Allelic Heterogeneity of Bovine Polledness species conservation calculation for the candidate region is shown in plot d. The candidate mutation PC1768587A is highlighted within a red frame and stars in red and black indicate identity among bovid and all animals, respectively. doi:10.1371/journal.pone.0039477.g002 comprised 89 Pp bulls of ten breeds (Table S1). Moreover, we complemented the sample of the homozygous case group by additional PP bulls of Holstein-Friesian (HF, including Red- Holsteins (RH)), German Fleckvieh (FV) and Jersey (JY). The P202ID allele was in complete LD to the POLLED allele in all breeds but HF, JY and WTG which turned out to carry the P202ID allele only sporadically. Polled HF, JY and WTG animals declared as Pp but missing the P202ID allele were carriers of the haplotype block of seven candidates detected by sequencing of HF animals (P5ID, PG1654405A, PC1655463T, PT1671849G, PT1680646C, PC1768587A and P80kbID). Furthermore, HF and JY animals declared as PP and missing P202ID were carriers of two copies of the haplotype detected by sequencing of polled HF animals. To further proof the hypothesis of allelic heterogeneity of POLLED, we randomly sampled 400 animals in South-Germany complemented by random samples from target breeds. This semi- random sample comprised sets of 238 random HF/RH, 293 HF- FV crosses, 52 random JY, 50 random PNZ, and 211 animals from our cattle diversity panel (Table S1). These 1,262 DNA samples as well as the case and the carrier group were chosen for genotyping for the candidate variants: P202ID, P5ID, P80kbID PG1654405A, PC1655463T, PT1671849G, PT1680646C, and PC1768587A. Two SNPs PT1671849G and PT1680646C were excluded due to sporadic and solitary occurrence of the candidate mutation on RefSeq background in horned animals. So far, we do not observe recombination within PF haplotype block (from P5ID to P80kbID) and consequently do not exclude any candidate mutations by recombination. As soon as one candidate was excluded by confirmed genotyping we cancelled further genotyping of the respective marker. The breeding records, including polled certificates of the genotyped animals and their ancestors as well as their breed affiliation, were used to confirm the congruence Figure 3. DNA sequence variants in diverse polled and horned cattle breeds. (a) SNP genotypes. The genomic position of DNA sequence variants (DSV) called by high-throughput sequencing of the target region are displayed as bars (SNPs) or triangles (InDels), in blue (homozygote) and red (heterozygote). Homozygous polled bulls are PP-DAN1, PP-GLW1, PP-GLW2 and PP-DFV1 and horned bulls are pp-FGV, pp-DFV2, pp-DFV3 and pp-MWF1. The candidate duplication P202ID is highlighted by an inverted triangle. The annotated genes and ESTs in this region are displayed in the green area. (b) Within-species conservation. The bovine variant density from dbSNP is shown in the re-sequenced region per kb. (c) Multi-species alignment. The genomic multi-species alignment around the candidate duplication P202ID was built with MULTIZ. Sequence identities among bovid animals are displayed with red stars whereas black stars denote the identity among all animals. The duplicated region is highlighted by the red shaded area and replaces the blue shaded nucleotides in polled animals. doi:10.1371/journal.pone.0039477.g003 PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39477