Screening for Causative Mutations of Major Prolificacy
Genes in Iranian Fat-Tailed Sheep
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 BM- PR1B, 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.
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.
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.
Reproductive traits such as ovulation rate and litter size
are genetically influenced by several minor genes as well
as some major genes, called fecundity (Fec) genes (1).
Three major genes including bone morphogenetic protein
receptor 1B (
Different causative mutations in the exon 8 of
Lori-Bakhtiari sheep is an important heavyweight indigenous breeds, mainly raised in a wide range of Zagrous Mountains in southwestern part of Iran, with a current census over 1.7 million heads. This breed has the largest fat-tail size among all Iranian sheep breeds. Lori-Bakhtiari sheep is well known for providing a major source of meat with an average litter size of 1.17 ± 0.38 at birth and a conception rate of more than 90 percent (6). Considering that increasing the reproductive ability of the Lori-Bakhtiari sheep has always been an important breeding goal, genetic strategies have currently focused on reproduction traits to improve the profitability of sheep operations.
The aim of this study was to identify the possible presence of known main mutations in
Materials and Methods
Experimental animals and DNA isolation
In the present experimental study, a total of 10 prolific Lori-Bakhtiari ewes with at least two twinning records in the first four parities were selected from different half-sib families at Sholi Sheep Breeding Station, Charmahal and Bakhtiari province, Iran. From the prolificacy point of view, the average of twinning rate in the studied population was about 18%. Blood samples were collected from jugular vein (5 ml per ewe) by venoject tubes contained ethylene diamine tetra acetic acid (EDTA) and immediately transported to the laboratory with ice before DNA isolation. Genomic DNA was extracted from whole blood by the CinnaGen DNP kit (CinnaGen Co, Iran).
All protocols were adhered in accordance with the ethical standards of the National Research Council’s 2011 guideline for the care and use of animals, approved by the research Ethics Committees of University of Guilan (Guilan, Iran) and Bu-Ali Sina University (Hamedan, Iran).
Polymerase chain reaction amplification and sequence analysis
Five pairs of primer (Table 1,) were designed to amplify exon 8 of
|Gene||Region||Primer sequence (5ʹ-3ʹ)||Product size (bp)|
|Exon 8||F: CCAGAGGACGATAGCAAAGCAA||190|
|Exon 1||F: AAGCGTTATCCTTTGGGCTT||380|
|Exon 2||F: CGCTTTGCTCTTGTTCCCTC||906|
|Exon 1||F: GAAGACTGGTATGGGGAAATG||462|
|Exon 2||F: TGGCATTACTGTTGGATTGTTT||1019|
Polymerase chain reaction (PCR) amplification protocols were similar in designated regions and they were carried out in a 50 μl volume, consisting of 25 μl Taq DNA Polymerase Master Mix 2X (CinnaGen, Iran), 10 pM of each primer (OD:2), 50-100 ng of DNA template and distilled water. PCR reactions were run in Applied Biosystems thermal cycler (Life technologies, USA) under the following thermal condition: Initial denaturation at 95°C for 5 minutes, followed by 35 cycles consisting of denaturation at 95°C for 1 minute, annealing at 60°C for 45 seconds, extension at 72°C for 1 minute and a final extension at 72°C for 5 minutes.
Individual fragments were distinguished by electrophoresis of PCR products in 2% agarose gel (CinnaGen, Iran). The gels were stained with ethidium bromide and photographed under UV light (BTS-20.M model, UVItec Ltd, UK) (Fig .1,).
All 50 samples (five fragments from each animal) were submitted to DNA sequencing. The purified PCR products were sequenced on both strands by Bioneer Co., Korea. The identified single nucleotide polymorphisms (SNPs) were compared to the referring sequences at NCBI database using BLAST (7). Ultimately, any potential effect of the identified mutations, in terms of the structure and function of codified polypeptides, was predicted using PolyPhen-2 online software tool (8).
Sequence analysis of the
BMPR1B gene exon 8
The sequence of exon 8 for
|Gene||Variation||Amino acid substitution||Frequency||Type of mutation|
||g.66496G>A||-||2 out of 10||Synonymous|
||g.656_658delTTC||p.Leu19del||1 out of 10||Non-synonymous|
||g.2118G>A||p.87Arg>His||2 out of 10||Non-synonymous|
||g.3451T>C||-||2 out of 10||Synonymous|
|g.3457A>G||-||2 out of 10||Synonymous|
|g.3701G>A||p.241Glu>Lys||2 out of 10||Non-synonymous|
Sequence analysis of
BMP15 gene exons 1 and 2
The sequence of exon 1 of
Sequence analysis of
GDF9 gene exons 1 and 2
Four single nucleotide polymorphisms (g.2118G>A, g.3451T>C, g.3457A>G and g.3701G>A), respectively known as G1-G4, were detected in the exons 1 and 2 of
Considering the hotspot regions involved in prolificacy of sheep, selection of a limited number of high prolific ewes and sequencing of their
Evidences show that Booroola Merino ewes have high ovulation rate and litter size, due to the effects of a missense mutation (FecB) in the exon 8 of
In the present study, analysis of the sequences for
Eight different causative mutations in ovine
In the present study, sequencing of the exon 1 of
Hanrahan et al. (10) reported eight point mutations (i.e. G1-G8) in the
Regarding the absence of known main causative mutations of
We can state that major prolificacy genes (i.e.
The authors are grateful to Agricultural Jihad Institutes of Charmahal and Bakhtiari Province for their kindly cooperation. The authors also would like to thank to University of Guilan for financial supporting the costs of this study. The authors declare that there is no conflict of interest for this article.
R.A.; Is the first author and participated in study design, data collection and evaluation. S.Z.M.; Is the corresponding author and performed scientific editing and writing. N.G.H.-Z., P.Z.; Co-edited the article. All authors read and approved the final manuscript.