Genetic Diversity And Wing Differentiation Of Tea Aphid (Toxoptera Aurantii Boyer)

Tea aphid Toxoptera aurantii Boyer is widely distributed in major tea gardens around the world and is one of the most destructive pests in tea gardens,causing economic losses.Besides harming tea trees,it also harms plants such as camellia oleifera,coffee,citrus,gardenia,fusmulberry,cocoa and figs.The polymorph of wing differentiation is one of the typical biological characteristics of tea aphid,which is also one of the important reasons for its population expansion,gene exchange and disaster caused by flow.In this study,we analyzed the geographic population diversity of tea aphid in key tea-producing regions of Guizhou and other provinces by genomic resequencing.Through genome size estimation,transcriptome and whole genome sequencing,the tea aphid genome research platform was constructed,and the comparative genomics and phylogenetic analysis of Hemiptera were carried out.The main results were as follows:1.Assembly and annotation of reference genomes of tea aphidOxford Nanopore long-read third-generation sequencing and Illumina short-read second-generation sequencing technologies were used to sequence the genome of tea aphid.The total sequencing volume was 46.51G and the coverage depth was 134.84X.The total sequence length of the tea aphid genome assembly draft was 318.95Mb,containing 228 scaffolding and 363 Contig（Scaffold N50=85.97Mb,Contig N50=15.19Mb）.It is 7.5 percent smaller than the genome predicted by K-mer（344.92Mb）.Combined with high-throughput chromosome conformation capture technology（Hi-C）assisted genome assembly,a total of 4 sequences were assembled to the chromosome level,and the length of scaffold N50 was 15.19 Mb.The total sequencing volume was 22.2Gb and the coverage depth was 64.36x.The genome mount rate was97.91%.The average GC content of the assembled genomes was 27.22%.The integrity of aphid genome assembly was evaluated using 1658 single-copy lineologous gene banks of BUSCO.Results showed that 92.7%of the complete single-copy genes could be assembled.Combined with transcriptome data,the aphid genome was annotated by Nr,Swiss-Prot,KOG,Inter Pro,GO and KEGG databases.It was found that 95.6%of the genes could be annotated to function in at least one of the databases.In total,12162genes have been annotated in the aphid genome.These results indicated that the tea aphid genome obtained in this study had high integrity and accuracy.2.Comparative genome and Phylogenetic analysis of tea aphidBy obtaining single-copy gene families and multi-copy gene families of tea aphid and selecting 12 published insect genomes,the tea aphid gene families were constructed by using whole-genome alignment,gene family clustering and phylogenetic tree construction.The genome of tea aphid（T.aurantii）,cotton aphid（A.gossypii）,corn aphid（R.maidis）,sugarcane yellow aphid（Sipha flava）,pea aphid（A.pisum）and other similar species were extracted for clustering,and the Venn diagram was drawn.The 13species had a total of 7640 gene families.231（295 genes）specific gene families were identified in tea aphid,among which 1382 genes existed in single copy form.The phylogenetic tree of 1382 single copy homologous sequences of 13 insect species were constructed by RAx ML.The results showed that cotton aphid and tea aphid were closely related.Using Mc Mctree in PAML software package to estimate the divergence time,it was found that tea aphid and cotton aphid began to diverge in about 7.6 million years.In addition,the collinearity analysis results of tea aphid and corn aphid showed that most of the chromosomes between them had corresponding collinearity,but the sequence of the two chromosomes was different,indicating that both tea aphid and corn aphid may have experienced chromosome rupture and fusion events.3.Genetic diversity analysis of tea aphid groups based on whole genome resequencingA total of 1223740 SNPs of 25 tea aphid groups from different regions of China were detected by genome resequencing,34 SNPs were synonymous mutations and 100SNPs were missense mutations.Principal component analysis and population genetic structure showed that LSK（Duyun）and LZ（Liuzhi）were far away from other populations,and there was no mixed ancestry of LSK and LZ at the same time.It was speculated that LSK and LZ populations were primitive and had less genetic variation.Phylogenetic evolution binary tree of 25 tea aphid groups is divided into three main groups.The Fst value of the three groups showed that,the third group>the second group>the first group,the degree of differentiation of the third group>the second group>the first group,and the third group Tajima’D more volatile,deviation value is bigger,stronger by natural selection and artificial selection,combined with population genetic structure,The third group had a large number of mixed ancestors,which were radially distributed around the southwest of China.Based on this,we speculated that the aphid of the third group were more strongly selected and more closely differentiated.From the perspective of origin and evolution,the aphids of the first group were more primitive.We speculated that the LSK and LZ groups were the most primitive aphid populations among the selected tea aphid populations,and the southwest region of China may be the origin center of tea aphid.4.Effects of phytopesticide matrine on wing differentiation of tea aphid.Immersion method was used to measure the toxicity of matrine to tea aphid,the influence of sub-dead concentration matrine on the developmental duration of tea aphid and the proportion of winged tea aphid.Scanning electron microscope was used to observe the external morphology of tea aphid in different periods.The sublethal concentration(LC₂₀)and sublethal concentration(LC₃₀)of matrine were 2.423mg/L（0.612-3.863）and 3.161mg/L（1.106-4.789）respectively;LC₅₀ was 4.905mg/L（2.675-7.493）.The growth duration of tea aphid were all prolonged at sub-dead concentration matrine,and the winged proportion of tea aphid was significantly increased by matrine,especially at LC₅₀ concentration,reaching 95.6%,while the proportion of winged aphid in control group was only 0.5%.On external form,the age of 1 year if age aphid cannot distinguish between its wings,but if age aphid and compared if aphid,the age of 1backboard has visible is obvious uplift,to the age of 3,from the form have been able to distinguish if the aphid wing type,form visible wing buds on winged type,4 age if winged aphid wing buds to further expand,ligulate.5.Key genes analysis of wing type differentiation of tea aphid based on transcriptomeThe transcriptome information of the tea aphid was obtained by analyzing the transcriptome difference between the tea aphid and the pterygogoid aphid by high-throughput sequencing technology.The sequencing results showed that the high quality data（Q20）of all test samples were higher than 95.74%,indicating that the sequencing quality was good.Analysis of the differentially expressed genes of winged adults and aphids revealed that peptide hormones and genes related to wing growth and development were differentially expressed,and genes related to wing formation were significantly enriched,suggesting that these genes were related to wing development and flight of tea aphid.Homeobox gene is a kind of gene specially regulating biological body in organisms.Once these genes are mutated,part of the body will be deformed.Homeobox gene is the main regulator of the development of various insect appendages and other parts,and its normal expression is crucial to determine the shape and function of insect organs.The results showed that the expression of Antp genes in the winged population was significantly higher than that in the wingless group of tea aphid,Ubx genes were lower,which further proved that the expression of Ubx and Antp genes may inhibit the formation of insect wings,laying a foundation for further identifying the key genes of tea aphid wing formation.BLAST was conducted using Drosophila Homeobox gene sequence to further verify its protein domain,and 31 Homeobox genes were screened from tea aphid.Further transcriptomic analysis showed that Homeobox gene expression patterns were significantly different in different wing types.The expression level of evm.model.HIC＿ASM＿0.2706 gene was 4 times that of winged tea aphid,and the expression level of evm.model.HIC＿ASM＿0.2710 gene was 2 times that of winged tea aphid.The expression patterns of evm.model.HIC＿ASM＿0.2713.1 and evm.model.HIC＿ASM＿0.1723.1 genes were on the contrary,and the expression levels of evm.model.hic＿asm＿0.1723.1 genes were significantly higher in winged type than in non-winged type.Therefore,we predict that evm.model.HIC＿ASM＿0.2706 and evm.model.HIC＿ASM＿0.2710 belong to Ubx gene.evm.model.HIC＿ASM＿0.1723.1belongs to Antp gene.In summary,this study completed the whole genome sequencing of tea aphid,obtained high-quality genome at chromosome level,clarified the phylogenetic position of tea aphid,identified the key genes affecting the wing type differentiation of tea aphid,and preliminarily revealed the wing type differentiation mechanism of tea aphid.The genetic diversity of geographical populations of Aphid tea in Sichuan,Zhejiang,Guangdong and Guizhou provinces was analyzed by whole genome resequencing.We speculated that the LSK and LZ groups were the most primitive aphid populations among the selected tea aphid populations,and the southwest region of China may be the origin center of tea aphid.The results provided a theoretical basis for the migration and gene exchange of tea aphid,and provide genetic information resources for further molecular genetic research and biological control of tea aphid in the future.