| 1.We generated a microsatellite-enriched genomic DNA library of Stichopus japonicus using the FIASCO method. Result showed that the enriched efficiency of the genomic DNA library was 56.3%. Of the sequenced clones, 110 microsatellite DNA were identified, among them 83 were perfect(70.4%), 30 were imperfect(25.4%) and 5 were compound(4.2%). Number of locus whose repeat number ranged from 5 to 9 was 88(73.5%), the number of locus repeat number more than 9 was 29(24.8). 84 pairs of Apostichopus japonicus microsatellite DNA markers were designed. Results showed that the FIASCO method can be well used to isolate microsatellite DNA from Apostichopus japonicus genomic DNA. These novel microsatellite DNA markers can be used as efficient molecular markers for population analysis, genetic linkage building, and marker-assisted selection in Apostichopus japonicus.2. Twenty pairs of primers were designed and used to investigate the polymorphism and genetic structure of wild Apostichopus japonicus collected from China(CH), Korea(KW, KE), Japan(JA) and Russia(RU), cross amplification of twenty loci were also tested in Parastichopus parvimensis which were collected in American(AM). The results demonstrated that all the markers were polymorphic and 16 loci had high polymorphism (PIC>0.5). The average observed and expected heterozygosities of the 20 loci were 0.39 and 0.69, respectively. In addition, a total of 231 alleles (102 effective alleles) were detected at 20 loci in the total sample. The Hardy-Weinberg equilibrium (HWE) analysis showed that all the loci deviated from genetic equilibrium to different extent in the six populations and heterozygote deficiency occurred in total samples (Fis>0). Genetic identity of the five Apostichopus japonicus populations was 0.71, while the genetic identity between Apostichopus japonicus and Parastichopus parvimensis were quiet low. In the UPMGA tree, CH and KW populations formed a cluster, RU, JA and KE populations formed another cluster, while AM felt into an independent cluster. The result of cluster analysis indicated that the clustering order had significiant correlation with geographic distance. The polymorphic microsatellite DNA markers can be used as efficient molecular markers for population structure analysis, genetic linkage building, and marker-assisted selection in Apostichopus japonicus. Besides, the genetic structure analysis results can be basic data in seed-breeding research.3.By using artificially infection, 15 disease-resistant Apostichopus japonicus samples were selected to compose the disease-resistant population, 15 susceptible samples were selected to compose the susceptible population. Twenty pairs of microsatellite primers were used to analyze those samples, afterχc2 analysis, we obtained 3 distinctive genetic markers with disease resistant trait and 3 distinctive markers with disease susceptible trait. Thirteen polymorphic primers were selected and used to analyze the genetic diversity and genetic structure in the two populations. Results showed that the two populations both had high level genetic diversity, and that the disease resistant population had a higher one than the disease susceptible population. Fst between the two populations was 0.0478, there were moderate genetic differentiation between two populations. The genetic distance was 0.1685, and the genetic identity index was 0.8449. These genetic markers can be useful in marker-assisted selection.4. Apostichopus japonicus(AJ) samples were in the breeding season, and were studied on the histologic level. The body wall of AJ was taken for control, the muscle tissue around the genital pores of male and female AJ were scanned by paraffin section. Finally, genital ducts were observed in both male and female pore area. Between the male and the female genital duct, we found difference in three aspects, for example, the tube diameter, the transaction shape, the organization structure around it. In coutrast, the diameter of genital pore in male AJ was much smaller, the transection shape was round or heart-shape, and no fibrous tissue arounded it; The diameter of genital pore in female AJ was larger, the transection shape was spindle, and some fibrous tissue arounded it. The discovery in this study can be useful basic data in research to find the sex determination, the formation of genital pore and sex differentiation mechanism. 5.AFLP technique was used to screen the sex-related DNA markers through analyzing DNA differences between male and female of sea cucumber(Apostichopus japonicus).96 pairs of AFLP primer were selected to detect the genome DNA of the male and female individuals by AFLP analysis. The results showed that the amplified results from electrophoresis were clear and rich. Totally 2753 bands were obtained, 212 of the loci showed apparently different distributions between female and male groups, the cluster analysis indicated that the female and male clustered respectively, no sex-specific loci was found. The data obtained by the study provided a base for the further research of sex determination in Apostichopus japonicus. |
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