Mutation analysis and genotyping

Highest association with polledness in Holsteins was identified for polymorphisms within the interferon gamma receptor 2 gene (IFNGR2). In total, 41 SNPs and two indel mutations were detected within IFNGR2 by sequencing of PCR products (Table S1 Figure S1). Using next generation sequencing technique, further 18 SNPs and five indels were discovered within IFNGR2 (Table S2 Figure S1). The SNPs AC000158: g.1390505T>C, AC000158: g.1390528T>C, AC000158: g.1376880T>A, AC000158: g.1376884T>C, AC000158: g.1376932A>G and AC000158: g.1376977A>G were localised within the coding sequence of exon 3 and 7 of IFNGR2, respectively (Figure 1). AC000158: g.1376520T>C was located within the 3’ UTR (Figure 1). All other polymorphisms were intronic. The indels AC000158: g.1394768_771delCTGC and AC000158: g.1400851_852delCT were not observed in breeds other than Holstein. We genotyped all SNPs and indels with high association to polledness in further 11 homozygous polled, 61 heterozygous polled and 336 horned Holsteins. Although most Holsteins were born in Germany or male animals had a German herdbook number, their genetic origin was highly diverse (Figure S2), as crossing with animals from different countries is common in Holstein breeding to improve milk yield. The ancestry is known for 82 of the 86 polled animals. Five of these animals were female. Two of these female Holsteins were born in Germany and Spain, respectively, and one female Holstein was born in the United Kingdom. Of the male Holsteins, 57% were born in Germany, 14% in the United States of America, 14% in the Netherlands, 5% in Canada, 4% in the Czech Republic, 3% in the United Kingdom and 1% in Spain and Belgium, respectively (Figure S3). Of these animals, 87% have ancestors from other than their native country. Parents came from Germany, the United States of America, the Netherlands, Canada or France and grandparents from Germany, the United States of America, the Netherlands, Canada, Italy or Austria. This high diversity of ancestry assures representation of many important polled lineages including animals from Aggravation, Burket-Falls, Hickorymea, Dansire, Sandy-Valley, West Port Holsteins, Arron Doon Holsteins, Golden Oaks, Baldus Holsteins and Hollysprings.

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Figure 1. Gene structure of IFNGR2.

Rectangles indicate exons. Coding sequence is filled with black, the 3’ UTR is filled with white. The size of exons and introns is specified in number of base pairs (bp). The black line below illustrates the sections analysed by PCR in Holsteins. Each PCR-product is pictured separately with start and end position. Position of start and stop codon is stated in bp. All detected exonic polymorphisms and intronic polymorphisms showing association with polledness in Holsteins are given with name and position above the gene model. Each position is given with accordance to Bos taurus assembly UMD3.1.

https://doi.org/10.1371/journal.pone.0067992.g001

Polymorphisms that showed association in all Holsteins were genotyped in further 21 homozygous polled, 43 heterozygous polled and 28 horned German Fleckvieh and five homozygous polled, nine heterozygous polled and eight horned Charolais.

Association analysis

The SNP AC000158: g.1390292G>A, within intron 3 showed a highly significant association with the polled genotype (χ^²=423, -log₁₀P=91.85) (Table 1 Figure 1) and perfect co-segregation in all 443 Holsteins. All horned Holsteins showed the genotype G/G, all heterozygous polled Holsteins the genotype G/A, and all homozygous polled Holsteins the genotype A/A. However, association could not be confirmed for the tested animals of the breeds Charolais and German Fleckvieh, as they had the genotype G/G in either case, whether they were horned or polled.

There was one progeny-proven pure-bred heterozygous polled Holstein sire used in artificial insemination with a polled mother and polled grandmother that was heterozygous for the SNP AC000158: g.1390292G>A. This animal carried neither the previously published 202-bp-indel associated with polledness in cattle [15] nor the polled-associated alleles from three tested markers (P5ID, PC1768587A and P80kbID) belonging to the previously published polled-associated haplotype of seven polymorphisms [15]. Thus, this sire would not have been identified as genetically polled without the SNP AC000158: g.1390292G>A. Furthermore, there was one homozygous polled pure-bred Holstein that was a compound heterozygote for the polled and horned alleles at both the seven polymorphism haplotype and 202 bp indel loci predictive of polled [15]. However, this animal was homozygous A/A for the SNP AC000158: g.1390292G>A, and was therefore identified reliably as homozygous polled by only genotyping this SNP.

The SNP AC000158: g.1385927G>A in intron 4 was also significantly associated with the horned and polled genotypes in the initially analysed animals (χ^²=13, -log₁₀P=2.82). Association of this SNP could not be confirmed analysing an extended number of Holsteins. All three genotypes were found in the analysed homozygous polled animals. This SNP was thus not genotyped in all horned animals.

The indels AC000158: g.1394768_771delCTGC and AC000158: g.1400851_852delCT showed significant association (Table 1 Figure 1) but since they did not perfectly co-segregate with the horned and polled phenotype as did the SNP AC000158: g.1390292G>A, they were genotyped in all polled but not all of the horned animals.

SNPs within the coding sequence of exons 3 and 7 (AC000158: g.1376880T>A, AC000158: g.1376884T>C, AC000158: g.1376932A>G, AC000158: g.1390505T>C and AC000158: g.1390528T>C) and the SNP AC000158: g.1376520T>C within the 3’ UTR were not associated with polledness in Holsteins (Table 1).

The r²-values indicating correlation among mutated alleles showed that the three polymorphisms AC000158: g.1390292G>A, AC000158: g.1394768_771delCTGC and AC000158: g.1400851_852delCT are in a strong linkage disequilibrium (LD) (Figure 2). Although these three mutations are located over 200 kb proximal on BTA1 to the three markers P5ID, PC1768587A and P80kbID [15], all six polymorphisms form a haplotype block of 519 kb length (Figure 2).

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Figure 2. Linkage disequilibrium among markers within the 1 Mb BTA1 region associated with polledness in Holsteins.

AC000158: g.1390292G>A, AC000158: g.1394768_771delCTGC and AC000158: g.1400851_852delCT are newly discovered polymorphisms located in the IFNGR2 gene. P5ID, PC1768587A and P80kbID represent three markers of the polled-haplotype previously published for Holstein [15]. LD coefficients (r²) between pairs of polymorphisms are indicated as black letters in red fields. All pictured polymorphisms share one common haplotype block of 519 kb length.

https://doi.org/10.1371/journal.pone.0067992.g002

Ethics statement

All animal work has been conducted according to the national and international guidelines for animal welfare. The Lower Saxony state veterinary office at the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany, was the responsible Institutional Animal Care and Use Committee (IACUC) for this specific study. The EDTA-blood and semen sampling for the present study had been approved by the IACUC of Lower Saxony, the state veterinary office at the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany (registration number 02A/101).

Animals, phenotypic data and DNA preparation

EDTA preserved blood samples of three male and one female homozygous polled, five male and five female heterozygous polled and two male and 19 female horned, not closely related pure-bred Holsteins were used for the detection of new polymorphisms. The genotypes of the homozygous polled animals were proved by progeny-testing. The heterozygous polled animals were each offspring of a polled sire and a horned dam.

For genotyping of polymorphisms with highest association with polledness we used further EDTA preserved blood samples and semen samples of 11 homozygous polled, 61 heterozygous and 336 horned unrelated Holsteins of highly diverse origin (Figures S2-S3).

In addition, we used EDTA preserved blood samples of 21 homozygous polled, 43 heterozygous polled and 28 horned German Fleckvieh and five homozygous polled, nine heterozygous polled and eight horned Charolais. Bovine genomic DNA was isolated from EDTA preserved blood using the NucleoSpin 96 Blood Quick Pure Kit (Macherey Nagel, Düren, Germany) or from EDTA preserved blood samples or semen samples following a standard protocol with ethanol-precipitation.

Mutation analyses

Primers for PCR were designed using primer3 (http://frodo.wi.mit.edu/) covering intergenic regions and the genes MRPS6, SLC5A3, ATP5O, ITSN1, CRYZL1, DONSON, SON, GART, LOC784171, TMEM50B, IFNGR2, IFNAR1, IL10RB, IFNAR2, HIST1H4C and OLIG1 and the pseudogenes LOC782947 and OLIG2-like (Table S1). Repetitive regions in sequence-data were masked using RepeatMasker (http://www.repeatmasker.org/) before developing the primers. In addition, we applied Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast) to ensure that the designed primer sequences were unique within the bovine genome. PCR-products were directly sequenced. PCR reactions were performed in a total volume of 32 μl using 2 μl (~ 20 ng/µl) genomic DNA, 3.2 μl 10x PCR buffer, 0.62 μl DMSO, 0.62 μl dNTPs (10 mM each), 0.18 μl (100 pmol/μl) of each primer and 0.18 μl Taq Polymerase (MP Biomedicals, Eschwege, Germany). The PCR conditions were as follows: 5 min initial denaturation at 95°C, followed by 36 cycles at 95°C for 30 sec, optimal annealing temperature (T_a, differed between 57°C and 60°C, according to the primer) for 1 min and extension at 72°C for 45 sec. The PCR was completed with a final cooling at 4°C for 10 min.

The amplicons were purified using the MinElute 96 UF Plate (Qiagen, Hilden, Germany) or ExoSAP-IT (Affymetrix, München, Germany) and directly sequenced with the DYEnamic ET Terminator Cycle Sequencing kit (GE Healthcare, Freiburg, Germany) on a MegaBACE 1000 capillary sequencer (GE Healthcare) or an ABI Prism 3500 capillary sequencer (Applied Biosystems, Darmstadt, Germany). Determined sequence data was analyzed using the Sequencher 4.8 program (GeneCodes, Ann Arbor, MI) and screened for polymorphisms.

Additionally, the genomes of one homozygous polled and three horned Holsteins and two DNA-pools, respectively, with six horned Holsteins each were sequenced using paired-end sequencing on the Illumina HiSeq 2000 (LGC Genomics, Berlin, Germany). On the average, an eight-fold coverage per sample was accomplished.

Genotyping

Genotyping of SNPs was performed by sequencing as described above. SNPs AC000158: g.1390292G>A and AC000158: g.1385927G>A were genotyped by PCR-RFLP (restriction fragment length polymorphism) or mismatch PCR-RFLP [17], respectively. The restriction enzymes were selected from the NEBcutter V 2.0 (http://tools.neb.com/NEBcutter2/). For SNP AC000158: g.1390292G>A the restriction enzyme Hha1 was used. For genotyping of SNP AC000158: g.1385927G>A we used the restriction enzyme DdeI. Digested products were size-fractionated by gel electrophoresis on 2% to 4% agarose gels (peqGOLD Universal Agarose, Peqlab Biotechnologie, Erlangen, Germany).

Indel mutations were amplified by using DY-682-tagged primers (Eurofins MWG Operon, Ebersberg, Germany). PCR reactions were performed in a total volume of 12 μl using 2 μl (~ 20 ng/µl) genomic DNA, 1.2 μl 10x PCR buffer, 0.4 μl DMSO, 0.2 μl dNTPs (10mM each), 0.5 μl (10 pmol/μl) of each primer and 0.2 μl Taq Polymerase (MP Biomedicals). The PCR conditions were as follows: 5 min initial denaturation at 94°C, followed by 36 cycles at 94°C for 30 sec, 58°C to 60°C (optimal annealing temperature according to the primer) for 1 min and extension at 72°C for 30 sec. The PCR was completed with a final cooling at 4°C for 10 min. Afterwards the PCR products were analysed by gel electrophoresis on the automated sequencer LI-COR 4300 (LI-COR, Bad Homburg, Germany) using 4% to 6% polyacrylamide denaturing gels (Rotiphorese Gel40, Carl Roth, Karlsruhe, Germany). Differences in allele sizes caused by deleted or inserted bases were defined by visual examination.

Statistical analyses

Association analysis with polledness in cattle was carried out for the detected polymorphisms. The markers have been tested for their polymorphism information content, Hardy-Weinberg-Equilibrium (HWE) and association with the polled phenotype and the polled genotype using the procedures ALLELE and CASECONTROL of SAS/Genetics, Version 9.3 (Statistical Analysis System, Cary, NC, 2012). Statistical calculation of pairwise linkage disequilibrium (LD) was performed and pictured using HAPLOVIEW 4.2 (http://www.broad.mit.edu/mpg/haploview/). For detection of SNPs with strong LD among the alleles we used the tagger algorithm r²≥0.8[18]. .

In silico screening for binding sites of gene-regulating elements

Polymorphisms associated with polledness and mutations located within the UTR-regions were screened for effects on binding sites for gene regulating elements using AliBaba 2.1 (http://www.gene-regulation.com/pub/programs/alibaba2/index.html).

SNPs localised in coding sequence were examined for causing amino acid changes. Discovered changes in amino acid order were analysed using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2) with regard to their influence on protein function.

The expression of genes can also be regulated post-transcriptionally by microRNAs (small non-coding RNAs) [19]. In vertebrates 40-70% of microRNAs are expressed from introns [20]. The database miRBase (http://www.mirbase.org/) was therefore scanned for microRNAs contained inside the IFNGR2-gene in human and mouse.