between the obtained genotyping results at six remaining candidate mutations and polled status. The results are presented in Fig. 4 and summarised below. The single candidate variant P202ID detected by the sequencing of eight non HF sires and the block of five candidates detected by sequencing of eight HF animals (P5ID, PG1654405A, PC1655463T, PC1768587A and P80kbID) segregate independently without evidence for any interference or recombination. As all animals carrying one or two copies of the P202ID allele are polled (Pp or PP, respectively) and belong to breeds originating from geographical areas with Celtic culture [17] we hereafter figuratively and provisory called this allele PC, indicating polledness of Celtic origin (see Fig. 4). Appropriately, the haplotype block (260 kb long) detected by sequencing of HF animals is hereafter called PF, indicating polledness of Friesian origin. Both PC and PF, independently and in combination, were in perfect association with POLLED locus. There were two HF and one JY bull declared as PP which we found to be heterogeneous polled at candidate level, i.e. PC/PF. These three bulls were progeny-tested and mated to a large number of horned cows, permitting an official declaration as PP by the respective breeder associations. The pedigrees chart and genotype analyses of the PC/ PF bulls and their sampled relatives (Fig. S3 and S4) clearly support allelic heterogeneity at the POLLED locus. Our diversity panel completed the semi-random sample (Table S1) and both together cover the European cattle population from the domes- tication centre, over the most likely dispersal routes for this species to the western- and northernmost parts of Europe (Fig. 4). This spatial well distributed panel comprises breeds with very small horns as well as extremely large horns [5] and with high genetic diversity between as well as within breeds [18]. In these horned breeds, only wild-type alleles (prs) were detected for all six candidate mutations, hindering further sieving of causative variants from passenger variants in this design. Sequence Conservation Among Horned Ruminants and Other Mammals Allows Scoring of Candidate Causal Mutations The DSVs PG1654405A and P5ID are part of repeat-masked regions (MLT1B and LTR13BT), hence no conservation score (PHASTCONS scores) could be computed. Local blat searches against the selected species revealed only short multiple alignments to various genomic locations; in consequence no local alignments could be built. This however makes it unlikely that these two DSVs are linked to horn development as the flanking sequence is not conserved among horned ruminants. Moreover, these DSVs are found in a region with a high density of SNPs (Fig. 2B), indicating little selective pressure and making it less likely to encode for a functional element important for horn development. The sequence flanking PC1655463T has a low conservation score, even among ruminants, and the local alignment (Fig. S5) shows that the corresponding sequence in the goat genome harbours a T, the same base as the variant allele found in polled Holstein cows. Thus, PC1655463T is less likely being causative for the polled phenotype. The RefSeq nucleotide C at SNP PC1768587A is conserved only among bovid ruminants, where the variant allele is at the central position of a 24 nucleotide block that is identical in all four available Bovidae genomes. Also, the surrounding sequence is highly conserved among Bovidae but far less among other mammals (Fig. 2D and E). The SNP density of the whole region (Fig. 3B and 3C) including PC shows a contiguous area with high sequence conservation within cattle, making it a likely candidate for the presence of an un-annotated functional element. Allelic Heterogeneity of Bovine Polledness Both PT1909354A, and P1909396D2, nested within the 80 kb duplication, affect elements that are conserved only among the bovid ruminants (Fig. S6). However, the 80 kb duplication can be causative itself, and sequence conservation of single elements within these 80 kb does not necessarily reflect the consequence this duplication may induce. Sequence conservation across and within species thus allows ranking of the candidate mutations within PF haplotype, resulting in the P80kbID duplication (including the two variations PT1909354A and P1909396D2) and the SNP PC1768587A as two most plausible candidates. The candidate PC1655463T is considered as least likely as it coincides with sequence variation within different members of the Bovidae family suggesting a lack of functional constraint at this position. Discussion The polled phenotype in cattle has been a target of selection and research since long. Large economic value of the phenotype [19], the simple dominant inheritance known since 1936 [6] and the mapping position known since 1993 [9] provided hope for an early and easy detection of the causal variant. This, however, was not the case due to special features of the phenotype, underlying causal mutations and livestock genetics: (i) the identification of functional candidate genes by the usual comparative trajectory with humans or mouse is hardly possible for this phenotype specific to Bovidae, (ii) the lack of functional and even positional candidate genes in large fractions of candidate region hindered identification of mutations conferring the polled phenotype and (iii) possible allelic heterogeneity of the phenotype investigated in a highly mobile species (world-wide and across-breeds use of some founders by artificial insemination) can reduce the ability to exploit haplotype diversity between breeds. In this study, we used approaches beyond LD-based fine mapping to reduce the list of candidate causative genes without relying on any a priori assumptions of gene function. According to the contemporary trajectory of fine-mapping by a relatively dense SNP marker panel, high-throughput re-sequencing and associa- tion proven in a large panel of individuals sampled across the European continent we detected a single complex insertion- deletion event (P202ID) perfectly associated with the POLLED gene in most European cattle breeds. The absence of any other congruent candidate variant in the entire chromosome segment detected by fine-mapping, as well as perfect association with the phenotype, high sequence conservation of the candidate fragment among horned ruminants and absence of the appropriate conservation in un-horned mammals (Fig. 3C), clearly suggest P202ID as most probably being the causal mutation for polledness in most Bos taurus breeds. The fact that InDel P202ID resides in a region without known function is neither a clear argument against nor in favour of the candidate but is rather a proof of current rudimentary knowledge about gene function or presumed effects of candidate causal variants. The plausible causal mutation must fall within the limits of the dominant mode of inheritance. One possibility is haploinsufficiency which unlike in type 2 scurs [2] should not result in an enhancement of the effect in homozygotes. The next possibility is a gain-of-function mutation which would be characterized by a complete inhibition of the separate ossification centre in the tissues above the periosteum [3]. Such new regulatory elements with abnormal function may fall within the class of non-coding RNA and micro RNA (miRNA) or promoter sequences. However, their de novo prediction from un-annotated sequence of a non-model organism is highly error-prone and was not used for speculation about the mechanisms causing polledness. PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39477