Screening for Causative Mutations of Major Prolificacy Genes in Iranian Fat-Tailed Sheep

Abstract

Background: The presence of different missense mutations in sheep breeds have shown that the bone morphogenetic protein receptor 1B (BMPR1B), bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) genes play a vital role in ovulation rate and prolificacy in ewes. Therefore, the present study aims to investigate BMPR1B, BMP15 and GDF9 gene mutations in prolific ewes of Iranian fat-tailed Lori-Bakhtiari sheep.

Materials and methods: In the present experimental study, genomic DNA was extracted from whole blood of 10 prolific Lori-Bakhtiari ewes with at least two twinning records in the first four parities to identify point mutations of the BMPR1B, BMP15 and GDF9 genes, using DNA sequencing.

Results: The results obtained from DNA sequencing showed a new synonymous mutation (g.66496G>A) in exon 8 of the BMPR1B gene, without any amino acid change. Sequencing of the BMP15 gene revealed a deletion of 3 bp (g.656_658delTTC) in exon 1, leading to an amino acid deletion (p.Leu19del). Four single nucleotide polymorphisms (G1:g.2118G>A, G2:g.3451T>C, G3:g.3457A>G and G4:g.3701G>A), were detected in exons 1 and 2 of the GDF9 gene, two of which caused amino acid substitutions (G1: p.87Arg>His and G4: p.241Glu>Lys). These amino acid alterations are proposed to have a benign impact on structure and function of the GDF9 polypeptide sequence.

Conclusion: Three major prolificacy genes (BMPR1B, BMP15 and GDF9) were polymorphic in Lori-Bakhtiari sheep, although none of the major causative mutation was detected in this sheep type. Further studies using high throughput methods such as genome-wide association study (GWAS) and evaluation of other candidate genes are necessary in the future.

Keywords: BMP15; BMPR1B; Fertility; GDF9; RFamide-Related Peptide-3; Rats; Sheep.

Fig.1

Polymerase chain reaction (PCR) products of BMPR1B, BMP15 and GDF9 genes bands. M; DNA molecular weight marker (Orange Ruler 50 bp DNA Ladder, CinnaGen, Iran), 1; 380 bp fragment of BMP15 exon 1, 2; 906 bp fragment of BMP15 exon 2, 3; 462 bp fragment of GDF9 exon 1, 4; 1019 bp fragment of GDF9 exon 2, and 5; 190 bp fragment of BMPR1B exon 8.

Fig.2

Sequencing chromatograms of the detected mutations of major prolificacy genes in Lori-Bakhtiari sheep. A. The identified transition (g.66496G>A) in BMPR1B exon 8, B. The identified 3bp deletion (g.656_658delTTC) in BMP15 gene exon 1, C. The identified polymorphism in exon 1 of the GDF9 gene (G1:g.2118G>A), and D. The identified polymorphisms in exon 2 of the GDF9 gene (G2:g.3451T>C, G3:g.3457A>G and G4: g.3701G>A). Positions of the mutations are based on the full sequences of BMPR1B, BMP15 and GDF9 genes (Gene IDs; 443454, 100141303 and 100217402, respectively).

Fig.3

Prediction of the amino acid substitutions impact (G1: P.87Arg>His and G4: P.241Glu>Lys) on structure and function of the GDF9 codified polypeptide.