Identification of Estrogen Receptor 2 Gene Variants and its Association with LitterSize in Lori Bakhtiari Ewes × Romanov Rams Crossbred

Litter size is a key economic trait in sheep, significantly influencing the reproductive efficiency of flocks. This study aimed to refine the genetic understanding of the ESR2 gene using a combination of ARMS-PCR and PCR-sequencing methods in a cross between Lori Bakhtiari ewes and Romanov rams. The primary objective was to identify beneficial mutations in the ESR2 gene associated with litter size in these crossbred lambs. A total of 114 whole blood samples were collected from the crossbred population. The study was conducted in two phases. Initially, a 500-bp region of the ESR2 gene was amplified via PCR, followed by molecular analysis using ARMS-PCR and direct sequencing according to standard laboratory protocols. Genotypic and allelic frequencies were analyzed using software tools such as POPGene, FinchTV, and MAFFT to confirm the presence of mutations and assess alignment consistency. For the first time, two specific primer sets were designed to amplify overlapping regions of the ESR2 gene. Two distinct bands (500 bp and 300 bp) were successfully observed, and the larger band was sequenced. Sequencing data were aligned using MAFFT software, confirming the presence of a nucleotide mutation, g.73383249T>C, in the intergenic variant region of the ESR2 gene. This mutation involves a transition from the wild-type T allele to the mutant C allele, previously associated with litter size in sheep. The study achieved two major outcomes: (1) the successful conversion of a GWAS-reported marker into a simple PCR-sequencing marker and (2) the development of specific primers enabling the detection of the mutation without the need for PCR sequencing. Genotypic analysis revealed frequencies of 0.74 for the TT genotype and 0.26 for the TC genotype. A significant association was found between genotype and multiple births (P < 0.001). The additive substitution effect of the C allele for the T allele on litter size was estimated at 0.904 ± 0.0771 (P < 0.001). In conclusion, the use of this marker for pre-selection of Romanov rams carrying the CC allele associated with increased litter size represents a practical strategy for improving reproductive performance in sheep. This approach could help farmers increase economic returns by enhancing lambing rates.