Establishment Of Molecular Identification Methed Of Releasing-recapture Individuals For Siganus Guttatus And Siganus Oramin

In recent years,China has carried out a large number of activities of Enhancement and Releasing.But,In the process of evaluation of the effect of releasing the proliferation of marine life,a key problem,that was how to accurately identify artificial released individuals and wild individuals.Genetic identification technology,as an effective identification method,had its unique advantage.The microsatellite markers,as a kind of genetic identification technology,had wide range application.In this paper,we used Siganus guttatus and Siganus oramin as model organisms,developed and screened microsatellite markers.At the same time,established the research of genetic identification techniques of releasing groups and wild populations,in order to establish theoretical basis in the future research for Siganus guttatus and Siganus oramin.This paper described the content as follows:（1）The development and characterization of microsatellite markers.In the process of building a microsatellite library,we obtained 1116 sequences of Siganus guttatus by using the"high-throughput sequencing method".PCR primer pairs were designed to amplify 167 microsatellite loci with suitable flanking regions.We use a wild population（N=38）to screen and test those primers.20 loci were reliably amplified and showed higher polymorphism.Finally,14 microsatellite markers were selected as a"molecular marker group".Among these 14 microsatellite loci,the numbers of alleles per locus ranged from 6 to 14,with an average of 9.5.The observed and expected heterozygosities ranged from 0.676 to 0.892 and from 0.671 to 0.912,with an average of0.803,respectively.PIC ranged from 0.620 to 0.891,with an average of 0.767.All the markers are in accordance with the"Hardy-Weinberg"balance test,and there is no chain imbalance between the markers.Two thousand and seven hundred microsatellite sequences of Siganus oramin were obtained.PCR primer pairs were designed to amplify 220 microsatellite loci with suitable flanking regions.We use a wild population（N=48）to screen and test those primers.19 loci were reliably amplified and showed higher polymorphism.Finally,12 microsatellite markers were selected as a"molecular marker group".Among these 12 microsatellite loci,the numbers of alleles per locus ranged from 7 to 14,with an average of 10.33.The observed heterozygosities ranged from 0.604 to 0.830,with an average of 0.715.The expected heterozygosities ranged from 0.637 to 0.870,with an average of 0.790.PIC ranged from0.565 to 0.846,with an average of 0.755.All the markers are in accordance with the"Hardy-Weinberg"balance test,and there is no chain imbalance between the markers.（2）The establishment of the parentage identification method of Siganus guttatus.There were 38 individuals of Parent population,100 individuals of breeding population and 92 individuals of Wild population.The genotyping data of 38 individuals of Parent population was inputted to the parentage analysis software CREVUS 3.0.2.Finally,the typing error rate is 4%,and the confidence level is 98%.The critical LOD value is obtained by simulation analysis,which was 4.75.The distribution rate of the analogue progeny was96.6%.The correctly identified breeding population number was 92,correct rate was 92%.The correctly identified breeding population number was zero,correct rate was 100%.The correctly identified mixed population correct rate was 95.3%.The accuracy of this identification method could distinguish between wild and farmed populations,there was a strong feasibility,but also had a large space can be adjusted.（3）The establishment of the parentage identification method of Siganus oramin.We analyzed variation at 12 microsatellite loci among 4 groups of Siganus oramin in the Daya Bay,Dongshan Bay,Dianbai and Yangjiang.After calculated F_ST values between four groups,the results demonstrated that there was no significant difference in genetic polymorphism among populations.Then,the group of Daya bay,Dongshan bay and Yangjiang group were mixed（N=157）as the parents of simulation analysis.Finally the critical LOD value was 8.19.In order to determine the maximum parental number pressure of this"molecular marker group",the results showed that when the parental number was increased to 800,the simulated subgeneration rate was less than 95%.Therefore,this tag group has sufficient discriminant ability in the current individual identification.