Establishment And Application Of DNA Polymorphism Detection Method For Isolated Guinea Pigs And Rabbits

The closed colony animals have heterozygosity on the genetic composition and are widely used in pharmaceutical experiments and tests for biological products. But so far, genetics quality control standards for experimental animals are mainly for inbred animals, due to the lack of unified genetic test methods and standards for closed group animals, which leads to different results when using different batches of closed group animals and affects animals tests and inspection.The closed colony guinea pigs, such as commonly used Hartley guinea pigs, are widely used in various fields of biomedical research because of its particular biological characteristics and physiological and anatomical characteristics. New Zealand rabbits, Japanese white rabbits and Chinchilla rabbits which are commonly used in our country, are complex genetic background closed group animals. Due to the lack of effective genetic monitoring, genetic quality control of closed group animals is mainly in the breeding process, which leads to the actual genetic background and severely restrict the application of closed group animals. But genetic monitoring is an essential measure to ensure and evaluate the genetic quality of closed colony animals. In this study, based on population genetics theory, we established microsatellite marker detection method for closed colony guinea pigs genetic monitoring by using polymorphism microsatellite markers obtained before, application of the method to analyze the population genetics of closed colony guinea pigs from two institutions; established SNP genetic monitoring method for closed colony rabbits, application of the method to analyze the population genetics of three closed group of rabbits to provide a new genetic test method for genetic quality control of experimental animals.The study is divided into two parts: 1.Establishing microsatellite DNA markers genetic testing method for closed colony guinea pigs and preliminary applicationFirstly, Comparison of three methods(fresh anticoagulant Na I manual method, Blood clot manual method and DNA extraction kit method) for blood genomic DNA extraction provide a suitable DNA extration method for the follow-up experiment. 40 guinea pigs microsatellite loci with polymorphisms were selected from the related references, 25 microsatellite loci with a clear amplification band and high polymorphism were chosen by optimization of PCR condition, 3% agarose gel electrophoresis and STR scanning. Secondly, using the 25 polymorphic microsatellite markers to detect genetic quality of the two group of guinea pigs, and the population genetic parameters were 5 calculated, including the average observed number of alleles, average expected heterozygosity, average polymorphism information content, Hardy Weinberg Equilibrium(HWE) test and heterozygote deficiency test. The results show that the average observed number of alleles of colony A and colony B were 4.12 and 4.64; The average effective number of alleles were 2.4232 and 2.7947, respectively; The average observed heterozygosity was 0.4467 and 0.5062, respectively; The average expected heterozygosity was 0.5195 and 0.5838, respectively; The average polymorphism information content was 0.459 and 0.518, respectively; Five loci and six loci showed significant deviation from Hardy Weinberg Equilibrium(P<0.05) in the two populations, respectively; The majority of loci which deviated from Hardy Weinberg Equilibrium showed significant heterozygote deficiency(P<0.05) in the two populations; The average Fst of all loci was 0.1056, which implied genetic variation between populations account for 10.56% of the total genetic variation; Nei’(1972) genetic distance and Nei ’(1978) unbiased genetic distance between the two populations were 0.3302 and 0.3204, respectively. The results show that genetic diversity of colony B is slightly higher than colony A. 2. Establishing SNP genetic testing method for closed colony rabbits and preliminary applicationEstablished genetic detection methods by direct sequencing of closed colony rabbits, and selected rich polymorphism regions to design primers, and then aligned sequence after direct sequenced tatget fragment. The results showed 9 polymorphic SNP loci were obtained; established high-fidelity enzyme specific SNP detection methods by using Pfu DNA polymerase extend phosphorothioate modified primers in a single tube bidirectional allele specific amplification, 20 loci with a clear amplification band were chosen, including 7 polymorphic loci; the last, using 29 SNP loci to detect genetic diversity of Japanese white rabbits, New Zealand rabbits and Chinchilla rabbits, and the population genetic parameters were calculated, including the average observed heterozygosity, average expected heterozygosity, average polymorphism information content. The average expected heterozygosity were 0.1453, 0.1696 and 0.1731, the average polymorphism information content were 0.1152, 0.1313 and 0.1354, respectively. The degree of the genetic varation of the 3 groups is Chinchilla rabbits >New Zealand rabbits >Japanese white rabbits, these results are identical with that of previously reported microsatellite method, but the population genetic parameters are lower than those got by microsatellite method.In conclusion, this study finally choosed fresh anticoagulant Na I manual method to extract blood genomic DNA; Established a microsatellite DNA genetic quality test method of guinea pigs. Test results of 2 guinea pigs closed colony showed that the 2 populations were in line with the population genetic characteristics of closed colony, but loci of individual deviated from genetic equilibrium, which speculated that there was a certain degree of inbreeding phenomenon in the process of feeding and breeding; established a rabbits SNP genetic quality test method by using direct sequencing technique and high fidelity enzyme specific detection technique and then use this method to analysis genetic quality of 3 rabbit closed colonies. The results showed consistent with the results of previously reported microsatellite which verified the validity of established SNP methods. These studies provide a basis for the establishment of genetic detection methods and standards of guinea pigs and rabbits, it’s important for improving the detection level of experimental animals.