A 1.8-kb insertion in the 3'-UTR of RXFP2 is associated with polledness in sheep

Abstract

Sheep breeds show a broad spectrum of different horn phenotypes. In most modern production breeds, sheep are polled (absence of horns), whereas horns occur mainly in indigenous breeds. Previous studies mapped the responsible locus to the region of the RXFP2 gene on ovine chromosome 10. A 4-kb region of the 3'-end of RXFP2 was amplified in horned and polled animals from seven Swiss sheep breeds. Sequence analysis identified a 1833-bp genomic insertion located in the 3'-UTR region of RXFP2 present in polled animals only. An efficient PCR-based genotyping method to determine the polled genotype of individual sheep is presented. Comparative sequence analyses revealed evidence that the polled-associated insertion adds a potential antisense RNA sequence of EEF1A1 to the 3'-end of RXFP2 transcripts.

Keywords: antisense RNA; horn growth; polledness; relaxin/insulin-like family peptide receptor 2; sheep.

Figure 1

A 1.8‐kb insertion in the 3′‐UTR of ovine RXFP2. (a) The gray bar represents the ovine reference sequence of the 3′‐flanking region of RXFP2. The annotated genes are displayed on top of the bar. Note the gap in the reference sequence marked with the red ‘N’. The second bar represents the respective bovine regions obtained by blasting the sheep sequence with the bovine genome. The matches to bovine chromosome 12 are displayed in green and the matches to chromosome 18 in yellow. (b) The 3′‐flanking region of RXFP2 in polled and horned sheep. The 1833‐bp insertion is displayed in blue and marked with an arrow. It is present in polled and absent in horned sheep. (c) PCR products used for screening the region. Product numbers 2, 3 and 5 (marked with red dashed lines) do not amplify DNA of the horned sheep as they span the breakpoints of the insertion. (d) PCR products used for genotyping the insertion. One forward primer (fwd) is combined with two reverse primers (rev). The first reverse primer binds within the insertion and results in amplification of a PCR product of 428 bp. The other reverse primer binds after the insertion and the respective PCR product of 466 bp is amplified in the absence of the insertion. (e) Image of gel electrophoresis of the described PCR products used for genotyping of the insertion. The first two PCR products were obtained from two horned animals, which do not carry the insertion. The two following PCR products are from animals carrying the insertion in heterozygous state; these animals show a polled phenotype. The PCR products to the right were obtained from polled animals carrying the insertion in homozygous state. (f) Exemplary phenotypes matching with the gel image: A horned Bündner Oberländer sheep is shown on the left. A polled Bündner Oberländer sheep, which is heterozygous for the insertion, is shown in the center, and a polled Swiss Mirror sheep, which is homozygous for the insertion, is shown on the right.