A Comparative Polymorphism Study On Mitochondrial Genome And Endosymbiotic Microbiota In Natural Populations Of Laodelphax Striatellus And The Factors Affecting Diversity

Small brown planthopper（SBPH,Laodelphax striatellus）,belonging to Hemiptera,Delphacidae,is widely distributed from the Philippines to Siberia and Europe.It is widely distributed in all parts of China,but the damage is serious in the Yangtze River valley and the rice growing areas in the north.L.striatellus can bite,lay eggs and transmit virus to harm crops.Despite its important economic status,the research on genetic diversity and genetic structure of L.striatellus populations is relatively backward.Previous population genetic studies have used mitochondria genes（part of the COⅠ and COⅡ genes）to mark natural populations of L.striatellus,and such results cannot accurately show the full range of mitochondrial genetic differences between different populations.The interaction between L.striatellus and symbiotic microorganisms will affect its rapid adaptation,colonization and diffusion,suggesting that its microbial community is associated with its ecological adaptability.However,there are few reports on the microbial community of natural populations of L.striatellus.To this,this dissertation study by more sampling from many sites in China and Japan,and by using a new generation of high-throughput sequencing technologies,investigates mitochondrial genomes and endosymbiotic microorganism diversity of natural populations,explores the factors affecting the diversity and biological characteristics of genotype haploid,and interprets natural populations within the mitochondrial genome and polymorphism,providing a new understanding on the formation of mitochondrial genome and endosymbiotic polymorphism in the natural populations.The main results and conclusions are as follows.1.Study on mitochondrial genome diversity of natural populations of L.striatellus and its influencing factors（1）Mitochondrial genome polymorphism and evolutionIn order to study mitochondrial genome polymorphism in natural populations of L.striatellus in China and Japan,samples were collected from 17 sites in China and 6 sites in Japan from 2010 to 2017,and 81 mitochondrial genomes were sequenced.Based on the tandem sequence of 13 mitochondrial genes encoding proteins,SNPs were found at 246 sites with a nucleotide diversity of 0.286%.Phylogenetic analysis showed that two major haplogroups（HGⅠ and HGⅡ）constituted a monophyletic group,and that HGⅡ was nested within HGⅠ,suggesting that HGⅡ evolved from HGⅠ,and that HGⅠ was more closely related to Nilaparvata lugens and Sogatella furcifera than HGⅡ.Three non-synonymous mutations were found in ND2,ND5 and CYTB genes,which were closely related to the differentiation between the two haplogroups.Based on three nonsynonymous mutations,we analyzed the topological results of the haplotype network and found that the HGⅠ subgroup presented a star-like pattern dominated by a single haplogroup.In contrast,the HGⅡ subgroup consists of several branches,suggesting that the HGⅡ haplotype has a more active evolutionary state than the HGⅠ haplotype.（2）The influence of environmental factors on the mitochondrial diversity of the natural population of L.striatellusPartial Mantel test and structural equation model（SEM）were used to analyze the correlation among the frequency of non-synonymous mutations,the genetic differentiation index and environmental factors.The results showed that the frequency of non-synonymous mutations was significantly correlated with the mean temperature in the coldest season（Bio11）,suggesting that Bio11 affected the geographical distribution of non-synonymous mutations.Except that the ND5 gene was not correlated with geographical distance in the partial Mantel analysis,other results showed that the frequency of non-synonymous mutations was correlated with geographical distance.These results suggested that geographical distance also played an important role in the geographical distribution of non-synonymous mutations.In addition to affecting the frequency of non-synonymous mutations,Bio11 and geographical distance also affected the population genetic differentiation.In particular,when analyzing HGⅡ haplotypes separately,partial Mantel test and SEM test showed that Bio11 affected the population genetic differentiation,which provided evidence for the effect of climate on the genetic differentiation of HGⅡ haplotype mitochondrial gene.2.Homology modeling of nonsynonymous mutant proteins and comparison of biological characteristics in different haplotypes of L.striatellus In order to distinguish the role of mtDNA from that of nuclear genes,introgression was used in this study to construct L.striatellus mitochondrial haplotype strains with similar nuclear genetic background.This part of the experiment used two geographically different populations in southern China（Chuxiong City,CX,Yunnan Province）and northern China（Harbin City,YS,Heilongjiang Province）.For each population,two mitochondrial types infiltrate into each other’s nuclear genetic background,resulting in a total of eight strains of L.striatellus,Ys-mitⅠnuⅠ,Ys-mit ⅡnuⅠ,Ys-mitⅠnu Ⅱ,Ys-mit Ⅱnu Ⅱ,Cx-mitⅠnuⅠ,Cx-mit ⅡnuⅠ,Cx-mitⅠnu Ⅱ,and Cx-mit Ⅱnu Ⅱ.By comparing the recovery time of cold coma of different haplotypes,it was found that HGⅡ haplotype had an advantage in cold resistance compared with HGⅠ haplotype.At the same time,mitochondrial transmission electron microscopy（TEM）results also showed that the HGⅡ haplotype mitochondria have structural defects,which may lead to changes in mitochondrial phenotypes.Based on homology modeling of non-synonymous mutant proteins,we hypothesized that the M114T amino acid alterations involved in ND2 may have played an important role in this evolutionary process.In addition,mitonuclear epistatic interactions were detected,suggesting that they played a role in maintaining mtDNA diversity.3.Study on the diversity of symbiotic microbiota in natural population of L.striatellus and its influencing factors（1）Diversity of endosymbiotic microbial communityUsing 16S rRNA amplicons sequencing technology,we analyzed 17 L.striatellus populations collected from 5 climatic zones in China and Japan.Since only the SAP population contained individuals uninfected with Wolbachia,the following analysis removed the SAP population.A total of 314 OTUs were identified by RDP classification software from 48 samples of the remaining 16 L.striatellus populations.Wolbachia was the most abundant bacteria,accounting for 87.9%of all 16S rRNA gene sequences in the Chinese population and 66.4%of all 16S rRNA sequences in the Japanese population.The dominant genera in the Chinese population were Spiroplasma（3.55%）,Asaia（2.47%）,Pantoea（1.04%）,and Herbaspirillum（1.03%）,while the dominant genera in the Japanese population were Diplorickettsia（10.9%）,Asaia（5.56%）,Spiroplasma（5.00%）,and Pantoea（2.08%）.With the exception of Wolbachia,the Japanese population was more abundant than the Chinese population on the other genera.These results indicated that the microbial structure of the L.striatellus was very different between the two countries.Based on the Bray-Curtis dissimilarity metrics（Adonis,r=0.428,p=0.001）,we found that the microbial communities of the 16 populations were significantly different.（2）Diversity of endosymbiotic microbial communityIn order to reveal effects of host genetic background and environmental factors on microbial community structure of natural populations of L.striatellus,we used SEM test and partial Mantel test to analyse the relationships between microbial community structure and five putative predictor variables,which are population genetic differentiation(F_ST),the annual average temperature（Bio1）,the average annual precipitation（Bio12）,latitude and longitude.Our results suggested that the formation of microbial community structure may be related to the changes of longitude,Bio12 and mitochondrial DNA,providing new evidence for the influence of climatic and genetic factors on the formation of microbial community structure.4.Effects of Wolbachia on the microbial community structure of natural populations of L.striatellusBy testing the infection rate of Wolbachia,it was found that only the northernmost population in Japan（SAP population）had individuals uninfected with Wolbachia,and the infection rate of Wolbachia in this population was 50%.In order to reveal the influence of Wolbachia on the microbial community structure natural population of L.striatellus,we randomly selected 9 individuals uninfected with Wolbachia and 10 individuals infected with Wolbachia from the SAP population for 16S rRNA amplification sequencing.Then we analyze the influence of Wolbachia on L.striatellus microbial community.Analysis of the original and removed Wolbachia sequences both showed that the northerly spread of Wolbachia seemed to have a greater influence on the microbial community than nonbiological factors,resulting in a decrease in the diversity and abundance of the host-associated microbial community.By comparing the RNA-seq of Wolbachia infected and uninfected individuals,Wolbachia did not seem to affect L.striatellus immune response.We speculated that Wolbachia might regulate L.striatellus microbial community structure by affecting the expression of metabolic genes.