Markers significantly associated with prolificacy.

a<p>: Location of markers are based on the OARv2.0 assembly available on <a href="http://www.livestockgenomics.csiro.au/sheep/" target="_blank">http://www.livestockgenomics.csiro.au/sheep/</a> website.</p>b<p>: MAF for Minor Allele Frequency.</p>c<p>: p _Unadjusted corresponds to exact p for the Fisher's test.</p>d<p>: p _{Genome-wide corrected} corresponds to p after genome-wide Bonferroni correction.</p>e<p>: p _{Chromosome-wide corrected} corresponds to p after chromosome-wide Bonferroni correction.</p

Functional effects of <b><i>FecX^Gr</i></b><b> and </b><b><i>FecX^O</i></b><b> mutations on the BMP15 activity.</b>

<p><i>In vitro</i> reporter luciferase assay from COV434 granulosa cells transiently transfected with empty vector +/− 100 ng of recombinant human BMP15 (Control +/− rhBMP15) or wild-type human BMP15 expressing vector (WT) or the 2 different BMP15 variant vectors (<i>BMP15^T317I (FecX^Gr)</i>; <i>BMP15^N337H (FecX^O)</i>) obtained by directed-mutagenesis. Results are expressed as Means±SD of the relative light unit (RLU) from 3 independent experiments in triplicate for each condition. Pairwise statistical comparisons using a one-way ANOVA test between means were performed and results of statistic test are symbolized by stars: * = p<5E⁻⁰²; ** = p<1E⁻⁰² and *** = p<1E⁻⁰³.</p

Genotypic distributions of <b><i>FecX^Gr</i></b><b> and </b><b><i>FecX^O</i></b><b> mutations.</b>

<p>(A) Genotypic distribution of the <i>BMP15^T317I (FecX^Gr)</i> in the French Grivette sheep population for the litter size phenotype. (B) Genotypic distribution of the <i>BMP15^N337H (FecX^O)</i> in the Polish Olkuska sheep population for the ovulation rate phenotype. (C) Genotypic distribution of the <i>BMP15^T317I (FecX^Gr)</i> in the French Grivette sheep population for the ovulation rate phenotype. (D) Genotypic distribution of the <i>BMP15^N337H (FecX^O)</i> in the Polish Olkuska sheep population for the litter size phenotype. The means LS or OR in breeds are firstly presented then ewes were ordered according to their genotype at the mutation of interest. Means±SD for prolificacy were calculated for the 3 groups of genotype and are noted into each histogram bar. Number of ewes counted per group of genotype is mentioned (n). Pairwise statistical comparisons using a one-way ANOVA test between means of genotype's clades were performed and results of statistic test are symbolized by stars. p: * = p<5E⁻⁰²; ** = p<1E⁻⁰² and *** = p<1E⁻⁰³.</p

Clusterization of haplotypes reconstructed at the OARX locus.

<p>(A) Haplotypes determined in the French Grivette sheep population. (B) Haplotypes determined in the Polish Olkuska sheep population. 87 markers located in the interest OARX region (45 Mb–55 Mb) were selected to reconstruct haplotypes. Each column represents one SNP and each line represents one haplotype. For one marker (<i>i</i>) allele 1 is in red (Grivette) or green (Olkuska) in controls, respectively or black in cases, (<i>ii</i>) allele 2 is in white when the phase was unambiguous and (<i>iii</i>) dark grey colour represents unphased SNP. Haplotypes were ordered to distinguish controls versus cases and clusterized to classify similar clades of haplotypes. Markers with evidence of association at significance levels are marked with a star or a hash in French Grivette and Polish Olkuska sheep populations, respectively. In both breeds, the specific haplotype preferentially selected in highly prolific ewes (cases) is symbolized by red (Grivette) and green (Olkuska) boxes. The <i>BMP15</i> gene (48140251 bp–48146740 bp) is located between markers named OARX_6082722 and OARX_56342973.</p

Hypothesized overview of ovulation quota control by the various Fecundity mutations.

<p>(A) The mono ovulation quota is tightly controlled by the integrative action of <i>i)</i> BMP15 homodimer through BMPR1B, BMPR2 and SMAD1/5/8, <i>ii)</i> BMP15/GDF9 heterodimer through SMAD2/3 and <i>iii)</i> GDF9 homodimer through TBR1, BMPR2 and SMAD2/3. (B) Increased of ovulation rate due to <i>FecX</i> mutations. This drawing represents an overview of hyperprolificacy dependent of BMP15 variants. Heterozygous or homozygous <i>FecX</i> mutations leading to an increased OR affect either the signaling pathways of BMP15 homodimer, BMP15/GDF9 heterodimer or both whereas the GDF9 homodimer signaling pathway remains stable. (C) Increased of ovulation rate due to <i>FecG</i> mutations. This drawing represents an overview of hyperprolificacy dependent of GDF9 variants. Heterozygous or homozygous <i>FecG</i> mutations leading to an increased OR impair either the signaling pathways of GDF9 homodimer, BMP15/GDF9 heterodimer or both whereas the BMP15 homodimer signaling pathway remains stable. (D) Dose sensitive effect of each <i>FecX</i> and <i>FecG</i> mutations on the BMP15, GDF9 homodimers and BMP15/GDF9 heterodimer signaling pathways. Based on the various <i>in vitro</i> tests performed, we assigned a dose (n = 0, 1 or 2) to each signaling pathway in an attempt to explain the prolificacy phenotype observed for all the <i>FecX</i> and <i>FecG</i> mutations. When the ovulation rate is normal ( = 1), i.e. the WT situation, 2 doses of BMP15 homodimer (blue), GDF9 homodimer (purple) and BMP15/GDF9 heterodimer (pink) were considered. As example, in the case of the <i>FecX^Gr</i> mutation where the BMP15 homodimer signaling pathway seemed clearly affected at homozygous and heterozygous status (as shown on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003482#pgen-1003482-g005" target="_blank">Figure 5</a>), we assumed that the BMP15/GDF9 heterodimer signaling pathway remained totally active.</p

Genetics variants causing prolificacy phenotypes in sheep.

<p>Genetics variants causing prolificacy phenotypes in sheep.</p

Polymorphisms within the minimal <i>FecL</i> locus and allele sharing.

<p>Polymorphism positions are given relative to OAR11 v3.1 and <i>FecL</i> locus (Genbank:KC352617). Polymorphism type, single nucleotide polymorphism (SNP), microsatellite (Ms) or insertion/deletion (ins/del) referred to <i>+/+</i> vs. <i>L/L</i> sequence. Allele sharing indicated number of (<i>+</i>) chromosomes carrying the (<i>L</i>) allele relative to the number of (<i>+</i>) chromosomes tested.</p>a<p>SNP corresponding to the interval borders and therefore excluded from the minimal locus.</p>b<p>Allele segregating as the <i>FecL^L</i> mutation.</p>c<p>Ms marker genotyped by fluorescent SSCP, no information on the dinucleotide repeat variation.</p><p>NA, non-available coordinate on OARv3.1.</p

B4GALNT2 transferase activity revealed by DBA lectin after in vitro overexpression of <i>B4GALNT2</i> in ovine granulosa cells.

<p>Primary ovine granulosa cells from <i>+/+</i> small antral follicles were transiently transfected with either the pCDNA-hB4GALNT2 expressing the human form of B4GALNT2 or the empty pCDNA3.1 vector. Twenty-four hours after transfection, cells were stained with biotinylated-DBA lectin (500 ng/ml). Arrows indicated DBA positive staining only in B4GALNT2 transfected cells. Cells were counterstained with hematoxylin. A black bar indicates the microscopy magnification scale.</p

Western immunoblotting analysis of B4GALNT2 transferase activity in Lacaune sheep granulosa cells and follicular fluids.

<p>Granulosa cell protein extracts (50 µg) and follicular fluids (200 µg) from <i>+/+</i> and <i>L/L</i> large antral follicles were precipitated (P) by agarose-DBA lectin or sepharose-protein A-KM694 monoclonal antibody. The resulting purified glycoproteins were separated on SDS-PAGE, transferred on nitrocellulose membrane and revealed after blotting (B) using biotinylated-DBA lectin or KM694 monoclonal antibody.</p

DBA-purified proteins present in L/L follicular fluid identified by mass spectrometry.

<p>DBA-purified proteins present in L/L follicular fluid identified by mass spectrometry.</p