| DNA molecular markers are new genetic markers with rich polymorphism and co-dominant,based on nucleotide sequence genetic variation of individuals.Because these DNA molecular markers have many advantages,there are wide range of applications in the aspects of genetics and breeding,genome mapping,association analysis of traits,evolution and parentage identification.In the past,although some research for DNA molecular markers of the Chinese mitten crab(Eriocheir sinensis)have been reported,most of them focused on the development of DNA molecular markers,the genetic diversity and mapping of populations.What more,the developed DNA markers were lack of experimental verification.There are few reports on the development of microsatellite multiplex PCR systems and paternity testing in E.sinensis,which is disadvantageous to the large-scale breeding process of E.sinensis.In this study,the transcriptome of E.sinensis were explored and a large number of microsatellite markers and SNPs markers were developed.At the same time,multiplex PCR systems were constructed by using the developed microsatellite markers.Finally,the microsatellite multiplex PCR systems were used in parentage assignment of E.sinensis.The purpose of this study is providing basic information for evaluation of germplasm resources,and conservation of resources,and sustainable development of resources and breeding of E.sinensis.Mainly included the following contents:1.The development of microsatellite markers and SNPs markers in E.sinensis.In order to develop microsatellite markers and SNPs markers with polymorphism and practicality.The transcriptome of different developmental stages of ovary of E.sinensis were sequenced,and obtained 1,521,819 microsatellite markers.Among them,272 pairs microsatellite primers were randomly designed and verified by 45 individuals of E.sinensis.The results showed that 205 pairs were verified to have clear bands and 100 pairs of microsatellite loci were highly polymorphic.At the same time,the transcriptome of hepatopancreas of different feeding modes of E.sinensis were sequenced,and 1,286 SNPs related to lipid metabolism genes were obtained.Among them,60 pairs of primers were randomly designed and verified by 45 individuals of E.sinensis.The results showed that there were 42 polymorphic SNPs loci across 17 contigs,and the minimum allele frequencies of 42 SNPs loci ranged from 0.0111 to 0.4778.The observed heterozygosity and the expected Heterozygosity ranged from 0.0222 to 0.6000 and 0.1061 to 0.5461,respectively.These newly developed DNA molecular markers can be used for future germplasm evaluation and molecular marker assisted breeding of E.sinensis.2.The development of microsatellite multiplex PCR system in E.sinensis.In order to improve the efficiency of microsatellite marker analysis of E.sinensis,the microsatellite markers with better polymorphic were selected to further experiments.After optimization the site composition and the concentration of PCR primers,finally 8 groups of multiplex PCR systems were obtained,and each group of multiplex PCR system contains four sites.Further tests,8 groups of microsatellite multiplex PCR systems were used to assess the genetic diversity of 95 individuals of E.sinensis.The results showed that the number of alleles(Na)ranged from 5 to 21 and the average number of alleles was 10.34375.The observed heterozygosity(Ho)and expected heterozygosity(He)ranged from 0.2970 to 0.9697 and from 0.7240 to 0.9090 respectively.The average observed heterozygosity was 0.6884 and the expected heterozygosity was 0.8127.The polymorphism information content(PIC)ranged from 0.6330 to 0.9000,the average polymorphism information content was 0.7870,and all sites were highly polymorphic(PIC > 0.5).These microsatellite multiplex PCR systems for the Chinese mitten crab can provide convenient methods for parentage assignment,and management of families and population genetic diversity assessment of E.sinensis,it can improve work efficiency and reduce experimental costs.3.Application of microsatellite multiplex PCR systems in E.sinensis of family parentage assignment.For aquatic animals,parentage assignment is an effective way to understand the accurate pedigree information and analyze the genetic relationship of different families.It can effectively prevent inbreeding recession and germplasm degeneration and ensure germplasm vigor.In the present study,we conducted a paternity analysis of six families in E.sinensis by selecting four microsatellite multiplex PCR system.The confidence level was set to 95%,and the Cervus3.0 was used for paternity assessment.The results showed that the correct identification rates of the four microsatellite multiplex PCR systems were 70.05%,41.55%,46.38% and 36.23%,respectively.Simulated correct identification rates were 79.71%,50.07% and 41.55%,54.59%,respectively.According to the actual identification rate of each multiplex PCR systems,the cumulative paternity test was performed from high to low.The results showed that the cumulative identification rate was over 90.34% when using two multiplex PCR systems with the highest identification rate.When using three or four multiplex PCR System,all offspring can correctly correspond to their parents,the correct identification rate can reach 100%.In this study,we screened 4 groups of microsatellite multiplex PCR system,which have high identification efficiency in the Chinese mitten crab,it can quickly and effectively distinguish the corresponding progeny of each family,and it has great value in the Chinese mitten crab. |
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