IGF2 is crucial for the regulation of cell proliferation, growth, differentiation and survival. Several studies have shown association of the IGF2 gene with milk production traits (Flisikowski et al. Reference Flisikowski, Adamowicz, Strabel, Jankowski, Switoński and Zwierzchowski2007; Bagnicka et al. Reference Bagnicka, Siadkowska, Strzałkowska, Żelazowska, Flisikowski, Krzyżewski and Zwierzchowski2010; Berkowicz et al. Reference Berkowicz, Magee, Sikora, Berry, Howard, Mullen, Evans, Spillane and MacHugh2011). Using comparative mapping of genes differentially expressed in mouse mammary gland with their bovine map positions for previously identified QTLs, Ron et al. (Reference Ron, Israeli, Seroussi, Weller, Gregg, Shani and Medrano2007) identified the IGF2 gene as a candidate affecting milk production traits in cattle. IGF2 binds to the non-signalling IGF type 2 receptor (IGF2R), a multifunctional, transmembrane glycoprotein that has binding domains for IGF2 and mannose-6-phosphate (M6P). IGF2R functions as a regulator of IGF2 level, sequestering excess circulating IGF2 and thereby influencing its bioactivity (Kornfeld, Reference Kornfeld1992). Dehoff et al. (Reference Dehoff, Elgin, Collier and Clemmons1998) showed that lactation in the bovine mammary gland is associated with increased IGF2R concentration and suggested that IGF2R regulates lactation, possibly via an effect on circulating or local IGF2 concentration.
Here we describe two novel genetic polymorphisms of bovine IGF2R: a TG-repeat variation in intron 23, and a G > A transition (SNP) in exon 24, and investigate their association with milk production traits in Friesian-Holstein cows.
Material and methods
Animals and management
All experimental procedures involving animals were conducted in accordance with ‘The Guiding Principles for the Care and Use of Research Animals’ and were approved by the Local Ethics Commission (permissions No 84/2006 and 27/2009).
The study was conducted on 283 Polish Holstein-Friesian (PHF) cows, daughters of 68 sires. The pedigree file covered information about two generations, i.e. parents and grandparents. They were maintained in the dairy herd of the IGAB PAS farm in Jastrzębiec. During the test period animals were fed a complete total mixed ration (TMR) consisting of corn silage, wilted grass silage and concentrates, supplemented with mineral and vitamin mixture, according to the INRA system.
Identification of IGF2R polymorphisms
Eight-ml blood samples from each cow were collected by an authorised veterinarian into test tubes containing K2EDTA and stored at −80 °C till further analysis. DNA was isolated as previously described (Bagnicka et al. Reference Bagnicka, Siadkowska, Strzałkowska, Żelazowska, Flisikowski, Krzyżewski and Zwierzchowski2010). Primers for PCR amplification of overlapping fragments of the bovine IGF2R covering introns 23, 24, 42, 43, 44 and exons 24, 43, 44 (Supplementary Table S2) were designed based on the sequence of the bovine IGF2R (bovine chromosome 9 genomic scaffold; GenBank accession no: NW_001495620.3) using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/). PCR was performed using a mix containing approximately 100 ng of bovine genomic DNA, 10 µM each primer, 5 µl REDTaq® ReadyMix™ (Sigma-Aldrich) and H2O to a total volume of 10 µl. The following PCR protocol was used: initial denaturation for 3 min at 95 °C, followed by 30 cycles of 55 s at 94 °C, 57 °C, 72 °C, and a final elongation step – 5 min at 72 °C. The PCR reactions were carried out in a MJ TETRAD thermocycler (Bio-Rad Laboratories, Hercules, USA). The amplification products were purified with a GenElute PCR DNA Purification Kit (Sigma–Aldrich Corporation, St. Louis, MO, USA) and directly sequenced in an ABI 377 sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed using Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA) software. Polymorphisms were detected using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Genotyping
All samples were genotyped for the g.72479 G > A SNP in exon 24 using RFLP-StyI. The gene fragment containing the mutation was amplified using the primers for exon 24: IGF2R24B and IGF2R24R2 (Supplementary Table S2) in the conditions described above. The 254-bp PCR product was then digested with restriction endonuclease StyI (Biolabs, Ipswich, MA, USA). The restriction fragments were separated on a 2% agarose gel with ethidium bromide.
For the analysis of the TG-repeat (microsatellite) length polymorphism, a fragment of IGF2R gene intron 23 (15-67 TG-repeats) was PCR amplified using a pair of primers flanking the polymorphic site: IGF2R23F and IGF2R23R (Supplementary Table S2), with the forward primer carrying the Cys-5 dye at the 5′ end (the same PCR conditions as above). PCR fluorescent products were denatured in a 50% formamide solution containing blue dextran, and then separated in 6% denaturing polyacrylamide gels in an ALFexpress DNA Sequencer (Amersham Biosciences Corporation, Piscataway, NJ, USA) as previously described (Maj et al. Reference Maj, Korczak, Bagnicka, Zwierzchowski and Pierzchała2007). In each lane, 1 µl PCR products were resolved together with a size marker. Allele Links 1·01 software (Amersham Biosciences) was used for automated allele calling, individual genotypes were checked by manual inspection and then exported as Excel files.
Analysis of milk composition
The cows were milked mechanically two times a day in a standard herringbone milking parlour. Representative milk samples were taken using milk meter each month in lactation parallel to official milk recording (A4 method).
Milk samples from 283 cows were analysed in the IGAB PAS laboratory. The daily milk yield during whole lactations (determined eight times per lactation) of studied animals for 3 years was determined and fat, protein and lactose content estimated in fresh milk samples using MilkoScan 104A/B (FOSS A/S, Hillerød, Denmark). Then, daily fat, protein and lactose yield were calculated. Somatic cells were counted using a Fossomatic apparatus (FOSS A/S). A total of 6,900 records were collected.
Statistical analysis
As in earlier studies (Bagnicka et al. Reference Bagnicka, Strzałkowska, Flisikowski, Szreder, Jóźwik, Prusak, Krzyżewski and Zwierzchowski2007, Reference Bagnicka, Siadkowska, Strzałkowska, Żelazowska, Flisikowski, Krzyżewski and Zwierzchowski2010) to determine the influence of the IGF2R STR and SNP genotypes on the milk traits investigated, we used the AI-REML (average information restricted maximum likelihood) method with repeatability, multi-trait animal model based on test-day information (Supplementary File S1). Combined effect of year-season of calving, parity, and animal IGF2R genotype as fixed effects and animal additive genetic and its permanent environment, dates of the milking tests as well as error as random effects were included in the model. To determine the effect of year-season of calving (6 classes) effect, two seasons of calving were established (October-March and April-September) with each year of calving as a separate subclass. According to parity, the information was divided into three classes, with the third class covering parities more than second. The DMU program was used for computation (Madsen & Jensen, Reference Madsen and Jensen2000). Somatic cell count was standardised using natural logarithm (lnSCC). Lactation days were adapted as Legendre polynomial (Brotherstone et al. Reference Brotherstone, White and Meyer2000) and fitted as fixed covariates within each parity subclass in order to represent changes in considered traits due to the stage of lactation. The differences between means for different genotypes were estimated by Duncan's test (P < 0·05). Statistical significance of differences between observed and expected frequency of genotypes was estimated using the χ2 test (P < 0·05).
Results and discussion
Two novel polymorphisms were identified in bovine IGF2R: a g.72479 G > A transition in exon 24, and a polymorphic TG-repeat in intron 23 starting at position 72 389 upstream of the putative transcription initiation site. The frequency of the exon 24 genotypes in the animals studied was: GG (0·52), GA (0·45), and AA (0·03) (Supplementary File S3). Analysis of this polymorphism with the Transeq program (http://www.ebi.ac.uk/Tools/st/embosstranseq/) revealed that the G/A transition is a synonymous mutation encoding arginine at position 1241 of the IGF2R protein. For all the milk traits tested, other than fat content, the GG and GA vs. AA genotypes showed statistically significant differences (P ≤ 0·01) (Table 1). Cows with the GG genotype produced about 1·7 kg of milk daily and 0·035 kg protein more than AA animals. Moreover, milk of the AA genotype cows contained more lactose and fewer somatic cells than those with GG and AG genotypes.
Table 1. Association between genotype g.72479 G > A in exon 24 of Igf2r gene and dairy production traits of HF cows
Values with various letters A, B within columns are significantly different at P ≤ 0·01. Values in brackets – Standard Error (se).
Regarding the intron 23 TGn polymorphism, twenty two alleles forming 64 genotypes were detected in 279 HF cows (Supplementary File S4 and S5). The most frequent allele was TG28 (0·213); the rarest were TG32 and TG65 (0·001). The most frequent genotype was TG28/TG25 (22 animals; frequency 0·078) followed by TG28/TG27 (20 animals; frequency 0·071). 29 genotypes were each represented by a single animal (frequency 0·003).
Association analysis was carried out between TGn genotypes, represented by at least 3 animals (n = 238), and milk production traits in HF cows (Table 2). The highest milk yield was observed in cows with the 29/22, 28/22, 28/29 and 28/28 genotypes, and the lowest milk yield in 28/30 and 29/33 genotype cows. Cows with 28/23 and 28/22 genotypes had the highest milk fat; and those with 28/30 and 20/33 the lowest milk fat. Cows with 28/23, 23/30 and 28/22 genotypes produced the highest daily amount of milk protein. The highest milk lactose was found with genotypes 25/23, 33/30, 25/22 and 25/20; and the lowest lactose with genotypes 22/30, 28/26 and 29/20. The somatic cell count was the lowest in the milk of cows with 28/28, 23/30 and 29/33 genotypes, and the highest in 23/30, 22/30, 20/19 and 22/33 genotypes.
Table 2. Association between g.72389(TG)15–67 genotype in intron 23 of Igf2r gene and dairy production traits of HF cows (summarised results from Supplementary Files S6–S10)
Values with various letters within columns are significantly different; A, B – with P ≤ 0·01; a, b – with P ≤ 0·05. Values in brackets – Standard Error (se).
The genetic parameters obtained in statistical analysis did not differ from those obtained in big population of Polish H-F dairy cattle (Rzewuska & Strabel, Reference Rzewuska and Strabel2013). This means that despite the small number of animals included in the analysis the results are not biased with big errors. Standard errors of estimates (Table 1) proved this statement.
The IGF2R gene is located on bovine chromosome 9 at position 97 638–97 741 Kbp, near the QTL region for fat yield (98 cM) and for SSC (103 cM) (Smaragdov et al. Reference Smaragdov, Prinzenberg and Zwierzchowski2006). Therefore, considering its location in the BTA9 QTL region, the functions of IGF2R in the controlling IGF2 level, and its interaction with other proteins, the IGF2R has been postulated as a molecular marker for production traits in cattle. So far, only a few experiments have been conducted to identify possible associations between IGF2R polymorphisms and cattle production traits. Dunner et al. (Reference Dunner, Sevane, Garcia, Leveziel, Williams, Mangin and Valentini2013) identified five IGF2R polymorphisms that affect meat texture, flavour and lipid composition. Berkowicz et al. (Reference Berkowicz, Magee, Berry, Sikora, Howard, Mullen, Evans, Spillane and MacHugh2012) reported associations between three SNPs located in the introns of bovine IGF2R and growth performance traits in Irish Holstein-Friesian sires, but none were associated with the milk traits they examined including milk, fat, protein yield and somatic cell count (SCC). As shown by Abu El-Magd et al. (Reference Abu El-Magd, Abo-Al-Ela, El-Nahas, Saleh and Mansour2014) in Egyptian Buffalo a nonsynonymous A/C SNP in exon 23 of the IGF2R gene was associated with the average daily gain during the early stages of life and this effect could be caused by increased expression levels of the IGF2 gene.
Our findings reveal an association between the synonymous g.72479 G > A SNP in IGF2R exon 24 with milk production traits in Polish HF cows. The GG and GA genotype cows showed higher milk yield and protein yield than those with AA genotype. As the AA genotype is very rare, the usefulness of the g.72479 G > A SNP for marker-assisted dairy cattle selection requires further study with larger populations of dairy cattle.
The TGn STR in IGF2R intron 23 (g.72389(TG)15-67) was also associated with dairy production traits. The effects of various genotypes were considered on the basis of several criteria: (1) those showing the most evident differences between milk traits; (2) positively or adversely affecting at least two dairy traits; (3) occurring at least 6 times in the population studied (frequency 0·021); and (4) differing significantly from at least 9 other genotypes (Supplementary File S6–S8). Our analysis revealed that the 28/28, 28/22 and 28/23 TGn genotypes had the highest values of milk, protein and fat yield, and are thus the most desirable from the breeder's perspective.
To our knowledge, ours is the first study showing the influence of IGF2R polymorphisms on milk production traits in cattle.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029918000110.
Supported by the Polish National Science Centre, Grants 2012/05/|B/NZ9/03425, 2014/13B/NZ9/02509 and the IGAB PAS project no. STAT/LECZWI/2017/02.
