A Profile of Single Nucleotide Polymorphisms in Fecundity Genes Among Iranian Sheep Breeds by Using Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) Method

Iranian Journal of Applied Animal Science

مقاله 9، دوره 10، شماره 2، شهریور 2020، صفحه 265-285   

نویسندگان

P. Potki^{* 1}؛ S.Z. Mirhoseini²؛ F. Afraz³؛ S.M.F. Vahidi³

¹Department of Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII), North Branch, Rasht, Iran

²Department of Animal Science, Faculty of Agricultural Science, University of Guilan, Rasht, Iran

³Department of Animal Biotechnology, Agricultural Biotechnology Research Institute of Iran (ABRII), North Branch, Rasht, Iran

چکیده

Ovulation rate and litter size are traits of fecundity and have a very remarkable economic value that leads to an increase in the production of meat, wool and milk. Therefore, exploring on the candidate and functional genes for these traits is very important. Growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and bone morphogenetic protein receptor-1B (BMPR1B) are the most important of these genes containing single nucleotide polymorphisms (SNPs) markers that are associated with high fertility in sheeps. The aim of this study was to determine the allelic and genotypic frequencies of seven of these SNPs including; G1, G4, G7 and G8 in GDF9 gene, B2 and B4 in BMP15 gene and FecB in BMPR1B gene among 532 sheeps from eleven of Iranian native breeds using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method. For each mutation region, polymerase chain reactions and digestion reactions with specific endonuclease enzymes, were performed. The results showed that G1 mutation presents among ten breeds except Zandi, G4 mutation in all breeds, and the FecB mutation in two breeds; Kalakuei and Zandi. The G7, G8, B2 and B4 mutations were not observed in any of breeds, and all sheeps were homozygote wild type for these four positions.

کلیدواژه‌ها

PCR-RFLP؛ SNP؛ GDF9؛ BMP15؛ BMPR1B؛ sheep

اصل مقاله

INTRODUCTION

The domesticated sheep breeds are probably originated from the wild sheep species such as Asian mouflons (Ovis orientalis) and Asiatic urial (Ovis vignei), which are still found in Iran National Parks and protected natural areas (Valizadeh, 2010). Iran has 28 native sheep breeds and in terms of breed diversity in sheep, has occupied the first rank in the world (‎Akbarinejad et al. 2014). According to the latest livestock census, conducted in 2017 by Ministry of Jihad-e-Agriculture of Iran (Agricultural Statistics of Iran, 2017), the Iranian sheep population is 47638600 animals that encompass 63.60% of Iranian animals (sheeps/lambs, goats/kids, cattles/calves, buffalos and camels). The meat production trait has taken into account in most previous Iranian sheep breeding program and fertility traits have played the main role to improve lamb and mutton production. Based on previous investigations, different mutations in the transforming growth factor-β (TGFβ) superfamily genes have been reported to affect ovulation rate and litter size in sheep. These genes include bone morphogenetic protein receptor, type 1B (BMPR-1B), bone morphogenetic protein 15 (BMP15), and growth differentiation factor 9 (GDF9) (‎Jansson, 2014). The Booroola's ancestor is Garole breed sheep that is indigenous to Bengal area in India (‎Abraham and Thomas, 2012). The Garole is a small sheep that have a mean litter size of 1.74 to 2.27 (Davis et al. 2002). The Booroola phenotype, which increased the litter size, was originally selected from special lines of Australian merino sheep. Booroola fecundity gene is the first major gene for prolificacy identified in sheep ‎(Wilson et al. 2001). This gene is located on chromosome 6 of sheep and is effective on the maturation of the granulosa cells, the development of the immature oocyte and its function (Abraham and Thomas, 2012). This gene is known as the activin receptor-like kinase (ALK6) and its mutation called FecB. A change from adenine (in wild type) to guanine (in mutant types) at the nucleotide position 746, results in a change from glutamine amino acid (in wild type) to arginine amino acid (in mutant types) at coding region 249 (Mulsant et al. 2001). This single autosomal locus can improve the ovulation rate and litter size. The FecB mutation, in addition to the Merino Booroola breed, also presents in some other sheep breeds such as; Hu ‎(Chu et al. 2011; Wang et al. 2018‎), Small Tailed Han (Chu et al. 2006; Zhang et al. 2011‎) and Bayanbulak in China (‎Zuo et al. 2013), Kendrapada in India (‎Dash et al. 2017) and etc and effects on high fertility in these breeds. Bone morphogenetic protein 15 (BMP15) (FecX) gene is located on the chromosome X of sheep and has two exons separated by an intron, which is about 5400 nucleotides. The protein encoded by this gene has 1179 nucleotides and its immature peptide has 393 amino acids. The mature peptide has 125 amino acids (Galloway et al. 2000). The gene, which was first found in Romney sheep, is also called GDF9B or FecX. This gene, and also GDF9, encode separate proteins that express in ovarian tissue. This mechanism is very effective in mammalian fertilization (‎Hanrahan et al. 2004). Until now, several mutations of this gene including Inverdale (FecX^I), Hanna (FecX^H), Belclare (FecX^B), Galway (FecX^G), Lacaune (FecX^L), Rasa aragonesa (FecX^R), Grivette (FecX^Gr) and Olkuska (FecX^O) have been identified in different sheep breeds. The first six mutations increase the rate of ovulation in heterozygous individuals (‎Qiuyue et al. 2014). For instance, the estimated effect of FecX^G is ranged from 0.77 in Belclare ewes to 1.18 in Cambridge ewes, and for FecX^B is 2.38 in Belclare ewes (‎‎Hanrahan et al. 2004). Growth differentiation factor 9 (GDF9) (FecG) is essential for folliculogenesis in sheep ovary and has a highly significant role from the first stage of follicular growth until ovulation. GDF9 gene is located on chromosome 5 of sheep and has about 2500 nucleotides. Two exons are separated by an intron with 1126 nucleotides. The two exons encode an immature peptide with 453 amino acids. The mature peptide has 135 residues (‎‎Hanrahan et al. 2004). FecG^H or G8 is one of eight mutations (G1 to G8) in this gene that has been identified in Cambridge and Belclare sheeps and led to an increase in ovulation rates in heterozygotes and infertility in mutant homozygotes ewes. The effect of that is 1.79 in Belclare ewes and 2.35 in Cambridge ewes. Up to now, the FecG^H has been reported in breeds of sheep like as Chios and Karagouniki in Greece too (‎Liandris et al. 2012). So far, in addition to the FecG^H, some other mutations in this gene such as; FecG^V in Ile de France, FecG^E in Embrapa and FecG^T in Icelandic Thoka sheep breeds have also been identified that demonstrated an association with high ovulation rate and litter size (‎Qiuyue et al. 2014). The purpose of this study was to investigate the presence or absence of seven SNPs in the three mentioned genes and to determine their allelic and genotypic frequencies among these eleven breeds.

 

MATERIALS AND METHODS

Blood samples were collected from 532 individuals belong to 11 Iranian native sheep breeds including; Afshari (N=55), Farahani (N=55), Grey Shiraz (N=55), Turki-Ghashghaei (N=55), Naeini (N=48), Kalakuei (N=47), Moghani (N=46), Shal (N=46), Taleshi (N=45), Makuei (N=41) and Zandi (N=39). The detailed information are given in Table 1. The detailed information of SNPs (Table 2) and the position of amino acid changes on amino acid sequences in genes (Figure 1) are given. The blood samples was collected between 2005 and 2007 according to breed distribution areas over a large geographic area. During sampling, attempts were made to collect animals as unrelative as possible. A modified protocol was applied to isolate genomic DNA from blood samples using GPP kit (Gene Pajoohan Pooya). The extracted DNA was qualified and quantified using NanoDrop (2000) and agarose gel electrophoresis methods. Subsequently, polymerase chain reactions were performed. Some of the primers were deliberately introduced a point mismatch (shown by underlines in Table 3) resulting in PCR products containing restriction site for each endonuclease enzyme (‎Davis et al. 2002; ‎‎‎Hanrahan et al. 2004). Details of each primer and PCR products (Table 3), PCR reactions (Table 4) and the temperature program for SNPs (Table 5) are given. The total volume of each reaction was considered 15 µL and the applied biosystems (AB) thermal cycler (model 9700) was used for amplification.

 

Table 1 Introduction of eleven breeds in this study

 

 

Table 2 Informations of the seven single nucleotide polymorphisms (SNPs)

 

 

Figure 1 a: four amino acid changes in GDF9; b: two amino acid changes in BMP15 and c: one amino acid change in BMPR1B

 

 

Table 3 details of primers

 

SNP: single nucleotide polymorphism and PCR:polymerase chain reaction.

 

Table 4 Polymerase chain reaction reaction components

 

 

Table 5 Polymerase chain reaction (PCR) program in thermal cycler

 

SNP: single nucleotide polymorphism.

 

Figure 2 indicates the sequences of seven PCR products. The sequences highlighted in red are annealing sites of the primers, the green nucleotide is the position of mutation and the underlined sequences are cutting sites of each enzyme. Restriction digestion process was carried out by using a specific restriction enzymes for each mutation. Information of each restriction enzyme and the size of fragments obtained from the products of the enzymes are given in Tables 6 and 7, respectively. The digested products were electrophoresed in 2% agarose gel and the DNA bands were visualized by ethidium bromide staining technique. Existence or absence of a restriction site is the source of polymorphism. For genotyping, each band represents an allele having a specific size in base pairs (bp). The data were subjected to appropriate analyses using POPGENE software (ver. 1.31).

 

RESULTS AND DISCUSSION

A total of 532 individuals from the 11 Iranian sheep breeds were genotyped with the PCR-RFLP approach. The electrophoretic profiles of genomic DNA and PCR products of seven loci are shown in Figures 3 to 10. Digestion with restriction enzymes in seven loci on agarose geles (2%) have been shown in Figures 11 to 17. Subsequently, statistical analaysis were carried out. Frequencies of genotypes and allelesfor G1 mutation (G→ A), G4 mutation (G→A), FecB mutation (A→G) and other SNPs are given in Tables 8 to 11, respectively. Due to the fact that polymorphism was observed only in the G1, G4 and FecB loci, the Hardy-Weinberg equilibrium for these three positions was examined based on the chi-square test. The HWE test showed that all three polymorphic loci did not deviate from HWE (P>0.05) while considering each breed individually.

 

Figure 2 The PCR product sequences

a: 462 bp; b: 161 bp; c: 156 bp and d: 139 bp in GDF9; e: 141 bp and f: 153 bp in BMP15; g: 190 bp in BMPR1B

 

 

Table 6 Information of restriction enzymes

 

 

Table 7 Digestion reaction components

 

 

But when all the breeds were combined into one population, deviation from HWE happened only in FecB mutation (P<0.05). Table 12 showes the p-value of HWE test for each mutation. Genotyping of G1 showed that there was no recessive homozygote individuals in all breeds. The highest and lowest frequencies of heterozygotes (GA) obtained in Taleshi and Zandi breeds with 0.156 and 0.000, respectively. The mean frequency of heterozygotes in all sheeps as one population was 0.068. The highest and lowest frequency of mutant allele (A) was observed in Taleshi and Zandi breeds with 0.078 and 0.000, respectively. Mean of mutant allele frequency per all animals was 0.034. Further details about this mutation to be extracted from different references are given in Table 13. The G1 mutation was identified in Cambridge and Belclare sheeps for the first time but without any association with ovulation rate (‎Hanrahan et al. 2004). With looking at the Table 13, it can be seen that, in other studies such as ours, which aimed only at evaluating allelic and genotypic frequencies, no sample of mutant homozygote is reported. It indicates that the frequency of this genotype is very low in the main populations of these breeds. In other studies with sheep fecundity records (and probably, the samples have also been selected based on their records), the number of mutant homozygous is low.

 

Figure 3 Some of genomic DNA in this study

 

 

Figure 4 G1 locus; a 462 bp band

 

 

Figure 5 G4 locus; a 161 bp band

 

 

Figure 6 G7 locus; a 156 bp band

 

 

The highest frequency of mutant homozygotes has been reported in the Greek Chios breed with 0.087 (‎Liandris et al. 2012). The homozygous (AA) ewes in this breed displayed litter size of 2.25 implying a significant effect of the A allele, as high as 0.33 lambs. The heterozygous ewes showed negative dominance deviation with a statistically significant decrease in litter size of 0.47 lambs.

 

Figure 7 G8 locus; a 139 bp band

 

 

Figure 8 B2 locus; a 141 bp band

 

 

Figure 9 B4 locus; a 153 bp band

 

 

Figure 10 FecB locus; a 190 bp band

 

 

The G1 has a major effect on litter size in Baluchi sheep breed of Iran. The heterozygous and wild homozygous ewes had 0.35 and 0.21 more lambs than the homozygous mutant ewes, respectively (‎Moradband et al. 2011). Another report says this mutation leads to increased litter size in heterozygotes (1.56) compared with the wild homozygotes (1.25) in Chilota sheeps in Chile (‎Paz et al. 2015). However, some other studies did not report any association between this mutation and high fertility in sheep. Genotyping of G4 revealed that there was no recessive homozygote in all studied breeds.

 

Figure 11 Polymorphism in G1 locus

 

 

Figure 12 Polymorphism in G4 locus

 

 

Figure 13 Monomorphism in G7 locus

 

 

Figure 14 Monomorphism in G8 locus

 

 

The highest and lowest frequency of heterozygotes (GA) obtained in Taleshi and Shal breeds with 0.156 and 0.043, respectively. The mean frequency of heterozygotes in all animals was 0.115.

 

Figure 15 Monomorphism in B2 locus

 

 

Figure 16 Monomorphism in B4 locus

 

 

Figure 17 Polymorphism in FecB locus

 

 

The highest and lowest frequencies of the mutant allele (A) were observed in Taleshi and Shal breeds with 0.078 and 0.022 respectively. Mean frequency of the mutant allele in all animals was 0.057. This mutation has not been reported in Garole sheep of India (‎Polley et al. 2010) but has been identified in some other breeds around the world. More details are given in Table 14. For the first time, it had been reported that 13 of 29 Belclare sheep, and 1 of 26 Cambridge sheep, had at least one copy of the G4 mutation with no association to fertility (‎‎Hanrahan et al. 2004). In Greek Chios sheep breed, the highest litter size (2.17) was observed in heterozygotes and a significant positive dominance deviation as high as 0.61 lambs was obtained (Liandris et al. 2012).

 

Table 8 Frequencies of genotypes and alleles for G1 mutation

 

 

Table 9 Frequencies of genotypes and alleles for G4 mutation

 

 

Table 10 Frequencies of genotypes and alleles for FecB mutation

 

 

Likewise, another report from India, indicates that the average litter size was 1.63, 2.00 and 1.91 for wild homozygous, heterozygous and mutant homozygous of Kendrapada ewes, respectively (‎Dash et al. 2017). Genotyping of FecB revealed that this mutation only finds in Kalakuei and Zandi breeds. The frequencies of wild homozygote (AA), heterozygote (AG) and mutant homozygote (GG) obtained 0.809, 0.170 and 0.021 respectively, for Kalakuei breed and 0.949, 0.051 and 0.000 for Zandi breed. The frequencies of the wild and mutant alleles were 0.894 and 0.106 respectively, in Kalakuei and 0.974 and 0.026 in Zandi. Mean frequency of the mutant allele in all samples was calculated as 0.011. More details about presence and absence of this mutation in many sheep breeds has been brought in Table 15. As seen in the Table 15, all reports of the occurrence of this mutation in sheeps show a significant relationship between that and higher fertility traits, except for the Bonapala breed of India.

 

Table 11 Frequencies of genotypes and alleles for G7, G8, B2 and B4 mutations

 

 

Table 12 Hardy-Weinberg equilibrium in three polymorphic sites¹

 

* (P<0.05).

NS: non significant.

 

In this sheep, although the litter size in mutant homozygotes (1.71) was more than heterozygotes (1.64) and both were more than wild homozygotes (1.50), but the statistical analysis didn’t reveal a significant association (‎Roy et al. 2011). FecB mutation showed an additive effect on litter size in Hu sheep in China. Mutant homozygote and heterozygote ewes significantly have 0.713 and 0.419 more lambs than non carrier homozygotes, respectively (‎Wang et al. 2018). In an investigation, this mutation showed an additive effect on ovulation rate and partially dominant effect on litter size in Small Tailed Han sheep of China. In this study, mean of litter size in homozygote mutant and heterozygote ewes was 2.65 and 2.16 respectively, which were significantly more than 1.14 of wild homozygotes (‎Chu et al. 2011). Another report from China clarified a significant effect on litter size in Small Tailed Han, Poll Dorset and their F1 generation, in total (Jia et al. 2005). The ewes with BB and B+ genotypes had about 1.04 and 0.74 lambs more than wild non-carriers ewes. Also, the mean of litter size in BB ewes was significantly more than ++ ewes in both breeds. FecB mutation also showed a phenotypic effect on litter size in Indian Kendrapada sheep breed. The average litter size of non-carriers, heterozygotes and homozygote carriers were 1.61, 1.80 and 2.06 respectively (‎Dash et al. 2017). Another report suggests a highly significant difference in the average of litter size in different genotypes of Indonesian fat Tailed sheep (‎Maskur et al. 2016). Mutant homozygous ewes had the highest average (1.685) compared with heterozygotes (1.455) and wild types (1.145). In Iranian Kalehkoohi sheep, genotypes BB (1.902) and B+ (1.725) had 0.52 and 0.35 lambs, more than ++ type (1.379), respectively and that was a statistically significant difference (P<0.01). But no significant difference between BB and B+ genotypes were observed (‎Mahdavi et al. 2014). Reffering to the Table 15, it can be seen that all breeds that carry the FecB allele, are distributed in different parts of Asia (China, Indonesia, India and Iran). Most likely, the Merino Booroola breed in Australia also received this gene from the Indian Garole sheep in the 18^th century (‎‎Davis et al. 2002). The effect of FecB is additive for ovulation rate and can increase by about 1.6 corpora lutea per cycle for each copy in Booroola sheep (‎Wilson et al. 2001).

 

Table 13 The polymorphisms of G1 reported in some studies¹

 

¹ This table is arranged based on the frequency of the mutant allele, from the highest to the lowest.

SAWF: significant association with fecundity.

 

BMPRIB gene would be inactivated partially, leading to an advanced differentiation of granulosa cells and also an advanced maturation of ovulatory follicles in homozygous mutant ewes of Australian Booroola merino (‎Mulsant et al. 2001). Genotyping of G7 revealed that this SNP didn’t occur in any of these eleven breeds and all sheeps have dominant homozygote genotypes. Our report about the absence of this mutation is consistent with several reports of other sheep breeds and of course, is not compatible with a few reports of other breeds. More details about this mutation in several sheep breeds are given in Table 16. For the first time, It had been reported that at least one copy of G7 mutation presents in 2 of 19 Belclare and 7 of 24 Cambridge sheeps with no linkage to high fertility ‎(‎‎Hanrahan et al. 2004). A similar study reported that there was no statistical significant association between mutation and ovulation rate in Finnish Landrace sheep, although a significant association was observed in the Belclare sheep. Heterozygote ewes had higher ovulation than wild homozygotes (0.17) and ovulation rates in heterozygotes were significantly lower than mean of homozygotes (‎Mullen and Hanrahan et al. 2014).

 

Table 14 The polymorphisms of G4 reported in some studies¹

 

¹ This table is arranged based on the frequency of the mutant allele, from the highest to the lowest.

SAWF: significant association with fecundity.

 

Based on the estimated breeding values for the Norwegian White sheeps, daughters of homozygous mutant rams produce additional lambs (0.46 to 0.57) compared to daughters of wild homozygous rams. Also, daughters of heterozygous rams produce more lambs (0.20 to 0.25) than daughters of homozygous wild rams (Vage et al. 2013). Also mentioned that the average litter size for ewes at first year of age is about 2.061 and at second year is about 2.671, showing that the average litter size is higher at the second year and the genotype showed a significant effect on litter size at both years (‎Siddiqua, 2013). In Olkuska sheep, only the presence of one copy of mutant allele, resulted in an increase (0.55 lamb) in litter size. Heterozygous ewes demonstrated high annual lamb production of 346%, with the same for dominant homozygous ewes at 236% . It is clear from Table 16, that those breeds which have this mutation are natives of the northern Europe or raised in that region. Genotyping of G8 (FecG^H) revealed that this mutation didn’t occur in any of the studied breeds and all sheeps were dominant homozygotes. Our report about the absence of this mutation is not compatible with some reports of other sheep breeds but is consistent with many reports of other sheep breeds. More details are given in Table 17. It has been identified that this mutation is essential for folliculogenesis and results in high fertility in heterozygotes and infertility in mutant homozygotes in Cambridge and Belclare sheeps. Estimation of the effect of the mutation on the ovulation rate showed that Belclare ewes had an increase of about 1.79 and Cambridge ewes had about 2.35. Based on progeny test data in Belclare sheeps, it has been shown that G8 can increase the ovulation rate of about 0.83 in ewe lambs and 1.75 in adult ewes, which both were significant. Overall, the mutation has led to an increase in ovulation rate about 1.39 in both breeds ‎(‎‎Hanrahan et al. 2004). Also, another study on Chios breed in Greece reported a statistically positive dominance deviation (0.44 lambs) for this mutation in heterozygous ewes but with no evidence of infertility in homozygous mutant ewes (‎Liandris et al. 2012). It has been suggested that the Lleyn sheep breed is the most likely source of this SNP (and also FecX^G) in Belclare and Cambridge sheeps (‎Mullen et al. 2013). Genotyping of B2 (FecX^G) revealed that this mutation did not occur in any of eleven breeds and all animals were dominant homozygotes. More details are given in Table 18. In Cambridge and Belclare breeds, the homozygous ewes for this mutation and also, heterozygous ewes for this mutation and B4 simultaneously, were sterile. The mutation significantly increases ovulation rate about 0.77 in Belclare and 1.18 in Cambridge ewes. Based on progeny test data in Belclare sheep, it has been shown that the mutation increases the ovulation rate of about 0.62 in ewe lambs and 0.72 in adult ewes, which both were significant. Overall, this mutation leads to an increase in ovulation rate of about 0.70 in both breeds. The effects of this mutation with B4 was about 0.28 in the Belclare breed ‎(‎‎Hanrahan et al. 2004). A study on Small Tailed Han sheep showed that the polymorphism of this mutation was not significantly associated with litter size but tended to be associated with that trait. In Hu sheep, the heterozygous ewes had 0.52 more lambs than the wild genotype ones. In total population, the heterozygous Hu and Small Tailed Han ewes showed 0.30 more lambs than those with wild genotype (‎Wang et al. 2015). Also, based on a study, the heterozygous Small Tailed Han ewes had 0.55 more lambs than wild homozygotes in China (‎Chu et al. 2006). However, no significant allele effects were obtained in this mutation polymorphisms in Chios and Karagouniki breeds of Greece )‎Liandris et al. 2012(. It has been suggested that the Lleyn sheep breed is the most likely source of this mutation (and FecG^H) in Belclare and Cambridge sheeps (Mullen et al. 2013). Genotyping of B4 (FecX^B) revealed that this SNP was absent in all of the eleven breeds and all sheeps were dominant homozygotes. More details of this mutation in some studies are given in Table 19. This SNP has been identified in Belclare sheepbut not in Cambridge sheep.

 

Table 15 The polymorphisms of FecB reported in some studies¹

 

Continued Table 15

 

¹ This table is arranged based on the frequency of the mutant allele, from the highest to the lowest.

SAWF: significant association with fecundity.

 

Table 16 The polymorphisms of G7 reported in some studies¹

 

¹ This table is arranged based on the frequency of the mutant allele, from the highest to the lowest.

SAWF: significant association with fecundity.

 

The mutation significantly increases ovulation rate and its effect was estimated about 2.38. Homozygous mutant ewes and also heterozygous ewes for this mutation and B2 simultaneously, were sterile. Based on progeny test data in Belclare sheep, this SNP increases the ovulation rate of about 0.76 in ewe lambs and 1.11 in adult ewes, which both were significant. Overall, the mutation leads to an increase in ovulation rate of about 0.97 in both breeds. The effect of this mutation with B2 was about 0.28 in the Belclare breed ‎(‎‎Hanrahan et al. 2004). Based on some finding, the FecX^B mutation that found among the hyper-prolific ewes in Ireland, has come from a high fertility line that had developed with the use of prolific ewes that choosed from commercial flocks in that country and then used for the genesis of the Belclare sheep breed (‎Mullen et al. 2013). In Greek Chios and Karagouniki breeds, no significant allele effect weas obtained in the polymorphisms analysis of this mutation (‎Liandris et al. 2012). Survey of linkage between G1 and G4 indicates that all sheeps that were heterozygous by G1, were also heterozygous by G4. On the contrary, sheeps that were heterozygous for G4 did not necessarily had the G1. Hence, the frequency of the G4 was more than the G1. It seems that there is a local haplotype for these SNPs in most breeds. As it is shown in Table 20, Taleshi, Shal and Naeini breeds have the highest linkage and the Zandi breed shows the lowest linkage in the nucleotide position of two mutations. Considering all animals as a population, three local haplotypes were identified. A similar report indicates a high degree of linkage disequilibrium between these SNPs in Mehraban sheep breed that has been confirmed by the high correlation coefficient values (r²=0.91) (‎Talebi et al. 2018). Based on the observed polymorphism in the G1, G4 and FecB mutations of GDF9 and BMPR1B genes, all animals used in the present study were classified in six genotypic groups (Table 21).

 

Table 17 The polymorphisms of G8 (FecG^H) reported in some studies¹

 

¹ This table is arranged based on the frequency of the mutant allele, from the highest to the lowest.

SAWF: significant association with fecundity.

 

Table 18 The polymorphisms of B2 (FecX^G) reported in some studies¹

 

¹ This table is arranged based on the frequency of the mutant allele, from the highest to the lowest.

SAWF: significant association with fecundity.

 

Table 19 The polymorphisms of B4 (FecX^B) reported in some studies¹

 

¹ This table is arranged based on the frequency of the mutant allele, from the highest to the lowest.

SAWF: significant association with fecundity.

 

Table 20 G1 and G4 mutations and three observed local haplotypes

 

 

Table 21 Genotypic classification of Iranian sheep breeds based on three point mutations at GDF9 and BMPR1B genes

 

 

CONCLUSION

Fertility is largely influenced by environmental conditions. Having larger populations, minimizing environmental factors, accurate, multiple and seasonal recordings and repeatability estimation are factors that can better determine the association or disassociation between mutants and the reproductive potential of sheeps. In the present study, the presence or absence of seven reported SNPs known as G1, G4, G7 and G8 in GDF9 gene, B2 and B4 in BMP15 gene and FecB in BMPR1B gene were examined among eleven Iranian native sheep breeds that are different in litter size. The results showed that G7, G8, B2, and B4 mutations were not in any of these breeds, and all of the sheeps were monomorphic with dominant homozygote genotype. FecB in the two breeds of Kalakuei and Zandi, G1 in all breeds except the Zandi and G4 mutation in all eleven breeds were identified. The absence of reported major mutations (such as G8, B2, B4, and FecB), suggests that minor mutations of fecundity genes and their cumulative effects may have pivotal role in monitoring of fertility traits among Iranian sheeps. It is suggested that to achieve a deep insight on how to control the fertility characteristics of the native sheep in Iran, exploring the major genes controlling fecundity trait in Iranian indigenous sheep breeds is performed through whole genome sequencing and SNP calling approaches.

 

ACKNOWLEDGEMENT

The authors are grateful to the local farmers of the target provinces to provide the herd for sampling.

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