Screening And Analysis Of Sex-Difference Genes Between Silkworm (Bombyx Mori) And Wild Silkworm (Bombyx Mandarina)

Silkworm is a major economic insect in China.The sex of male and female is one of the important factors that affect the efficiency of cocoon production.Studying the sex-differential and sex determination relate genes of silkworms is of theoretical and practical significance for the development of the silk industry and even understanding the basic knowledge of biological sex determination.Although the whole genome sequencing of Bombyx mori has been accomplished,due to the limitation of sequencing technology and objective conditions,the sequencing of the W-chromosome of Bombyx mori has not been totally obtained so far.In this study,the near-isogenic lines constructed by crossing between female wild silkworm and male silkworm(DZ)and continuous backcrossing with male silkworm were used as materials.Through comparative transcriptome analysis,the differential genes between male and female of silkworms and wild silkworms were screened to obtain sex determination related genes;At the same time,sex identification and W chromosome candidate sequences were screened by whole-genome resequencing method.1 Phenotype survey and analysis of near isogenic linesThe female wild silkworm and the male silkworm(DZ)were selected to cross,and the female offspring of F1 and the male silkworm(DZ)were backcrossed for more than10 generations to construct a near-isogenic silkworm(WS).In addition to the W chromosome from the wild silkworm,the overall characteristics of the WS were highly consistent with DZ,indicating that the near-isogenic line has been successfully constructed and can be used for subsequent analysis and research.Phenotype survey found that the incubation rate of WS was significantly greater than that of DZ.Analysis of the transcriptome data of DZ and WS gonadal revealed that the expression level of hatching enzyme-like II isoform X1 in gonad of WS group was significantly higher than that of DZ.It is speculated that the different expression levels of hatching enzyme-like II subtype X1 resulted in the difference in hatching rate;the result of HE staining of gonadal tissue sections showed that the development of ovarian germ cells in the WSgroup was faster than that in the DZ group,and the oocytes in the WS group contained more cells in the DZ group,suggesting that the difference was related to the high expression level of microvitellogenin(Vg)and PPO1 genes in the WS ovaries,as nutrients required for the growth and development of the eggs.2 Screening for sex-differential gene using gonadal transcriptome sequencing analysisIn order to research the strain specific feature of DZ and WS from the transcription level,the glands of female and male of DZ and WS at 5L3 d were selected for transcriptome analysis,and 2436 regulate genes in males and 1380 up regulate genes in females were selected.Pcs.Through KEGG enrichment analysis,it was found that these differentially expressed genes were significantly enriched in the translation and metabolism-related pathways,such as ribosome,oxidative phosphorylation,pyruvate metabolism,etc.These differentially expressed genes have constituent ribose structural functions,such as structural constituent of ribosome,oxidoreductase activity,nutrient reservoir activity,a large number of ribosomal proteins in the ribosomal pathway and various enzymes in the oxidative phosphorylation pathway,Regulates the normal development of the silkworm's gonads;the expressions of SP1 in B.mandarina,WS,and DZ ovaries gradually increased,indicating that WS is an intermediate strain between wild silkworms and silkworms;The RNA Interference experiments of SP1 were carried out on female and the results confirmed that the down-regulation of SP1 would reduce the expression of Vg,but the expression of key genes Siwi and Ago in PIWI pathway was not affected,suggesting that SP1 was mainly involved in the regulation of downstream genes of the female sex determination pathway in silkworm.3 Screening W chromosome candidate sequences using whole genome resequencing methodIn order to compare and analyze the difference between male and female at the genome level,the silk glands tissues of DZ and WS at 5L3 d were collected for re-sequencing mutation detection(a total of 12),and 19 previous re-sequencing data were downloaded from the NCBI SRA database.The silkworm re-sequencing data was de novo assembled,and Fem sequence was used as a marker for sex identification.Finally,183 W-chromosome candidate sequences were obtained.The results of PCR experiments showed that total candidate fsequences could be amplified in females.Two candidate sequence were also found in wild silkworm males,suggesting that this type ofcandidate sequences may originated from the Z chromosome or autosome of the wild silkworm.After the long-term evolution,they eventually evolved into the Wchromosome.