Allelic Heterogeneity of Bovine Polledness Figure 1. Homozygosity mapping in case-control design of polled and horned cattle animals. (a) Genome-wide association mapping of the POLLED mutation to the proximal end of bovine chromosome 1 (BTA1) with 162 affected animals and 162 controls (ASSHOM method, Charlier et al. 2008). Nominal ASSHOM statistic is presented chromosome by chromosome for all 29 bovine autosomes. Evidence for association (y axis) is measured as nominal ASSHOM statistic with genome-wide significance (P = 0.0002, grey line) at 3.315, being determined by 50,000 nominal ASSHOM statistics along chromosomes with randomly permutated markers. (b) Association mapping details for the region from 0 to 4 Mb on BTA1. Nine markers homozygous in all 162 cases covered a region from 1,760,113 to 1,983,902. Two informative SNP markers flanking this core (i.e. variable sites in the case group) are ARS-BFGL-NGS-39992 (1,668,494 bp) and ARS-BFGL-NGS-29653 (2,049,400 bp). (c) Candidate region of 547 kb (1.543–2.090 Mb (UMD3.1 genome build)) chosen for high-throughput sequencing and most likely encompassing the POLLED mutation. This interval is larger than homozygosity bracket detected by multi-breed design and nested five genes (IL10RB, IFNAR2, OLIG1, C1H21orf62 and GCFC1), one pseudo gene (OLIG2-like) and some not further annotated ESTs (e.g. BC122836, EV693397 and DT837875). doi:10.1371/journal.pone.0039477.g001 duplicated in the same direction and only differs from the RefSeq by one TRA transversion at the third position after the beginning of the duplicated sequence (PT1909354A) and by a two-base pair (TG) deletion at the 45th position in the duplicated sequence. This 2 bp deletion corresponds to position 1,909,396 in the original sequence and will be denoted as P1909396D2. Both variants close to the junction between the original and the duplicated sequence, PT1909354A and P1909396D2, allow for a PCR design for the detection of this 80 kb InDel (allele P80kbID). In addition to P5ID and P80kbID, both flanking the homozygosity region in polled HF, we detected five point mutations at the positions 1,654,405 (GRA), 1,655,463 (CRT), 1,671,849 (TRG), 1,680,646 (TRC) and 1,768,587 (CRA), respectively. In these five candidate SNPs (PG1654405A, PC1655463T, PT1671849G, PT1680646C, PC1768587A) mutant alleles determine the sequence in the polled HF animals. These seven candidate mutations do not include any known coding sequence, or splice site, or intronic region, or any known regulatory elements (Fig. 2). Furthermore, none of these variants were previously reported in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/). The remaining eight non HF sires (four PP and four pp) belong to spatial and genetic quite differentiated breeds: Angus, Galloway, Fleckvieh, Gelbvieh and Murnau-Werdenfelser. Nev- ertheless, both PP and pp sires were homozygous for the common haplotype that is always homozygous in PP animals [10]. Considering these eight sires separately paired-end sequencing identified 248 putative DSVs, or an average nucleotide diversity of ,0.08%. Applying the same filter to the 248 DSVs yielded only one complex insertion-deletion (InDel) event as the putative causative variant (Fig. 3), which was confirmed by visual inspection in a genome browser to be the only DSV concordant with the pp and PP genotypes. The sequence of 212 bp (1,705,834– 1,706,045 bp) is duplicated and replaces a sequence of 10 bp (1,706,051–1,706,060 bp). This InDel (P202ID) is between the genes IFNAR2 and OLIG1 (Fig. 3). The InDel P202ID again does not disrupt any known coding sequence or a splice site, or an intronic region, or any known regulatory regions. All candidate DSVs detected in HF subset were homozygous for RefSeq allele in all eight non HF sires, both in PP as well as in pp. Target Genotyping of Candidate DSVs in Polled Animals, Random Samples and Diversity Panel Supports Allelic Heterogeneity of the POLLED Phenotype In order to further investigate the presumed allelic heterogeneity of POLLED, we chose sperm samples of polled bulls of all of the most important cattle breeds with European origin. This PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39477