Figure 1.
Homozygosity mapping in case-control design of polled and horned cattle animals.
(a) Genome-wide association mapping of the POLLED mutation to the proximal end of bovine chromosome 1 (BTA1) with 162 affected animals and 162 controls (asshom method, Charlier et al. 2008). Nominal asshom statistic is presented chromosome by chromosome for all 29 bovine autosomes. Evidence for association (y axis) is measured as nominal asshom statistic with genome-wide significance (P = 0.0002, grey line) at 3.315, being determined by 50,000 nominal asshom statistics along chromosomes with randomly permutated markers. (b) Association mapping details for the region from 0 to 4 Mb on BTA1. Nine markers homozygous in all 162 cases covered a region from 1,760,113 to 1,983,902. Two informative SNP markers flanking this core (i.e. variable sites in the case group) are ARS-BFGL-NGS-39992 (1,668,494 bp) and ARS-BFGL-NGS-29653 (2,049,400 bp). (c) Candidate region of 547 kb (1.543–2.090 Mb (UMD3.1 genome build)) chosen for high-throughput sequencing and most likely encompassing the POLLED mutation. This interval is larger than homozygosity bracket detected by multi-breed design and nested five genes (IL10RB, IFNAR2, OLIG1, C1H21orf62 and GCFC1), one pseudo gene (OLIG2-like) and some not further annotated ESTs (e.g. BC122836, EV693397 and DT837875).
Figure 2.
DNA sequence variants in Polled and horned Holstein Friesian.
(a) Positions of DNA sequence variants (DSV) detected in the eight HF animals are presented by bars for SNPs and inverted triangles for InDels, with red symbols for heterozygous and blue symbols for homozygous differences from the reference sequence. Homozygous polled bulls are PP-HF1, PP-HF2 and PP-HF3. Pp-HF4 is declared as heterozygous polled and pp-HF5, pp-HF6, pp-HF7 and pp-HF8 are horned. Annotated genes and ESTs lying in the re-sequenced region are shown in the green shaded region. The five candidate mutations for polledness are superimposed: three SNPs were outlined by red crosses, 5 bp InDel and the duplicated region P80kbID were highlighted by inverted triangle. The red triangle area above P80kbID marks the duplicated sequence. (b) Within-species conservation. The nucleotide diversity in the re-sequenced region is shown by a density plot of bovine variants from dbSNP per kb. (c) Copy number variations. The ratio of mapped sequence reads between polled and horned animals is plotted with blue dots. The red line represents the result of segmentation analysis, showing the average ratio in the determined bins. (d) Across-species conservation. For each candidate mutation the surrounding base conservation for species without horn, bovid species and all aligned species was determined as PhastCons score calculated from multiz alignments. As an example, here we display the base conservation for the candidate mutation PC1768587A. (e) The underlying multi-species alignment for the across-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.
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.
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 (P5IDPG1654405APC1655463TPC1768587AP80kbID) 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.