Allelic Heterogeneity of Bovine Polledness Figure 4. Distribution of POLLED candidate variants in European cattle breeds. The largest proportion (shown in the row marked %) of genotyped animals (78.03%) were homozygous for the wild-type allele at all six candidate variants of the POLLED locus, i.e. they inherited two copies of the RefSeq haplotype prs presented by vertical gray bar. Plausibility analyses suggested all these 1261 (shown in the row marked with No) prs/prs animals as being horned, therefore pp at the POLLED locus. Five candidate mutations detected by sequencing of polled and horned Holstein-Friesian animals form a haplotype block (P5ID-PG1654405A-PC1655463T-PC1768587A-P80kbID) consisting of three SNPs flanked by two InDels and called PF signifying polledness of Friesian origin. The five candidate mutations of Friesian polledness were superimposed on RefSeq background (vertical gray bar): three SNPs and a 5 bp InDel were outlined by red horizontal bars and the duplicated region P80kbID by the red area. The PF block is in perfect association with POLLED genotype and segregate only in cattle breeds originating from north-western coast of continental Europe (HF, RH, JY and WTG). The candidate mutation P202ID is represented by black horizontal bar on RefSeq background. All animals carrying one (86) or two copies (192) of the P202ID are polled (Pp or PP, respectively) and belong to breeds originating from Scandinavia, Great Britain, France and South-Germany, hereafter figuratively called polledness of Celtic origin, PC. Three bulls with PP genotype determined by extensive progeny testing were found to be heterogeneous polled at candidate gene level, i.e. PC/PF. The genotyping of their sampled relatives as well as entire experimental design provide no evidence for recombination within PF haplotype block. The geographic origin of the sampled breeds is outlined by dots on Europe map. Breeds lacking polled samples are represented by gray dots. The breeds with P202ID as only causal variant or variant in perfect association with POLLED locus are marked by black dots. The breeds with PF as predominant variant in perfect association with POLLED locus are marked by red dots. The approximate distribution area of Celtic and Friesian polledness are highlighted as gray and red shading respectively. doi:10.1371/journal.pone.0039477.g004 The biology of horn development has not been studied in detail, partly also due to the obvious lack of model organisms. The development of a new ossification centre should probably engage the well characterized network of bone development with the RUNX2 master transcription factor and its known regulators including twist proteins [20] and several miRNAs [21]. Indeed, mapping of the type 2 scurs locus has identified a TWIST1 mutation as likely cause [2] and expression analysis of skin biopsies of the horn bud area in newborn polled and normal calves has shown evidence of endothelial-mesenchymal transition [22], a process involving bone morphogenetic protein signalling and twist protein activity. Genetic polledness is also known in goat and sheep, but the underlying mutations are mapped to chromosomes without synteny to the bovine polled locus investigated, here. Moreover, the polled/horned phenotype shows sexual dimor- phism in sheep [23] and in goat is even linked with impaired sexual development [24], so that it is assumed that polledness in these species is caused by different mechanisms. Including the cattle breeds originating from the North See area –HF, JY and WTG– led (Fig. 4) to the detection of a single haplotype block composed of three SNPs flanked by two InDels, all possible candidate causal variants for an independent mutation causing polledness. This Friesian haplotype, PF, segregates independently from the previously mentioned Celtic mutation, PC. Both are complementary and there is no evidence for any interference or recombination. Two Friesian and one Jersey bull were heterogenic at the POLLED locus (PF/PC) and declared as homozygous polled by progeny testing, i.e. all progeny from mating with horned dams are polled. All three bulls were intensively used for artificial insemination without any particular features and bred hundreds of exclusively polled offspring. Recombinant offspring of these bulls can supply important PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39477