Screening for Causative Mutations of Major Prolificacy Genes in Iranian Fat-Tailed Sheep

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 (BMPR1B) or FecB, bone morphogenetic protein 15 (BMP15) or FecX and growth differentiation factor 9 (GDF9) or FecG, belong to the transforming growth factor-beta (TGF-â) superfamily, located on ovine chromosomes 6, X and 5, respectively, and they affect the prolificacy (2).

Different causative mutations in the exon 8 of BMPR1B (FecB), both exons 1 and 2 of the BMP15 (FecX^G, FecX^B, FecX^I, FecX^H, FecX^L, FecX^R, FecX^O, FecX^Gr) and GDF9 (FecGH, FecGT, FecGE, FecGNW) with major effects on ovulation rate and litter size have thus far been distinguished in various sheep breeds around the world (3). In this regard, the importance of the BMP system as an intraovarian regulator of follicular growth and maturation has been described (4, 5).

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 BMPR1B, BMP15 and GDF9 genes affecting reproductive performance in Lori-Bakhtiari sheep.

Considering the hotspot regions involved in prolificacy of sheep, selection of a limited number of high prolific ewes and sequencing of their BMPR1B exon 8, in addition to the both exons 1 and 2 of BMP15 and GDF9 genes could be contemplated as an ideal approach to find related causative mutations. This approach would be less expensive than other methods and may lead to find new causative mutations in the indicated fragments. If this approach does not detect any important mutation, genome-wide association study (GWAS) implications would be a more appropriated method to determine causative mutations or candidate genes affecting prolificacy in the other parts of genome.

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 BMPR1B gene, located on chromosome 8 (9). This mutation has also been reported in Indian Garole and Kendrapada sheep breeds (11) as well as Chinese sheep breeds, Small Tail Han and Hu (12). The FecB was also found in Kalehkoohi sheep, as an Iranian breed (13).

In the present study, analysis of the sequences for BMPR1B gene exon 8 did not show the FecB alteration, as a mutation coordinating with increase of litter size and ovulation rate in the sheep. However, this analysis showed a new transition of g.66496G>A, compared to sequences reported for Garole (GenBank: GQ863576.1) and Booroola merino sheep, which is very close to the position of FecB mutation. This mutation causes a synonymous substitution which does not alter amino acid sequences. Western blot and qRT-PCR techniques could in future be used to verify possible effects of this SNP on translation and transcription aspects respectively. Moreover, linkage disequilibrium of this SNP with variants in other loci should be considered and investigated (14).

Eight different causative mutations in ovine BMP15 gene with major effects on ovulation rate and litter size have previously been reported in literature (3). Hence, the BMP15 gene could be considered as the most polymorphic locus among the major genes affecting prolificacy in sheep. As a case, evidences showed that a nonsense mutation, due to deletion of 17 bp nucleotides, altered the amino acid sequence and introduced a premature stop codon in this protein (15).

In the present study, sequencing of the exon 1 of BMP15 gene showed 3 bp nucleotides deletion (g.656_658delTTC) in one sample, whereby nine sheep had two leucine codons at this position while this sheep showed heterozygote mutation. This mutation has been reported for the first time in Cambridge and Belclare sheep, without any determined significant effect on prolificacy (10). However, because of low frequency of this deletion and missing the infertile or singleton-bearing ewes, investigation of this aberration was not feasible in the present study. Further investigations demonstrated no polymorphism in the exon 2 of BMP15 gene.

Hanrahan et al. (10) reported eight point mutations (i.e. G1-G8) in the GDF9 gene of Cambridge and Belclare sheep, five of which deduced amino acid changes (G1: p.87Arg>His; G4: p.241Glu>Lys; G6: p.332Val>Ile; G7: p.371Val>Met and G8: p.395Ser>Phe), while only one of them (G8; also known as FecGH) had additive effects on prolificacy. The first mutation (G1: g.2118G>A) was also reported to associate with an increased ovulation rate and litter size in some sheep breeds (16, 17). In the present study, the mutations of G1 (g.2118G>A), G2 (g.3451T>C), G3 (g.3457A>G) and G4 (g.3701G>A) were identified in exons 1 and 2 of the GDF9 gene. With exception for G1 mutation, which had an additive effect (16, 17), other mutations (G2, G3 and G4) did not carry any significant effect on litter size in some sheep breeds (18). The impacts of amino acid substitutions for two non-synonymous mutations (G1: p.87Arg>His and G4: p.241Glu>Lys) were also benign with scores of 0.00. In fact, the phenotypic expression of an allele, to some extent, depends on other alleles, mainly multiple interacting mutations, and thus, a phenotypic effect of an allele may be observed in one breed while being absent in the other (3, 19). For instance, a GWAS analysis showed a missense mutation in the GDF9 (FecG^NW) with strong association with litter size in Norwegian white sheep (20). This mutation, known as G7, has also been identified in Belclare and Cambridge sheep as well as G1, but without any phenotypic effects (10). Hence, more studies are still needed to determine the precise effects of this kind of mutations and epistatic effects evaluation.

Regarding the absence of known main causative mutations of BMPR1B, BMP15 and GDF9 genes in the studied population, high prolificacy could be attributed to the other genetic factors not studied here. Thus, seeking for other related major genes, such as a regulatory mutation in intron 7 of B4GALNT2 (Fec^L) largely affecting the respective gene expression in prolific ewes and most likely taking responsibility for high prolificacy in Lacaune sheep (1), could be considered as an interesting subject for similar studies in the future. However, regarding the polygenic control of reproductive traits, GWAS and evaluation of linkage disequilibrium with other mutations or transcriptome analysis are also necessary to understand the precise underlying pathway(s) of prolificacy in Lori-Bakhtiari sheep.

We can state that major prolificacy genes (i.e. BMPR1B, BMP15 and GDF9) were polymorphic in the studied population of Lori-Bakhtiari sheep, but none of the previously identified mutations contributing to prolificacy was detected in the studies genes. Moreover, no clear statistical association was determined among the observed polymorphisms and prolificacy. More studies on other candidate genes or use of high throughput methods such as GWAS are necessary in the future studies.