| Objectives:Oncomelania hupensis is the only intermediate host of Schistosoma japonicum,and plays a vital role in the life history and transmission of Schistosoma japonicum.At present,schistosomiasis endemic areas in China are mainly compressed in the middle and lower reaches of the Yangtze River.The total number of schistosomiasis cases in Jiangsu Province has reached 2,530,700,and the snail area is 1.473 billion square meters.Since 2014,many snail environments been found in Suzhou every year,and the growth rate of environments containing snail in 2018 was 350%.Because of the complexity of snail living environment,the snail is not well controlled.Understanding the genetic diversity of snail population in various environments is beneficial to the effective control of snail populations.In this study,based on microsatellite loci and mitochondrial COX1 gene sequencing,the genetic diversity of snail in Suzhou City,Jiangsu Province were analyzed the degree and distribution of genetic variation,and explored the relationship between geographical environment and genetic variationMethods:1.Snail samples were collected in environments containing snail of Dongshan Town(DS,2019)and Jinting Town(XS19)of Suzhou City in 2019,and were collected in environments containing snail of Jinting Town of Suzhou City in 2018(XS18).The Snail genomic DNA was extracted one by one,PCR amplified,tested,read alleles,and analyzed for population genetics,including genetic diversity,bottleneck effect,effective population size,variation composition,genetic differentiation,principal coordinate and phylogenetic tree.2.Snail samples were collected from four snail environments in Dongshan Town(DS),Jinting town(XS),Taicang district(TC)and Hengtang town(HT)of Suzhou City,and three snail environments in Shitai(ST),Hexian(HX)and Zongyang(ZY)of Anhui Province(for comparison).The Snail genomic DNA was extracted one by one,PCR amplify the COX1 gene of the snail samples,send the product to sequencing to obtain the gene sequence,and conduct population genetic analysis,including gene sequence characteristics,genetic diversity,neutrality tests,genetic differentiation,gene flow,molecular variance,haplotype network and phylogenetic tree.Results:1.Based on 9 microsatellite loci,we found that:(1)The genetic diversity in populations:Number of alleles of DS,XS18 and XS19 was 4.500,3.875 and 2.875.Number of effective alleles of DS,XS18 and XS19 was 2.754,2.340 and 1.964.The genetic diversity of DS snails was the highest,XS19 was the lowest.The inbreeding coefficient(Fis)of XS18 and XS19 were greater than 0.5.(2)The bottleneck effect:Under IAM model and TPM model,the three populations did not experience bottleneck event or expansion tendency(P>0.05),and were stable.(3)The effective population size:Based on the heterozygote excess method,the effective population size(Ne)of the three populations is infinite;based on the linkage disequilibrium method,the Ne of XS19 population is the largest(NeEst:inf;LDNe:44.1);the Ne of DS population is the second(NeEst:164.2;LDNe:223.2);the Ne of XS18 population is the smallest(NeEst:24.0;LDNe:39.5)(4)Variation composition analysis:The genetic variation mainly came from individuals within the population,accounting for 67.26%of the total variation;the total genetic differentiation index Fst among the three snail populations was 0.327,and the p value was less than 0.01,indicating that there were significant genetic differences between at least two populations.(5)Genetic differentiation among populations:The genetic differentiation index between DS and XS18 was 0.4132(P<0.01),that between DS and XS19 was 0.3096(P<0.01),and that between XS18 and XS19 was 0.2221(P<0.01).(6)Principal Coordinate Analysis and phylogenetic tree:The three snail populations were clustered into two groups,namely,DS population was one group,while XS18 and XS19 populations were one group.2.Based on the sequencing of mitochondrial coxl gene,we found that:(1)Gene sequence acquisition:The consistency of the detected gene sequence with coxl sequence of O.hupensis in GenBank was 98.75%;all the gene sequences were stored in GenBank database with the registration numbers of MW024950 to MW025080.(2)Gene sequence analysis:The A+T content(58.9%-60.0%)was significantly higher than C+G content(40.0%-41.2%),showing a preference for A and T base composition bias;there were 29 haplotypes,of which 5 public haplotypes(appeared in multiple snail populations),24 private haplotypes(only appeared in a single snail population);a total of 642 information sites were obtained,including 56 variation sites(8.72%);among the mutation sites,51 transitions,4 transversions and 1 multiple mutation occurred at codon 1 and codon 3.(3)The genetic diversity in populations:Among the four populations in Suzhou,the haplotype diversity of DS population(Hd=0.523)was the largest,followed by HT(Hd=0.343),XS(Hd=0.111)was smaller,TC was 0,while number of polymorphic sites(S=11),nucleotide diversity(π=0.00588)and average number of nucleotide differences(K=3.771)of HT population were the largest,followed by DS(S=3,π=0.00245,K=1.569)and XS(S=3,π=0.00052,K=0.333)was smaller,and the values of S,π and K of TC were all 0.Three populations in Anhui have high genetic diversity(S:14-32,Hd:0.800-0.917,π:0.00681-0.01226,K:0.00681-0.01226).(4)Neutrality tests:Based on Fu&Li’s D method,the test value of HT population is 1.44097(P<0.05),which may experience population contraction or balanced selection;based on Tajima’s D method,and the test value of DS population was 2.21387(P<0.05),which may experience population contraction or balanced selection.(5)Genetic differentiation and gene flow among populations:Except for between high gene flow(FST=-0.01158)between ZY and HX populations in Anhui,other populations showed significant genetic differentiation(Fst:0.4230-0.9851)and lower gene flow(Nm:0.0165-0.3620).(6)Analysis of molecular variance:The genetic variation mainly came from the population,accounting for 67.52%of the total variation;the total genetic differentiation index Fst was 0.67520,and the P value was less than 0.01,indicating that there were significant genetic differences between at least two populations.(7)Haplotype network and phylogenetic tree:all populations can be divided into three lineages.DS,XS and ST populations in Suzhou and Anhui were divided into one branch,which may be due to the fact that all the three areas are hilly endemic areas.HT and TC populations in Suzhou and HX and ZY populations in Anhui were divided into one clade,which may be due to the fact that these four areas belong to lake marsh type epidemic areas.Some of the three populations of Oncomelania hupensis in Anhui Province were divided into one clade.Conclusion:1.The snail population in Dongshan Town of Suzhou city(DS)was stable without bottleneck effect or expansion trend,and was not affected by inbreeding recession.However,it may have experienced balanced selection,maintained a large genetic diversity,and had a strong adaptability to environmental changes.It may be that a local snail in Dongshan is rapidly reproducing after surviving.2.The genetic diversity level of snail population in Jinting town of Suzhou City in 2018(XS18)is low,which may be affected by inbreeding recession,and its adaptability to the environment is weak.Theoretically,its potential threat is small.The O.hupensis population in 2019(XS19)did not detect bottleneck effect or expansion trend,the level of genetic diversity was low,and it had weak adaptability to the environment,but it had evolutionary potential,which provided potential for disease transmission.3.The population genetic diversity of snail in Hengtang area(HT)of Suzhou city is low,its adaptability to environmental changes is weak,and it has undergone population shrinkage and genetic vulnerability.4.The snail population in Taicang area(TC)of Suzhou city is a new snail environment.It is located in the lower reaches of the Yangtze River Basin and is vulnerable to floods.It may be due to the immigration of snail other places.5.Compared with the three snail populations(ST,HX,ZY)in Anhui Province,the genetic diversity of Suzhou snail populations was lower,showing that the genetic stability,haplotype number,polymorphic loci,nucleotide diversity and average nucleotide difference were lower.6.There was significant genetic differentiation samong the snail populations in Suzhou City.7.Oncomelania hupensis was divided into three branches,which were associated with locations and endemic types of schistosomiasis. |
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