| The eri-silkworm(Philosamia cynthia ricini,Lepidoptera),is native to India and is reared in Northeast,East and South China.Compared with silkworm(Bombyx mori,Lepidoptera),which only feeds on mulberry leaves,eri-silkworm is a polyphagous silkworm that feeds on castor leaf,cassava leaf,etc.As an important silk insect,eri-silk has excellent performance and unique feature,and has a wide prospect of development and application.The haploid chromosome number of common Lepidoptera insects is more than 20,while eri-silkworm is only 14,which is the least number of Lepidoptera insects reported.The male sex type of eri-silkworm is ZZ and female is ZO,and the sex determination mechanism is the deletion of W chromosome determinism,so it is an excellent material for molecular biology research.Based on the second-generation sequencing technology,this study used Illumina Hiseq-2000 sequencing platform and whole-genome shotgun sequencing strategy to conduct whole-genome sequencing on the cultivated variety Nan-yi eri-Silkworm in Nanning,and combined with bioinformatics methods,the genome of eri-Silkworm was preliminarily assembled.We evaluated the quality and annotated the genome on the preliminarily assembled genome,and assisted by the HI-C chromatin conformation capture technology,the genome of eri-silkworm was upgraded from scaffold level to chromosome level.Comparative genomics was used to compare the conserved regions on the genomes of eri-silkworm and other Lepidopteran insects to reconstruct the ancestor genome of Lepidopteran insects.Chromosome rearrangement and chromosomal fusion events in modern species were identified,and the relationship between repeats in different genomic regions and chromosomal variation events was explored.In this study,the genome of eri-silkworm at chromosome level was assembled and annotated,which provided support for further industrial development and scientific research of eri-silkworm.Moreover,comparative genomics analysis was carried out combining with other Lepidoptera genomes,which enriched the evolutionary theory of Lepidoptera chromosomes.The main results of this study are as follow:1.Whole genome sequencing and assembly and annotation of Eri-silkworm genome.In this study,the silk gland materials of the male eri-silkworm were used to extract their genomes.Seven Pair-End and Mate-Pair sequencing libraries were constructed,and then sequenced in sequence machine.After strict filtering and quality control of the original sequencing data,we obtained 426 GB of sequencing data for genome assembly.Using the De Brujin graph-based assembly method,the short read and long fragments of the sequencing data were assembled into a eri-silkworm genome consisting of 3612 scaffolds through contig and scaffold assembly stage.The contig-level genome size was460 M,and the scaffold-level genome size was 480 M.The contig N50 was 12322 bp and the Scaffold N50 was 5.83 Mbp,which could be compared with 95% of metazoan BUSCOs on the genome,indicating that the high quality assembly genome was obtained.Using the HI-C chromatin conformational capture technique,the scaffoldlevel genome was elevated to the chromosome level,and 95% of the sequences were assigned to 14 of the longest scaffolds corresponding to 14 chromosomes of the erisilkworm.The scaffold-level N50 was about 33 M,a fourfold increase compared with the previous one.The genome of eri-silkworm has expanded the reference genome resources of lepidopteran insects and contributed to the study of molecular biology and genetic improvement of eri-silkworm.Besides,the sequences of the least 14 erisilkworm chromosomes in the published Lepidoptera genome were analyzed,which play a fundamental influence in the further study of chromosome evolution.2.Genome annotation of castor silkwormBased on the genome sequence,we annotated the protein-coding genes and other genomic elements of the eri-silkworm genome.(1)Gene recognition and functional annotation.Combined with de novo prediction and homologous annotation,we identified 16,291 protein-coding genes in the genome of eri-silkworm,with an average of 5.3 exons per gene and an average of 221 bp,which was within the average level of lepidopteran insects.Moreover,the average length of introns showed great species specificity.Through comparison with protein function databases such as PFAM,TREMBL,Swiss-Prot,KEGG and GO,97.35% of these genes were annotated by at least one database.(2)Non-coding RNA annotations.The sequence of 818 kb noncoding RNA in the genome of eri-silkworm was annotated using database alignment and domain identification methods,and there was no significant difference in the average length of non-coding RNA among the lepidopteran species.(3)Repeat sequence annotation.In the genome of the Silkworm,we identified 1218498 repeat elements,which accounted for 43.76% of the genome.SINEs were 1.34%,DNA transposons(18.41%)and LINEs(14.23%)were more than SINEs,and LTRs were4.14%.3.Chromosome evolution of Lepidoptera.It is widely believed that the ancestor of Lepidoptera has a haploid chromosome number of 31.Modern Lepidoptera species inherited this karyotype and developed a variety of karyotypes through chromosomal fusion and fission events.In the synteny analysis of the genome of eri-silkworm and other chromosome-level genomes,the chromosomes of the mordern species with the number of chromosomes of 31 matched one to one.The larger chromosome of lepidopteron species with a chromosome number less than 31 can be formed by the fusion of the smaller chromosome of a species with a chromosome number of 31.In the genome of eri-silkworm,it was found that up to 4chromosomes could be fused into 1 chromosome,while in other species,2chromosomes were fused into 1 chromosome.By comparing Genomes of seven species,synteny blocks composed of common conserved regions were obtained,which were finally spliced into a 31-chromosome ancestor genome of Lepidoptera.Based on the orders of synteny blocks in ancestral genomes,the rearrangement events of chromosome segments from seven families of Lepidoptera were identified.Among them,the Capricornidae and Pteridae were considered to have experienced the most dramatic rearrangement changes.According to the sequence alignment result,genome can be divided into conservative region and the conservative region.Comparison of genomic elements in two regions showed that the distribution of repeat sequences was not uniform.In most species,the number of repeats in the non-conserved region was significantly higher than that in the conserved region,suggesting that the repeats in the non-conserved region of Lepidoptera may be an important driving force for the evolution of chromosomes.In this study,based on the second-generation high-throughput sequencing technology and whole-genome shotgun sequencing strategy,we sequenced the whole genome of Castor silkworm from Nanning,and assembled the genome of Castor silkworm with the highest chromosomal quality at present,which provides data support for the related basic research and genetic development.Our study verified the ancestor karyotype of lepidopteran at the DNA level and provided a new insight for the chromosomal evolution theory of lepidopteran. |
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