Genome Size And Repeat Sequence Analysis Of 18 Species Of Spotted Wing Locusts

[Objective]There are 40 genera and approximately 156 species of Oedipodidae in China,and are widely distributed throughout the country.Most of them inhabit in dry and temperate regions(Shaanxi,Ningxia,Inner Mongolia,Xinjiang,etc.).We found that the genome size inthis family were enlarged,and the variation mechanism of the genome size is related to the type and contents of the repeat sequences on the genome.Aftermeasuringthe genome size of 18 species in this family,the karyotypes of 8 species were determined.Then,types and contents of repetitive sequences in the genome of this family were analyzedby combining high-throughput sequencing with bioinformatics tools.The main repeat sequences such as the satellite DNA and LTR of Sphingonotus ningsianus were verified to reveal its distribution characteristics on the chromosomesby using fluorescence in situ hybridization.Finally,based on the complete mitochondrial genome data set,the maximum likelihood method was used to construct the phylogenetic relationships of 18 species of Oedipodidae.The phylogenetic signal of genome size and relative lengthof sex chromosomes were analyzed.[Methods](1)Male Locusta migratoria manilensis was selected as an internal reference.By using flow cytometry,the genome size of 18 species of Oedipodidae were determined.(2)Karyotype analysis of 8 species was performed by conventional cytogenetic method.(3)The Illumina platform was used for sequencing the DNA of 18species.We used the local installed RepeatExplorer 2 software and the webbasedGalaxy platformto perform bioinformatics analysis on the sequencing results,and obtained the content of repeat sequences in the genomes of each species.Then we did a comparative analysis for 18 speciesbased on the data above.An in-depth analysis of the repeat sequences of S.ningsianus was performed to find out the content and types of repetitive sequences in the genome.(4)Based on the complete mitochondrial genome sequence of each species,the Haplotropis brunneriana of Thrinchinae,the Calliptamus abbreviatus and Calliptamus barbarus of Calliptaminae,the Euchorthippus unicolor and the Pararcyptera microptera meridionalis of Gomphocerinaewas set as out groups.Maximum likelihood was used to construct a phylogenetic tree of 18 species of Oedipodidae.Evolutionary analysis of the phylogenetic signal of the genome size and the relative length of the sex chromosomes were carried out in R 3.5.3.In addition,MEGA X was used to construct a phylogenetic tree on 153 LTRs of S.ningsianus.(5)Based on the results of repeat sequences analysis,we selected 5 sequences,including 4 satellite DNAs and 1 LTR,and used SnapGene to design primers.Finally,fluorescence in situ hybridization experiment was carried out on the previously prepared male chromosome slides.[Results](1)In this study,we determined the genome size of 18 species of Oedipodidae.The average genome size is 12.006 pg.The genomes size of this family is quite different,with the smallest genome of Aiolopus tamulus(?9.177 pg and ?7.858 pg),and the largest genome of Bryodemella holdereri(? 18.041 pg and ?17.601 pg)respectively,both showed a two-fold difference between them.Comparing the genome sizes of male and female species,we found that the average genome size of female grasshoppers is 13.513 pg,and the male is 12.560 pg.The difference between the two is 0.953 pg.In general,the genomes of females are larger than that of males.(2)We measured the chromosomes data of 8 species of Oedipodidae in the experiment,and found that the number of chromosomes of all species is 2n(?)=23.Gastrimargus marmoratus has the largest sex chromosome,which is located at the second place in the whole chromosome group.S.ningsianus,Ai.tamulus,and Oedaleus asiaticus have medium-sized sex chromosomes.Oedaleus manjius,Angaracris rhodopa and Bryodema kozlovi have the smallest sex chromosomes,and located in the uttermost end of the whole chromosome set.Sex chromosome of B.holdereri is not the smallest in the whole chromosome group,but it is also a small chromosome.(3)There are 74.62%of repeat sequences in the genome of S.ningsianus,and 59.26%of the sequences are annotated as top clusters.Among the identified repetitive sequences,the content of satellite DNA is 2.94%,the content of rDNA is only 0.14%,and the content of mobile elements is the highest,which accounts for 19.33%,including 8.39%of Ty3_grpsy,7.17%of LINE,2.08%of Maverick,1.63%of Penelope,0.02%of Helitron,and 0.04%of unclassified retrotransposons.In addition,the top 110 clusters of the sequence were clustered for S.ningsianus,and found that LINE and LTR had the largest number of sequences,which were 27 and 26 respectively,followed by satellite DNA and Maverick,which were 7 and 8,and the content of Penelope,Helitron,and rDNA were relatively less.This result is consistent with the overall annotation of the repetitive sequence of the species.(4)Among the 18 species of Oedipodidae,the repeat sequences accounted for more than 50%of the genome,except for B.kozlovi(46.64%).The repetitive sequences of the An.rhodopa,S.ningsianus,O.manjjius,Epacromius coerulipes,and Angaracris barabensis have the most abundant repetitive sequences,which respectively account for 76.68%,74.62%,74.05%,71.52%,and 71.25%of the entire genome.In addition,a cluster analysis of top clusters of the repeating sequences was carried out for all species,and we found that G.marmoratus,O.decorus asiaticus,Ai.tamulus,Trilophidia annulata and E.coerulipes contained their own specific satellite DNA,S,ningsianus,Sphingonotus turcmenus and Leptopternis gracilis bunch the common satellite DNA,Ty3_gypsy,and LINE.CL58-148 and CL191-136 are repetitive sequences common to all species.What is noteworthy is that the 5S rDNA corresponding to CL131 and 45S_rDNA with CL148 have only been detected once and were present in genomes of all species.(5)The phylogenetic tree of 18 species of Oedipodidae was constructed and the genome size and relative length of sex chromosomes of insects were fitted to the phylogenetic tree.We found that there was no phylogenetic dependence on the genomic size and relative length of sex chromosomes.The phylogenetic relationship of LTR showed that Bel_Pao and Ty1_Copia were closely related,Ty1_Copia(6)and Ty3_Gypsy were sister groups,and Ty3_Gypsy could be divided into four branches.(6)Fluorescence in situ hybridization experiments revealed that HS1 had a hybridization signal on each chromosome arms,HS2 and HS3 had a clear and bright signal at the centromere of all chromosomes,and LS1 only showed some bright signal at the end of the chromosome,and the repeats corresponding to LTR 1 are located in different parts of the chromosome arms.