| Objective: Using DNA barcoding technology to explore different geographical strains of Aedes albopictus and Aedes albopictus subgroup on the molecular level, and enrich their DNA barcoding database.It has an important reference value to establish a molecular identification method for Aedes albopictus subgroups and Aedes albopictus in different geographical strains.Methods: 1.According to Aedes albopictus life habits to collect Aedes albopictus mosquitoes and larvae from Fujian Province, Guangdong Province, Hainan Province, Yunnan Province, Liaoning Province and Taiwan. Parts of adult mosquitoes were made in tag specimens,the remainder were kept in 75% ethanol. 2.Mosquito genomic DNA was extracted and amplified with universal primers.Then mitochondrial CO Ⅰgene sequences were cloned and sequenced.The obtain sequences were compared with the corresponding sequences in the NCBI database and chosen with homology of 98% or more. 3. Using MEGA software, Dna SP 5.0 software, Network4.6 software, IBDWS online software and Arlequin 3.1 software to analysis the CO genetic structure characteristics Ⅰof Aedes albopictus in different geographic strains. 4. According to the habits of Aedes albopictus subgroups to collect Aedes subalbopictus,Aedes flavopictus,Aedes pseudalbopictus,Aedes sibiricus, identified by morphology.Then we established the co I gene phylogenetic tree of Aedes albopictus subgroup,It used to enrich their DNA barcoding database and apply in molecular identification of Aedes albopictus and Aedes albopictus subgroups.Results: 1 COⅠ gene sequences of Aedes albopictus A total of 106 Aedes mosquito coⅠgene sequences : 20 from Fujian, 20 from Guangdong, 19 from Hainan, 20 from Yunnan, 22 from Taiwan and 5 from Liaoning. The sequence length was 709 bp. The content of A+T(67.7%) was more than G+C(32.3%). Mutation locus were 90(13.68%), of which 57 conversion(63.33%), 28 transversion(31.11%), 5 hypervariable sites(5.56%). 2 the relationship between different geographic strains of haplotypes(1)A total of 42 haplotypes. 4 haplotypes were shared between different geographic strains(9.52%),including h2, h6, h18, h19. h2 was shared by Fujian strain,Guangdong strain, Taiwan strain and Liaoning strain; h6 for Fujian strain and Guangdong strain; h18 and H19 for Hainan strain and Yunnan strain. The haplotype diversity of Aedes albopictus was 0.882, nucleotide diversity was 0.01017.(2) Haplotype family network diagram showed that most haplotypes were parallel evolution, which suggested that the local expansion of Aedes albopictus in history. The undetected haplotypes were small(just 5).The abundances of shared haplotype h2(34), h18(9) and h19(6) in the population were 32.08% and 8.49% and 5.66%.They might be the source of the expansion, which developed other haplotypes after 1 ~ 2 steps.(3) COI gene haplotypes Neighbor-Joining tree showed that some haplotypes from Fujian strain were clustered into one group with high degree of confidence.(Hainan strain+ Yunnan strain),(Guangdong strain + Taiwan strain + Liaoning strain+part of Fujian strain) each clustered in different groups, but the confidence was low. It suggested a certain correlation between clustered haplotypes and geographical distribution.Aedes albopictus in different geographic strains were different. 3 the genetic structure of Aedes albopictus population in different geographic strains(1) The genetic distance of COI gene sequences were 0.00091 ~ 0.02473 among populations. The genetic distance between Fujian strain and other geographical strains was more than 0.02(2) Genetic differentiation coefficient(FST) and gene flow(Nm) were 0.21871 and 0.893, which suggested that the gene flow level was failed to prevent genetic drift.The populations had a certain level of genetic differentiation. The Nm-value between Hainan strain and Yunnan strain was more than 1, which indicated this two strains had frequent gene exchange.The Nm-value between other strains and Hainan strain or Yunnan strain was less than 1, which indicated the gene exchange was limited.(3) The analysis of molecular showed the variation within population(78.13%)was more than that among populations(21.87%).It suggested that the main genetic variation existed among individuals within population rather than between populations. 4 Aedes albopictus population dynamics The result of neutrality test and mismatch analysis showed that the expansion in Aedes albopictus in history. From the perspective of geographical strains, Hainan strain, Yunnan strain, Taiwan strain were also in the expanded state. Estimation of effective female mosquitoes were about 3.4 ×104 ~ 7.6×109. The broad population expansion occurred 3.0 × 105 years ago within two generations of Aedes albopictus in one month. 5.the relationship between population genetic differentiation and geographic distance The correlation curve between the FST and geographic distance showed: r = 0.5789, P = 0.0120, which suggested there was a positive correlation between population genetic differentiation and geographic distance. 6. the mt DNA-COI gene structure of Aedes albopictus subgroup(1) A total of 15 Aedes albopictus subgroup COI gene sequences: 2 Aedes subalbopictus,3 Aedes flavopictus,7 Aedes pseudalbopictus,3 Aedes sibiricus.(2) The COI gene Neighbor-Joining tree of Aedes albopictus subgroup showed that Aedes albopictus and Aedes subalbopictus were clustered into one group, Aedes flavopictus,Aedes pseudalbopictus,Aedes sibiricus were each clustered in different groups.Conclusions: 1.There are genetic differences among different geographic strains of Aedes albopictus population.There is a positive correlation between population genetic differentiation and geographic distance.2 Part of Fujian unique haplotypes may be superiority population.The cause of this phenomenon may be associated with the landform and climate environment in Fujian. 3. The mitochondrial COI gene as the target gene for DNA barcoding can effectively analysis genetic structure of Aedes albopictus population and identify Aedes albopictus subgroup. 4.The molecular identification of Aedes albopictus subgroup with mt DNA-COI gene :Aedes albopictus and Aedes subalbopictus are clustered into one group, Aedes flavopictus,Aedes pseudalbopictus,Aedes sibiricus are each clustered in different groups. |
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