Variant GDF9 mRNA is likely not the main cause of larger litter size in Iranian Lori-Bakhtyari, Shal, Ghezel, and Afshari sheep breeds

Abstract. 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.