Independent polled mutations leading to complex gene expression differences in cattle

Abstract

The molecular regulation of horn growth in ruminants is still poorly understood. To investigate this process, we collected 1019 hornless (polled) animals from different cattle breeds. High-density SNP genotyping confirmed the presence of two different polled associated haplotypes in Simmental and Holstein cattle co-localized on BTA 1. We refined the critical region of the Simmental polled mutation to 212 kb and identified an overlapping region of 932 kb containing the Holstein polled mutation. Subsequently, whole genome sequencing of polled Simmental and Holstein cows was used to determine polled associated genomic variants. By genotyping larger cohorts of animals with known horn status we found a single perfectly associated insertion/deletion variant in Simmental and other beef cattle confirming the recently published possible Celtic polled mutation. We identified a total of 182 sequence variants as candidate mutations for polledness in Holstein cattle, including an 80 kb genomic duplication and three SNPs reported before. For the first time we showed that hornless cattle with scurs are obligate heterozygous for one of the polled mutations. This is in contrast to published complex inheritance models for the bovine scurs phenotype. Studying differential expression of the annotated genes and loci within the mapped region on BTA 1 revealed a locus (LOC100848215), known in cow and buffalo only, which is higher expressed in fetal tissue of wildtype horn buds compared to tissue of polled fetuses. This implicates that the presence of this long noncoding RNA is a prerequisite for horn bud formation. In addition, both transcripts associated with polledness in goat and sheep (FOXL2 and RXFP2), show an overexpression in horn buds confirming their importance during horn development in cattle.

Figure 1. Horn phenotypes in Simmental cattle.

Normally horned cow (A), cow with loosely attached small horns termed scurs (B), smooth polled cow showing the typical peaked shape of the proximal frontal bone (C).

Figure 2. Homozygosity mapping on BTA 1.

SNP genotypes of BTA 1 markers are presented as vertical bars. The dark grey segments represent homozygous blocks with shared alleles. A total of 28 progeny tested homozygous polled bulls belonging to beef and dual-purpose breeds of Celtic origin are shown on the left (SI: Simmental, LI: Limousin, CH: Charolais; HF: Hereford, PG: Pinzgauer; BA: Blonde d'Aquitaine, BV: Braunvieh). Some recombinations were observed in more than one animal, the number of animals is displayed above the chromosome bar. The haplotype analysis suggests the position of the Celtic polled mutation within a 212 kb interval shown in red. The annotated genes and loci on the BTA 1 segment (UMD3.1 assembly) are shown in the center. Three progeny tested homozygous polled bulls and a single heterozygous polled bull belonging to the Holstein (HO) breed of Friesian origin are shown on the right. The 932 kb critical region of the Friesian polled mutation is indicated in blue.

Figure 3. Gene expression study based on RT-PCR.

Relative expression of FOXL2 (A), RXFP2 (B), and LOC100848215 (C) transcripts in fetal skin and horn bud biopsies of different developmental stages and different polled genotypes. Different fetal stages are divides in eight groups of estimated age (d). Wildtype fetuses are marked with the shape of a horned cow head, fetuses carrying the polled mutation are marked with the shape of a polled cow head, whereas each icon designates one fetus. For each individual a biopsy of the horn bud area (H) and a biopsy of the frontal skin (S) were studied. Expression levels in wildtype fetuses are shown in blue, those of heterozygous Pp polled fetuses in orange and those of homozygous PP polled fetuses in dark red. Expression levels are normalized to the average of three control genes and shown as relative expression in relation to the frontal skin sample of the youngest horned fetus.