Association of IGF1R Polymorphisms with Growth Traits and its Expression Profiles in Different Pig Breeds

The importance of average daily gain (ADG) and body weight (BW) in pig breeding programs and in meat production industry motivate researchers and animal breeders to dissect the genetic architecture behind these growth traits (Ji et al., 2019). Single nucleotide polymorphisms (SNPs) in genomic regions, which accounting for significant portions of phenotypic variations for economically important traits, have been identified and implemented in marker assisted selection (Andersson et al., 1994; Blaj et al., 2018). A genome-wide scan through selection signatures in Chinese indigenous and commercial pig breeds reveals that IGF1R gene was associated with growth and development (Yang et al., 2014; Zhang et al., 2018).

Porcine IGF1R gene has been mapped to the chromosome 1, which contains 21 exons and 20 introns. IGF1R protein contains receptor L domain, furin-like cysteine rich region, fibronectin type 3 domain and the cytoplasmic tyrosine kinase domain, which is an unstable in domestic animals (Sahoo et al., 2019). Several reports indicate that numerous mutations in IGF1R affect late prenatal and early postnatal growth restriction, perinatal growth velocity and diminutive body size (Harris et al., 2014). IGF1R null mice die at birth of respiratory failure and exhibit only 45% of the body weight of their wide type littermates (Liu et al., 1993). The potential association between the polymorphisms in the IGF1R locus and traits with economic importance have been investigated (e.g., BW, ADG) in different species of animals, including cow (Leyva-Corona et al., 2018), buffalo (El-Magd et al., 2013) and chicken (Lei et al., 2008). In pigs, the role of IGF1R in the regulation of the ADG and BW is still intelligible.

The objectives of this study were to examine the associations of SNPs in IGF1R gene with growth traits in three pig populations, Duroc, Yorkshire and Landrace. Meanwhile, the expression pattern of IGF1R was detected in Yorkshire and Jinhua pig breeds.

Animal care
 
The experimental procedure was approved by the Institutional Animal Care and Use Committee at Hebei University of Engineering (AEEI-16015).
 
Animals and Trait Measurements
 
The experimental populations included 406 pigs, specifically 62 Duroc (male = 22, female = 40), 154 Landrace (male = 40, female = 114) and 190 Yorkshire (male = 60, female = 130) pigs. The pigs maintained at three different farms belonged to Yunnan Honghe Bangge Animal Husbandry Co., Ltd. under the same housing and feeding system. The feeding system contained the grower stage from 30kg to 50kg of body weight and the finisher stage from 50kg to 100kg of body weight. Body weights at 70 d of age as well as the age when the pigs reached the target weight were recorded. The target weight contained 30kg, 50kg and 100kg. We also measured the ADG from 30kg to 50kg, from 50kg to 100kg, from 30kg to 100kg and the backfat thickness when the body weight was 100kg. Longissimus muscle tissues were collected from six Yorkshire and six Jinhua pigs at each 30, 90 and 180 days of age. A total of 36 pigs were sampled. The samples were taken immediately after euthanasia, frozen in liquid nitrogen and stored at -80°C until use.
 
DNA extraction and polymorphism analysis
 
Genomic DNA was extracted from the ear tissues using a standard phenol-chloroform method then quantified using a NanoDrop 2000 spectrophotometer. IGF1R polymorphisms in all exons were screened. The PCR primers were designed basing on the sequence of pig IGF1R (Gene Bank accession. NC_010443.5) (Table 1). PCR amplifications were carried out in 20 μL reaction volumes containing at the following final concentrations: 50 ng of template DNA, 400 μM of dNTPs, 0.25 μM of primer and 1 unit of Taq polymerase. The PCR protocol consisted of an initial denaturation at 94°C for 5 min, followed by 36 cycles of denaturation at 94°C for 30 s, annealing at annealing temperature of each pair primer for 30 s, extending at 72°C for 30 s and then with a final extension at 72°C for 10 min. The PCR products were directly sequenced. Sequences were aligned for identification of mutations.



Based on the sequencing, a novel polymorphism was identified in the exon 20: c.3678C>T, which was genotyped by the Created Restriction Site PCR (CRS-PCR). The mismatched base in the primer enabled the application of restriction enzymes to discriminate the sequence variations. Meanwhile, the reported polymorphism in intron 9 was also genotyped using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), which was named as allele A (379 bp) or allele B (235bp and 144bp) (Kopecny et al., 2002). SacII and TaqI were two kinds of endonucleases detecting the SNP localized in intron 9 and exon 20, respectively. After PCR, 5 μL of PCR product, 0.2 μL of endonuclease, 1 μL 10×buffer and 3.8 μL ddH2O were mixed and placed in a 37°C or 65°C thermostatic water bath overnight. The enzyme-digest products were subsequently identified by electrophoresis on 1.5% agarose gels and genotyped according to the fragment length. The details of the obtained DNA fragment after endonuclease digestion are presented in Table 2.

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RNA isolation and Quantitative reverse transcription PCR (RT-qPCR)
 
Total RNA was isolated from porcine longissimus muscle tissues using TRIzol reagent. The quality of RNA was evaluated using a NanoDrop 2000 spectrophotometer. Only samples with an A260/A280 ratio of 1.8-2.0 and an A260/A230 ratio greater than 2.0 were used for subsequent analysis. Reverse transcription of RNA to cDNA was performed using an Improm-II Reverse Transcriptase kit following the manufacturer’s instructions. qPCR was performed using SYBR Green Universal Master mix, following the manufacturer’s recommendations in a CFX96 Real-Time system. The PCR program was 95°C for 5 min, 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, with a melting curve analysis (65-95°C) in the last cycle to evaluate amplification specificity. Beta-actin (ACTB) was used as an endogenous control. The primer sequences are shown in Table 3.

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Statistical analysis
 
Statistical analyses were performed using the GLM (General Linear Model) procedure of the SAS (v9.4; SAS Institute, Cary, NC). The LSM (Least Squares Means) method was used to determine the statistical significance between groups. The linear model was:
 

Y_ijklm = μ + A_i + B_j + Ck + R_l + e_ijklm

 
Where,
Y_ijklm is the observation of the trait; μ is the population mean; A_i is the fixed effect of the experimental farms, B_j is the fixed effect of breed, C_k is the fixed effect of sex, R₁ is the fixed effect of the haplotype and the e_ijklm is the random residual. The analyzed animals were not related. The results are presented as mean±standard error (SE). Significant and extreme differences were set at P<0.05 and P<0.01, respectively.

Gene frequencies were determined by direct counting. He (gene heterozygosity), Ne (effective number of alleles) and PIC (polymorphism information content) were calculated according to previous report (Kumar et al., 2020), respectively.
 
The formulas were

                                                                                   

 
Where
P_i and P_j are the frequency of the i and j allele and n is the number of allele.

In terms of the RT-qPCR, all samples were analyzed in triplicate and normalized to ACTB mRNA levels, with values expressed as means ± standard deviation (SD). Significance was determined using a Student’s unpaired t-test in SAS (v.9.4; SAS Institute, Cary, NC) with a significant
level of P<0.05.

A total of 21 exons of IGF1R gene were sequenced and only a novel polymorphism in exon 20 (c.3678C>T) were detected. PCR product digestion by endonuclease TaqI generated fragments with lengths of 200bp and 35bp for genotype CC, 235bp for genotype TT and 235bp, 200bp and 35bp for genotype CT (Table 2, Fig 1). Conflicting to a previous study reported by Harumi et al., (2001), who identified 12 silent sequence variants in coding regions based on RT-PCR derived IGF1R sequence in 12 unrelated pigs. The novel SNP in the exon 20 was a missense mutation and located in the cytoplasmic tyrosine kinase domain of IGF1R protein. The mutations in the kinase domain can cause conformational changes consequently affect the binding capability (Gately et al., 2015). Hence, the polymorphism in exon 20 (c.3678C>T) resulting in the amino acid changing might have serious consequences for ligand binding, which needed to be further verified.

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At the c.3678C>T site, the frequency of the T allele was greater than that of the C allele in the three pig breeds. The frequency of the TT genotype was higher than the other two genotypes. According to the c² test statistics, the SNP c.3678C>T in Duroc met Hardy-Weinberg equilibrium (P>0.05), while which in Landrace and Yorkshire breed deviated from the Hardy-Weinberg equilibrium (P < 0.01). The He of the mutant loci was 0.27, 0.40 and 0.17 in different pig breeds, respectively (Table 4). It was likely that allele frequencies at the QTL is different among breeds and thereby influence genetic and phenotypic variance. The association analysis of IGFR1 gene genotypes and recorded growth traits within the three different population were analyzed (Table 5). In Landrace breed, animals with the genotype CC had significantly higher ADG of 30-50kg than those with genotypes TT (P < 0.05). In Yorkshire breed, genotype CC showed differences in ADG of 30-50kg compared to genotypes TT (P<0.05) and also in the ADG of 50-100kg (P < 0.01). The CC individuals were characterized by great ADG of 30-50kg and ADG of 50-100kg. In Duroc breed, this SNP had no significant correlation with the recorded growth. Moreover, differences in growth traits between breeds have been reported (Lents et al., 2016; Tyra et al., 2019).

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In the present study, we also screened the SNP in intron 9 in the Duroc, Landrace and Yorkshire breeds. However, only the AA genotype was identified (Table 4), which was consistent with the previous study that the European pigs principally carried allele A and the pigs with AA genotype exhibited greater body weights (Kopecny et al., 2002; Wang et al., 2006).

Meanwhile, IGF1R has different influences at various stages of growth and its expression in regulated depending on developmental and functional status (Cheng et al., 2016; Yang et al., 2019). We detected the differently expressed pattern of IGF1R in muscle of Yorkshire and Jinhua pigs at different growth stages (Fig 2). In Jinhua pigs, IGF1R had significantly higher expression at 30-day than at 90-day and 180-day. Compared with Jinhua pigs, IGF1R showed lower expression at 30-day but higher expression level at 90-day in Yorkshire pigs (P<0.05). There were no significant differences at 180-day. We speculate that the dramatic difference in longissimus dorsi muscle growth between the two breeds may be partially regulated by IGF1R at early stage. It is consistent with the difference in longissimus dorsi muscle area observed after but not before day 90 in Jinhua and Landrace pigs, the latter of which are similar in growth to Landrace pigs (Wu et al., 2013).

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Overall, a novel SNP (c.3678G>A) was identified in the exon 20 of porcine IGF1R gene that encoded a portion of cytoplasmic tyrosine kinase domain and caused the amino acid changing. The association analysis showed that the SNP c.3678G>A had significant impact on ADG. Effects of CC, CT and TT genotypes on the ADG at early and late growth stages were different in the three commercial pig breeds. The novel IGF1R polymorphism may be useful as molecular markers in pig selection but needed to be future studied. Additionally, the IGF1R expression had different profiles in Yorkshire and Jinhua pigs. Despite the lack of the association of the novel SNP at exon 20 along with the IGF1R expression pattern, our results provide a basis for further studies on the genetic influence of IGF1R gene on pig growth and development.

This research was funded by the National Transgenic Major Project of China (2018ZX0800928B) and the Natural Science Foundation of Hebei Province of China (Youth Project, C2017402162).