Genomic Evolution And Environmental Adaptation Of Cotton Bollworm

The cotton bollworm,Helicoverpa armigera,is a worldwide pest.It can colonize in complex conditions due to high fecundity,long-range migration and facultative diapause.It has been reported that there are significant differences in insecticide resistance,pheromone response and climate adaptation among different cotton bollworm populations,yet the underlying molecular mechanisms are still unclear.In 2017,the published genome of H.a.conferta,csiro4 b,greatly advanced the progress in physiology,biochemistry and molecular ecology of cotton bollworm.However,the fragmentary nature and numerous assembly gaps severely limit its further applications.In this study,the reference genome of H.a.armigera was assembled using thirdgeneration sequencing and genetic mapping techniques.Genome collinearity analysis revealed the evolutionary relationships of chromosomes among lepidoptera insects.The P450 s,CCEs,GSTs and ABCs gene superfamilies were manually annotated in new genome,and the divergence of detoxification proteins were calculated between two cotton bollworm subspecies.The biological function of CYP337B29 was explored through in vitro expression and metabolism experiments.The genetic structure and diversity of cotton bollworm in the Changjiang River Region(CRR),Yellow River Region(YRR)and Northwestern Region(NR)were investigated on the basis of the dense variant map.The divergence landscape and evolutionary history within cotton bollworm were compared.This study supplies important molecular resources for further understanding the evolutionary pattern of cotton bollworm,and provides a foundation for formulating management strategies designed for cotton bollworm in China.The main results are as following:1.Genome assembly and annotation of cotton bollworm,Ha SCD2In this study,a male cotton bollworm(ZZ,H.a.armigera,2n=62)was selected for highdepth sequencing,and a total of 48.07 Gb Pac Bio sequences were obtained,with a coverage depth of 140.07 X.Genome survey estimated that the genomic size of cotton bollworm was about 352.30 Mb.By comparing the seven assemblies,two assemblies with the highest completeness(Canu and Falcon)were selected for downstream analysis.After merging,polishing and contamination removing,the total length of Ha SCD2 was about 356.67 Mb,with the BUSCO assessment of 97.70% and the N50 value of 7.34 Mb.Comparing to the published genome csiro4 b,the continuity of Ha SCD2 was improved by 313 times.Based on the resequencing data of the cotton bollworm full-sib family,a total of 425,788 female and756,597 male effective information markers were obtained,and an ultra-dense genetic map composed of 31 linkage groups was successfully constructed,spanning a genetic distance of1545.4 c M.Using the genetic map,353.56 Mb sequences of Ha SCD2 were anchered into 31 chromosomes,wherein 8 chromosomes were composed of a single contig and contained no gaps.Genome annotation showed that the repetitive sequences accounted for 27.04 % of the total length of Ha SCD2.On the basis of ab initio,protein and transcriptome evidence,18,668protein-coding genes were annotated,of which 95.37% were assigned with biological functions.45.02% gaps in the csiro4 b were successfully mapped to Ha SCD2 assembly,wherein 44.45% overlapped with repeat sequences,suggesting that repetitive sequences affecting the quality of genome assembly.The collinearity analysis revealed extensive interchromosomal collinearity in lepidopteran insects,yet there were three large genomic rearrangements on chromosome 9 between noctuidae and Bombyx mori.This study offers a dense genetic map and a high-quality reference genome for cotton bollworm,which fills in the knowledge gaps about genomic divergence within cotton bollworm,and shows that the high-quality reference genomes are important in revealing the evolutionary pattern of chromosomes.2.Identification and functional study of gene families involved in detoxificationDue to recent gene duplications,the multi-gene families generally share high sequence similarities,which makes automatic annotation pipeline error-prone.Thus,our study established a manual annotation platform GAWP(Gene Annotation Web Platform)for visualization of transcriptomic and protein sequences.320 detoxification genes were manually identified from the Ha SCD2 genome,including 117 P450 s,42 GSTs,105 CCEs and 56 ABCs.These genes are unevenly distributed across the 30 chromosomes of Ha SCD2.300 detoxification genes were determined to have 1:1 orthologous relationships between H.a.armigera and H.a.conferta,and their similarities ranged from 89.38% to 100%.In addition,27 detoxification genes were identified in a single assembly,of which 7 were from csiro4 b and 20 from Ha SCD2.Phylogenetic trees showed that these detoxification genes belonged to CYP321(2),CYP337(2),ABCC(2),ABCB(1),CCE001(10),CCE012(1)and CCE006(2)subfamilies,respectively.By comparing the depth ratios between target genes and their flanking sequences,CYP321A2,CYP337B28,CYP337B29,CYP6AE59,and CCE001u-like were inferred as the lineage-specific genes.Previous studies have proved that the resistance of cotton bollworm to pyrethroid insecticides was related to the CYP337 subfamily.Therefore,this study further studied the structure and biological function of CYP337B28 and CYP337B29,which were highly similar to CYP337B1.The gene structure of these two P450 genes was confirmed by PCR amplification,and their gene sequences were successfully cloned.Phylogenetic tree showed that CYP337B28 and CYP337B29 were derived from the gene duplication events of CYP337B1.In vitro expression experiments showed that CYP337B29 could correctly fold in Sf9 cells,while CYP337B28 was unstable.We found that CYP337B29 protein had the activity to metabolize esfenvalerate with metabolic activity of0.056 ± 0.002 pmol/min/pmol P450,and the major product was 4’-hydroxyesfenvalerate.In this study,we systematically compared the evolutionary patterns of four detoxification gene superfamilies between two subspecies of cotton bollworm,and revealed the metabolic activity of recent duplicate P450 genes to esfenvalerate.3.Population genomics of Chinese cotton bollwormIt is important for cotton bollworm to clarify the evolutionary dynamics,which is benefical for make the integrated management.From three major cotton growing regions in China,a total of 141 cotton bollworm were collected for whole-genome resequencing,with a average sequencing depth of 10.21 X.In addition,another 30 resequencing data of Helicoverpa complex were retrieved.Finally,a total of 41,091,024 bi-allelic SNP variants were obtained,covering more than 87.10% of Ha SCD2 assembly.Phylogenetic tree and principal component analysis(PCA)showed that cotton bollworm could be divided into three different lineages: H.a.armigera from CRR & YRR,unknown cotton bollworm lineage from NR,and H.a.conferta from Oceania.The gene flow were also identified from CRR & YRR to NR lineages.Population history analysis further revealed that two cotton bollworm lineages in China began to diverge during Marine Isotope Stage 5(MIS5),and the effective population size of NR and CRR & YRR sharply increased at the same time,yet there was a significant difference in the magnitude of increase.Through the sliding-window analysis,nine universally differentiated genes were identified from three cotton bollworm lineages,wherein per and clk genes were related to the differences in physiological rhythm of cotton bollworm.Transcriptome analysis showed that per and clk genes had high expression levels in different tissues and developmental stages of cotton bollworm,particularly in the abdomen,antennae and thorax.The selective sweep analysis identified a series of genes subjected to natural selection pressures.The enrichment results showed that these genes were related to host range,insecticide resistance evolution and environmental adaptation of cotton bollworm.In this study,we studied the evolutionary history and local adaptation mechanism of Helicoverpa armigera in China using population genomics,providing an important basis for understanding the mechanisms of regional disaster of cotton bollworm.