In sheep, improvement of reproduction characteristics is one of the main objectives of breeding programs throughout the world. Reproduction traits such as litter size can be genetically regulated not only by many genes with minor effects but also sometimes by major genes with large effects that called fecundity (Fec) genes. To date, three major genes have been identified to influence prolificacy in sheep, which they mainly belong to superfamily of transforming growth factor-beta (TGF-β), namely, growth differentiation factor 9 (GDF9), known as FecG (Hanrahan et al. 2004); bone morphogenetic protein 15 (BMP15), known as FecX (Galloway et al. 2000; Hanrahan et al. 2004); and bone morphogenetic protein receptor type 1B (BMPR1B), known as FecB (Mulsant et al. 2001; Souza et al. 2001; Wilson et al. 2001). GDF9 is an oocyte-derived component which is necessary for folliculogenesis, oogenesis, and ovulation which play a crucial role in fertility of female (De Castro et al. 2016). The gene encodes for ovine GDF9 map on chromosome 5 (Sadighi et al. 2002), and spans about 2.5 kilo base pairs (kb) which contain 2 exons interrupted by an intron with 1126 base pairs (bp). The gene encodes for 453 amino acid prepropeptide, while the long mature peptide is 135 amino acids. Single nucleotide polymorphisms (SNPs) on sheep GDF9 have been revealed to influence fecundity traits like ovulation rate and litter size. Several different SNPs in GDF9 have been reported to be associated with fertility in sheep viz. FecGH (G8) mutation in Cambridge and Belclare sheep (Hanrahan et al. 2004) and in Sangsari sheep (Kasiriyan et al. 2011), FecGE or FecGSI mutation in Santa Ines sheep (Silva et al. 2011), FecTT mutation in Thoka sheep (Nicol et al. 2009), G1 mutation in Moghani and Ghezel sheep (Barzegari et al. 2009), in Garole sheep (Polley et al. 2010) and in Bonpala sheep (Roy et al. 2011), FecGV mutation in Brazilian sheep (Souza et al. 2012; Souza et al. 2014), FecGI mutation in Baluchi sheep (Moradband et al. 2011) and G7 in Norwegian White Sheep (Souz et al. 2014). The FecGE and FecGF mutations have additive effects on ovulation rate and litter size, while FecGH, FecGT and FecGV causes increased ovulation rate and litter size in the heterozygote ewes and infertility in the homozygous carriers (Souza et al. 2014). Generally, most of the Iranian sheep breeds product single lamb per lambing. The Bahmaei and Lak Ghashghaei sheep are two local breeds of Kohkiloueh and Boyer-Ahmad province in Iran, which were reared for meat production. There is no closed nucleus center for breeding of these two breeds in Iran. Furthermore, the Bahmaei and Lak sheep are reared in nomadic conditions, thus, increase the amount of the litter size could provide better economic status for sheep breeder and lead to immigration preventing to the city. The average twin rate in Bahmaei and Lak Ghashghaei sheep breeds have been reported to be less than 10% in nomadic breeding conditions (Khaldari, 2005). To date, little is known about the genetic structure of this breed with regard to the fecundity genes. Therefore, the aim of this study was to identify the genetic variation in the GDF9 gene and to evaluate their possible associations with litter size in the Bahmaei and Lak Ghashghaei sheep breeds.
MATERIALS AND METHODS
In this study, based on the history of the last lambing, a total of 40 multiparous ewes (20 Bahmaei sheep and 20 Lak Ghashghaei sheep) were selected, including 10 single births and 10 twin births for each breed, at Kohkiloueh and Boyer-Ahmad province of Iran. Selected animals for Bahmaei and Lak breeds were taken from two different flocks at Dehdasht and Gachsaran regions, respectively. Blood samples were collected from the jugular vein using sample tubes containing ethylenediaminetetraacetic acid (EDTA), and genomic DNA was extracted using AccuPrep® genomic DNA extraction kit (BiONEER, South Korea), according to the manufacturer's instructions. To amplify complete region of exon 1 (397 bp) and 2 (1265 bp), three pairs of primer were designed according to ovine GDF9 sequence (GenBank accession number AF078545) using Primer3plus (Table 1). Polymerase chain reaction (PCR) was carried out in 25 μL of total volume containing 25 pm of each primer, 200 μM of each dNTP , 1.5 mM MgCl2, 2.5 mM10 × buffer, 1.0 U Taq DNA polymerase and 50 ng genomic DNA template. The amplification process was carried out by initial denaturation at 94 ˚C for 5 min and then, 35 cycles at 94 ˚C for 45 s, 63 ˚C for 60 s, and 72 ˚C for 30 s followed by 72 ˚C for 7 min. PCR products were checked by electrophoresis using 1.0% agarose gel in 1 × TBE buffer along with DNA ladder. The purified PCR products were commercially sequenced in one direction (two repeats) and the sequences were analyzed using MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/) and FinchTV 1.4.
Data analysis was performed using the general linear model (GLM) procedure of SAS (2012). Mean separation procedures were performed
using the least significant difference test.
Yijk= μ + Bi + Gj + eijk
Yijk: individual observation on litter size.
μ: overall population mean.
Bi: effect of breed.
Gj: effect of combined genotypes of identified SNPs.
eijk: random error.
RESULTS AND DISCUSSION
The entire length of exon 1 and 2 ovine GDF9 gene was amplified using one and two pairs of primer, respectively. Then, the PCR products were analyzed by Sanger sequencing method. The obtained nucleotide sequences of amplified fragments were aligned and compared using MUSCLE and FinchTV. Multiple sequence alignment showed that obtained sequences of P1 fragment were identical and share 100% homology with ovine GDF9 gene. It means that there was no SNP in this part of GDF9 gene in two studied breeds. The sequence analysis of P2 fragment revealed 4 transition mutations, at positions 3137G/A, 3375C/T, 3381G/A, and 3625G/A (Figure 1) according ovine GDF9 sequence (GenBank accession number AF078545). The first novel identified SNP (3137G>A) located at intronic part but the remaining mutations were in coding sequence of exon 2 at positions 471, 477 and 721 corresponded to G2, G3 and G4 mutations reported by Hanrahan et al. (2004), respectively.
Table 1 Primer sequence, product size and amplified region of ovine GDF9
Figure 1 Nucleotide changes of four identified SNPs in GDF9
a) G to A at position 3137 in intron; b) C to T at position 471; c) G to A at position 477 and d) G to A at position 721
SNPs 3137G > A, c477G > A and c721G > A were detected in both breeds while SNP c471C > T was identified only in Bahmaei sheep. In Bahmaei sheep, all three possible genotypes (GG, AG and, AA) were observed for SNPs 3137G > A, c477G > A and c721G > A, while SNP c471C > T revealed wild homozygous (CC) and heterozygous (CT) genotypes. The genetic composition of Lak sheep was different with regard to identified SNPs in P2 fragment. No mutant homozygous genotypes were observed for SNPs 3137G > A, c477G > A and c721G > A in Lak ewes. In addition, all Lak samples were wild homozygous genotype (CC) for SNP c471C > T. An interesting finding of the sequencing results was linkage between mutant alleles of four identified SNPs for P2 fragment in both breeds. The genotypes and alleles frequencies of four identified SNPs were presented in Table 2. In P3 fragment, that cover second half of exon 2, no genetic variation was found in two breeds. Combined genotype effects of identified SNPs on litter size were not significant; p-values were 0.72 and 0.55 for breed and combined genotype, respectively. Differences between least squares means are given in Table 3. As it shows, mutant homozygous genotypes were not observed in Lak ewes. In sheep, improvement of fecundity traits by traditional selective breeding has been confirmed to be difficult because of their low heritability. Therefore, use of marker-assisted selection (MAS) along with traditional selection methods could be effective for these traits. To date, several SNPs in fecundity genes have been identified that influence on reproduction traits like ovulation rate and litter size in sheep. So far, many SNPs have been reported for GDF9 gene in different sheep breeds throughout the world. For example, eight different SNPs (G1-G8) have been identified in Belclare and Cambridge prolific sheep breeds (Hanrahan et al. 2004) which G1 in exon 1, G4, G7 and G8 in exon 2 were the most commonly identified. In our study, four SNPs were detected in two small samples of Bahmaei and Lak sheep. Furthermore, substitutions of A to C in FecTT allele (Nicol et al. 2009), T to G in FecGE allele (Silva et al. 2011), C to T in FecGV allele (Souza et al. 2012), and G to A (Vage et al. 2013) were identified in different sheep breeds. In the present study, the three identified SNPs corresponded to G2, G3 and G4 alleles in exon 2 that were discovered in Belclare and Cambridge sheep breeds (Hanrahan et al. 2004). However, based on our results, exon 1 ovine GDF9 did not show any genetic variation in Bahmaei and Lak sheep, but a novel substitution of G to A was found in intron at position 3137 in Bahmaei ewes. In GDF9 gene, as a fecundity gene, some of identified SNPs can increase ovulation rate and litter size, while others have no effect on the ewes’ fecundity. In this study, no significant association was found between combined genotypes of identified SNPs and litter size in Bahmaei and Lak Ghashghaei breeds of Iran. However, in Bahmaei sheep, ewes with combined genotypes of AATCAAAA and AGCCAGAG showed lower and higher least square mean for litter size, respectively. The sequencing result of GDF9 cDNA revealed the G2, G3, and G4 mutations in Iranian Afshari sheep (Eghbalsaied et al. 2012) which is in agreement with our results. They also reported that the G4 mutation was only observed in ewes ovaries defined superficially as top quality. No significant difference was found between different genotypes of SNP c477G > A (G3) with litter size of ewes in Laticauda and Bagnolese sheep breeds (Albarella et al. 2015). However, with respect to SNP c750G > A, the GG homozygous ewes displayed a significantly higher value of litter size compared with GA heterozygous individuals (2.06 vs. 1.75) in Bagnolese sheep (Albarella et al. 2015). The SNPs G1, G4 and G8 of the ovine GDF9 gene were found to be heterozygous with no evidence of infertility for the homozygous individuals in the Chios sheep breed (Liandris et al. 2012). Two mutations G1 and G4 were observed in Indian Bonpala sheep but their associations with litter size were not significant (Roy et al. 2011). Four SNPs c.471C > T, c.477G > A, c.721G > A and c.978A > G were reported in GDF9 of Ashgar, Dubasi and Watish sheep breeds in Sudanese, but no significant differences were found in allele or genotype frequencies between the two groups differing in litter size for any of these SNPs (Ali et al. 2016). The G1 SNP was found in four fat-tailed sheep breeds of Iran, Afshari, Baluchi, Makui, Mehraban, and it had a litter size of 1.78 ± 0.07 in heterozygous sheep, while in homozygous wild type ewes, the average litter size was 1.16 ± 0.05.
Table 2 Allele and genotype frequencies at four identified mutations in two breeds
Table 3 Least squares means for litter size of ewes according to combined genotypes of GDF9 gene
None of the individuals analyzed showed homozygote for this SNP (Javanmard et al. 2011). The FecGV SNP was reported in Ill de France sheep in Brazil and contributed to an ovulation rate of 2.1 ± 0.1 in homozygous ewes of SNP, while the ovulation rate for ewes without this SNP was 1.2 ± 0.1 (De Soza et al. 2012). The FecGE variant was found in Santa Ines sheep. The homozygous ewes for this SNP had multiple ovulations approximately 96.3%, whereas multiple ovulations in heterozygous and homozygote non carrier’s ewes were approximately 31% and 14%, respectively, meaning the SNP has additive action (Silva et al. 2011). In another study, significant differences in allelic frequencies of FecGE were not observed between Elite/Superior and Regular/Inferior groups of ewes in Morada Nova sheep, but the effect of FecGE on litter size showed positive influence in profitability in medium to high input farm systems (Lacerda et al. 2016). The FecGF (G7) variation was the only polymorphism present on Finnish Landrace sheep. However, no significant effect was found between FecGF and reproduction performance of Finnish Landrace sheep. All ewes in the high selection group were homozygous for FecGF, in the low group but none of the ewes were homozygous for this SNP (Mullen and Hanrahan, 2014). Vage et al. (2013) showed that a polymorphism in the GDF9 that had previously been identified but not considered to have an effect on ovulation rate, is present in Norwegian white sheep and gives an increase in ovulation rate.
Polymorphism of GDF9 gene as a candidate gene for fecundity traits was studied in two sheep breeds of Kohkiloueh and Boyer-Ahmad province of Iran. The results display that genetic structure of two breeds was different with respect to GDF9 gene. However, three SNPs 3137G > A, c471C > T and c477G > A were observed in two breeds but their genotype frequencies were different. The Lak Ghashghaei ewes were all homozygous wild-type (CC) genotype for SNP c.471C > T, while Bahmaei samples revealed CC and CT genotypes with frequencies of 0.85 and 0.15, respectively. However, no significant association was found between the different combined genotypes of this candidate gene and litter size in both breeds, but further studies with more number of ewes are required to confirm the result of this research.
This study was supported by Yasouj University of Iran. The author thanks from farmer who provided samples for this study.