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J. Anim Sci. 2006. 84:2902-2906. doi:10.2527/jas.2006-144
© 2006 American Society of Animal Science
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ANIMAL GENETICS

Identification of a single nucleotide polymorphism of the insulin-like growth factor binding protein 2 gene and its association with growth and body composition traits in the chicken

Z. H. Li, H. Li1, H. Zhang, S. Z. Wang, Q. G. Wang and Y. X. Wang

College of Animal Science, Northeast Agricultural University, Harbin, 150030, People’s Republic of China


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
 Acknowledgements
 LITERATURE CITED
 
Insulin-like growth factor binding protein 2 regulates a broad spectrum of biological activities involved in growth, development, and differentiation. The current study was designed to investigate the associations of IGFBP2 gene polymorphisms with chicken growth and body composition traits. The Northeast Agricultural University Resource Population (NEAURP) was established by crossing broiler sires, derived from lines at Northeast Agricultural University, that were divergently selected for abdominal fat, with Baier layer dams, a Chinese local breed. The F1 birds were inter-crossed to produce an F2 population. Body weight and body composition traits were measured in the NEAURP. The PCR primers for the intron 2 region of IGFBP2 were designed based on chicken genomic sequence. Nucleotide polymorphisms between parental lines were detected by DNA sequencing. A C/T SNP in intron 2 was detected, and PCR-RFLP methods were then developed to genotype the F2 individuals. The results showed that the IGFBP2 SNP was associated with multiple traits, including BW, metatarsus length, shank length, femur length, shank weight, femur weight, metatarsus claw weight, and abdominal fat weight in the 1,028 NEAURP F2 individuals. This research suggests that IGFBP2 or a tightly linked gene has broad effects on growth and development in the chicken.

Key Words: insulin-like growth factor binding protein 2 gene • body composition • chicken • growth • quantitative trait locus


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
 Acknowledgements
 LITERATURE CITED
 
Selection for rapid growth in meat-type chickens results in an increase in physiological disorders such as obesity, ascites, sudden death syndrome, and leg problems, as well as a reduction in overall immunocompetence (Deeb and Lamont, 2002Go). Production performance and fitness traits are negatively correlated in chickens (Martin et al., 1990Go). Multiple-trait selection to simultaneously improve fitness traits and increase production performance is, therefore, difficult to achieve by direct phenotypic selection. Marker-assisted selection may be required to increase selection efficiency and make further improvements in production performance (Li et al., 2003Go).

Insulin-like growth factor-binding protein 2 is among the predominant IGFBP in serum of different species and binds IGF (Drop et al., 1992Go). The IGFBP2 gene is highly expressed in several tissues of embryos, such as eye, skeletal muscle, brain, and intestine (Schoen et al., 1995Go). The IGFBP2 has broad biological functions through coordinating and regulating the biological activities of IGF and transforming growth factor ß (TGF-ß; Rajaram et al., 1997Go; Hoeflich et al., 1999Go). Eckstein et al. (2002)Go reported that IGFBP2 level negatively affected bone size and mineral content in mice, which suggested that it was an important regulator of bone biology in vivo. In addition, IGFBP2 might indirectly affect adipocyte differentiation by controlling IGF (Richardson et al., 1998Go) and TGF-ß biological actions in fat tissue (Butterwith and Goddard, 1991Go).

The objectives of the current study were to identify SNP in the IGFBP2 gene, develop PCR-RFLP methods to detect those DNA polymorphisms in F2 resource populations, and evaluate associations between IGFBP2 SNP and traits of growth and body composition.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 IMPLICATIONS
 Acknowledgements
 LITERATURE CITED
 
Animal care and handling were conducted in accordance with policies on the care and use of animals promulgated by the ethical committee of Northeast Agricultural University.

Experimental Populations and Management

The Northeast Agricultural University Resource Population (NEAURP), a unique resource family, was used in the current study. The NEAURP was established by crossing broiler sires (4 males), derived from lines at NEAU divergently selected for abdominal fat, with Baier layer dams (1 male:5 females), a local breed from China. The F1 birds were intercrossed (1 male:5 females) to produce an F2 population. For the study, a total of 1,028 F2 individuals were produced from 12 F1 families.

All birds had ad libitum access to feed and water. Commercial corn-soybean-based diets that met all NRC requirements (National Research Council, 1994Go) were provided in the study. From hatch to 3 wk of age, birds received a starter feed (3,000 kcal of ME/kg and 210 g of CP/kg) and from 3 to 12 wk of age, birds were fed a grower diet (3,100 kcal of ME/kg and 190 g of CP/kg).

Phenotypic Measurements

Body weight was measured at hatch and weekly up to 12 wk of age. Body composition traits were recorded at 12 wk of age; these measurements included carcass weight, abdominal fat weight (AFW), heart weight, liver weight, spleen weight, metatarsus length (ML), keel length, shank length (SL), femur length (FL), shank weight (SW), femur weight (FW), metatarsus claw weight (MCW), breast muscle weight, and leg muscle weight. All traits were also expressed as a percentage of BW at 12 wk of age.

Development of PCR-RFLP Assays

Genomic DNA was isolated from venous blood collected in EDTA. A PCR was carried out with 50 ng of genomic DNA from the 2 grandsires and 2 granddams to investigate sequence polymorphisms of the intron 2 region of the IGFBP2 gene.

The PCR primers for the IGFBP2 intron 2 region (5'GTC CCA GAT AAA CCT TGC T 3'; 5'GCT GGC AAG GGG TCT G 3') were designed to amplify a 367-bp fragment by Primer 5.0 (Premier Biosoft International, Palo Alto, CA) according to the chicken genomic sequence in the GenBank database (accession number AY 326194). The reaction conditions were 94°C for 7 min, followed by 33 cycles of 94°C for 40 s, 58°C for 40 s, and 72°C for 40 s, and an extension at 72°C for 7 min. The 25-µL reaction volume included 50 ng of template, 1 x reaction buffer, 5 pmol of each primer, 0.16 mM dinucleotide triphosphate, 1.5 mM MgCl2, and 1 U of Taq polymerase.

Screening the F2 Population for Restriction Enzyme-Detectable SNP

A PCR of DNA from each F2 bird was performed using the conditions previously described. The PCR product was digested using 3 U of Eco72 I at 37°C overnight. The restriction digests were electrophoresed for 1.5 h at 100 V on a 3.0% agarose gel with ethidium bromide. Individual PCR-RFLP fragment sizes for the gene were determined by visualizing the band pattern under ultraviolet light.

Statistical Analysis

The association between the polymorphism and the phenotypic traits was analyzed using the GLM procedure (SAS Inst. Inc., Cary, NC). The model was fitted with the genotype (G; 3 levels), sex (S; 2 levels), and hatch (H; 2 levels) as fixed effects; family (F; 12 levels), dam nested within the family (D(F); 60 levels) as random effects, G*S and G*H as interaction terms, and BW at 12 wk (BW12) as a linear covariate (except for the % traits and the earlier BW traits), as follows:


Formula

where Y is the dependent variable, µ is population mean, and e is the random error. Sex x hatch and sex x family interactions were not significant for all traits and, therefore, were not included in the final model. Significant differences between least squares means of the different genotypes were calculated using a LSMEANS contrast procedure. Significance was determined as P < 0.05, unless otherwise specified.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 IMPLICATIONS
 Acknowledgements
 LITERATURE CITED
 
Sequence Variation and PCR-RFLP Analysis

The amplification product of the IGFBP2 intron 2 region was 367 bp in length. Sequencing of multiple individuals showed a C/T SNP at base 1032 (accession number AY 326194). The PCR-RFLP method was developed successfully for genotyping the C1032T SNP in intron 2 of the chicken IGFBP2 gene, and all of the F2 individuals of the NEAURP were screened. Three genotypes were detected and defined as AA, AB, and BB (Figure 1Go). The restriction enzyme Eco72 I-digested PCR products had fragment sizes of 367 bp for the AA genotype, 265 bp for the BB genotype, and a combination of 367 and 265 bp for AB genotype.


Figure 1
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  		Figure 1. The PCR-RFLP pattern for IGFBP2 gene intron 2 region with Eco72 I digestion. M = DL 2000 markers.

 
Association of IGFBP2 Gene SNP with Growth, Skeletal, and Body Composition Traits

The IGFBP2 polymorphism was generally significantly associated with growth, skeletal, and fatness traits (Table 1Go). There were significant associations between the IGFBP2 polymorphism and BW at 2 to 12 wk of age, skeletal traits (ML, SL, FL, SW, FW, MCW, %ML, %SL, %FL, %SW, %FW, %MCW), and fatness traits (AFW, %AFW) in the F2 population.


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  		Table 1. Effects (P values) of IGFBP2 polymorphism on chicken growth, and skeletal and body composition traits in F2 population
 
The BW2, BW3, BW4, BW5, BW6, BW7, BW8, BW9, BW10, BW11, and BW12 were significantly higher in F2 birds that were homozygous for the B allele (IGFBP2-BB birds) than in those homozygous for the A allele (IGFBP2-AA birds; Table 2Go). With skeletal traits, ML, SL, FL, SW, FW, MCW, %ML, %SL, %FL, %SW, %FW, and %MCW were significantly higher in F2 birds homozygous for the A allele than in those with homozygous for the B allele (Table 2Go). For fatness traits, there were significantly higher AFW and %AFW in F2 birds that were homozygous for the B allele than in those homozygous for the A allele (Table 2Go).


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  		Table 2. Effects of IGFBP2 genotype on growth, skeletal, and body composition traits (least squares means ± SE)
 

   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
IMPLICATIONS
 Acknowledgements
 LITERATURE CITED
 
The study of candidate genes is one of the primary methods to determine whether specific genes are related to economic traits in farm animals (Li et al., 2003Go). The IGFBP2 is capable of controlling the biological actions of IGF (Hoeflich et al., 1999Go) and TGF-ß (Rajaram et al., 1997Go) in vivo via endocrine, autocrine, or paracrine mechanisms and further affects the growth and development of animals. The IGFBP2 gene is expressed in many locations in the early avian embryo (Schoen et al., 1995Go) and may be involved in the regulation of phenotypic transformation, matrix deposition, and cell proliferation.

The current study found a C/T SNP at base 1032 (accession number AY 326194) in the second intron of the IGFBP2 gene. The polymorphism was associated with growth, fatness, and skeletal and body composition traits of growing birds. In an F2 cross of divergent lines, however, linkage disequilibrium was substantial. The examined SNP, therefore, may either be causal or linked with functional polymorphisms of any region of the IGFBP2 gene or other nearby genes.

Effects of IGFBP2 Gene Polymorphisms on Phenotypic Traits

Body Weight. Growth is a comprehensive reflection of development of various parts of the body, and its final expression is the result of interaction among genetic, nutritional, and environmental factors (Scanes et al., 1984Go). Growth rate, especially in meat-type chickens, has been intensely selected for more than a half century and will continue to be one of the most important economic traits in broiler chicken breeding programs. Growth is under complex genetic control, and uncovering the molecular mechanism of growth will contribute to more efficient selection for growth in broiler chickens (Deeb and Lamont, 2002Go). Studies of IGFBP2 biological function showed that reduced growth of mice selected for low BW was associated with increased hepatic IGFBP2 mRNA expression and elevated serum IGFBP2 levels (Hoeflich et al., 1999Go), further suggesting IGFBP2 as a negative growth regulator in vivo. Increased IGFBP2 expression was found in several experimental models of growth retardation in rat and swine (Price et al., 1992Go; Kampman et al., 1993Go; Tapanainen et al., 1994Go). From the present results, the C1032T SNP was associated with BW of chickens at 2 to 12 wk of chickens (Table 1Go). DeKoning et al. (2003)Go reported that a QTL for carcass weight was mapped between marker brackets MCW0030 and MCW0236 (about 2.3 to 29 Mb) on GGA7, a region that contains chicken IGFBP2 gene (23 to 24 Mb). The current study thus identifies IGFBP2 as a candidate gene of QTL for growth, which may be used to increase growth rate or market weight in marker-assisted selection programs.

Skeletal Traits. Leg problems in broilers may result from a lack of coordination of development and growth between whole body mass and the skeleton system (Julian, 1998Go). Increasing bone strength and keeping proper skeletal proportions are objectives in breeding of heavy-bodied poultry (Li et al., 2003Go). In the current study, ML, SL, FL, SW, FW, and MCW were measured as indicators of leg growth. Many investigations have shown that IGFBP2 is involved in human and mouse bone growth, maintenance, and bone mineral density (Kim and Lee, 1996Go; Eckstein et al., 2002Go). Our research found that chicken IGFBP2 is highly expressed in bone (unpublished data). It suggested that the IGFBP2 gene might affect bone growth of chickens.

We did not found any reports in the literature concerning about the association between the IGFBP2 gene and skeletal traits in chickens. In the present research, the C1032T SNP of the IGFBP2 gene was all significantly associated with ML, SL, FL, SW, FW, MCW, %ML, %SL, %FL, %SW, %FW, and %MCW in the NEAURP (P < 0.05). The results, therefore, pointed to IGFBP2 as a good candidate gene for skeletal growth in chickens.

Abdominal Fat. Excessive fat in chickens should be avoided in order to enhance production efficiency and product quality. The IGFBP2 could inhibit the biological actions of IGF in vivo via endocrine or paracrine mechanisms (Hoeflich et al., 1999Go) and indirectly control adipocyte differentiation by regulating the actions of IGF (Richardson et al., 1998Go). Recently, a QTL for fat deposition was mapped between marker brackets LEI0064 and ROS0019 (75 kb to 27 Mb) on GGA7 in the chicken linkage map (Ikeobi et al., 2002Go), which covers the chicken IGFBP2 gene (23 to 24 Mb). Our research found that the IGFBP2 gene was highly expressed in abdominal fat (unpublished data). Therefore, extensive study on chicken IGFBP2 gene may lead to breakthroughs in the understanding of regulation of body fatness in poultry.

In the current study, F2 birds homozygous for the B allele (IGFBP2-BB birds) had higher AFW and percentage AFW than birds of the other 2 genotypes. On average, F2 birds with the IGFBP2-BB genotype grew faster and simultaneously deposited more fat in the body. The results point to the possible identification of IGFBP2 as a candidate gene useful for altering growth rate and abdominal fat.


   IMPLICATIONS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
Acknowledgements
 LITERATURE CITED
 
The results from the current study indicated that the IGFBP2 was associated with growth rate, skeletal development, and amount of abdominal fat in chickens growing to market weight. The IGFBP2 gene is, therefore, a potential marker for use in marker-assisted selection programs. The work presented here is just a preliminary to a long period of research into the effects of IGFBP2 gene for chicken growth and body composition. To make further progress, there is a need to 1) evaluate associations between IGFBP2 C1032T single nucleotide polymorphism and traits of growth and body composition in other different breeds and lines of chicken, and 2) conduct further function studies of defining the effect of C1032T mutation at a molecular level.


   Acknowledgements
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
 Acknowledgements
LITERATURE CITED
 
The authors gratefully acknowledge the members of the Poultry Breeding group of the College of Animal Science and Technology in the Northeast Agricultural University for help in managing the birds and collecting the data. This research was supported by National Natural Science Foundation Key Project (No. 30430510) Program for New Century Excellent Talents in University (No. NCET-04-0343) and the Excellent Young Teachers Program of MOE. People’s Republic of China (No. 1985).

1 Corresponding author: lihui{at}neau.edu.cn or lihui645{at}hotmail.com

Received for publication March 14, 2006. Accepted for publication June 26, 2006.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 IMPLICATIONS
 Acknowledgements
 LITERATURE CITED
 


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Drop, S. L., A. G. Schuller, D. J. Lindenbergh-Kortleve, C. Groffen, A. Brinkman, and E. C. Zwarthoff. 1992. Structural aspects of the IGFBP family. Growth Regul. 2:69–79.[Medline]

Eckstein, F., T. Pavicic, S. Nedbal, C. Schmidt, U. Wehr, W. Rambeck, E. Wolf, and A. Hoeflich. 2002. Insulin-like growth factor-binding protein-2 (IGFBP-2) overexpression negatively regulates bone size and mass, but not density, in the absence and presence of growth hormone/IGF-I excess in transgenic mice. Anat. Embryol. (Berl.) 206:139–148.[CrossRef][Medline]

Hoeflich, A., M. Wu, S. Mohan, J. Foll, R. Wanke, T. Froehlich, G. J. Arnold, H. Lahm, H. J. Kolb, and E. Wolf. 1999. Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal BW gain. Endocrinology 140:5488–5496.[Abstract/Free Full Text]

Ikeobi, C. O., J. A. Woolliams, D. R. Morrice, A. Law, D. Windsor, D. W. Burt, and P. M. Hocking. 2002. Quantitative trait loci affecting fatness in the chicken. Anim. Genet. 33:428–435.[CrossRef][Medline]

Julian, R. J. 1998. Rapid growth problems: Ascites and skeletal deformities in broilers. Poult. Sci. 77:1773–1780.[Abstract/Free Full Text]

Kampman, K. A., T. G. Ramsay, and M. E. White. 1993. Developmental changes in hepatic IGF-2 and IGFBP-2 mRNA levels in intrauterine growth-retarded and control swine. Comp. Biochem. Physiol. B 104:415–421.[CrossRef][Medline]

Kim, J. G., and J. Y. Lee. 1996. Serum insulin-like growth factor binding protein profiles in postmenopausal women: Their correlation with bone mineral density. Am. J. Obstet. Gynecol. 174:1511–1517.[CrossRef][Medline]

Li, H., N. Deeb, H. Zhou, A. D. Mitchell, C. M. Ashwell, and S. J. Lamont. 2003. Chicken quantitative trait loci for growth and body composition associated with transforming growth factor-ß genes. Poult. Sci. 82:347–356.[Abstract/Free Full Text]

Martin, A., E. A. Dunnington, W. B. Gross, W. E. Briles, R. W. Briles, and P. B. Siegel. 1990. Production traits and alloantigen systems in lines of chickens selected for high or low antibody responses to sheep erythrocytes. Poult. Sci. 69:871–878.[Medline]

National Research Council. 1994. Nutrient Requirements of Poultry. Natl. Acad. Press, Washington, DC.

Price, W. A., A. D. Stiles, B. M. Moats-Staats, and A. J. D’Ercole. 1992. Gene expression of insulin-like growth factors (IGFs), the type 1 IGF receptor, and IGF-binding proteins in dexamethasone-induced fetal growth retardation. Endocrinology 130:1424–1432.[Abstract/Free Full Text]

Rajaram, S., D. J. Baylink, and S. Mohan. 1997. Insulin-like growth factor-binding proteins in serum and other biological fluids: Regulation and functions. Endocr. Rev. 18:801–831.[Abstract/Free Full Text]

Richardson, R. L., G. J. Hausman, and J. T. Wright. 1998. Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcinestromal-vascular cell cultures. Growth Dev. Aging 62:3–12.[Medline]

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