Exploring the genetic variants of insulin-like growth factor II gene and their associations with two production traits in Langshan chicken

ABSTRACT

Insulin-like growth factor II (IGF-II) is one of the members of the somatotropic axis which controls the growth and development of animals. This study was conducted to identify single-nucleotide polymorphisms in the IGF2 gene and study their associations with two production traits in a Chinese indigenous chicken (Langshan chicken) population. Genetic variants within the IGF2 gene were screened through PCR-SSCP and DNA sequencing methods. A C6707G mutation (Chromosome 5 position 13,161,038 build 4) was identified. Associations between the mutations of the gene with two production traits were analysed. The results showed that individuals within the GG genotype had significantly higher 16-week-old body weight than those within the CG genotype. In addition, the GG individuals also had significantly higher 300 days egg numbers than individuals within CC or CG genotype. Ratios of dominance effects to absolute values of additive effects were less than 0 for both traits implying that Langshan chickens with homozygous genotypes for this locus had higher performance than individuals with heterozygous genotypes.

KEYWORDS:

• Association
• IGF2 gene
• Langshan chicken
• PCR-SSCP
• SNP

View correction statement:

Erratum

1. Introduction

As in mammals, the growth and development of chickens is primarily regulated by the somatotropic axis. The somatotropic axis, also named as neurocrine axis or hypothalamus-pituitary growth axis, consists of essential biological factors, such as growth hormone, growth hormone-releasing hormone, insulin-like growth factors (IGF-I and -II), somatostatin, their associated carrier proteins and receptors, and other hormones such as insulin, leptin and glucocorticoids or thyroid hormones (Buyse & Decuypere 1999; Renaville et al. 2002). Insulin-like growth factor II (IGF-II) promotes cell growth, survival, migration and differentiation via the IGF1 receptor tyrosine kinase (Chao & D'Amore 2008). In addition, it stimulates mitogenic responses via the insulin receptor isoform-A and it is important for foetal growth and development. In mice, the IGF2 gene knockout leads to a significant reduction in body size (about 60% of normal) (Gicquel & Le 2006), and placental specific knockout demonstrated a critical role in that tissue's development and function (Constância et al. 2002). There is evidence suggesting that IGF-II may influence growth rate, body composition and lipid metabolism in poultry (McMurtry 1998; Tomas et al. 1998; Beccavin et al. 2001). Spencer et al. (1996) implanted osmotic mini pumps containing either saline or recombinant human IGF-II into 4-week-old female broiler chickens such that the treated chickens received 0.5 mg IGF-II/kg body weight per day. They found that IGF-II can affect the relative weight of the abdominal fat pads of chicken.

The chicken's IGF2 gene is mapped on chromosome 5 and contains three exons (Darling & Brickell 1996; Yokomine et al. 2001). In all eutherians examined, IGF2 gene has been shown to be imprinted (O'Neill et al. 2000; Nolan et al. 2001). However, IGF2 is not an imprinting gene and displays an allelic pattern of expression in chicken (O'Neill et al. 2000; Nolan et al. 2001; Yokomine et al. 2001; Wang et al. 2005). The IGF2 gene polymorphisms were associated with growth or production traits in cattle (Bagnicka et al. 2010; Huang et al. 2014), pig (Hou et al. 2010) and various chicken breeds (Amills et al. 2003; Wang et al. 2005; Tang et al. 2010). However, their studies did not include Langshan chicken, a well-known indigenous chicken breed in China. Further, association studies between mutations at the IGF2 gene and production traits are still scarce in chicken. Thus, the objective of this study was to identify single-nucleotide polymorphisms (SNPs) in the IGF2 gene and analyse associations between the IGF2 gene polymorphisms and two production traits in Langshan chicken.

2. Materials and methods

2.1. Sampling and DNA extraction

Blood samples were obtained from 300 female Langshan chickens. Langshan chicken is one of the most famous Chinese local chicken breeds for its superior meat and egg qualities. Phenotypic records for body weight at 16-week old of age and total egg numbers of 300 days were collected on these 300 hens. These chickens had access to feed (commercial corn–soybean diets meeting the National Research Council's requirements) and water ad libitum. They were kept in a farm located in Nantong, a medium-sized city in the Jiangsu Province of China. Two millilitres of blood was collected aseptically in a tube containing the anticoagulant ACD (citric acid:sodium citrate:dextrose – 10:27:38) from the Winged vein. All samples were taken back to the laboratory in an ice box. The genomic DNA was extracted from whole blood using a salt–chloroform DNA extraction procedure (Müllenbach et al. 1989). The DNA samples were dissolved in a Tris-EDTA buffer made from 10 mM Tris–Cl (pH 7.5) and 1 mM EDTA (pH 8.0) and stored at −20°C.

2.2. Primer design and polymerase chain reaction amplification

Four pairs of polymerase chain reaction (PCR) primers were designed by the Primer Premier 5 software (Premier Biosoft International, Palo Alto, CA, USA) based on the Gallus gallus IGF2 gene sequence (GenBank accession number: NM_001030342.1) to amplify the coding regions of the IGF2 gene in all the studied population (Table S1). The 25 µL reaction mixture contained 50 ng genomic DNA, 1 µM of each primer, 1.5 mM MgCl₂, 200 µM dNTPs (dATP, dTTP, dCTP and dGTP) and 0.6 units of Taq DNA polymerase (MBI, Shanghai, China). The cycling protocol involved 5 min at 94°C, 35 cycles of denaturing at 94°C for 45 s, annealing at X°C (Table S1) for 1 min, extension at 72°C for 45 s and a final extension at 72°C for 10 min.

2.3. Genotype determination by single-strand conformation polymorphism and DNA sequencing

The single-strand conformation polymorphism (SSCP) method was used to scan mutations within the amplified regions (Zhang et al. 2007). Aliquots of 5 µL PCR products were mixed with 5 µL denaturing solution (95% formamide, 25 mM EDTA, 0.025% xylene–cyanole and 0.025% bromophenol blue), heated for 10 min at 98°C and chilled in ice immediately. Denatured DNA was subjected to 10% polyacrylamide gel electrophoresis in Tris-Borate-EDTA buffer and constant voltage (130 V) for 15 h at a constant temperature of 4°C, and then gels were stained with 0.1% silver nitrate and visualized with 2% NaOH solution (containing 0.1% formaldehyde). Then, the PCR products that represented different electrophoresis patterns were sequenced in both directions at a commercial laboratory (Sangon Biotech, Shanghai, China) in an ABI 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA) and the sequences were analysed with DNAMAN software (version 5.2.2, Lynnon Biosoft, 1994).

2.4. Statistical analysis

Genotypic frequencies, allelic frequencies and gene heterozygosity observed (Ho) were computed for the population. Genotype frequencies were tested for Hardy–Weinberg equilibrium (HWE) using a χ² test. Expected gene heterozygosity (He), effective allele numbers (N_e) and polymorphism information content (PIC) were computed using the following formulas (Nei & Roychoudhury 1974; Nei & Li 1979; Botstein et al. 1980):where p_i and p_j are the frequencies of the ith and jth allele, respectively, and m is the number of alleles.

Statistical analyses to determine the significance of IGF-II genotypic effects were performed using the general linear model procedure of SAS (SAS Institute Inc., Cary, NC, USA). The following model was used to analyse the association of different genotypes with the production traits: y_ij = u + G_i + H_j + e, where y_ij was a trait measurement, u was the overall population mean, G_i was the fixed effect associated with ith genotype of the IGF2 gene, H_j was the fixed effect of hatch and e was the residual.

Least squares means for each genotype and their corresponding standard errors were computed. When a statistical significance was detected (p < .05), comparisons between means were carried out using Duncan's multiple-range test. Additive and dominance effects within the IGF-II locus were estimated using the following expressions: where CC, CG and GG are the least square means of the CC, CG and GG genotype groups, respectively. Finally, the ratios of dominance effects to absolute values of additive effects were calculated for all traits.

3. Results and discussion

3.1. Detection of polymorphisms of the IGF-II gene by PCR-SSCP

Because IGF-II is one of the important compounds of the somatotropic axis in animals, the IGF2 gene has been frequently chosen to study associations between variants of this gene and production traits. In this study, the genetic variability of the IGF2 gene was explored in a well-known Chinese indigenous chicken breed (Langshan) using the PCR-SSCP method, a fast, economic and accurate strategy to scan genetic polymorphisms in an animal population. Results here showed that there were no polymorphisms in I1, I3 and I4 loci. In I2 locus, three SSCP patterns (CC, CG and GG) were identified and sequenced (Figure S1). When compared with the sequence of the chicken IGF2 gene at GenBank (GenBank Accession no. NM_001030342.1), the sequencing analysis of the samples in this population revealed only one SNP: g.6707C > G (Figure S2) located in exon 2 of the IGF2 gene. This SNP caused a synonymous mutation TCC (Ser) > TCG (Ser) at the 95th aa position of IGF-II (187aa). Similar results were found by other researches. Wang et al. (2005) found a C > G mutation in exon 2 of the IGF2 gene in F₂ chicken derived from French Broilers crossed with Tauhe Silky. The same results were found in Beijing You Chickens, Silkie Chickens and a reciprocally crossbred population of Silky and Broiler chickens (Wang et al. 2005; Tang et al. 2010). However, Amills et al. (2003) found two different SNPs in the Black Penedesenca chicken strain. One of them was a neutral substitution C > T in exon 3 and the other was a G > A substitution at intron 2. We inferred that the C > G mutation in exon 2 of the IGF2 gene is widespread in chickens except for some special breeds, and that the IGF2 gene could be a suitable marker gene for marker-assisted selection in chickens.

3.2. Genotypic frequencies, allelic frequencies and population genetic indexes

Genotypic and allelic frequencies of the IGF2 gene I2 locus are presented in Table 1. Allele C was the major allele. The χ² test showed that the genotype distribution of the IGF2 gene was not in HWE in the Langshan chicken population (p < .05). Subsequent sequencing showed that the badly skewed HWE was not due to a technical error. Factors that may have contributed to the lack of HWE are: (1) The Langshan chicken population has been under high selection pressure in recent years. The government increased the selection pressure on Langshan chicken to improve its marketability and increase their numbers. The sustained artificial selection eventually led to an increase in gene frequency of alleles favoured by selection, and the IGF2 gene appears to have been one of them; (2) the Langshan chicken population is small and (3) mating between individuals was subjected to human control.

Table 1. Genotypic and allelic frequencies of the IGF2 gene I2 locus in Langshan chicken.

Genotypic frequencies (observed genotype numbers)			Allelic frequencies
CC	CG	GG	C	G	Locus equilibrium χ^2 test (p value)
0.467 (140)	0.343 (103)	0.190 (57)	0.638	0.362	χ^2 = 19.990 (p = .000008)

Ho, He, N_e and PIC values for the IGF2 gene in this population are presented in Table 2. He and PIC are two measures to calculate the quality or informativeness of a polymorphism as a genetic marker. The PIC has become the most widely applied formula to measure the information content of molecular markers in genetic studies since its first application (Nagy et al. 2012). According to the classification of PIC (low polymorphism if PIC value < 0.25, medium polymorphism if 0.25 < PIC value < 0.5 and high polymorphism if PIC value > 0.5), the Langshan chicken possessed medium genetic diversity. This suggested that, as a candidate gene, the IGF2 gene could be an effective selection tool for the Langshan chicken population. In addition, the observed heterozygosity was lower than the expected one. This may be due to the limited size of the studied population. Another possible reason is that CG individuals may have been inadvertently discarded by the breeding researchers, suggesting that homozygous genotypes may have positive effects on production traits.

Table 2. Population genetic parameters for the IGF2 gene I2 locus in Langshan chicken.

Observed heterozygosity (Ho)	Expected heterozygosity (He)	Effective allele numbers (Ne)	Polymorphic information content (PIC)
0.3433	0.4617	1.8579	0.3551

3.3. Associations between the polymorphisms and two production traits, genetic effects

Several studies (Amills et al. 2003; Wang et al. 2005; Tang et al. 2010) indicated the existence of associations between IGF2 gene polymorphisms and the egg production or growth traits in various chicken breeds. In our study, associations between the IGF2 gene polymorphisms and two production traits of Langshan chicken were analysed. Least squares means for genotypic effects, standard errors and pairwise comparisons between least squares means for the three genotypes are presented in Table 3. Langshan chickens with the GG genotype had significantly higher 16-week-old body weight than those with the CG genotype (p < .05). The GG individuals also had higher 300 days egg numbers than individuals with the CC or CG genotype (p < .01). The 16-week-old body weight of GG individuals was not significantly different from CC chickens. The C6707G mutation did not affect the amino acid sequence of the IGF-II protein. These associations may be the result of linkage disequilibrium between this mutation and other genes on the same chromosome which have a significant effect on egg production and growth traits. Cis acting regulatory mutations may be another reason. Further studies are warranted. However, because of the significant associations between its polymorphisms with two production traits, the IGF2 gene was a suitable candidate gene for marker-assisted selection. Further, considering that the GG individuals had not only the highest body weight but also the highest egg numbers, individuals with homozygous genotype GG may be preferentially chosen as parents of the next generation of the Langshan commercial chickens.

Table 3. Least squares means of genotypic effects and additive and dominance effects for two production traits.

Genotype	Number	Traits
		Body weight at 16-week old (kg)	Egg numbers of 300 days
CC	140	1.553^ab ± 0.013	85.060^B ± 1.542
CG	103	1.522^b ± 0.015	89.660^B ± 1.948
GG	57	1.593^a ± 0.017	100.810^A ± 2.455
Additive effect		0.020	7.875
Dominance effect		−0.051	−3.275
Ratio		−2.550	−0.416

Note: Different small letters (a and b) in the same column indicated significant deviation (p < .05), while different capital letters (A and B) meant highly significant deviation (p < .01); Ratio is the ratio of the dominance effect to the absolute value of the additive effect.

Additive effects, dominance effects and the ratio of dominance to the absolute value of the additive effect are also presented in Table 3. The additive effect is an estimate of the quantitative change in a trait that is associated with substituting one allele with another allele in an interbreeding population. And, dominance is a relationship between alleles of a single gene in which one allele masks the phenotypic expression of another allele within a locus. Theoretically, a ratio of estimated dominance to the absolute value of additive effect larger than unity is regarded as exhibiting overdominance, a ratio between 0 and 1 represents partial dominance and a ratio less than 0 is regarded as underdominance (Hua et al. 2003). Underdominance can also be described as homozygote advantage, wherein homozygous individuals have a higher performance than heterozygous individuals. Here, ratios were less than 0 for both traits suggesting that homozygous Langshan chickens had higher performance than those with heterozygous genotypes and those homozygous individuals should be preferentially selected as parents of the next generation. This result was consistent with the results of the association analysis presented above.

In conclusion, a synonymous mutation g.6707C > G in exon 2 of the IGF2 gene was detected and demonstrated to have significant effects on body weight and egg production traits. Homozygous GG Langshan chickens had the highest 16-week-old body weight and 300 days egg numbers. However, the reason why the synonymous mutation can result in significant effects on Langshan chicken's production traits is not clear. Further research into the validation of these associations using a different, larger population, as well as functional validation is warranted.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by the Science and Technology Industrialization (Agriculture) Program of Nantong [No. CL2009002], Qing Lan Project of Jiangsu Province, Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents, A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).