| Spodoptera litura is a polyphagous pest widely distributed in the world,posing a great threat to agricultural production.Therefore,how to effectively control S.litura has become a hotspot for researchers.Benefiting from the rapid development of sequencing technology and bioinformatics technology,the research on the relationship between genome variation and environmental adaptability of S.litura has become a new direction.In recent years,there has been increasing evidence that genomic variation has an important impact on the evolution of organisms and environmental adaptability.Copy number variation(CNV),a type of genomic variation,refers to changes in the copy number of a DNA sequence from Kb to Mb length.Compared to single nucleotide polymorphisms,CNVs have a wider range of biological effects because they cover longer sequences.In recent years,researches on CNV has become a hot direction for genomic variation and biological evolution.In this study,we used genomic resequencing data and partial transcriptome data from S.litura in 14 regions of three countries,combined with bioinformatics and statistical methods to detect CNVs at the genome-wide level.We analyzed the number,length,and frequency of all CNVs;performed functional analysis of CNVs related genes;studied the relationship between CNVs and environmental adaptability of different populations;and explored the effects of CNVs on gene expression levels.This study is helpful to understand the genome-wide copy number variation pattern of S.litura,and also provides a richer content for the genome variation of S.litura,which helps to understand the relationship between S.litura and environmental adaptability.Furthermore,it provides ideas for the biological control of S.litura.The main findings are as follows:1.Identification of CNVs in S.litura at the genome-wide levelIn this study,55 genomes of S.litura larvae from 14 regions in three countries(China,Japan,and India)were extracted to construct genome resequencing libraries,which were then sequenced using Hiseq2000.A total of 558.2 Gb of sequence was obtained with an average depth of 11.4X.After filtering out the low quality sequences,the remaining sequences were aligned to the reference genome.Finally,the CNVnator software detected a total of 3,976 CNVs,and the number of non-repetitive CNVs was 3,096.Analysis of each sample CNVs showed that the number of repeated CNVs and deleted CNVs in most samples were less than 200,and the deleted type CNVs were more than the repeated CNVs.The length analysis showed that the CNVs of the three samples including Delhi,Hyderabad,and Okinawa covered the longest genomic sequence,accounting for 81% of the total CNVs sequences.The results of chromosome distribution showed that the high-frequency chromosome of CNV in the Chinese region was chromosome 8,and Japan and India were chromosome 2.Next,we merged CNVs that overlapped more than 1 bp in all samples into non-overlapping CNV regions(CNVRs)and constructed the genome-wide CNVR map of S.litura.The length of CNVRs was mainly between 5 and 500 Kb,54.6% of CNVRs were present in only one sample,and 3.1% of CNVRs were present in more than 10 samples.Ten genes related to CNVRs were selected for real-time PCR,and the success rate of 6 genes exceeded 67%.2.CNVs and environmental adaptability in S.lituraAdaptive evolution is often achieved through genomic variation.In insects,there have been many reports about CNVs and environmental adaptability.We first constructed a 0-1 matrix based on whether CNVR exists in the sample,and then performed cluster analysis based on this matrix.The clustering results showed that 14 samples were divided into 4 clusters.Further,according to the clustering result,we corresponded the four clusters into four groups,which were named group 1,group 2,group 3,and group 4.We then used the statistic Vito measure the CNVs population difference and calculated the Vivalue for each CNVR.The larger the Vivalue,the greater the impact of the CNVR on a particular population in evolution.To screen for population-specific CNVRs,we removed the CNVR present in only one sample and analyzed the top 5%Vivalues of the remaining 717 CNVRs.The results showed that the number of CNVRs present in all four populations was zero and that 19 CNVRs were present in at least two populations.Functionally annotate the genes related to CNVRs in each population,and found that the genes in group 1 were related to detoxification and immunity;the genes in group 2 were related to growth and development;the genes in group 3 were related to resistance;the genes in group 4 were mostly related to receptors This result suggested that CNVs of different populations were subject to different natural selections.3.Effect of CNVs on gene expression in S.lituraStudies have reported that CNVs are associated with gene expression levels.Therefore,we investigated the specific effect of S.litura CNVs on gene expression levels.A total of 5,527 genes related to CNVRs were detected.Functional annotation and enrichment analysis showed that the enriched genes were associated with the growth and development of S.litura,such as regulation of apoptosis,gene silencing,and metabolic processes.Then we selected transcriptome sequencing(RNA-seq)data from different tissues and different samples to study the relationship between CNVs and gene expression in S.litura.Sequence alignment was performed using Tophat,and the expression level of each gene was calculated using Cufflinks software.The results showed that the expression of repeated CNV-related genes was the highest in the different tissues of the same sample,and the expression of the deleted CNV-related genes was the lowest.We also compared the gene expression levels of the CNV sample with the reference genomic sample and found differences between the two samples.Among them,the expression levels of the deleted CNV-related genes and the repeated CNV-related genes in the CNV sample were lower than those in the reference genomic sample,and the expression levels of the related genes in the non-CNV regions were not significantly different between the two samples.Further differential analysis was performed on these two samples.There were 180 genes with a coefficient of variation greater than or equal to 2,and 29 genes with a coefficient of variation greater than or equal to 4.Among them,there were 12 up-regulated genes and 17 down-regulated genes Functional annotation of the up-regulated gene revealed that there were genes such as P450,and larval epidermal protein;functional annotation of the down-regulated gene,and genes such as lipase and carboxypeptidase were found.These significantly altered genes can be used as targets for further functional analysis.In order to study the specific mechanism of action of CNVs on gene expression,we analyzed 180 genes with a coefficient of variation greater than or equal to 2.It was found that among the 125 genes of the gain type,there were 58 genes whose expression levels were up-regulated with the increase of the copy number,and the expression levels of 67 genes were down-regulated with the increase of the copy number Among the 39 genes of loss type,the expression level of 23 genes was down-regulated with the decrease of copy number,and the expression level of 16 genes was up-regulated with the increase of copy number.These results suggest that S.litura CNVs not only affect the expression of genes by the dose effect of copy number,but may also affect the expression level of genes by affecting transcriptional regulation or more complex methods. |
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