity of frozen thawed chinchilla lanigera spermatozoa cryopreserved with glycerol or ethylene glycol. Reproduction in Domestic Animals, 43 (2), 228–233.
27. Socha S. & Olechno A., 2000. Analysis of changeability of features in chinchillas (Chinchilla velligera m.). Electronic Journal of Polish Agricultural Universities, 3(2).
28. Takada D., Ezura Y., Ono S., Iino Y., Katayama Y., Xin Y., Wu L.L., Larringa-Shum S., Stephenson S.H., Hunt S.C., Hopkins P.N., Emi M., 2003. Growth hormone receptor variant (L526I) modifies plasma HDL cholesterol phenotype in familial hypercholesterolemia: intra-familial association study in an eight-generation hyperlipidemic kindred. American Journal of Medical Geneticspart A, 121 (2), 136–140.
29. Tiulpakov A., Rubtsov P., Dedov I., Peterkova V., Bezlepkina O., Chrousos G.P., Hochberg Z., 2005. A novel C-terminal growth hormone receptor (GHR) mutation results in impaired GHR-STAT5 but normal STAT-3 signaling. The Journal of Clinical Endocrinology & Metabolizm, 90 (1), 542–547.
30. Zhou J., Lu Y., Bai Y.X., Wang Y.P., Shen Y., Wang B.K., 2004. Single nucleotide polymorphisms of growth hormone receptor gene in Chinese Han ethnic population. Chinese Journal of Stomatology, 39 (2), 97–99.
31. Zhou J., Lu Y., Gao X.H., Chen Y.C., Lu J.J., Bai Y.X., Shen Y., Wang B.K., 2005. The growth hormone receptor gene is associated with mandibular height in a Chinese population, Journal of Dental Research, 84 (11), 1052–1056.

Accepted for print: 11.10.2013

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