| Schistosomiasis, the zoonotic infectious parasitic disease, is one of the major public health problems greatly threatening human health in China. Oncomelania hupensis, distributing in the southern areas to the Yangtze River, including marshland, mountainous and hilly regions, is the sole intermediate host of Schistosoma japonicum. Therefore O. hupensis plays a key role in the transmission of schistosomiasis japonica. Due to different geographical distribution, variation in ecological environment and natural factors, distinct genetic evaluation has occurred among O. hupensis generations. In view of Significance in the research of epidemiology, control and prevention of schistosomiasis, the investigation on genetic diversity of O. hupensis has attracted extensive attention. However, the studies and techniques applied in this field seem too far from adequacy to coping with the rapid development of bioinformatics in addressing these problems. Therefore, it is necessary to study on the population genetics and subspecies differentiation of O. hupensis in China.In this study, O. hupensis was sampled based on different landscape distribution, and geospatial database on bioinformatics of O. hupensis were established firstly. Secondly, after the establishment of microsatellite DNA database of O. hupensis, populations genetic structure of O. hupensis from middle-lower reaches of the Yangtze valley was analyzed based on its hybridization between restriction fragments of genomic DNA and oligonucleotide probe including (AAT)17, (GA)25 (CCT)17, (AC)25,(CAG)17, (CA)18, (CAC)5, (TC)10, (GT)8 and (TG)18 marked with biotin. Thirdly, the complete sequence of mtDNA from Hunan isolate of O. hupensis was detected by application of Long PCR and walking sequencing technology as well as SubPCR and clone sequencing. Additionally, the relationship between genetic variation of O. hupensis from different landscapes and geographic isolation on basis of mtDNA (16S) detection and ribosomal fragment (ITS1-ITS2) sequencing was explored in the line with the theory of landscape genetics.1. Establishment of management system on geospatial genetic information of O. hupensis1.1 In the line with the theory of landscape genetics, and aiming to investigate geospatial distribution and population genetics of O. hupensis, a management system on geospatial genetic information of O. hupensis was established in computer language. The system composed of 2 parts, one was the basic database which made up of 3 sub-datasets, i.e. collection sites, samples and genetic information datasets; the other was information management system which provide functions on accessing the datasets.1.2 Tthe database was primarily founded including 73 collection sites, 676 sample records and relevant genetic information of collected O. hupensis. Through second endoding and effective indexing, functions on accessing datasets, such as data query, filtration, amending, import and export, can be carried out which preserve further space for data amplification and online filling out. The system provides query service which facilitates study design and statistic analysis. Therefore, the system worth application in the study of distribution and population genetics in other samples in addition to O. hupensis.2. Genetic diversity of O. hupensis based on microsatellite DNA 2.1 A total of 209 effective sequences were attained from our study, of which 79 were completely repeated (37.8%),101 were incompletely repeated (48.33%) and 29 were combined (13.88%). Among microsatellite DNA, double-nucleotides took the major part and treple-nucleotides took the second place following mutiple-nucleotides. In addition, the number of (CA)n and (GT)n ranked the first place, of which that of (CA)n repeated 98 times.2.2 Based on the classification of microsatellite DNA sequences,16 out of 20 selected primer pairs from 67 designed ones resulted in obvious specific amplification in accordance with expected bands, among which 14 sites were polymorphic accounting for 70% of the total polymorphism. After gene scanning on 7 sites randomly selected,6 ones, i.e., P84, T5-13, T5-11, T4-22, T6-27 and P82 were found to attain good signals. Of these 6 sites, only P84 showed low observed heterozygosis and polymorphism information content (PIC) value, with 0.1667 and 0.1813, respectively. For others, observed heterozygosis and PIC value were between 0.36-0.8929 and 0.8437-0.9289, respectively, which showed good polymorphism.2.3 In application with 6 microsatellite DNA sites, genetic diversity in 5 populations of O. hupensis were detected. Among 6 microsatellite DNA sites, P84,T5-11 and T4-22 were unbalanced to some degree. A total of 188 alleles genes were detected, of which the average number of sites among different populations was 15.83 without obvious central tendency. Analysis of population genetics revealed that the observed and expected heterozygosis, PIC value of all sites equaled to 0.637,0.811 and 0.777, respectively. It was found that genetic variation of O. hupensis was the highest in Jiangsu population while was the lowest in Hubei population. Results from analysis of population genetics showed that genetic differentiation was high between Jiangsu and Jiangxi populations, while low between Anhui and Hunan populations. As a result, gene exchanges were not frequent among population and species caused high heterozygosis. However, low differentiation coefficient showed that genetic variation mostly resulted from that of individuals.3. Landscape genetics of O. hupensis based on mitochondrial genomes3.1 The 15 182 bp-long complete sequence of O. hupensis mtDNA (Genbank registration No.:FJ997214) was sequenced, and it is a closed circular molecular with 67.32% AT content which encoded 37 genes, including 13 protein genes,2 RNA genes and AT Rich Region, of which 8 tRNA genes were light chain coded and the others were heavy chain coded.3.2 All of 13 protein-coding genes were found with ATG as promoter and TAA or TAG as terminator, among which potential T was the terminator of ND1. All of those protein genes coded had the same transcription direction with strong AT preference of codon base. The length of 21 total intergenic region of mtDNA was 145bp ranging from 1-30bp with 2 short gene overlapping with length of 4bp and 7bp, respectively. Totally 22 transferring RNA were found in mtDNA, all of which were typical cloverleaf structure with specific tRNA (tRNASeC) except for 2 tRNASer (AGN), tRNAGIn and tRNAIle.3.3 The genetic diversity of landscape populations were analysised based on ITS1-ITS2 of ribosomal DNA and mtDNA-16S sequences, O. hupensis in Mainland China could be divided into 4 populations, i.e., population in the middle-lower reaches of Yangtze Valley, mountainous population in Yunnan and Sichuan, inland hilly population in Guangxi and coastal hilly population in Fujian, all of which were in accordance with landscape ecological types. Obvious geographical aggregation of genetic diversity was observed between of 2 DNA molecular colleted in different point showed. As a result, there was a significant positive correlation (RITS1-ITS2= 0.784,R16S=0.717, P<0.01) between geographical distance and genetic variation which showed that population genetics distribution were in accordance with the Isolation-by-distance Model.
|
PDF Full Text Request