Study On The Genetic Monitoring And Its Methods Of Silkworm Inbred Strains

Silkworm (Bombyx mori), which is an important economical insect, has been used as an experimental animal for hundreds years. It has a clear genetic background and rich variety of mutant characters. And it has the genetic characteristics to be an experimental animal such as small size, short breeding cycle, high reproductive coefficient and easy to breeding. Breeding high-inbreeding animals can not only decrease experimental animal amount and experimental repetition times, but also improve accuracy, comparability and repetition of experimental result. The remarkable characteristics of experimental animals are gene purity and gene stability. There are probabilities of genetic variation or genetic contamination during breeding, breeding conservation and production. Therefore, a strict and timed genetic monitoring for inbred strains is needed. The research of highly inbreeding in silkworm, which is also the purpose of the breeding of the ideal experimental animals, is still in its infancy. Thus, the genetic testing of inbred strains is also a part of the inbreeding development job. The national standards for experimental animals ruled that the skin grafting, immune marker of gene detection and biochemical maker of gene detection can be used to monitor conventionally. With the rapid development of molecular biology techniques in recent years, we can monitor the change of nucleic acid in animal genome directly. It provides a direct and objective way for the silkworm inbred strains of the genetic monitoring.RAPD and/or SSR methods were used to monitor the genetic purities of full-sib mating inbred stains BmIS-C108, BmIS-Dazao and 28 congenic inbred strains in this study. We hoped to explore and establish a technical system of monitoring the genetic purity of silkworm inbred strains initially, and make a foundation for the related researches, which the silkworm would be used as experimental animals and model organisms. The main results are as follows.1. RAPD monitoring of inbred strain C108 in Bombyx mori28 random primers and RAPD-PCR technique were used to monitor the genetic purities of the 20th generation of full-sib mating inbred strain C108, which were three batches. Thirty individuals were selected in each of IS-C108₂₀-1, IS-C108₂₀-2, and IS-C108₂₀-3. The strain 19-100 was the parents of inbred strain C108 and used as the control.183, 182, and 182 loci were amplified in each batch of the inbred strain C108, which had 7, 5, and 5 polymorphic bands, respectively. The frequency of polymorphic bands in the three batches of the inbred strain C108 were 3.825%, 2.747% and 2.747%, respectively, and the average was 3.106%. The frequency of polymorphic bands was 6.486% between batches of the inbred strain C108, which was lower than the frequency of polymorphic bands in parents 19-100 (8.995%). The frequency of polymorphic bands was 22.751% between inbred strain C108 and 19-100. The genetic purities in the three batches IS-C108₂₀-l, IS-C108₂₀-2 and IS-C108₂₀-3 were 0.9976, 0.9982, and 0.9982, the average was 0.9980, while the parent 19-100 was 0.8994.2. SSR monitoring of inbred strain C108 in Bombyx moriSSR-PCR technique was used to monitoring the aforesaid materials.28 pairs of SSR primers were belonged to 28 linkage groups in SSR linkage map of the silkworm. 61 loci and one polymorphic band were amplified in each batch of inbred strain C108, and the frequency of polymorphic bands was 6.849%. The frequency of polymorphic bands was 3.279% between batches of the inbred strain C108, while it was 20.548% between inbred strain C108 and 19-100. The Ne, Ho, He, and D were all 2.179, 0.016, 0.016, and 0 among the three batches of IS-C108,and they were 2.964, 0.068, 0.046, and 0.478 in 19-100.The Hardy-Weinberg balance deflection index among the three batches of IS-C108 was 0, it revealed that the genotype distribution of IS-C108 was in balance condition. And it was 0.478 in 19-100, which indicated that the heterozygote in 19-100 was luxuriant.The genetic purities in the three batches IS-C108₂₀-1, IS-C108₂₀-2 and IS-C108₂₀-3 were 0.9984, 0.9989, and 0.9984, its average was 0.9986. While the genetic purity in the 19-100 was only 0.9185.3. SSR monitoring of inbred strain Dazao in Bombyx mori20 pairs of SSR primers were used to monitor the genetic purities of the 20th generation of full-sib mating inbred strain Dazao. Thirty individuals were selected in each of the IS-Dazao₂₀-1, IS-Dazao₂₀-2 and IS-Dazao₂₀-3. The strain 19-200 was the parents of inbred strain Dazao and used as the control. 51 loci and one polymorphic band were amplified in each batch of inbred strain Dazao, and the frequency of polymorphic bands was 1.961%. The frequency of polymorphic bands was 3.846% among the batches of the inbred strain C108, which it was equal to the frequency of polymorphic bands in 19-200. The Ne, Ho, He, and D were 2.550, 0.020, 0.020, and 0, their genetic purities were 0.9993, 0.9987, and 0.9993, respectively, which the average was 0.9991 in three batches of inbred strain Dazao. For the 19-200 strain, the Ne, Ho, He, D, and genetic purity were 2.600, 0.038, 0.038, 0,and 0.9980, respectively.The results indicated that the genotype distribution of inbred strain Dazao and its parents 19-200 was in balance condition.The average genetic purity of inbred strain Dazao was 99.91% which was very high.4. SSR monitoring of congenic inbred strains in Bombyx moriSSR-PCR technique was used to monitoring 28 congenic inbred strains in Bombyx mori. Twenty-eight pairs of SSR primers were belonged to 28 linkage groups in SSR linkage map of the silkworm. The result is that genetic similarity coefficient was all 100%.The result indicated that the genetic construction of congenic inbred strains was similar with the parents through more than the 20th generation back cross mating except for destination gene. At the same time, the genotype and genetic purity of Dazao K congenic inbred strain was monitored, the experimental value was 100%. The result indicated that the genetic purity of congenic inbred strains was very high.5. Comparison of two molecular techniques in monitoring the same inbred strain.RAPD and SSR molecular techniques were used to monitor the genetic purity ofthe 20th generation of full-sib mating inbred strain C108. Polymorphic level: the amplified bands and the frequency of polymorphic band between each batch and each strain of RAPD molecular technique was higher than SSR molecular technique. Genetic purity: the average genetic purity was 0.9980 by RAPD, and it was 0.9986 by SSR, the difference was only 0.0006.The result revealed that the genetic purity of inbred strain C108 was higher than the expectation value, 0.99, which was the similar with the theoretical anticipation of inbred strain during 20 generations full-sib mating. It also explained that the two molecular techniques were suitable for genetic monitoring in inbred strain of the silkworm. By comparison, one random primer can monitor several loci. so at the same experimental size, the circumscription of monitored loci cover genome was more widespread. The PAPD molecular technique was an economical and convenient method. Meanwhile, SSR molecular technique was an ideal monitoring method, which had many good qualities such as monitoring one locus, stabile results, convenient operation. The experimental reproducibility of SSR was better than RAPD. The experimental flow-sheet of SSR and RAPD was similar.