Chromosome-level Genome Of Three Species Of Planthoppers And MiR-34 Regulation Of Wing Dimorphism In The Brown Planthopper

Rice planthoppers,including brown planthopper（Nilaparvata lugens,BPH）,white-backed planthopper（Sogatella furcifera,WBPH）and small brown planthopper（Laodelphax striatellus,SBPH）,are the most notorious pests in China and other Asian countries,which cause huge losses of rice production.They are typical insects with wing dimorphism.Long-wing individuals with fully developed wings and flight muscles are capable of dispersing,while short-wing individuals allocate resources to reproduction rather than to flight.Therefore,it is meaningful to explore genetic background and the molecular mechanism of wing dimorphism for pest control.However,previous genomes of three planthoppers were fragmented and incomplete,which cannot satisfy current demand of biological analysis.Here,we generated chromosome-level scaffold assemblies of three planthoppers,and analyzed the evolution of chromosomes of rice planthoppers.and took in-depth study of miRNA regulation the mechanism of the wing dimorphism in brown planthoppers.1.The analysis of the chromosome-level scaffold assemblies of three planthoppersThe previous version of BPH genome assembly was sequenced in 2014 and the quality of genome assembly need to be improved.Using PacBio and Illumina sequencing platforms,we generated a new genome assembly for BPH,with genome size of 1,087 Mb.The contig N50 and BUSCO score of this new assembly were 589.46Kb and 95.9%,which were significantly higher when compared to those in previous version.The draft assembly of WBPH and SBPH were used previous version.Next,we used Hi-C scaffolding technology to improve the draft assemblies of three planthoppers to chromosome-level.The genome sizes of BPH,WBPH,and SBPH were 1,088Mb,656.76 Mb,and 541.15 Mb,containing 15,14,and 14 chromosomes,respectively.24,901 protein coding genes in BPH genome,15,929 genes in WBPH genome and16,412 genes in SBPH genome were annotated.The large size of BPH genome might due to its higher repeat sequence proportion（56.87%）compared with other two planthoppers genomes.Moreover,BPH genome had larger numbers of species-specific genes（8,595）,which may play important roles in its adaptations to specific environments.Synteny analysis showed that three planthoppers shared high level genome syntenies,with identification of some small fusion and fission events.This work provided chromosome-level assemblies of three planthoppers and analyzed the chromosome evolution of these planthoppers genomes,providing several valuable resources of future studies on biology of planthoppers and Hemipteran genome evolution.2.The analysis of sex chromosome evolution in planthoppers.Three rice planthoppers belong to Auchenorrhyncha,a large suborder in Hemiptera.The sex determining system in WBPH and SBPH is XX/X0,while BPH is in XX/XY system.Using the re-sequencing data of males and females,we identified sex chromosomes of three planthoppers by calculating the reads coverage of each chromosome.Further functional enrichments revealed the functions of the genes in each sex chromosome.To obtain complete Y chromosome sequence in BPH,we also assembled a Y chromosome of BPH using PacBio long reads by Redkmer pipeline.The size of newly assembled Y chromosome was 15.3 Mb,which is longer than that of Hi-C version（10.5 Mb）,suggesting that the newly assembled Y chromosome was more complete.According to their phylogeny positions,the XX/X0 sex determining system is regarded as the ancestral trait of three planthoppers,and BPH obtained the Y chromosome after divergent from the common ancestor of WBPH and SBPH.Based on the theory,canonical Y chromosome evolved from autosomes,it shared some homologous region with the X chromosome or autosomes.While in BPH,the Y chromosome lacked homology with other chromosomes.It seems that Y chromosome of BPH did not originated from autosome.This work identified sex chromosomes in three planthoppers genomes,analyzed the origin of Y chromosome in BPH,and provided a useful strategy to analyze the evolution of sex chromosome in planthoppers and Auchenorrhynchan insects.3.The mechanism of wing dimorphism in brown planthopper by miRNA.The wing dimorphism is an important type of phenotypic plasticity,controlled by multiple regulators,such as insulin,juvenile hormone and so on.However,the potential roles of miRNAs in modulating wing dimorphism remain largely elusive.To identify wing dimorphism-related miRNAs,we isolated and sequenced small RNAs from 1^st to5^th instar nymphs of long-wing（LW）and short-wing（SW）strains of BPH.503 miRNAs were annotated in BPH genome and 122 of them were differentially expressed between two strains.Specifically,94,5,56 and 66 miRNAs were differentially expressed in the1^st,3^rd,4^th,and 5^th instars,respectively.We found that several pathways might be involved in wing form determination with the Gene Ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway analysis of these miRNA targets.We also found Nlu-miR-34-5p can inhibit the expression of NlInR1,the key gene regulating wing morph switch.In 3^rd-instar nymphs,overexpression of Nlu-miR-34-5p induced the development towards SW BPHs.With the inhibition of Nlu-miR-34-5p,we did not observe a decrease of SW morphs,whereas knocking down Nlu-miR-34-5p resulted in the higher ratio of LW BPHs when another NlInR1 suppressor,NlInR2,is also suppressed simultaneously.In all,we uncovered the regulation mechanism of miRNA controlling wing dimorphism in planthoppers and provided a strategy to analyze the molecular mechanism of phenotypic plasticity.