Bone morphogenetic protein 15 (BMP15), member of TGF-β family, expressed in oocyte is essential for ovulation. The BMP15 gene of sheep links to the X chromosome so it is also known as FecX gene. The FecXG mutation in BMP15 gene is characterized by an C>T substitution (Q239Ter) at coding position 718 in exon 2 region that results in the insertion of a premature stopping point in the transcription of the protein, associated with increase in ovulation in sheep. In the present study, DNA was isolated from blood samples collected from the Muzzafarnagari sheep breeds (n = 200) maintained at LFC (Livestock farm complex) of DUVASU, Mathura, U.P. The 141 bp amplified fragments of the of BMP15 gene were digested with HinfI restriction enzyme. The HinfI/PCR-RFLP assay of BMP15 gene revealed only GG genotype (141 bp uncut, 100%) with G allele (1.0) in screened sheep population. The studied region of the BMP15 gene showed monomorphic pattern. It revealed that FecX G allele seems to be fixed in screened sheep population.
Bone morphogenetic protein 15 (BMP15), also known as FecX is a member of the TGF-β superfamily. It expresses in oocytes and has potent role in prolificacy of sheep that affect the ovulation rate. The ovine BMP15 gene links to the X chromosome (FecX locus) and consists a coding 1179 nucleotide sequence in two exons, separated by a 5.4 kb intron, encoding a 393 amino acid residue precursor and a 125 amino acid mature peptide (Galloway et al., 2000). BMP15 regulates granulosa cell proliferation and differentiation by promoting granulosa cell mitosis, suppressing follicle-stimulating hormone receptor expression and stimulating kit ligand expression, all of which play a pivotal role in female fertility in mammals (Juengel et al., 2002; Moore et al., 2004; Chu et al., 2007). Five different BMP15 mutated alleles, called FecX alleles led to increased ovulation rates in heterozygous ewes and sterility in homozygous ewes (Hanrahan et al., 2004). In previous studies, heterozygous ewes with FecXI (Inverdale), FecXH (Hanna), FecXB (Belclare), FecXG (Galway) or FecXL (Lacaune) mutations exhibited one to two additional ovulations compared with non-carriers, whereas homozygous ewes were sterile (McNatty et al., 2005). This finding indicates the critical impact of BMP15 or FecX on ovarian function in mammals (Bodin et al., 2007). Among them, FecXG mutation is characterized by the C to T transition at nucleotide 718 position in exon 2 region. This introduces a premature stop codon in the place of glutamic acid at amino acid residue 239 (Q239Ter) of the unprocessed protein, which presumably resulted in complete loss of BMP15 function. The FecXG mutation was associated with increased ovulation rate and sterility in Cambridge and Belclare sheep (Hanrahan et al., 2004).
SNP C>T has been reported in exon 2 of the BMP15 gene and associated with prolificacy in several exotic (Chu et al., 2007; Javanmard et al., 2011; Moradband et al., 2011 and Elkorshy et al., 2013) and few Indian sheep breeds (Kumar et al., 2008). However, scanty information is available on FecXG polymorphism in Indian sheep breeds including Muzzafarnagari breed. Therefore, the present study was undertaken to investigate the status of FecXG (C>T) polymorphism in exon 2 region of BMP15 gene in Indian Muzzafarnagari sheep breed.
Materials and Methods
Animals Source, DNA Extraction and HinfI/PCR-RFLP
A total of 200 adult females (2-5 years of age) of Muzzafarnagari sheep maintained at Livestock Farm Complex (LFC), DUVASU, Mathura (U.P.), were utilized in the present investigation. Genomic DNA was extracted from venous blood using the standard protocol of Sambrook and Russel (2001). The PCR cycle conditions and primers (F: 5′- CACTGTCTTCTTGTTACTGTATTTCAATGAGAC -3′ and ‘R: 5′- GATGCAATACTGCCTGCTTG -3′) were used for amplification of BMP15 exon 2 region as per method described by Moradband et al. (2011). The PCR-RFLP was carried out overnight at 37oC with HinfI restriction enzyme in a total volume of 15μl containing 5.0 µl of PCR product, 1.5 µl of 10X RE buffer and 10 units (1.0 µl) of enzyme.
The data was generated by estimating the frequency of different BMP15 genotypes. The allelic and genotypic frequencies were estimated by standard procedure (Falconer and Mackay, 1996).
Results and Discussion
The amplified fragments of the BMP15 exon 2 region revealed 141 bp product (Fig. 1). The results revealed that all the screened samples of Muzzafarnagari sheep were found monomorphic for FecXG gene. The HinfI PCR-RFLP assay revealed only one type of banding pattern GG genotype, which was of 141 bp (uncut) in all the screened samples.
|Fig. 1: HinfI/PCR-RFLP assay of BMP15 (FecX) gene revealed by 1.5% agarose gel electrophoresis in Muzaffarnagri sheep, M1: Marker (100 bp DNA ladder, New England Biolabs, Cat No. N3231S), Lane 1: 141 bp PCR product Lane 2-4: RFLP product of 141bp showing uncut 141 bp; GG genotype.
The genotypic frequencies of G+, ++ and GG genotypes were 0.0%, 0.0% and 100%, respectively in all the screened animals and the allelic frequency of allele + and G was observed as 0.0 and 1.0, respectively. These findings were similar to the report of Kasiriyan et al. (2011) in Sangsari sheep breed while were in contrast with the findings of Polley et al. (2010) and Kumar et al. (2015) in Garole. Dincel et al. (2018) in Chios and Pineda et al. (2018) in Colombian Creole sheep breeds wherein ++ (wild type) genotype was observed with fixed wild allele (+). However, the genotypic frequencies of ++, G+ and GG genotype as observed by Elkorshy et al. (2013) in Barki, Ossimi, and Rahmani sheep breeds, Chu et al. (2014) in Small Tailed Han sheep breed and Barakat et al. (2017) in Barki, Ossimi, and Rahmani sheep breeds were not in accordance with the findings of the present study as they have observed polymorphic pattern in these genotypes with varying degree of frequency of mutant allele G (Table 1).
Table 1: Genotypic frequencies of BMP15 (FecX)/HinfI gene in different sheep breeds as observed by other authors
|Breed||Genotypic Frequency||Allelic Frequency||References|
|GG (%)||G+ (%)||++ (%)||G||+|
|Garole||0||0||100||0||1||Polley et al., 2010|
|Sangsari||100||0||0||1||0||Kasiriyan et al., 2011|
|Barki||0||68||32||0.66||0.34||Elkorshy et al., 2013|
|Small Tailed Han||0||60||40||0.3||0.7||Chu et al., 2014|
|Garole||0||0||100||0||1||Kumar et al., 2015|
|Mehraban||25.9||74.1||0||62.9||37.1||Nadri et al., 2016|
|Ossimi||0||72||28||0.36||0.64||Brakat et al., 2017|
|Kenguri||0||100||0||0.5||0.5||Asharani et al., 2018|
|Kenguri X NARI Swarna||0||96.7||3.3||0.484||0.516|
|Chios||0||0||100||0||1||Dincel et al., 2018|
|Colombian Creole||0||0||100||0||1||Pineda et al., 2018|
In the present study, we observed monomorphic pattern of FecXG mutation in BMP15 gene in screened Muzzafarnagari breed, consequently we could not perform association study with reproduction trait because in these screened sheep FecXG allele was found fixed. However, all the screened animals were fertile and 27% twinning rate was found in our Muzzafarnagari flock. These results may be due to small sample size and close organized herd from where the samples were taken for study making it imminent to further investigate this SNP along with other fecundity genes on large diversified population for better understanding of functioning of reproduction traits in sheep.
The assistance of Livestock Farm Complex (LFC), DUVASU Mathura (U.P.) for providing blood samples of Muzzafarnagari sheep and data is duly acknowledged. The authors also acknowledge the funding agencies (Department Animal Husbandry, Dairy and Fisheries; DADF, Government of India) for providing financial assistance under Muzzafarnagari conservation project during 2012-2018.