GAAAGG-39 and 59-TGTTCTGTGTGGGTTTGAGG-39). PCR amplification (32 cycles 94uC 30 sec, 59uC 60 sec, 72uC 60 sec) resulted in a RefSeq related product (prs) which was obtained in all animals, whether horned or polled. Additionally, a second PF related product, containing the two-base pair (TG) deletion was observed in all animals bearing one or two copies of the PF haplotype. Discrimination of the two products differing 2 bp in size was performed on an ABI PrismH 3130XL DNA sequencer. Animals bearing PF/PF were distinguished from PF/prs by quantitative evaluation of the obtained signals. PF/PF animals yielded signals of similar peak heights for the PF and prs products, while a heterozygous constellation (PF/prs) resulted in signal intensities of approximately double height for the prs product when compared to the PF specific product. SNP variants PG1654405A and PC1655463T were genotyped by competitive allele-specific PCR using commercially available kits (KASParH, KBioscience). PCR was performed as recommended by the manufacturer, while subsequent discrimination of the obtained fluorescent-labelled (FAM and CAL Fluor Orange 560) allele-specific products was performed on an ABI PrismH 3130XL DNA sequencer. SNP variant PC1768587A was analysed by PCR-RFLP. Genomic DNA was amplified (32 cycles 94uC 30 s, 58uC 60 s, 72uC 60 s; 59-CTGGAACCACGGATTACACAG-39 and 59-ACAGT- TATGGTCAGGAGGCAAA-39). Subsequently, three ml of the PCR product were treated with three units of TspRI (65uC for a minimum of 3.5 h). Obtained fragments were size-separated and visualized by 2% ethidium-bromide stained agarose gel electro- phoresis. Targeted re-sequencing One mg of Genomic DNA was randomly sheared by sonication (Bioruptor, Diagenode, Liege, Belgium) for 25 cycles (30 sec on/ off, ‘‘low’’ intensity). Sheared DNA with a median size of 200– 300 bp was rendered blunt-ended and 59-phosphorylated (NEB- next end repair module, New England Biolabs Inc, Ipswich, USA). After addition of a single non-templated 39-A (NEBnext A-tailing module) fragments were ligated to Illumina compatible adapters that carried a sample-specific 3 nt barcode and a 39-T overhang. The ligated library was size-selected on a 2% agarose gel and amplified by PCR with Illumina PE1 and PE2 primers. Equimolar amounts of the eight samples were pooled for array-capture (Agilent 244 k capture Array, Agilent, Santa Clara, USA; custom designed by e-array, repeat-masked, 3 bp tiling). Briefly, the libraries were hybridised for 65 h at 65uC, washed and eluted with nuclease-free water for 10 min at 95uC. The eluted DNA was concentrated in a vacuum centrifuge, amplified by PCR (10 cycles 98uC 15 s, 65uC 30 s, 72uC 30 s) and purified with Ampure XP beads. Mapping and Variant Calling Sequence reads from the Illumina Genome Analyzer IIx were aligned to the bovine reference genome (UMD3.1) using BWA [28]. The mapped reads were filtered for PCR duplicates with SAMTOOLS [29] and only uniquely mapping reads were retained. A pileup of the mapped reads was created for each animal using SAMTOOLS and variants were detected with VARSCAN (v.2.2.7) [30] at a minimal coverage of 20 and a minimal variant frequency of 0.01. The applied filters for the detection of potentially causative variants were: Homozygosity in the PP sires with a variant frequency range of 95 to 100%. For the heterozygote carrier Pp they must fall within a range of 40 to 60%, while the resulting candidate variants must be undetected in the pp sires. The putatively causative variants must be different from the UMD3.1 Allelic Heterogeneity of Bovine Polledness genome sequence, which was derived from a horned Hereford dam and therefore cannot carry the polled allele. Copy Number Variation For the identification of large insertion-deletion events between polled and horned animals we calculated the coverage ratio between the two groups in dynamical bin sizes. Each bin size was calculated by iterating over the mapped reads in the reference group until a determined number of reads (400) was reached and the start position of the last read was taken as bin size [31]. This step was repeated until the whole target region was processed. Then, for the target group the number of reads falling in each bin were counted and used for the calculation of the log2 ratios between reference and target group. The log2 ratios were plotted using R [32] and the average ratios were segmented using the circular binary segmentation implementation in the DNACOPY package (v1.14.0) from BIOCONDUCTOR [33]. Multi-species Alignment and Conservation Scores For the re-sequenced target region (chr1:1,543,412-2,089,648) we created multiple alignments for bovid ruminants (cattle, sheep and water buffalo), for unhorned mammals (horse, dog, human, mouse, pig, elephant, alpaca and dolphin) and for all selected species. The repeat-masked bovine genome sequence of the target region was obtained from UCSC genome browser [34] and used as reference for each of the pair-wise LASTZ genome alignments [35]. The resulting alignments were chained and netted to obtain best matching hits [36] and multi-species alignments were generated with MULTIZ [37] projected to the bovine reference. Additionally, short read sequences from the goat genome project (SRA Accession number: SRX016522) were added to the multi- species alignments only in the neighborhood of candidate mutations. To do so we aligned the short read to the bovine reference using BLAT [38] and manually selected the best alignments to the bovine reference. The reference genomes of sheep (Ovis aries 1.0), human (hg19), mouse (mm9), dog (canFam2), horse (EquCab2) and pig (Sscrofa9.1) were obtained from the FTP-site at UCSC. The draft genome sequences from alpaca, elephant and dolphin were obtained from the Broad Institute [39] and the water buffalo genome sequence was taken from the Indian Buffalo Genome Project (http://210.212.93.84). From the multiple species alignments we calculated the base conservation among horned animals, unhorned mammals and for all species using the PHAST package. For that purpose, we estimated a phylogenetic tree with PHYLOFIT from which the conservation score for each base was calculated with PHASTCONS. Supporting Information Figure S1 The pedigree chart of most polled Braunvieh. All declared polled Braunvieh bulls are descendents of the well- known American Brown-Swiss bull BS1 which is founder of polledness in Brown-Swiss/Braunvieh cattle population. The case individuals (PP) are represented by solid circles (females) and squares (males); declared carriers by half-filled symbols; not sampled individuals are marked with a diagonal line. The haplotype associated with polledness (red letters) of the four genome-wide genotyped bulls can be traced back to the same carrier bull BS1. This pedigree includes also four polled Braunvieh animals (*) genotyped only for candidate mutations. All sampled PP animals were genotyped as PC/PC and all Pp as PC/prs. (TIF) PLoS ONE | www.plosone.org 9 June 2012 | Volume 7 | Issue 6 | e39477