The Establishment And Preliminary Application Of A Genetic Detection Method For Experimental Cat SNP Loci And Microsatellite Markers

Although cats were used as experimental animals in biomedical research,pharmaceutical industry and other fields as early as the end of the 19 th century,there have been few reports on the genetic quality of experimental cats.Establishing appropriate genetic testing methods and establishing relevant standards is essential to ensure the quality of experimental cats and the accuracy of experimental data.However,China has not yet issued a national standard for testing the genetic quality of experimental cats,and there is no a local standard for the genetic quality of experimental cats in any place.With the increase of the use of experimental cats by universities,pharmaceutical manufacturers and verification institutions,it is extremely urgent to establish experimental cat genetic quality standards and genetic quality testing methods in line with China's current research level and application requirements.This study established two methods for detecting the genetic quality of experimental cats: The first is to establish a STR markers genetic test method for experimental cats.This study obtained 74 candidate microsatellite loci by searching the GeneBank database and referring to relevant literatures.In this experiment,agarose gel electrophoresis and STR scan results were used as the basis for screening microsatellites.A total of 41 microsatellite loci with good amplification and high polymorphism were obtained.Further,considering the distribution of these loci on cat chromosome,one or two polymorphic loci were appropriately selected on the autosomes of the cat(except from the A1 and B1 chromosomes).Finally,31 STR loci were screened for detecting the genetic quality of experimental cats.The genetic structure of three experimental cat populations(Siamese cat,Domestic cat,Orange tabby cat)was analyzed initially using 31 microsatellite loci.In this study,the average heterozygosity of Siamese cats,domestic cats,and Orange tabby cats was 0.655,0.7516,and 0.5474,respectively.The results showed that Siamese cats and Orange tabby cats had a close relationship among the groups,and they were consistent with the genetic characteristics of closed animals.Domestic cats had a far relative relationship and became wild.Except from the PIC values of the Orange tabby cats at FCA736,FCA920 and FCA987,the PIC values of other microsatellite loci selected in this study were all greater than 0.25,belonging to moderate or highly polymorphic loci.The average polymorphism information content of Siamese cats,Domestic cats,and Orange tabby cats was 0.6081,0.7174,and 0.4957,respectively.The STR locus provided highly abundant genetic information in three populations.The average inter-group differentiation coefficient of the three cat populations at 31 microsatellite loci was 0.167.That is,the genetic variation among the populations accounted for 16.7% of the total genetic variation,indicating that there was a higher genetic differentiation among the three populations.The genetic distance between Siamese cat and Orange tabby cat is 1.0415;the genetic distance between Orange tabby cat and Domestic cat is 0.8459;the genetic distance between Siamese cat and Domestic cat is 0.5202,which is closely related..The second is to establish a SNP markers genetic quality test method for experimental cat.Based on the base sequence of 340 SNP loci,the SNP information was retrieved using the Ensemble database,and the primers were designed by Beacon Designer 7 software.Finally,78 candidate SNP loci with RS numbers and meeting PCR-HRM requirements were obtained.Then,HRM genotyping was performed on some samples with 78 SNPs,and 27 SNPs with good polymorphism and significant typing were screened.The genetic structure of three experimental cat populations(Siamese cat,Domestic cat,Orange tabby cat)was analyzed initially using 27 SNP loci.The PIC values of SNPs in this experiment were all less than 0.05,and none of them belonged to highly polymorphic loci.The average polymorphic information content of Siamese cats,Domestic cats and Orange tabby cats was 0.2901,0.3186 and 0.3345.The average heterozygosity of Siamese cats,Domestic cats and Orange tabby cats was 0.3695,0.4083,and 0.4309,respectively.SNP loci provide moderately rich genetic information in three populations,and every cat population has a close genetic relationship and tends to be inbred.The results are inconsistent with the trend of the results measured by STR.Whether the results of the two methods are relevant are still to be further verified.The genetic distance between Siamese cat and Orange tabby cat is 0.2489;the distance between Orange tabby cat and Domestic cat is 0.1223;the distance between Siamese cat and Domestic cat is 0.0717.The genetic relationship is similar to that of STR.Siamese cats and Orange tabby cats have the longest genetic distance,followed by the genetic distance between Domestic cats and Orange tabby cats.The genetic distance between Siamese cats and domestic cats is the closest.When detecting the same genetic parameters of the same population,it was found that the results measured by SNP were lower than those obtained by STR.The reason may be that the SNPs polymorphism screened in this experiment are not rich enough.In summary,31 microsatellite loci and 27 SNP loci were successfully screened in this study,and the STR and SNP methods for genetic quality testing of cat population were established.The two methods were used to analyze the genetic structure and genetic relationship of three experimental cat populations of Siamese cat,Domestic cat and Orange tabby cat The research results provide data support for the development of genetic quality control standards for experimental cats in China.