Abstract: The purpose of this study was to verify the accuracy of specific SNPs of red deer based on genotyping sequencing (GBS) technology,and provide a reliable basis for the identification of sika deer,red deer,and their hybrid offspring.Thirty specific SNPs of red deer were randomly selected,based on the sequences of 200 bp each before and after the SNPs,Primer Premier 6.0 software was used to design specific primers,and the randomly selected DNA of the validation samples was used as a template for amplification,PCR was performed,and the amplified products were sequenced in one generation.The sequences obtained by sequencing were analyzed by Mega 6.0 software,and the genotypes of each SNP site in different validation populations were observed.The results showed that 28 out of 30 specific loci were consistent with the results of the previous study,and the gene frequency of the G allele at one of SNPs (SNP3) was 0.05 in sika deer,while the gene frequency of the G allele was 1 in red deer.The loci showed significant differences in genotype distribution between red deer and sika deer (P<0.05).The gene frequency of the T allele in the other SNP (SNP5) was 0.15 in sika deer,while the gene frequency of the T allele was 1 in red deer,and the genotype distribution of this SNP in sika deer and red deer was significantly different (P<0.05).So these two loci could still be used as specific SNPs of red deer.The results showed that the specific SNPs of red deer selected by GBS sequencing could be used as molecular markers for identification,and laid a theoretical foundation for the identification of sika deer,red deer and their hybrid offspring.

Key words: red; deer:specific; SNPs:Sanger; sequencing:validation