A 17 bp deletion in the Bone Morphogenetic Protein 15 (BMP15) gene is associated to increased prolificacy in the Rasa Aragonesa sheep breed

Abstract

Different mutations in the Bone Morphogenetic Protein 15 (BMP15) and the Growth Differentiation Factor 9 (GDF9) genes cause increased ovulation rate and infertility in a dosage-sensitive manner in sheep. They cause increased ovulation rate and twin and triplet births in heterozygotes, and complete primary ovarian failure in homozygotes resulting in total infertility.

We are here presenting a novel mutation in the second exon of the ovine BMP15 gene, found in the Spanish breed Rasa Aragonesa. It consists of a 17 bp deletion resulting in displacement of the open reading frame and premature stop codons. As a consequence, nearly 85% of the sequence of the wild type aminoacidic chain in the second exon of the BMP15 pro-protein is modified or suppressed as only the first 45 amino acids are conserved of the 245 original. The mature peptide is lost. The ewes heterozygous for this deletion present very high prolificacy (2.66 lambs/birth) when compared to a mean flock prolificacy of 1.36 lambs. The deletion causes a complete lack of functionality of the second exon of BMP15, comparable to the effect of premature stop codons in other mutations. Therefore, homozygous females for the deletion are expected to present primary ovarian failure.

DNA sequence analysis of the GDF9 coding regions detected only a synonymous Single Nucleotide Polymorphism (SNP), apparently not linked to changes in prolificacy.

Introduction

The biological roles of Bone Morphogenetic Protein 15 (BMP15) and Growth Differentiation Factor 9 (GDF9) are not completely understood even if both genes co-operate to regulate granulosa cells function (McNatty et al., 2005).

BMP15 is located in the X chromosome. Prior to the present report, five mutations in this gene affecting prolificacy have been described. They originate either nonconservative aminoacidic substitutions (FecX^I, FecX^B and FecX^L) or premature stop codons (FecX^G and FecX^H) having a dosage-dependent effect: While ovulation rates are highly increased in the heterozygotes, the homozygotes show a primary ovarian failure resulting in complete sterility (Galloway et al., 2000, Montgomery et al., 2001, Hanrahan et al., 2004, Davis, 2005, Bodin et al., 2007).

The GDF9 gene maps to sheep chromosome 5, and a mutation in its sequence (FecG^H) causing an aminoacidic substitution and having a similar dosage-dependent effect has been described (Hanrahan et al., 2004).

The Rasa Aragonesa is the dominant breed found in the basin of the river Ebro in Spain (2.5 million adult individuals) and its mean prolificacy is around 1.2–1.5 lambs/birth (Sierra, 1992). The National Association of Rasa Aragonesa Breeders (ANGRA) is currently developing a genetic improvement programme, with increased prolificacy as one of the important targets for improvement.

An initial aspect of this objective has been to monitor reproductive performance in different flocks throughout the Aragón region since 1990. A resulting database of more than 300,000 births has been obtained to clearly characterise the breed.

The goal of the present work was to search for possible mutations in GDF9 and BMP15 associated with increased prolificacy in Rasa Aragonesa. The experience in the French Lacaune breed was a clear inspiration for this research, as after several decades of genetic improvement, a high proportion of the most prolific Lacaune ewes were found to carry the FecX^L mutation in BMP15 (Bodin et al., 2007).

For this search of mutations, 12 highly prolific ewes and 5 rams were originally selected. This group included the female showing the maximum prolificacy in the history of the ANGRA breeding programme (a mean of 3.67 lambs/birth over three parturitions), and four of her daughters.

A deletion in the second exon of the BMP15 precursor having dramatic effects on translation was detected in the hyperprolific ewe, three of their daughters and one of her grandsons. The screening was then extended to the rest of the flock. As a result, three unrelated ewes not previously included in the official control programme were also found to bear the deletion in heterozygous condition. A male lamb born from one these two sheep was also found to carry the deletion.

We are here describing this new BMP15 variant. Following the previous nomenclature for BMP15 mutations, we have named it FecX^R as it is described in the Rasa Aragonesa breed.