In cattle, as in other Bovidae, horn development and morphology are characterised by a substantial degree of polymorphism. Even if only European cattle breeds (Bos taurus) are considered, there are completely hornless cattle breeds like Angus, and breeds with very short horns like Busˇa as well as breeds with very long and lyra- shaped horns like the Pannonian Podolian cattle [5]. The polled phenotype has been considered to be caused by a dominant inhibitor [3] that is characterised by the complete absence of corneous appendices [6]. In addition to diverse forms of cattle horns, abnormal types of horns called scurs frequently occur, i.e. incompletely developed horns that are not fused to the frontal bone [7]. Scurs can be considered as the intermediate phenotype between polled and horned because the horn core is not an outgrowth of the skull but originates from a separate ossification centre in the tissues located above the periosteum with subsequent fusion to the skull [2,3]. While the inheritance pattern and mapping position of the POLLED locus on Bos Taurus Autosome 1 (BTA1) [8,9,10] is beyond controversy, the scurs locus displays heterogeneity [2]’ [11]’ [12]. To avoid complications due to possible interference between POLLED locus and type 1 [11] or type 2 [2] scurs syndrome we exclusively focussed on polled and horned animals of 31 cattle breeds of European origin. This material exploited the full capacity of the population structure of European cattle and in combination with high-density SNP genotyping and high-through- put sequencing, was used to identify the genetic variation that causes the polled phenotype in Bos taurus. Results The POLLED Locus Maps to Chromosome 1 in Divergent European Cattle Breeds We collected DNA samples from twelve polled cattle breeds (Table S1). The applied mapping approach and major part (62%) of the samples has been presented in our previous study [10] and was now completed by additional 61 polled animals as well as by three additional polled breeds: Norwegian Red, Fjall cattle and Braunvieh. Case (homozygous polled, PP) and control (homozy- gous horned, pp) group each consisted of equal numbers of animals (162) from 18 breeds in total. As some breeds are consolidated either for the polled or horned phenotype uneven phenotype distribution was inevitable within some breeds. An improved case-control design accounting for this uneven distribu- tion did not refine the mapping (Fig. 1). We mapped the POLLED locus in the same 381 kb interval (1.668 Mb - 2.049 Mb; UMD3.1 genome build) we already found in our previous study [10]. The genome-wide significance (P#0.0002,) was determined by 50,000 random permutations of markers along chromosomes. The applied mapping procedure [13] performs searches for shared segments of homozygosity without the need of an identity by state and thus revealed two haplotypes. The most common haplotype (AGACAAGGA) was found in all but one case animals. Two copies of an alternative haplotype, with a variant allele at the sixth SNP (AGACAGGGA) were found in a homozygous polled Braunvieh bull (Fig. S1) that was one of the animals from the current addition to the mapping design. Genotyping of three related bulls revealed the same alternative haplotype associated with the polled phenotype in Braunvieh (Fig. S1). As the origin of polledness in Braunvieh is unknown and recombination within the homozygosity block of the available Braunvieh pedigrees could not be detected, the Braunvieh data did not refine the mapping. In order to confirm and possibly improve the mapping results we embedded 89 heterozygous polled animals (POLLED carriers; Pp) and 62 horned relatives into the analysis. These 151 animals Allelic Heterogeneity of Bovine Polledness were genotyped with the same Illumina BovineSNP50 assay [14] and have not been used in the case-control design. These 89 Pp bulls comprise ten breeds and are ancestors or descendents of PP bulls included in the case group, except for carriers from the Witrug breed (WTG) (Table S1). The haplotypes were manually traced through the pedigree charts. The result clearly confirmed the mapping result (Fig. S1, S2, S3 and S4), but the absence of any observable recombination within the candidate region hindered further improvement of fine-mapping by linkage analysis. Sequencing Eight Polled and Eight Horned Sires did not Detected a Common Allele Associated with Polled Status We selected seven PP, one Pp and eight pp bulls and performed high-throughput sequencing of a 547 kb interval (1.543–2.090 Mb (UMD3.1 genome build)) that nested the most likely location for the POLLED mutation [10]. The polled group was represented by four Holstein-Friesian (HF), two Galloway (GLW), one German Angus (DAN) and one Fleckvieh (FV) bull. One HF bull was declared as Pp while remaining seven bulls of polled group were progeny tested as PP. The horned group was represented by four HF, two FV, one Gelbvieh (FGV) and one Murnau-Werdenfelser (MWF) bull. Paired-end sequencing of the libraries enriched for a 547 kb region on BTA1 resulted in average sequence depth of non- repetitive sequences of ,406 per individual, with 98.9% of the non repeat-masked target region being covered. From these data, we identified 451 putative DNA sequence variants (DSVs), or an average nucleotide diversity of ,0.15%. Further analysis of the sequencing data (on a locally installed instance of GALAXY [15]) revealed almost complete absence of sequence variability in a region of 125 kb (1.697 to 1.822 Mb) between all 16 sequenced animals. Under the assumption that the POLLED locus is biallelic with dominant inheritance [7,9] and because the reference sequence (RefSeq) is based on a horned Hereford cow [16], the causative variant has to be homozygous in the seven PP sires, heterozygous in one Pp sire and not present in RefSeq or in the eight pp sires. Applying this filter to the 451 DSVs did not detect a common allele associated with polled status. Also a visual inspection in a genome browser did not yield DSV concordant with the POLLED genotypes. These results suggested at least two plausible explanations: (i) the targeted 547 kb interval that was covered to 98.9% for the non repeat region harbors the functional mutation(s) in the remaining 1.1% (ii) some of DSVs are causal but there is allelic heterogeneity at the POLLED locus and different alleles have been selected in different geographic regions or breeds. Dividing Sequenced Animals into Two Groups Suggests Allelic Heterogeneity of the POLLED Phenotype Eight of 16 sequenced sires are Holsteins and in the case of the allelic heterogeneity these could most probably harbour the same POLLED mutation. The average sequence depth of non-repetitive sequences in eight HF bulls was ,296 per individual, covering 98.5% of the non repeat-masked target region. From these data, we identified 312 putative DSVs, or an average nucleotide diversity of ,0.11% (Fig. 2). Applying the same consecutive filtering as above reduced the number of potential causal variants to seven. On the proximal end of the region homozygous in polled HF (1.648– 2.027 Mb; Fig. 2) we detected a complex InDel event, replacing 7 bp (cgcatca; RefSeq: 1,649,163–1,649,169) by 12 bp (ttctcagaa- tag) and thus resulting in a 5 bp longer sequence in polled HF animals (allele P5ID). On the distal end of the region of homozygosity in polled HF we detected a duplication of an 80,128 bp (1,909,352– 1,989,480 bp) sequence (Fig. 2). This large sequence is seamlessly PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39477