Abstract: [Objective] To explore the single nucleotide polymorphisms （SNPs） in the promoter region and the first exon of the growth differentiation factor 9（GDF9） gene in Sonid ewes and its association with litter size traits, and to screen molecular markers related to high fecundity of Sonid ewes, aiming to provide data support for genetic improvement of reproductive traits of Sonid ewes, molecular marker-assisted breeding and improvement of large-scale breeding efficiency of mutton sheep. [Methods] A total of 95 healthy Sonid ewes were selected as experimental animals, including 48 single-lambing individuals and 47 twin-lambing individuals. Blood samples were collected and total DNA was extracted using a blood genomic DNA extraction kit. Specific PCR primers were designed according to the GDF9 gene sequence of sheep. The promoter region and the first exon region of GDF9 gene were amplified using PCR, and the genotyping was performed using Sanger sequencing. MegAlign and Chromas software were used for sequence assembly, alignment and genotype determination. The genotype frequency and allele frequency of each mutation site were counted, and the population genetic parameters （homozygosity Ho, heterozygosity He, effective number of alleles Ne, polymorphism information content PIC） were calculated. Chi-square test was used to analyze the Hardy-Weinberg equilibrium state of each locus, and the association between GDF9 gene polymorphisms and litter size traits of Sonid ewes was analyzed. [Results] Five SNP polymorphic loci were successfully detected in the GDF9 gene of Sonid ewes, including chr5: 41769149, chr5: 41768729, chr5: 41768538, chr5: 41768521 and chr5: 41768463, respectively. The results of population genetics analysis showed that the polymorphic information content （PIC） of chr5: 41769149, chr5: 41768538, chr5: 41768521 and chr5: 41768463 was less than 0.25, which were low polymorphic loci; the PIC value of chr5: 41768729 locus was between 0.25 and 0.5, which was a moderately polymorphic locus. Chi-square equilibrium test showed that the loci chr5: 41768521 and chr5: 41768729 significantly deviated from Hardy-Weinberg equilibrium （P<0.05）, while the remaining three loci were in equilibrium （P>0.05）. Trait association analysis showed that the dominant genotype of chr5: 41768521 was CC and the dominant allele was C, and the allele frequency of twin-lambing ewes （0.93） was significantly higher than that of single-lambing ewes （0.84）. The dominant genotype of chr5: 41768729 locus was AA and the dominant allele was A, and the allele frequency of twin-lambing ewes （0.84） was significantly higher than that of single-lambing ewes （0.73）. The polymorphisms of the above two loci were significantly associated with litter size traits of Sonid ewes and the remaining three loci were not significantly associated with litter size traits. [Conclusion] The polymorphisms of chr5: 41768521 and chr5: 41768729 loci of GDF9 gene in Sonid ewes are significantly associated with litter size traits, which can be used as effective molecular markers for the breeding of high fecundity traits in Sonid ewes. The results enrich the molecular genetic research of sheep reproductive traits, and provide a reference for the improvement of reproductive performance of Sonid ewes, the construction of molecular breeding system and the high-quality development of mutton sheep industry.

Key words: Sonid ewes, GDF9 gene, litter size traits, SNP polymorphism, molecular markers