Screening And Typing Of Tahe Red Deer-specific SNP Sites Based On Resequencing Technology

Tahe red deer species are precious genetic resources in China and an important component of the genetic diversity of deer species.Due to the lack of safety awareness of the protection of red deer genetic resources,the phenomenon of crossbreeding is serious,and the genetic resources of Tahe red deer are seriously lost.In order to rescue the collection and safe preservation of Tahe red deer resources,analyze the unique biological characteristics of Tahe red deer.In this study,the whole genome of 32 blood DNA samples of Tahe red deer collected by the National Special Economic Animal Resource Sharing Platform in 1999 were resequenced,combined with the red deer resequencing data obtained earlier,All of the data was mapped to the chromosome-level reference genome of sika deer,to screen high quality single nucleotide polymorphic sites(SNP)of Tahe red deer,construct specific molecular genetic markers,provide reference for pure breed identification of Tahe red deer.The results of the study are as follows:1.The results of whole-genome resequencing showed that the effective data volume of the blood genomic DNA of 32 Tahe red deer that passed the quality inspection was 868 791 354600 bp,and the sequencing quality met the requirements of subsequent data analysis.The average mapping rate of the 32 samples sequencing data was 98.06%,the average coverage was 97.66%,and the average depth was 6.787×.After filtering,20 139 122 high-quality SNP were obtained,of which SNP were the most distributed on chromosome 4,and more than 90%of the mutations were located in the intergenic and exon regions.The number of candidate specific SNP sites was 12 050 781.544 717 after establishment selected by VCFTOOLS.The top 1 500 SNP of each chromosome were selected for PCA,the PCA results showed that when the number of SNP decreased from 49 500 to 100,the discrimination efficiency was not decreased.Finally,100 SNP with strong specificity and high stability were screened out.2.The specific SNP sites obtained by screening were used to genotype 210 Tahe red deer samples(30 antler-shaped standard Tahe red deer and 180 female deer)based on multiplex PCR technology,the detection rate of SNP sites is more than 95%,deer range between 2 to 3groups,locus detection rate and typing was well.3.94 randomly selected Tahe red deer and 20 hybrid deer samples were genotyped and verified by KASP typing platform,3 SNP sites selected for conversion into KASP markers based on typing data.The results showed that the sites were successfully amplified in most samples.Among the three KASP markers,most of the genotypes of Tahe red deer were homozygous reference types(AA genotype for SNP1,AA genotype for SNP2 and CC genotype for SNP3),the genotyping results of hybrid deer at SNP2 showed that the proportion of heterozygous and homozygous variants was significantly higher than that of the homozygous reference type.In summary,100 Tahe red deer-specific SNP were obtained through whole-genome resequencing technology and bioinformatics analysis,based on multiplex PCR technology and KASP typing,specific designed primers can amplify normally in Tahe red deer population,by calculating the IBS(Identity by State)genetic distance matrix for cluster analysis,the genetic relationship between red deer individuals is inferred,which build the foundation for the identification of the provenance of the red deer in the Tahe River,the establishment of core germplasm resources,and the selection and breeding of red deer.