| Objectives: Oncomelania hupensis is the only intermediate host of Schistosoma japonicum,and plays a key role in the transmission process of schistosomiasis.The establishment of microsatellite DNA Library of Oncomelania hupensis has provided abundant repository for subsequent microsatellite screening,but the loci in the library have not been fully utilized.Therefore,this paper uses bioinformatics methods to screen the polymorphic microsatellite loci,and design and synthesize the corresponding primers to provide effective molecular markers for the subsequent genetic analysis of population.The geographical distribution of Oncomelania hupensis is consistent with the epidemic area of schistosomiasis,mainly distributed in the Yangtze River Basin and the southern part of China.Due to the widespread distribution of Oncomelania hupensis,complex environment and geographical isolation,significant genetic differentiation and variation have occurred among different geographic populations of Oncomelania hupensis.Therefore,in this study,the population genetics of Oncomelania hupensis in different regions was studied in order to find the law of genetic variation,and to provide a scientific basis for the evaluation of the control measures of Oncomelania hupensis and the monitoring of the spread of Oncomelania hupensis in low epidemic level areas.Methods: Downloading the nucleotide sequences of the Oncomelania hupensis from the public database(Genbank),and searching the microsatellite sequences using SciRoKo 3.3 software.Then the microsatellite loci were selected(3 nucleotide repeat fragments),as well as the polymorphic loci were selected from the published literature.Software Primer premier 6 and Oligo7.0 were used to design and evaluate the primers,then these primers were delivered and synthetized.The effect of primers amplification was identified,and a multiplex reaction system was finally constructed.Collecting samples from Shitai,Hexian and Zongyang in Anhui and Hengtang,Taicang in Jiangsu,DNA were extracted.4 pairs of microsatellite primers(T1-10,T4-25,T4-22 and D11,multiple reaction system 2)were adopted from reported literatures.Thus,9 pairs of STR(2 multiple reaction systems)were used for PCR amplification,and the amplified products were sent out to be sequenced.The GenAIEx 6.5,Structure2.2,Bottleneck,MEGA,DIYABC and other softwares were used to analyze the microsatellite polymorphism,genetic diversity,genetic differentiation,population structure,bottleneck effect,phylogenetic tree and evolutionary history of Oncomelania hupensis and so on.Results: Finally 5 pairs of microsatellite primers was screened from the Oncomelania hupensis microsatellite DNA library and published literatures.The polymorphism information content(PIC)was 0.861,0.889,0.87,0.86 and 0.911,respectively,with an average of 0.878.All of them were higher than 0.50,indicating that the 5 pairs of primers were highly polymorphic.One multiple reaction system was constructed to provide effective tools for subsequent population genetics study.2 multiplex reaction systems consisting 9 STR primers were used to amplify PCR from 150 Oncomelania hupensis.Of them 146 snails were successfully typed,and the success rate was 97.3%.At 9 microsatellite loci,251 alleles were detected among 5 groups,with a range of 17(T1-10)~46(T4-25),with an average of 27.889,indicating that the amplification effect of all loci were well.The PIC range of the microsatellite loci was 0.810(T1-10)~0.947(B14 and T4-25),with an average of 0.905,indicating that the 9 microsatellite loci used in this study were highly polymorphic.The population diversity of Oncomelania hupensis: allele number(Na)was 4.556(Taicang)to 16.222(Zongyang),with an average of 11.689.The number of effective alleles(Ne)was 2.866(Taicang)to 10.849(Hexian and Zongyang),with an average of 6.964.Three populations in Anhui have more highly diversity than two populations in Jiangsu.Hardy-Weinberg balance detection did not conform to the equilibrium.In the 45 groups of data(5 groups,9 sites),32 were significantly deviated from the Hardy-Weinberg balance.Among them,Hengtang population from Jiangsu deviated from HWE at all sites(P<0.05).AMOVA results showed that the variation among populations,among individuals and in individuals accounted for 15%,47% and 38% of the total variation,and all were statistically significant.That is,among the 5 groups,there were significant genetic differences between at least 2 groups.There was a moderate genetic differentiation among 5 Oncomelania hupensis populations.The results of Population structure,principal component and phylogenetic tree analysis are the same.That is,the two groups in Anhui Hexian and Anhui Zongyang were gathered first,separately from the other 3 groups.The DIYABC analysis uses the logistic regression method to get the scene 1(sequential evolution),the posterior probability was 0.5523,and the confidence interval was [0.3490,0.7555].The scene 2(mixed evolution),the posterior probability was 0.4477,and the confidence interval was [0.2445,0.6510].In the 1000 simulation operations,543 times the result were scene 1,and 457 times the result was scene 2.Therefore,scenario 1 is most likely the evolution model of 5 regions in the study,that is,the evolution of Oncomelania hupensis was spreaded from top to bottom along the Yangtze River Basin.Conclusion: 1.DH01 and DH02(3-nucleotide repeat fragments)were designed and optimizedly synthesized the synthesis of B14,C22 and T6-17(2-nucleotide repeat fragments),and the polymorphism of each site was evaluated.A multiple reaction system of 1 was constructed,including 5 STR loci(B14,DH01,C22,DH02,T6-17).2.Combined with the other 4 pairs of microsatellite primers,9 microsatellite primers were applied to analyze the molecular biology of specimens from different regions of Oncomelania hupensis.1)Three populations in Anhui were highly diverse,while two populations in Jiangsu are lower in diversity,which may be related to historical epidemics and snail breeding environment.2)There is a moderate genetic differentiation among the Oncomelania hupensis populations,of which,the genetic differentiation between Jiangsu Hengtang and Jiangsu Taicang population was the largest.3)The two populations of Anhui Hexian and Anhui Zongyang were clustered together and separated from the other 3 groups,which may be accounted for that they were on the edge of the Yangtze River and belong to the environment of the lake area and closely to the lake area.4)The 2 groups of Jiangsu Hetang and Anhui Shitai did not experience bottleneck effect(P > 0.05),and the other 3 groups experienced a bottleneck effect(P < 0.05).5)The evolutionary order of Oncomelania hupensis was from top to bottom along the Yangtze River Basin,and with no hybrid evolutionary. |
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