information for a formal test of allelic heterogeneity of the POLLED phenotype. Not only economical reasons but also increasing serious animal welfare issues urge for a solution in the production of hornless cattle other than by dehorning. Even though the positive effects of genetic dehorning predominate, possible negative aspects should not be dismissed. In spite of a strong selection and serious attempts to improve breeding value of the polled sires these still lag behind in performance. For example, the best progeny tested polled (Pp) Fleckvieh bull currently ranks at position 1,724 of selection index, more than 1.3 standard deviations worse than the top 100 bulls. We find a similar situation in the Holstein breed. The inferior dairy breeding values of polled bulls might be caused simply by long-standing neglect of polled animals within sophisticated breeding programs or by multiple pleiotropic effects. It is known that some domestication traits result from mutations that cause large phenotypic effects but include deleterious pleiotropic effects [25]. Short-termed agricultural interests can obviously overcome the negative pleiotropic effects of some large phenotypic effects like in myostatin mutations in cattle [26]. The recently mapped type 2 scurs syndrome locus with its underlying most probably causal mutation in the TWIST1 gene [2] represents another breeder- selected trait that is negatively correlated with fitness due to embryonic lethality for the homozygous state. To avoid possible negative long-term impacts, possible deleterious pleiotropic effects should be closely investigated before the massive amplification of the POLLED gene in large cattle populations. In conclusion, we describe the mapping as well as the perfect association of one InDel and one short haplotype causing polledness in cattle and suggest the conservation score as prioritising criterion of the most probable causal mutations. The distribution of the two putative POLLED alleles across the European continent supports the hypothesis of two independent mutations. Traits with allelic heterogeneity are no exception but tedious to decipher. We presented research strategies which could be more widely applicable for deciphering the molecular mechanisms of phenotypes with allelic heterogeneity. Materials and Methods Ethics Statement Collection of blood samples was conducted exclusively by local veterinarians during regular health inspection and quality control of breeding records on the farms, so that randomness of the sample was assured and no ethical approval was required for this study. The regular health inspection includes annual IBR diagnostics (BGBl. I Nr. 74 S. 3520 ff) and ruminant metabolisms control at Lehr- und Versuchsgut, Oberschleißheim, Germany (Dec. 55.2-1- 54-2531.3-80-10). The regular quality control of breeding records includes paternity testing organised by the respective breeding associations. Paternity testing involves blood, semen and hair root samples. Blood sampling by veterinarians with state examination avoids unnecessary pain, suffering and damage and is in accordance with the German Animal Welfare Act. Semen samples were collected by approved commercial artificial insemination stations as part of their regular breeding and reproduction measures in cattle industry. Hair roots samples were collected by breeders themselves (hair plucking from pinna or tail-tassel) as part of regular sampling for parentage control. As acknowledged below and specified in Table S2 we used these regularly sampled blood, semen and hair roots samples for DNA preparation necessary for this study. Allelic Heterogeneity of Bovine Polledness Animals All animals sampled and genotyped in this study are presented in Table S1 according to their breed affiliations and purpose in the experimental design. These 1,675 animals originate from 31 European cattle breeds belonging to the Bos taurus subspecies. The DNA samples of all 162 cases, 162 controls and 89 carriers were genotyped genome-wide with the Illumina BovineSNP50 Bead- Chip [14]. All 162 cases, 89 carriers and 1,262 semi random animals were genotyped for the revealed candidate mutations. Phenotypes and Declaration of the Underlying POLLED Genotype Polledness or complete absence of horns is a visible phenotype that can be identified at relatively young animals (four to six months). Because the growth of scurs occurs later in life than horns, phenotyping of some scurred animals will not be possible until nine to eighteen months of age [12]. Polled animals, especially breeding bulls and important dams, are declared as polled (name suffix P) in their pedigree certificate and/or other records. One P designates polled animals with the PP or Pp genotype at underlying POLLED locus. Breeding animals with 12 to 15 (depending on breeding organisation) consecutively polled offspring originating from horned mates are declared as homozy- gous polled and get a name suffix PP. One horned offspring with confirmed paternity is sufficient to declare a polled animal as Pp. Homozygosity Mapping DNA samples of 162 case and 162 controls were genotyped with the Illumina BovineSNP50 BeadChip [14]. Marker order was based on release UMD3.1 of the Bos taurus genome (http://www. cbcb.umd.edu/research/bos_taurus_assembly.shtml). The SNP haplotypes were inferred and missing genotypes imputed using hidden Markov models (software package BEAGLE [27]). Three cohorts, namely trios (two parents, one offspring), pairs (one parent, one offspring) and unrelated animals were formed, including those animals that turned out not to be relevant for this study (2,721 animals in total). Genome-wide homozygosity mapping in polled animals and controls was performed using the ASSHOM procedure [13]. Case and control genotypes served as input data after completion of haplotype-inference and imputation by the BEAGLE package. To determine the statistical significance of each summary score, we permutated (50,000 permutations) the complete markers along the chromosomes [10] and estimated the summary score as harmonic mean across all cases. Thus, the corresponding P values were corrected for multiple testing and accounted for the level of inbreeding within the cases [13]. The statistical significance based on 50,000 permutations is comparable to the number of markers used. Genotyping of the Candidate Causal Mutations In general, 30–60 ng of genomic DNA were used for genotyping of the candidate mutations. InDel variants P202ID and P5ID were PCR amplified (94uC 30 sec, 58uC 60 sec, 72uC 60 sec for 31 or 35 cycles, respectively) using primer binding sites flanking the InDel events (59-TCAAGAAGGCGGCACTATCT- 39 and 59-TGATAAACTGACCCTCTGCCTATA-39 for P202ID and 59-FAM-CCTTGTCACGTTAGATGTATGTCC-39 and 59-TCAATCTCTAATAAGGAACAGAAGAAA-39 for P5ID). PCR products were size-separated and visualized by 2% ethidium-bromide stained agarose gel electrophoresis (P202ID) or analysed on an ABI PrismH 3130XL DNA sequencer (P5ID). Genotyping of the P80kbID was performed by use of two primers flanking the variable site P1909396D2 (59-GAAGTCGGTGGTCT- PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39477