The insulin-like growth factor (IGF) family includes three ligands, three cell membrane receptors, six binding proteins, and a number of other associated proteins. In mammals, insulin regulates cell metabolism, whereas insulin-like growth factor 1 (IGF-1) is an important regulator of cell growth (Ullrich et al. 1986). The majority of circulating IGF-1 is produced by the liver, where its expression is stimulated by growth hormone (GH). However, other organs can also produce IGF-1 in an autocrine and paracrine manner, including bovine mammary gland and muscle (Plath-Gabler et al. 2001). The insulin-like growth factor 1 receptor (IGF-1R) is a receptor-tyrosine kinase that plays a crucial role in signaling that is important for cell survival and proliferation. After binding to the primary ligand (IGF-1), signal cascade activates the phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK), and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins (Galvan et al. 2003).

In view of the important role of genes coding for components of the somatotropic axis (GH-GHR-IGF1-IGF1R) in growth regulation, the objectives of the present study were to estimate the allele and genotype frequencies of the two SNPs (rs41640706 and rs41960562) within the coding sequence of the bovine IGF1R gene in beef cattle and to determine the effects of these markers on growth traits in animals under an intensive production system. Both of these SNPs were submitted in the year 2006, but they have not been validated so far. The study involved 310 purebred Angus female calves of 54 sires kept in the years 2005–2010 on a single farm located in the West Pomeranian province, Poland. The DNA was isolated using the MasterPure™ DNA Purification Kit (Epicentre Technologies), according to the manufacturer’s instructions.

The first polymorphism, a silent transition in the third nucleotide in the codon coding for proline (CCA → CCG), is localized within exon 12 of the Bos taurus IGF1R gene and is annotated as rs41640706 in the dbSNP database (http://www.ncbi.nlm.nih.gov/snp/). The MspI restriction endonuclease is able to recognize guanine at this position and, therefore, this SNP was identified using the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method. To genotype the IGF1R/e12/MspI polymorphism, a pair of primers (Ta = 59.5 °C; forward: 5′-ttcttgcctgtttcaattgttg-3′; reverse: 5′-ctcgacttgggatccatatttt-3′) was used to amplify a 164-bp DNA fragment.

The second polymorphic site (rs41960562; A<->G transition) is localized within the 3′ untranslated region (3′UTR) of the Bos taurus IGF1R gene. Due to the lack of commercial endonucleases that would be able to identify this SNP, the amplification-created restriction site (ACRS) (Haliassos et al. 1989) protocol was developed to introduce an artificial TaqI recognition site. The TaqI recognition site (t↓cga) was engineered by enforcing a base change within a new front primer by replacing T with C (Ta = 62 °C; forward: 5′-gccggtcaccataggtctCg-3′; reverse: 5′-agtgggggttttggcagaat-3′; mismatch is underlined). A 163-bp DNA fragment was then amplified.

PCR was carried out in a total volume of 20 μl containing 60–80 ng of DNA template, 0.5 U of Taq DNA polymerase, buffer with (NH4)2SO4, MgCl2, dNTP mix (MBI Fermentas/ABO, Gdansk, Poland), 10 pmol of each primer (Oligo, IBB PAN, Warsaw), and nuclease-free deionized water (Epicentre Technologies™, AKOR Gdansk, Poland). The reaction conditions were as follows: an initial DNA template denaturation at 94 °C for 5 min, followed by 33 cycles of: denaturation of DNA template at 94 °C for 50 s, annealing (Ta) at 59.5 °C or 62 °C for 50 s, extension at 72 °C for 50 s, and the final elongation at 72 °C for 7 min in the Biometra TPersonal™ thermal cycler (Biometra, Goettingen, Germany). Two specific PCR products were obtained. The 164-bp fragment was digested with 5 units of the MspI endonuclease (10 U/μl, C↓CGG; MBI Fermentas/ABO, Gdansk, Poland) at 37 °C for 3 h. In the case of the 163-bp fragment, the PCR product was digested for 2 h at 65 °C with 5 units of the TaqI restriction enzyme (10 U/μl, T↓CGA; MBI Fermentas/ABO, Gdansk, Poland). After digestion, 10 μl of each of the products was then separated by electrophoresis in 2 % ethidium bromide-stained agarose gels (Basica Prona™ Agarose, ABO, Gdansk, Poland).

The association of birth weight (BWT), weaning weight adjusted to 210 days of age (WWT210), as well as average daily gains between birth and weaning (ADG), with the selected SNPs within the bovine IGF1R gene was analyzed based on the data obtained from the official recordings.

Statistical calculations were performed using a general linear model (GLM) incorporated in the procedure of the STATISTICA 9.0 PL software package (StatSoft, Inc. 2009). The following statistical model was used:

$$ {Y}_{ijkl}=\mu +{G}_i+{s}_j+ BY{S}_k+{e}_{ijkl} $$

where Y ijkl is the analyzed trait, μ the overall mean, G i the fixed effect of the IGF1R genotype or combination of genotypes (i = 1,…2, or 1,…, 4), s j the random effect of the sire (j = 1,…, 43), BYS k the fixed effect of the year/season of birth (k = 1,…, 16) and e ijkl is the random error.

The 164-bp fragment amplified to identify rs41640706 SNP contained a single MspI recognition site. The digestion resulted in two fragments (51 and 113 bp) in the case of the IGF1R G allele and one uncut 164-bp-long fragment carrying the IGF1R A allele (Fig. 1). The GG genotype had the highest frequency (0.593), followed by the AG genotype (0.371). The least frequent genotype was AA (n = 11, only 3.6 % of the analyzed population). The frequency of the rare IGF1R A allele was 0.22.

Fig. 1
figure 1

Ethidium bromide-stained PCR products after digestion and gel electrophoresis. Lanes 1, 5, and 9: M, the DNA length marker pUC19/MspI. Lanes 2–4: the IGF1R/e12/MspI polymorphism. Lanes 6–8: the IGF1R/3′UTR/TaqI polymorphism (the small 18-bp fragment is not visible in the gel)

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The 3′UTR PCR product carrying the rs41960562 polymorphism was cleaved by TaqI in the presence of the IGF1R A allele into two fragments of 145 bp and 18 bp; when the IGF1R G allele was present, no cleavage was observed (163 bp) (Fig. 1). Among the 310 calves that were genotyped for this polymorphism, 196 (63.2 %) were of the AA genotype, 95 (30.7 %) were of the AG genotype, and only 19 individuals (6.1 %) possessed the GG genotype (allele IGF1R A: 79 %; IGF1R G: 21 %).

Table 1 shows the effect of the RFLP-MspI and ACRS-TaqI polymorphisms in the IGF1R gene on growth performance. The IGF1R/e12/MspI polymorphism had a significant effect only on WWT210 (P ≤ 0.05). The GG genotype was associated with a higher WWT210 (+5.06 kg) when compared to the AG genotype. Because of the low number of AA individuals, this genotype group was excluded from the association study. In the case of the polymorphism within the 3′UTR, no significant differences were observed when a small genotype group of GG individuals was omitted.

Table 1 Mean values of growth traits of Angus cows with the different IGF1R gene variants (standard errors in parentheses)
Full size table

The relatively high frequencies of the GG (IGF1R/MspI), AA (IGF1R/TaqI), and heterozygous genotypes enabled analysis of the association between their combinations (as combined genotypes) and growth performance. The model was similar to that of a single-marker association analysis, except that the interaction between the two SNPs was included as a fixed effect. A total of four combinations showed a frequency higher than 0.1 (Table 1). Due to the low number of individuals in the groups, five rare combinations (GG/GG, n = 15; AA/AA, n = 9; AG/GG, n = 3; AA/AG, n = 1; and AA/GG, n = 1) were excluded from further analysis. The means for all the analyzed traits are given in Table 1, including a comparison of their combinations. As shown, the GG/AA and GG/AG combined genotypes were associated with superior growth trait in regard to WWT210. Calves carrying the above-mentioned combinations were heavier than calves with the AG/AA and AG/AG combined genotypes (P ≤ 0.05). The association analysis suggested that no significant differences were detected between the combined genotypes and BWT and ADG.

The bovine IGF1R gene was mapped to chromosome 21 (Moody et al. 1996). It consists of at least 21 exons encoding 1367 aa of unprocessed protein chain, including a 30-residue signal peptide. Like in humans, gene and protein organization is structurally related to the insulin receptor (Ullrich et al. 1986). Davis et al. (1998) and Casas et al. (2003) reported significant quantitative trait loci (QTL) for birth weight in the centromeric region of BTA21. The most significant QTL was at 4 centimorgans (cM) from the beginning of the linkage group on chromosome 21. Therefore, the gene(s) of interest should be located in the candidate region up to 10 cM. With respect to this criterion, we have identified several genes located in the candidate region, based on the Bos_taurus_UMD_3.1/Btau_4.6.1 genome assemblies (http://www.ncbi.nlm.nih.gov/assembly/; RefSeqID AC_000178.1/NC_007319.5). One of the most promising candidates is the IGF1R gene (relative position: 8.278 cM), closely related to IGF1, located in the QTL for birth weight on BTA5 (Kim et al. 2003).

Little is known about the associations between the polymorphisms within the bovine IGF1R gene and meat production traits. The IGF1R/TaqI polymorphism within intron 12, detected originally by Moody et al. (1996), seems to be exclusive to Bos indicus. To date, very few data are available regarding the effect of this polymorphism on growth and carcass traits in Bos indicus (Curi et al. 2005) and Chinese indigenous beef cattle (Zhang and Li 2011).

In the present study, we described a novel protocol to detect silent mutation within exon 12 (rs41640706; GenBank accession no. JQ924783) that does not affect the protein sequence. The proline-807+30 residue is localized exactly in the middle of the extracellular sequence of the IGF-1R β subunit (residues 711 to 905 upstream from the transmembrane domain; amino acids of the pro-receptor are numbered starting at Glu 1, preceded by a 30-residue signal peptide). The second polymorphic site, annotated as rs41960562 (see also GenBank accession no. JQ957797), is one of the several SNPs detected within the long 3′UTR (exon 21) and, therefore, it is not related to the amino acid substitution either. However, point mutations within the 3′UTR can be associated with the determination of mRNA stability/instability and, in special cases, with diseases. MicroRNAs (miRNAs) function post-transcriptionally by base-pairing to the mRNA 3′UTRs to repress protein synthesis by mechanisms that have not yet been fully understood. Current evidence suggests that the target mRNA degradation provides a major contribution to silencing by miRNAs (Huntzinger and Izaurralde 2011). To evaluate the potential role of the aforementioned SNP, we compared the nucleotide sequences of a full-length IGF1R 3′UTR (about 6.75 kb) of several species (incl. Bos taurus, Homo sapiens, Mus musculus, and Rattus norvegicus; TargetScanHuman rel. 6.2; http://www.targetscan.org/), and then the location of the JQ957797 sequence with the rs41960562 SNP was established. A total of 66 miRNAs families conserved only among mammals were used to predict the biological targets of these miRNAs by searching for the presence of conserved 8- and 7-mer sites within the 3′ UTR regions that would match the seed region of each miRNA (Lewis et al. 2005). The results did not indicate the presence of any conserved miRNAs overlapping the rs41960562 polymorphism. The nearest miRNA 7-mer (bta-miR-328) was located just 80-bp downstream.

The somatotropic axis contains the most promising candidate genes associated with meat production traits. Based on the knowledge of the polygenetic control of the studied physiological processes, we agree that further studies investigating the functional biology of the bovine IGF1R gene are necessary, in order to be able to consider the polymorphisms studied in this work as the causative mutations.