Development And Validation Of A Novel 13-plex STR System For Parentage Testing And Genetic Analysis In Donkeys (Equus Asinus)

Short tandem repeats（STRs）tend to be the most important molecular markers in as-sessing the genetic diversity of populations and the paternity testing,which could help people to conserve breeds or select the evolution direction for donkeys.Previous studies investigat-ing donkey parentage and genetic diversity used horse-specific multiplex systems.However,several low amplification efficiency and poor specificity issues have found with some of the horse primers when used in the donkey.In 2017,the International Society for Animal Genet-ics（ISAG）recommended the use of 13 autosomal di-nucleotide STRs（AHT4,ASB23,HMS2,HMS3,HMS6,HMS7,HMS18,HTG7,HTG10,TKY297,TKY312,TKY337 and TKY343loci）for individual identification and parentage testing in donkeys.To date,no single multi-plex STR typing system containing all 13 donkey STRs recommended by the ISAG has been reported.We have established a novel and donkey-specific multiplex STR typing system containing all 13 recommended STRs via redesigning the partial primers,optimizing the reac-tion system and conditions of this system.（1）This system can efficiently obtain excellent genotype profiles via using the low-cost polymerase,which the PCR reaction can complete within 73 minutes.The optimal volume for our system was determined to be 15μL.The sequencing of STR amplicons has became fast and simple with the primers labeled with fluorescent dyes.（2）The sensitivity of this system was tested.A complete and accurate genotyping profile could be obtained from as little as 5 ng of DNA template.（3）The species-specificity of this system was tested.No full genotype profile generated with these primers when DNA from humans or 12 other species used.（4）This study provides an allele collection table and stutter analysis for donkeys in the first time,which should help researchers obtain accurate results for STRs genotyping in don-keys.（5）In this study,we calculated the population statistical parameters values for the 150donkeys.The high heterozygosity and polymorphic information content indicated that this system applied to genetic diversity for donkeys.The results of CPE_trio was 0.999665018.Our results indicate that this donkey-specific 13-plex STR typing system is fast,high-efficiency,sensitive,species-specific,and robust for individual identification,paternity testing,and population genetic analysis in donkeys,and has potential in forensic applications.