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Advances in Environmental Biology, 8(16) Special 2014, Pages: 67-71
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Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Polymorphisms of Insulin-Like Growth Factor 2 Gene of West Azerbaijan Native
Chicken and its Associations with Performance Traits Using PCR-RFLP
Naser Mahmoudi Aghdam and Karim Mardani
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
ARTICLE INFO
Article history:
Received 25 June 2014
Received in revised form
8 July 2014
Accepted 14 October 2014
Available online 16 November 2014
Keywords:
IGF2; polymorphism; PCR; chicken
ABSTRACT
The polymorphisms of native chicken insulin-like growth factor 2 gene (IGF2) in West
Azerbaijan province of Iran was investigated. Blood samples were collected from a
number of 100 birds. Genomic DNA was extracted by rapid salt-extraction method.
1146 bp fragments of the gene were amplified using polymerase chain reaction (PCR).
The amplified fragments were subjected to restriction enzyme digestion by HinfI
endonuclease enzyme, and then the digested products were run on 2% agarose gel. The
results revealed the existence of two alleles A and B for the target locus with
frequencies of 73.3% and 26.7% respectively. A number of three different genotype
variants including AA, AB, BB were identified with genotype frequencies of 59.1%,
28.4% and 12.5% respectively. The chi square test on the basis of observed and
expected frequencies of different genotypic variances of IGF2 gene showed that
deviation from Hardy-Weinberg equilibrium was not significant for West Azerbaijan
native chicken (p<0.01). It was also revealed that birds with genotype AB had higher
body weight at 8th week old and higher weight at their sexual maturation time
comparing with birds having AA and BB genotypes. It was concluded that IGF2 gene
had the high degree of polymorphism which could be considered as a candidate genetic
marker in selection and breeding programs in poultry.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Naser Mahmoudi Aghdam and Karim Mardani., Polymorphisms of Insulin-Like Growth Factor 2 Gene of West
Azerbaijan Native Chicken and its Associations with Performance Traits Using PCR-RFLP. Adv. Environ. Biol., 8(16), 67-71, 2014
INTRODUCTION
IGF2 is a mitogenic polypeptide with insulin-like structure modulating primary growth in chickens. Insulinlike growth factor genes in chickens’ cells are located on chromosome 5 which is composed of two intron and
three exon zones [6,24]. Introns and exons of chicken IGF2 gene is structurally similar to mouse and human
being IGF2 genes, [6]. Being composed of 187 amino-acids, including 24 signal peptides, 67 IGF2 peptides, and
96 amino-acids for its C-terminal part, chicken IGF2 is similar to its rat counterpart in 33 amino-acids as well as
its human counterpart in 82 amino-acids [6]. Many researches on different mammals have demonstrated broad
impact of IGF-1 on growth [18]. The general function of IGF system in chickens is similar to mammals [18,10].
Insulin-like growth factors cause growth and differentiation of chickens’ tissues. These factors mainly affect
protein synthesis speed, DNA synthesis, and substrate transition. Insulin-like growth factors can also affect rates
of ovulation and extension of ovarian follicles in addition to modulating body and muscle growth in chickens
[10]. The gene besides acts among insulin-like receptors and causes decrease in blood glucose [12]. Gene
expression in chicken embryo demonstrated that there are much lower levels of IGF2 peptides comparing with
embryonic IGF2 gene transcripts [21].
In mammals, there are 10 types of binding proteins to IGF1, two types of which are highly expressed in
chickens. In vitro studies have demonstrated that chicken embryo myoblasts and satellite cells secrete binding
proteins [9]. Chicken insulin-like growth factors 1, 2 are polypeptide hormones contributing their role through
binding to specific type 1 receptors [25]. There is evidence in some placental mammals that IGF2 gene heredity
is paternal [8,15]. IGF2 affects growth rate, body composition, and lipid metabolism of chickens [18,4,20].
Insulin-like growth factors also have great impact on embryonic growth and differentiation of various
animals. Researches have also indicated that the IGF is the principal gene affecting chickens overweight
[10,17]. IGFs act according to the body paracrine system [14]. The system of IGFs is a complex system
Corresponding Author: Naser Mahmoudi Aghdam, DVM., Department of Food Hygiene and Quality Control, Faculty of
Veterinary Medicine, Urmia University, Sero Avenue, Urmia, West Azerbaijan, Iran
Ph: +98 482 58593; Fax: 04826227084; Ph: +98 914 6356562
E-mail: [email protected]
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Naser Mahmoudi Aghdam and Karim Mardani, 2014
Advances in Environmental Biology, 8(16) Special 2014, Pages: 67-71
composed of peptide hormones, cell surface receptors, and binding proteins. IGF-1 & IGF2 hormones are bound
to insulin-like growth factors receptor 1 and insulin receptor, respectively, and activate tyrosine kinas principal
and intrinsic activities [1].
It is also revealed that IGF2 has an impact on wasted muscles of rat, pigs, and cows as well as lipid
metabolism in poultry [13]. IGF2 as the principal gene affecting chickens overweight is a molecular marker of
selecting chickens with low abdominal fat [17].
In this research the polymorphism of the promoter region of Insulin-like growth factor was investigated
using PCR-RFLP method in west Azerbaijan native chickens, and determination of allele and genotype
frequency as well as the association of these polymorphisms with chicken’s growth traits were studied.
MATERIALS AND METHODS
Blood sample collection and DNA extraction:
A number of 100 native chicken were randomly chosen from native chicken breeding center of west
Azerbaijan. The performance trait records of selected chickens including their weight in week 8 th and 12th of
their age were obtained from the breeding center. An amount of 1.5 ml blood were collected from wing vein of
chickens and stored in sterile tubes coated with EDTA. The collected blood samples were transferred to central
laboratory, faculty of veterinary medicine. An amount of 200 µl of blood sample was used for genomic DNA
extraction. DNA was extracted according to the rapid salt-extraction method described by Aljanabi and
Martinez [2].
Amplification of IGF2 gene:
A primer pair including,(IGF2-F) 5'-CCA GTG GGA CGA AAT AAC AGG AGG A -3' and (IGF2-R) 5'TTC CTG GGG GCC GGT CGC TTC A-3' was used for amplification of 1146 bp fragment of IGF2 gene
(Amills et al. 2003). The PCR was crried out in 25 µl reaction volume containing 50 mM each of dATP, dTTP,
dCTP and dGTP, 0.5 mM each primers, 2.5 µl of 10X PCR buffer (Cinnagen, Tehran, Iran), 2 mM magnesium
chloride, 2.5 U Taq DNA polymerase (Cinnagen) and 50 ng extracted DNA as template. The amplification was
performed in 35 cycles including denaturation at 94◦C for 1 minute, annealing at 67◦C for 3 minutes, elongation
at 72◦C for 3 minutes, and final extension at 72 ◦C for 5 minutes. The resultant PCR products were separated in a
1.5% agarose gel and the gel photographed using ultraviolet trans-illumination.
Restriction Fragment Length Polymorphism (RFLP):
PCR products were digested with the HinfI restriction endonuclease. Digestion reaction was carried out in
15 µl mixtures containing 5 µl of PCR product, 5U Hinf I endonuclease (Cinnagen) and 1.5 µl Hinf I buffer. The
mixtures were incubated at 37 °C for 2 hours. After that, the digested DNA fragments were run on the 1.5%
agarose gel and photographed using UV trans-illumination.
Data analysis:
The allelic and genotypic frequencies and observed and expected Nei’s heterozygosities were estimated
using PopGene32 version 1.31 [23]. PopGene 32 was also used to perform the Hardy-Weinberg equilibrium
test. Homozygosity and heterozygosity percentages were defined.
Results:
IGF2 gene amplification:
PCR products of 1146 bp in size were successfully amplified using described primers. All extracted
genomic DNAs from chicken blood samples yielded a specific, single-band PCR product without nonspecific
bands. Therefore, the PCR products were directly used for RFLP analysis.
RFLP:
RFLP patterns produced from IGF2 gene PCR products by digestion with HinfI are shown in figure 1. Two
alleles A and B with frequencies of 73.3% and 26.7%, respectively and three genotypes including AA, AB and
BB with frequencies of 59.1%, 28.4% and 12.5% respectively were observed (Table 1). It was revealed that the
AA homozygous genotype was the most frequent genotype.
Table 1: Genetic diversity of exon 2 and 3 of IGF-2 gene in native chicken of West Azerbaijan, Iran.
Genetic diversity statistics
Value
Allele frequencies
Value
Genotype Frequencies
NA
2
A
0.733
AA
NE
1.4
B
0.260
AB
Observed homozygosity
0.7159
BB
Observed heterozygosity
0.2841
Value
0.591
0.284
0.125
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Naser Mahmoudi Aghdam and Karim Mardani, 2014
Advances in Environmental Biology, 8(16) Special 2014, Pages: 67-71
Expected homozygosity
0.6063
Expected heterozygosity
0.3937
Average heterozygosity
0.3915
Nei Heterozygosity
0.3915
NA = observed number of alleles, NE = effective number of alleles (Kimura and Crow, 1964).
Fig. 1: It illustrates RFLP patterns produced by Hinf1 enzyme cutting, electrophoresed on 2% agarose gel. Sink
1 is the molecular marker of German Fermentas (50 bp plus), sinks 2, 3, 4, 5, 6, 9, 11, 12, 13, 14 are AA
genotype, sinks 8, 10 are AB genotype, and sinks 7, 16, 17 are BB genotype.
Allele heterozygosity and allele effective size were 0.39 and 1.64, respectively. Chi square test expressed
lack of Hardy-Weinberg equation (P<0.01), indicating the presence of allele and genotype frequency
transformers among generations.
Association of observed genotypes with performance traits:
The association of studied chicken IGF-2 genotypes with registered record of growth traits [8th week
weight and puberty (12th week) weight] is showed in table 2. It was revealed that among the genotypes of the
studied population, BB genotype significantly excelled AA and AB in terms of 8th week weight and puberty
weight.
Table 2: Association of IGF2 gene genotypes with values of growth traits as well as 8th and 12th week weight trait in the whole study
population.
Study traits
AA genotype
AB genotype
BB genotype
8th weight
842.33
844.71
873.33
12th weight
1607.86
1573.75
1700.00
Discussion:
Growth trait is in among of important economic traits in poultry and recognizing genetic data of growthrelated genes in domestic animals are so valuable for genetic selection and improvement via marker assisted
selection. IGF system stimulates liver glycogenesis and also increases DNA synthesis and tissue growth in
chickens [18]. Affecting division and differentiation of embryonic cells, IGF2 is a polypeptide hormone which
is of high importance in embryonic development. During development, the gene is expressed in many species.
IGF2 gene transcription activity is persistent during the primary embryonic phase, then decreases in many
tissues till puberty in which has influence on growth [22].
Using candidate genes is a powerful method for studying associations between gene polymorphism and
important economic features of domestic animals [19]. In this study the polymorphism of IGF2 gene and its
association with growth trait in native chicken of West Azerbaijan province is investigated. It’s revealed that
IGF2 gene can be a candidate gene for growth and body features, and its genotype is associated with phenotype,
indicating it’s high influence on growth and development, which is confirmed previously [22].
The results of our research also confirm Amills et al. [3] study, in which they didn’t find any significant
association between the single nucleotide polymorphism of IGF2 gene and growth and feeding trait in Black
Penedesenca breed. In the other hand, they found a significant association between these polymorphism and
average daily gain at a specific age. In another study by Li et al., [17] polymorphism was detected in exon 2
region of the chicken IGF2 gene using PCR-RFLP and DNA sequencing. We demonstrated that BB genotype
had better growth records of 8th week and puberty weight traits, comparing to chickens with AA and AB
genotypes. Therefore, the present study and previous researches demonstrated that IGF2 could be a principal
gene affecting chickens overweight and might be used as a genetic marker in selection process.
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Advances in Environmental Biology, 8(16) Special 2014, Pages: 67-71
Conclusion:
On the basis of PCR-RFLP method, it was concluded that exons 2 and 3 regions of IGF2 gene are highly
polymorph. Although there’s not significant association between identified polymorphisms and growth traits,
however, there is a strong correlation between these polymorphisms and daily weight gain at puberty age.
Therefore the IGF2 gene can be used as an appropriate selection marker in order to improvement of economical
traits in carcass.
REFERENCES
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
Adam D., L.J. Cosgrove, G.W. Booker, J.C. Wallace, B.E. Forbes, 2005. Molecular interactions of the IGF
system. Cytokine & Growth Factor Reviews, 16: 421-439.
Aljanabi, S.M., L. Martinez, 1997. Universal and rapid salt-extraction of high quality genomic DNA for
PCR-based techniques. Nucleic Acids Research, 25: 4692-4693.
Amills, M., N. Jimenez, D. Villalba, M. Tor, E. Molina, D. Cubilo, C. Marcos, A. Francesch, A. Sanchez,
J. Estany, 2003. Identification of three single nucleotide polymorphisms in the chicken insulin-like growth
factor 1 and 2 genes and their associations with growth and feeding traits. Poultry Science, 82: 1485-1493.
Beccavin, C., B. Chevalier, L.A. Cogburn, J. Simon, J.M. Duclos, 2001. Insulin-like growth factors and
body growth in chickens divergently selected for high or low growth rate. Journal of Endocrinology, 168:
297-306.
Bloom, S.E., M.E. Delany, D.E. Muscarella, 1993. Constant and variable features of avian chromosomes.
Pages 39-59. In R. J. Etches and A.M. Gibbins (eds.): Manipulation of the avian genome. 1th ed. CRC
Press, Boca Raton, USA.
Darling, D.C., P.M. Brickell, 1996. Nucleotide sequence and genomic structure of the chicken insulin-like
growth factor II (IGF-II) coding region. General and Comparative Endocrinology, 102: 283-287.
Deeb, N., S.L. Lamont, 2002. Genetic architecture of growth and body composition in unique chicken
populations. Journal of Heredity, 93: 107-118.
DeChiara, T.M., A. Efstradiatis, E.J. Robertson, 1991. A growth deficiency phenotype in heterozygous
mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature, 345: 78-80.
Duclos, M.J., 2005. Insulin-like growth factor-I (IGF-1) mRNA levels and chicken muscle growth. Journal
of physiology and pharmacology, 56(3): 25-35.
Duclos, M.J., C. Beccavin, J. Simon, 1999. Genetic models for the study of insulin-like growth factors
(IGF) and muscle development in birds compared to mammals. Domestic Animal Endocrinology, 17: 231243.
Duclos, M.J., R.S. Wilkie, C. Goddard, 1991. Stimulation of DNA synthesis in chicken muscle satellite
cells by insulin and insulin-like growth factors: evidence for exclusive mediation by a type-insulin-like
growth factor receptor. Journal of Endocrinology, 128: 35-42.
Froesch, E.R., J. Zapf, 1985. Insulin-like growth factors and insulin: comparative aspects. Diabetologia,
28: 485-493.
Goodall, J.J., S.M. Schmutz, 2007. IGF2 gene characterization and association with rib eye area in beef
cattle. Saskatoon, SK S7N 5A8.
Jones, J.I., D.R. Clemmons, 1995. Insulin-like growth factors and their binding proteins: biological
actions. Endocrine Reviews, 16: 3-34.
Killian, J.K., C.M. Nolan, N. Stewart, B.L. Munday, N.A. Andersen, S. Nicol, R.L. Jirtle, 2001.
Monotreme IGF2 expression and ancestral origin of genomic imprinting. Journal of Experimental
Zoology, 291: 205-212.
Kimura, M., J.F. Crow, 1964. The number of alleles that can be maintained in a finite population.
Genetics, 49: 725-738.
Li, Z.H., H. Li, Q.G. Wang, J.G. Zhao, Y.X. Wang, 2004. The Study on Correlation analysis of Single
Nucleotide Polymorphism of IGF2 Gene and Body Fatness Traits in Chicken. Agricultural Sciences in
China, 3: 789-794.
McMurtry, J.P., 1998. Nutritional and developmental roles of insulin-like growth factors in poultry.
Journal of Nutrition, 128: 302-305.
Rothschild, M.F., M. Soller, 1997. Candidate gene analysis to controlling traits of economic importance in
detect genes. Probe, 8(1): 13-20.
Tomas, F.M., R.A. Pym, J.P. McMurtry, G.L. Francis, 1998. Insulin-like growth factor (IGF)-I but not
IGF-II promotes lean growth and feed efficiency in broiler chickens. General and Comparative
Endocrinology, 110: 262-275.
Tylor, E.R., E.A. Seleiro, P.M. Brickell, 1991. Identification of antisense transcripts of the chicken insulinlike growth factor-II gene. Journal of Molecular Endocrinology, 7(2): 145-54.
71
Naser Mahmoudi Aghdam and Karim Mardani, 2014
Advances in Environmental Biology, 8(16) Special 2014, Pages: 67-71
[22] Wang, G., B. Yan, X. Deng, C. Li, X. Hu, N. Li, 2005. Insulin-like growth factor 2 as a candidate gene
influencing growth and carcass traits and its bi-allelic expression in chicken. Science in China Series C:
Life Sciences, 48(2): 187-194.
[23] Yeh, F.C., T. Boyle, R. Yang, 1997. POPGENE Version 1.31. Microsoft Window-Based Freeware for
Population Genetic Analysis. University of Alberta, Edmonton.
[24] Yokomine, T., A. Kuroiwa, K. Tanaka, M. Tsudzuki, Y. Matsuda, H. Sasaki, 2001. Sequence
polymorphisms, allelic expression status and chromosome locations of the chicken IGF2 and MPR1 genes.
Cytogenetics and Cell Genetics, 93: 109-113.
[25] Zhou, M., Z. Ma, W.S. Sly, 1995. Cloning and expression of the cDNA of the chicken cation-independent
mannose-6-phosphate receptor. Proceedings of the National Academy of Sciences, 92: 9762-9766.