AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus PublicationsGöttingen, Germany10.5194/aab-60-119-2017Variant GDF9 mRNA is likely not the main cause of larger litter size in
Iranian Lori-Bakhtyari, Shal, Ghezel, and Afshari sheep breedsEghbalsaiedShahinshahin.eghbal@khuisf.ac.irhttps://orcid.org/0000-0002-9030-8679KhorasganiFarzad RashidiAminiHamid-RezaFarahiMajidDavariMaryamPiraliAhmadPouraliSheilaVatankhahMahmoodRostamiMahmudAtashiHadiTransgenesis Center of Excellence, Isfahan (Khorasgan) branch, Islamic
Azad University, Isfahan, IranDepartment of Animal Science, Faculty of Agriculture, Isfahan
(Khorasgan) branch, Islamic Azad University, Isfahan, IranDepartment of Animal Science, Chaharmahal va Bakhtiary Agricultural
and Natural Resources Research and Education Center, AREEO, Shahrekord, IranDepartment of Animal Science, Shiraz University, Shiraz, IranShahin Eghbalsaied (shahin.eghbal@khuisf.ac.ir)17May201760211912921January20176April201715April2017This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/This article is available from https://aab.copernicus.org/articles/60/119/2017/aab-60-119-2017.htmlThe full text article is available as a PDF file from https://aab.copernicus.org/articles/60/119/2017/aab-60-119-2017.pdf
This study was carried out to screen the GDF9 gene and evaluate the
polymorphism effect on litter size of four Iranian sheep breeds using
the PCR-RFLP and PCR-SSCP methods. First, sequencing of the GDF9 gene in
16
twin-birth, 4 triplet-birth, and 2 infertile ewes showed that, in addition to G2,
G3, G4, G5, and G6 mutations that have been previously reported in other breeds,
a new G0 mutation, called C25T, exists in the GDF9 sequence of 1 out of 22 ewes and causes L9F substitution in the signal peptide region. None of
the triplet-birth or infertile ewes carried G1, G4, G7, FecGE, G8,
or FecGT mutations. In the second experiment, a large dataset was used:
605 individuals including 496 ewes (145 Afshari, 54 Shal, 126 Ghezel, and 171 Lori-Bakhtyari sheep), and 109 rams (26 Afshari,
23 Shal, 10 Ghezel, and 50 Lori-Bakhtyari sheep. There were no
sheep carrying the G7, G8, or Thoka mutations. Among all 109 rams that were
used in this study, none of them were homozygous for the G1 mutation.
Moreover, abundance of heterozygote rams (G1/G+) varied from 0.0 (Afshari)
to 28.6 % (Lori-Bakhtyari and Ghezel). The highest and the lowest
frequencies of the G4 mutation were 30.6 and 3.0 % in Shal and Afshari breeds,
respectively. Moreover, G4 abundance varied from 0.0 to 42.3 %, from 3.0
to 26.9, and from 3.0 to 30.6 % in rams, ewes, and overall, respectively.
There was a significant difference in the abundance of G1 and G4 mutations
between breeds. However, neither the G1 nor the G4 mutation was associated with litter
size in Afshari, Ghezel, Lori-Bakhtyari, or Shal sheep breeds. In conclusion, the
results of this study showed that GDF9 G1 and G4 mutations are not the reason for
higher litter size in Iranian sheep. Moreover, the GDF9 G0 and G6
mutations do not cause triplet births or infertility in Iranian ewes.
Therefore, it is unlikely that variant GDF9 mRNA induces larger litter size or
infertility in Iranian ewes.
Introduction
In mammals, ovulation rate and fetus survival are decisive managerial
attributes. It has been well-documented that single-nucleotide polymorphism
(SNP) in a narrow assembly of genes, including growth differentiation factor
9 (GDF9), bone morphogenetic protein 15 (BMP15), bone morphogenetic protein
receptor 1 (BMPR1B), and leptin, can increase ovulation rate, multiple-lamb
births, and fecundity in sheep (Souza et al., 2001; Wilson
et al., 2001; Hanrahan et al., 2004; Juengel et al., 2004, 2015). These genes belong to the transforming growth
factor β (TGFβ) superfamily, which consists of 50 physiologically important macromolecules that regulate fertility and growth
attributes, as well as cellular differentiation processes (Dong et al.,
1996; Yan et al., 2001).
A summary of known mutations that have been detected in the GDF9 gene
of sheep species around the world.
ReferenceProtein segmentAmino acid changeDNA base changeSNPThis paperSignal peptideL9FC25TG0Hanrahan et al. (2004)PropeptideR87HG260AG1Hanrahan et al. (2004)PropeptideUnchanged VC471TG2Hanrahan et al. (2004)PropeptideUnchanged LG477AG3Hanrahan et al. (2004)PropeptideK241EA721GG4Chu et al. (2011)PropeptideQ243HG729TFecG-HanNicol et al. (2009)PropeptideUnchanged RG750AG-CJuengel et al. (2011)Mature peptideR286SG858TG-DHanrahan et al. (2004)Mature peptideUnchanged EA978GG5Hanrahan et al. (2004)Mature peptideV332IG994AG6Silva et al. (2011)Mature peptideF345CT1034GFecGEHanrahan et al. (2004)Mature peptideV371MG1111AG7Hanrahan et al. (2004)Mature peptideS395FC1184TG8 (FecGH)Nicol et al. (2009)Mature peptideS427RA1279CFecGT
The importance of GDF9 protein on oocyte and follicular growth and function
was defined by Dong et al. (1996) and McGrath et al. (1995), and
afterward the ovine GDF9 gene was mapped on chromosome 5 (Sadighi et al.,
2002). Even though the GDF9 gene is expressed in the oocyte of cumulus-oocyte complex
(COC) (McGrath et al., 1995; Laitinen et al., 1998), its
higher expression in the cellular layers around the antral follicles has
recently been identified as key during the follicular phase of ewes
(Foroughinia et al., 2017). As summarized in Table 1, following the
first publication on discovering eight mutations, designated as G1 to G8, in
the GDF9 gene of Belclare and Cambridge breeds (Hanrahan et al., 2004),
other mutations were discovered in Thoka (FecGT) (Nicol et al., 2009),
Han (FecG-Han) (Chu et al., 2011), and various Spanish (FecGE) (Silva et
al., 2011) sheep breeds. Among these mutations, G2, G3, G5 (Chu et al.,
2011), and G-C (Nicol et al., 2009) are ineffective mutations without
amino acid codon alteration. However, G1, G4, G6 (Chu et al., 2011), and
FecGH (López-Ramírez et al., 2014) modify the GDF9 propeptide,
while G7, G8 (Chu et al., 2011), G-D (Juengel et al., 2011), and
FecGT (Chu et al., 2011) mutations cause variation in the mature
peptide. Two of these SNPs, GDF9 G8 and FecGT, show over-dominant
inheritance for ovulation rate and twin birth but an infertility event at
a homozygous mutant state (Hanrahan et al., 2004; Nicol et al., 2009). Moreover, the FecGE (Silva et
al., 2011), G1 (Barzegari et al., 2010; Javanmard et al., 2011), G4 (Eghbalsaied et al., 2012, 2014), and G7
(Våge et al., 2013) mutations were reported as ovulation inducers
without showing sterility. It has been proven that the origin of
FecGH in Belclare and Cambridge sheep is the highly prolific Lleyn breed
(Mullen et al., 2013). However, a large proportion of rams and high-fecundity ewes from the Lleyn breed and other highly prolific ewe breeds
that
had records of triplet births did not carry the known significant mutations
(Mullen et al., 2013). This might indicate that other mutations in
the detected major genes or other genes from the transforming growth family
could affect the ovulation rate in ewes.
Mutation in the GDF9 gene has been detected in Iranian sheep breeds, i.e. Zel
(Ghaderi et al., 2010; Javanmard et al., 2011; Nassiry et al., 2006),
Lori-Bakhtyari, Sangesari (Hafezian, 2011), Moghani (Barzegari et al.,
2010), Ghezel (Akbarpour et al., 2008; Barzegari et al., 2010;
Eghbalsaied et al., 2014), Shal (Ghaffari et al., 2009), Kurdi, Arabi
(Ghaderi et al., 2010), Baluchi (Moradband et al., 2011), Afshari
(Eghbalsaied et al., 2012), Mehraban (Zamani et al., 2015),
and Lori (Zamani et al., 2015). However, all of these SNPs moderately
modify the signal peptide or GDF9 propeptide. Conversely, neither the
major known mutations that can change the mature peptide nor the ewe
sterility that is the main side effect of ewe homozygosity for these major
SNPs have been observed in Iranian ewes (Akbarpour et al., 2008;
Eghbalsaied et al., 2012, 2014; Ghaffari et al., 2009;
Moradband et al., 2011; Nejhad and Ahmadi, 2012). Even though the GDF9 G1 mutation was
not considered as an effective mutation for sheep prolificacy by
Hanrahan et al. (2004), it was suggested as an effective source for
inducing twin birth in Iranian Ghezel and Moghani breeds (Barzegari
et al., 2010; Javanmard et al., 2011). However, further research on
the Mehraban breed (Abdoli et al., 2013) could not prove the significant
effect of the GDF9 G1 SNP on Iranian sheep flocks. Also, the effect of other
mutations, such as GDF9 G4 (Eghbalsaied et al., 2012) and GDF9
G6
(Khodabakhshzadeh et al., 2016), needs to be evaluated in
highly fertile Iranian ewes. Therefor, evaluation of GDF9 mutations in
twin births of Iranian ewes remains to be explored in a large
dataset of Iranian sheep breeds. The aim of this study was to screen and
analyse GDF9 polymorphism effects on twin births of Iranian sheep
breeds, including Lori-Bakhtyari, Shal, Ghezel, and Afshari.
Distribution of collected samples from ewes and rams belonging to
Iranian sheep breeds.
BreedEwe RamLocationTotal numberTriplets Twins Single Infertile Shal33019223Pir Yusefiyan (Bu'in Zahra)77Ghezel–1030–10Miandoab Research Center136–5234––Semmeneh Rud (Boukan)Afshari–7030–6Khatoon Abad (Isfahan)1714520IsfahanLori-Bakhtiyari111060–50Shahrekord, Lordegan, Farsan221Total number42722182109Iran605Material and methodsExperiment 1Screening of the GDF9 gene in a sample of twin-birth ewes
To search for possible mutations that are segregated in ewes with larger
litter size sheep, a random sample of 16 ewes with twin births,
4 ewes with triplet births, and 2 ewes with infertility were
selected from Shal, Ghezel, Afshari, and Lori-Bakhtyari breeds. The blood
was immediately mixed with 50 mM EDTA, transported to the laboratory, and
stored at -20 ∘C for further analysis. Genomic DNA was
extracted from the whole blood using the standard phenol–chloroform method. The
quantity and quality of extracted DNA was measured by spectrophotometer and
agarose gel electrophoresis, respectively. Based on the Ovis aries breed Texel chromosome
5, Oar_v3.1, whole genome shotgun sequence on the National Center for Biotechnology Information (NCBI) website,
three primer pairs were designed by Oligo6 software to cover the 5' UTR and
complete sequence of exon 1, complete sequence of exon 2, a part of exon 2,
and the 3' UTR of the ovine GDF9 gene (Table 2). Polymerase chain reactions (PCR)
were carried out in 25 µL volume, included 1X Buffer, 250 mM dNTP, 5 mM
MgCl2, 5 µM primers, 50–100 ng template DNA, and 1 IU Taq DNA
Polymerase (SinaClon, Iran). Except for the annealing temperature, PCR
conditions were similar for all reactions, with initial denaturation at
94 ∘C for 4 min, 35 cycles of denaturation at 94 ∘C for
30 s, annealing at 55–60 ∘C for 30 s, and extension at
72 ∘C for 30 s, and finished by a final extension at 72 ∘C
for 4 min. The PCR amplicons of the 16 ewes were sent for sequencing
(Bio Basic Inc., Canada) and aligned with the Ovis ariesGDF9 mRNA; sequence ID is gb|AF078545.2|AF078545.
Primer pairs for mutation detection in the GDF9 gene of Iranian sheep
based on NCBI accession reference AF078545.
In this experiment, the detected mutations in Experiment 1 were assessed
using
twin births of four main breeds of Iranian sheep. Five millilitres of a blood
sample was collected from the jugular vein of 605 sheep, including 77, 136,
171, and 221 Shal, Ghezel, Afshari, and Lori-Bakhtyari breeds, respectively (Table 3).
Among these animals, four triplet-birth and two infertile ewes were also
included. Five primer pairs were used for detection of G0, G1, G4, G6, and
G8 mutations in GDF9 gene (Table 2) (Polley et al., 2010). The PCR
conditions were similar to Experiment 1.
Restriction fragment length polymorphism (RFLP)
For detection of G1 and G8 mutations, 1 µg of the PCR product was
digested with HhaI and DdeI restriction enzyme, respectively (Takara Bio Inc., Japan), at
37 ∘C for 1 h. Then the digested solution was loaded on a 1.5 %
agarose gel containing GelRed (Biotium, USA) and screened using gel
documentation (Uvitech, UK) merchandiser.
Single-strand conformation polymorphism (SSCP)
Detection of the G0, G4, G6, and G8 mutations as well as the G0 mutation that was
newly detected in Experiment 1, was carried out using the SSCP procedure
(Orita et al., 1989). In summary, 5 µL of the PCR product was
transferred to an Eppendorf tube and mixed with 7.0 µL of a gel loading
solution that contained 99 % formamide, 0.05 % bromophenol blue,
0.05 % xylene cyanol, and 20 mM EDTA (pH = 8.0). The mixture was
incubated and denatured at 96 ∘C for 10 min, chilled on ice for 5 min, and loaded onto 12 % neutral polyacrylamide gels. Electrophoresis was
performed at 130 volt for 18 h at 10–12 ∘C. Afterward, the single-strand DNA was made visible using silver staining (Bassam et al., 1991).
Statistical analysis
The following generalized linear model (GLM) was used for evaluation of the
detected polymorphisms in ewe twin births.
Twinbirth=mean+breed+G1+G4+E,
where mean, breed, G1, G4, and E were the average of twin births in the
whole population. Breed and genotype of ewes for the G1 and G4 mutations were
fixed effects, and unknown residual effects were random effect.
The statistical analysis was carried out using SAS 9.2 software. Mean of
litter size was compared between genotype categories using the least significant difference test
(p value < 0.05). Genetic analysis was carried out using the Popgene
1.32 software (Francis and Yang, 2000) and genotypic and allelic
frequencies were compared using the Chi-square test (p value < 0.05).
Graphical representation of detected mutations in GDF9 DNA of Iranian
twin-birth ewes compared to sequence ID gb|AF078545.2|
AF078545. G0 (C25T) is the newly detected mutation; G2–G6 were previously
defined by Hanrahan et al. (2004) .
ResultsDetection of mutations converting L9F (G0) and V332I (G6)
In the first part of this study, the pair-wise alignment results of the
sequenced fragments and the Ovis ariesGDF9 mRNA were used to determine possible
mutations in the surveyed samples from twin-birth, triplet-birth, and
infertile ewes. As depicted in Fig. 1, results showed that along with
the G1 mutation, five previously reported mutations, namely the GDF9 G2, G3, G4, G5,
and G6 (Hanrahan et al., 2004), are present in highly
prolific Iranian ewes with 3.1 (1 out of 16), 3.1 (1 out of 16), 7.1 (3 out of 21),
30.0 (6 out of 20), and 30.0 % (6 out of 20) frequency, respectively.
Furthermore, we detected a new mutation, designated as G0, in one out of the
22 sequenced samples. This mutation exists in exon 1 of the GDF9 gene, causing
C25T to shift and subsequently L9F to convert into the amino acid polypeptide.
This amino acid change occurs in signal peptide of GDF9 protein (Senta
et al., 2009) and is completely conserved among sheep [NP_001136360.2], goats [NP_001272637.1], cattle
[NP_777106.1], dogs [NP_001161485.1], wild boar
[NP_001001909.1], humans [NP_001275754.1], and
mice [NP_032136.2] (Fig. 2). The GDF9 G6 mutation was the only
detected mutation that affected the active polypeptide sequence. We did not
detect G7, G8, FecGT, and FecGE mutations in the evaluated
samples.
Multiple alignment of GDF9 propeptide sequence among sheep
[NP_001136360.2], goats [NP_001272637.1],
cattle [NP_777106.1], dogs [NP_001161485.1],
wild boar [NP_001001909.1], humans [NP_001275754.1], and mice [NP_032136.2]. The amino acid changes
due to the detected GDF9 mutations in sheep are illustrated by arrows.
G1 and G4 SNPs were unimportant for twin births in Iranian
ewes
In this study, we collected a large sample size of sheep from four
main breeds in Iran, including Afshari, Ghezel, Lori-Bakhtyari, and Shal.
Four primer pairs were used to amplify DNA sequences that cover the G0, G1, G4,
and G6 mutations. We used a SSCP approach for discriminating the possible
alleles in the studied population. However, amplicons that contain G0 or G6 mutations were not distinguishable using the SSCP method, although we included
the animals that were previously verified as heterozygous genotypes for
these mutations.
Allelic and genotypic frequencies of the GDF9 G1 mutation (G260A) in
Iranian Shal, Ghezel, Afshari, and Lori-Bakhtyari sheep breeds.
BreedSexGenotype frequency (%) Allelic frequency (%) G+/G+G+/G1G1/G1G+G1Male93.76.30.096.93.1ShalFemale81.89.19.186.413.7Overall85.78.26.189.810.2Male71.428.60.085.714.3GhezelFemale74.024.02.086.014.0Overall73.724.61.786.014.0Male100.00.00.0100.00.0AfshariFemale81.414.04.688.411.6Overall85.411.03.690.99.1Male71.428.60.085.714.3Lori-BakhtyariFemale89.310.70.094.75.4Overall88.211.80.094.15.9Total83.913.82.390.89.2
Results of this study for genotypic and allelic frequencies for the G1
mutation (G260A) in Iranian sheep breeds are presented in Table 4. Among all 109 rams that were used in this study, none of them were homozygous for the G1
mutation. Moreover, abundance of heterozygote rams (G1/G+) varied from 0.0 (Afshari) to 28.6 % (Lori-Bakhtyari and Ghezel). Analysis of pooled data
from ewes and rams indicated that there was a high difference in G1 allelic
frequency as well as genotypic distribution among the four mentioned breeds
(p value = 0.046). Although this allele was not detected in the Lori-Bakhtyari
breed, its abundance was 14.0 % in the Ghezel breed. Our results based on the
large dataset comprised of Afshari, Ghezel, Lori-Bakhtyari, and Shal breeds
could not detect any significant effect of the G1 mutation on the litter size of
ewes (Fig. 3) (p value = 0.991). In addition, neither the two infertile
ewes nor the four triplet-birth ewes carried the G1 mutation.
Litter size of Iranian ewes carrying the G1 mutation in the GDF9 gene.
Allelic and genotypic frequencies of the GDF9 G4 mutation (G721A) in
Iranian Shal, Ghezel, Afshari, and Lori-Bakhtiari sheep breeds.
BreedSexGenotype frequency (%) Allelic frequency (%) G+/G+G+/G4G4/G4G+G4Male30.853.815.457.742.3ShalFemale51.243.94.973.226.9Overall46.346.37.469.530.6Male75.012.512.581.318.8GhezelFemale62.530.07.577.522.5Overall64.627.18.378.221.9Male0.00.00.00.00.0AfshariFemale94.15.90.097.13.0Overall94.15.90.097.13.0Male66.616.716.775.025.1Lori-BakhtyariFemale61.428.410.275.624.4Overall61.727.610.775.524.5Total63.528.77.877.922.2
The highest and the lowest frequencies of the G4 mutation were detected in the Shal
and Afshari breeds, respectively (Table 5). G4 mutation frequency varied from 0.0 to 42.3 %,
from 3.0 to 26.9, and from 3.0 to 30.6 % in rams, ewes, and overall,
respectively. There was a significant difference in the abundance of the G4
mutation between breeds (p value = 0.003). However, no homozygote G4/G4
was observed in ewes and rams of the Afshari breed. Moreover, using this large
dataset in the current study showed that the G4 mutation did not have a significant effect on ewe twin births (Fig. 4) (p value = 0.864).
Discussion
A high number of mutations were reported by Hanrahan et al. (2004)
in Belclare and Cambridge sheep. The GDF9 G1 mutation was previously detected in
the Iranian Ghezel, Moghani, and Afshari breeds (Abdoli et al., 2016;
Eghbalsaied et al., 2014; Noshahr and Rafat, 2014). Additionally, the
presence of GDF9 G2, G3, and G4 SNPs was confirmed in the Iranian Afshari
breed (Eghbalsaied et al., 2012). Moreover, existence of G5 and G6
mutations in the GDF9 gene of Iranian ewes has also been recently reported
(Khodabakhshzadeh et al., 2016). The GDF9 G1 and G4 mutations convert arginine to
histidine and glutamic acid to lysine in the pre-peptide but not
the matured polypeptide. The G6 mutation converts valine to isoleucine
in the active GDF9 protein. Both valine and isoleucine are
classified into hydrophobic side-chain amino acids. It should be noteworthy
that all significant mutations in the ovine GDF9 gene, including G7, G8, FecGE, and
FecGT, occur in a completely conserved region of the protein. Results of a
recent publication indicated that unlike the suggestion by Hanrahan
et al. (2004), the G7 missense mutation that causes valine to convert to methionine
(both have a hydrophobic side-chain amino acid) does affect the
twin birth rate in Norwegian white sheep (Våge et al., 2013). The
existence of the G0 mutation, C25T, has recently been reported by the Ensembl
website using the next generation sequencing data from Iranian (IROV) and
Moroccan Ovis aries sheep (MOOV) with 2.5 and 0.6 % frequency, respectively
(rs605683468). In agreement with this only sequencing report, the frequency
of this mutation was 2.3 % with no homozygote genotype in the sequenced
samples. We need to consider the point that only 1 out of 16 twin-birth
ewes carried this mutation, while 94 % of twin-birth ewes, all four
triplet-birth ewes, and two infertile ewes did not carry this mutation.
Therefore, the C25T or G0 mutation is not likely the reason for twin births in Iranian ewes, although valine is conserved among sheep, goats,
cattle and wild boar. However, the G6 mutation was observed in 6 out of 14
sequenced samples from twin-birth records, all in a heterozygote state.
However, neither the ewes with triplet births nor the infertile ewes
carried this mutation. Although the SSCP technique could not differentiate
the mutation in this study, we cannot rule out the possible partial effect
of this mutation in litter size of Iranian ewes. Moreover, the G1 mutation
alters a non-conserved region, even in ruminants, and the G4 mutation also
supports conservancy rather than decreasing it. Moreover, both mutations
occur in the pre-peptide region. Thus, these mutations might be less likely to
be effective in altering GDF9 activity. In the sequenced sample, we could not
detect any homozygous genotype for these mutations, although, for example, the
moderate frequency of G6 mutations indicated that we could expect around
4 % homozygosity for GDF9 G6. This may be due to the fact that such
mutations are segregated at very low frequency and thus they are highly
affected by the sampling method. Therefore, using a larger sample size will
be required for evaluation of the allelic and genotypic frequencies as well
as the corresponding effect of these SNPs. Similarly, further mutations
are expected to be detected by increasing the number of highly fertile ewes.
Litter size of Iranian ewes carrying the G4 mutation in the GDF9 gene.
In the second phase of this study, we collected a large sample size of
Iranian sheep. We tried to genotype the animals for G0, G1, G4, and G6
mutations. However, the SSCP approach could not detect either G0 or G6
mutations. This could be due to insensitivity of the SSCP procedure in
detecting these mutations. There are several parameters such as type of
filtering matrix, type of additive, wall coating, temperature, and voltage
that can significantly alter the SSCP banding pattern (for review see
Sinville and Soper, 2007). Insensitivity or low sensitivity of SSCP
compared to other SNP detection technologies was also reported (Low et
al., 2000). Although we used a standard range of parameters for SSCP
optimization (Eghbalsaied et al., 2016; Khodabakhshzadeh et al., 2016),
implementing this technology was not helpful for distinguishing possible
mutations in these amplicons.
Using the RFLP and SSCP methods, the G1 and G4 mutations were distinguished,
respectively. A considerable difference was observed between genotypic
frequencies in different breeds so that this allele was not detected in
the Lori-Bakhtyari breed, while it was present in 14.0 % of the Ghezel breed. Also, none of
the sires in this study were homozygous for the G1 mutation. In the literature,
the frequency of the G1 mutation in Iranian breeds has been reported as follows:
8.7–15.7 % in the Ghezel breed (Barzegari et al., 2010; Eghbalsaied et
al., 2014), 2.7–24.0 % in the Afshari breed (Eghbalsaied et al., 2014;
Javanmard et al., 2011), 0.0 % in the Shal breed (Eghbalsaied et al.,
2014), 15.7 % in the Moghani breed (Barzegari et al., 2010), 18.0–23.0 % in the Baluchi breed (Javanmard et al., 2011; Moradband et al.,
2011), 19.8 % in the Sangsari breed (Hafezian, 2011), 22.5 % in the Makui
breed (Javanmard et al., 2011), and 18.0–40.6 % in the Mehraban breed (Abdoli et al., 2013; Javanmard et al., 2011). These genetic
polymorphisms in Iranian sheep corresponded to a 24.0 % frequency in the Chios
and Karagouniko breeds in Greece (Liandris et al., 2012), and 5.0–20.0 % in the Sal'skaya and Romanov breeds in Russia (Kolosov Yu et al.,
2015).
The importance of the G1 mutation on ewe prolificacy is controversial in the
literature. Although it has been suggested as an important mutation that
increases twin births in the Ghezel and Moghani breeds in Iran (Barzegari
et al., 2010) as well as the Chios and Karagouniko breeds in Greece
(Liandris et al., 2012), an adverse effect of this mutation on litter
size was reported in Iranian Baluchi sheep (Moradband et al., 2011).
Interestingly, an over-dominant effect of this gene was also reported for
litter size of Iranian Mehraban, Afshari, Baluchi, and Makui breeds
(Javanmard et al., 2011), while no important effect of the G1 mutation on
litter size was reported in Mehraban ewes (Abdoli et al., 2013).
Differences in the sampling size and the power of the SSCP vs. RFLP techniques
may be the reason for these large discrepancies in the literature. Our
results with a large sample size showed that the distribution of genotypic
frequencies among single-birth and twin-birth ewes was not different in all
the studied breeds. This clearly showed that the G1 mutation is not responsible
for infertility or multiple-lamb births in Iranian ewes. It was
suggested that G1 mutation (G260A) causes R87H changes in the amino acid
chain,
leading to replaced arginine instead of histidine in the pre-peptide region
of the GDF9 protein and therefore it has no effect on ewe litter size
(Hanrahan et al., 2004). The results of our study support the
hypothesis of Hanrahan et al. (2004) and the findings of Abdoli
et al. (2013) for Iranian sheep. In addition, lack of homozygous mutant rams
in all four breeds in the current study indicated that this mutation does
not favour twin births in
Iranian ewes under natural or artificial selection.
The other mutation that was screened for in the current study on Iranian sheep
breeds was (G721A) G4 (Hanrahan et al., 2004). The G4 mutation
frequency varied significantly between breeds, so that it was estimated at 3.0 % in the Shal breed and 30.6 % in the Afshari breed. It is evident that the G4
mutation is present in the Iranian Afshari, Shal, and Sangsari breeds. The
frequency of this mutation was estimated at 7.9 % in the Afshari breed (Eghbalsaied et al., 2014); 6.25 % in Shal (Eghbalsaied et al.,
2014);
10.0 % in the Ghezel breed (Eghbalsaied et al., 2014); 19.0 % in Greek sheep
breeds (Liandris et al., 2012); 44.8 and 3.8 % in the Belclare and
Cambridge breeds of Ireland, respectively (Hanrahan et al., 2004);
and 95.0 and 80.0 % in the Sal'skaya and Romanov sheep breeds of Russia
(Kolosov Yu et al., 2015).
The GDF9 G4 mutation was not significantly important for ewe twin births.
The effect of the G4 mutation on twin births in Greek ewes was also not
significant (Liandris et al., 2012). There are limited publications
that address the frequency and the importance of the G4 mutation. There was one
ewe, which was homozygous for G4
mutation, with a very high number of antral follicles (Eghbalsaied et al., 2012). The G4 mutation (G721A) causes
glutamine–241–lysine conversion, which takes place in the GDF9 pre-peptide and is
unlikely to affect GDF9 functional activity (Hanrahan et al.,
2004). More importantly, none of the four ewes with triplet births or the two
infertile ewes carried the G1 or G4 mutations.
Screening the GDF9, BMP15, and BMPR1B genes in Davisdale sheep indicated
that several mutations in GDF9 and BMP15 segregate in these breeds. However,
none of these mutations were responsible for higher ovulation rate and
litter size in these breeds (Juengel et al., 2011). Instead, mutation
in the leptin receptor gene was significantly associated with delayed onset of
puberty and a decrease in ovulation rate and litter size in these breeds
(Haldar et al., 2014). Therefore, it is possible that mutations in
other genes, such as BMP15, BMPR1B (Eghbalsaied et al., 2016), leptin, or
leptin receptor, are responsible for higher antral follicle count, ovulation
rate, and litter size in Iranian sheep breeds. A recent study showed that
in addition to the bone morphogenetic protein signaling pathway, expression of genes involved in estrogen
and AMPK synthesis can be important factors in antral follicle count in
ewes (Foroughinia et al., 2017). Therefore, more comprehensive studies
are required to determine effective genetic mechanism controlling ewe
twin births in Iranian sheep breeds.
Conclusion
In this study, a new mutation was detected in the early pre-peptide region of
the GDF9 gene. In addition, the G1 and G4 mutations were highly variable among different
breeds so that they were not observed in homozygote mutants in rams of some
breeds. Moreover, neither the G1 nor the G4 mutation had an effect on ewe litter
size. This clearly indicated that the selected rams in these flocks were neither naturally nor artificially selected for these mutations. In addition,
the G8 mutation was not observed in these sheep breeds. None of
triplet-birth or infertile ewes carried the G0, G1, G4, G6, G8, and Thoka
SNPs. In conclusion, the results of this study suggest that the GDF9 G1 and G4
mutations are not the reason for higher litter size or fecundity in Iranian sheep.
Moreover, neither GDF9 G0 nor G6 mutations cause triplet births or
infertility in Iranian ewes. Therefore, it is unlikely that variant
GDF9
SNPs, which currently segregate in Iranian ewes, induce larger litter size or
infertility.
The data (sequencing
data and SSCP results) used in this article can be found in the Supplement.
The Supplement related to this article is available online at doi:10.5194/aab-60-119-2017-supplement.
The authors declare that they have no conflict of interest.
Acknowledgements
The authors would like to thank Alan McNeilly for his invaluable
collaboration in conducting this project. This project was supported by the Iran
National Science Foundation (INSF) through grant no. 90002710.
Edited by: S. Maak
Reviewed by: P. Zamani and M. Mohammadabadi
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