Research On Genetic Diversity Among Different Geographical Populations Of Neopsylla Specialis In Northwestern Yunnan

ObjectiveThe molecular markers that Mitochondrial CO? Gene and Ribosomal 18S rDNA and 28S rDNA were used to analyze the genetic diversity and genetic differentiation of the Neopsylla specialis from seven different geographic populations in the northwestern Yunnan,It can not only reveal the genetic differences of different geographic populations of Neopsylla specialis,and explore initially the molecular mechanisms and taxonomic status of the subspecies differentiation,but also built a database of biological and genetic information of Neopsylla specialis,which will provide a molecular biological evidence for the further study of the system classification and control of the plague.Method1.Sample Collection:The Neopsylla specialis were collected from 7 geographical regions of De Qin,Wei Xi,Xiang Gelila,Yun Long,Jian Chuan,Yu Long and Gong Shan in the northwest Yunnan,and the specimens were identified according to their morphological characteristics.2.Isolation DNA:The Fleas was cut into three sections,head,thorax,and abdomen.The procedure was performed according to The QIANEN bacterial kit instructions.After the DNA was extracted,the chitin shells that have not been digested was retained.3.PCR amplification:Amplification of Mitochondrial DNA CO? Gene and Ribosomal DNA 18S rDNA and 28S rDNA.4.agarose gel electrophoresis:PCR products was detected by 1.2%agarose gel electrophoresis and was visualized by UV transilluminator.5.Sequencing and analysis:Sequences of successful sequencing were manually calibrated and spliced.Sequence analysis was performed using Clustal X1.8 software,MEGA6.0 software,DnaSP5.0 software,Network 4.6 software and Arlequin v3.1 software.Results1.A total of 61 mitochondrial COII gene sequences of Fleas samples were obtained.There were 8 samples had large differences in bases.A total of 193 mutation sites were detected,43 haplotypes were defined,and the NJ phylogenetic tree of the COII gene haplotype was shown.The haplotypes Hap11,Hap13,Hap14,Hap24,and outgroup broad new ticks were clearly clustered into one branch.Hap40 Hap40,Hap25,and Hap41 clustered into one branch,and the remaining haplotypes clustered into one large branch.The genetic distance was 0.00190-0.02126,and the genetic distance between the branches was 0.08578-0.17654,which was greater than 0.02.2.A total of 73 mutation sites were detected in the 53 mitochondrial COII gene sequences of the Neopsylla specialis,and 36 haplotypes were defined.The haplotype diversity index Hd>0.5 and the nucleotide diversity index Pi>0.005 were observed in the population and most of the geographic populations.The population showed a pattern of high haplotype diversity and nucleotide diversity.3.The genetic distance within 7 geographical populations of the Neopsylla specialis was less than 0.02,and the average genetic distance was 0.01986.The genetic distance between most geographical populations was greater than 0.02,and the genetic differentiation factor Fst was greater than 0.25,the overall Fst value was 0.35491,and the Nm value was 0.4544,the genetic variation of each geographic population is large.4.In the haplotype network map and the NJ tree species,the three subspecies of the Neopsylla specialis did not form corresponding subspecies clusters,and there was no obvious correspondence between the haplotype distribution of each geographic population and the geographical population in which it was located.The relationship is in a more mixed distribution pattern and does not form a clear system geography.5.The AMOVA analysis results showed that the intra-group variation(63.53%)of the geographical populations of the Neopsylla specialis was greater than that between the populations(31.58%)and the variation between subspecies(4.89%).The Nm value between the three subspecies was 4.86561,the Nm value among the subspecies within the subspecies was 0.50283,and the Nm value within the population was 0.43546.The mismatch analysis calculated the P values of the SSD and HR parameters were all greater than 0.05,and both the Tajima'D value(-0.4078)and the Fu's Fs value(-9.8681)were negative.6.The genetic diversity of 18S rDNA and 28S rDNA sequences of the seven geographical populations was only 4 and 5 base pairs.The genetic variation was small.The 18S rDNA and 28S rDNA gene sequences can distinguish between the Neopsylla specialis and the Frontopsylla spadix.Conclusion1.The mitochondrial COII gene is an ideal molecular marker of the Neopsylla specialis.This sequence not only can provide abundant genetic diversity information,but also can effectively identify the Neopsylla specialis and perform intra-specific genetic analysis.2.Based on the mitochondrial COII gene analysis,There is a large genetic variation among the Neopsylla specialis of different geographical populations in northwestern Yunnan,and the population is rich in genetic diversity.3.Based on the mitochondrial COII gene research analysis and the morphological identification based on the three subspecies of the Neopsylla specialis,the classification status does not match.Whether there are three subspecies,further research and analysis are needed.4.The ribosomal DNA 18S rDNA and 28S rDNA sequences are relatively conserved.It is not suitable to analysis the genetic diversity of different geographical populations of the Neopsylla specialis.