The Genetic Mapping Of Cocoon Morphology And The Influence Of Candidate Gene LOC101743693 On Cocoon Shape In Silkworm,bombyx Mori

Cocoon spinning is a unique behavior of silk-producing insects,and cocoons are formed in various shapes.Following the long-term domestication of silkworm(Bombyx mori),their cocoon shape has become an economically important trait because the different shapes affect cocoon reelability,wrinkle,and other features.Differences in cocooning behavior of silkworms is an important factor in determining cocoon shape.Although the description of this behavior has been studied based on behavioral observations,less is known of the genetic and molecular regulation mechanism of silkworm cocoon shape and cocooning behavior.In this study,the aspect rate was used as the investigation index of cocoon shape based on differences between the ellipse and waist cocoon shapes produced by Chinese and Japanese silkworms.First,the genetics determining cocoon shape was established and the trait was then genetically mapped by bulk segregation analysis sequencing(BSA-seq).Combined with brain transcriptome analy sis during the early stage of silk spinning,candidate genes that alter the cocoon shape by influencing the cocooning behavior of silkworm were screened out.Further analyses determined the structure,evolution,and expression patterns of candidate genes.The gene-knockout strains was developed to preliminarily explore how the candidate genes affect cocoon shape and cocooning behavior.(1)The genetic patterns of ellipse and waist-shaped cocoon are inherited as quantitative charactersUsing Chinese varieties L6J5 cocoon(ellipse-shaped)and Japanese varieties J8 cocoon(waist-shaped)as the parent material,F1 and F2 generations were bred by hybridization and selfing.Cocoon shape genetic analyses were carried out using the aspect ratio of cocoons as the test index.The aspect ratio of the F1 generation cocoon shape was between a waist and an ellipse shape.The frequency of the aspect ratio in the F2 cocoon generation showed a normal distribution,indicating that ellipse and waist-shaped cocoons are quantitative characteristics.(2)BSA-seq combined with transcriptomics analysis revealed candidate genes that influence cocoon shape through behaviorA backcross system BC1 was constructed by backcrossing F1 individuals with their parents.A mixed population of individuals with extreme cocoon-shaped differences was selected for BSA-Seq.Five candidate regions were obtained,with a total length of 6.46 Mb and an interval containing 269 genes.The candidate genes were enriched in the GO and KEGG databases and found to map to pathways that included metal ion binding,mTOR signaling,FoxO signaling,and purine metabolism,among others.The candidate gene LOC101443693 affecting cocoon shape was further screened through joint analysis with brain transcriptomics during the early stage of silk spinning.Phylogenetic analysis of LOC101743693 showed that the species with high homology to silkworm were all silkspinning insects;Transcriptomics and qPCR results showed that the expression of LOC101743693 in J8 brains was significantly higher than in L6J5 brains during the early stage of silk spinning;Spatiotemporal expression analysis showed that LOC101743693 was highly expressed in testis and brain tissues,with expression levels in the brain during hibernation and silking being significantly higher than those during consumption of mulberry.(3)LOC101743693 knockout mutant was constructed to explore the effect of this gene on cocoon shapeBased on the structural analysis of LOC101743693,a LOC101743693-knockout mutant was constructed using CRISPR/Cas9 technology from the DaZao variety of silkworm.After fluorescence screening and purification selection,genome sequencing verification and gene expression verification,dual fluorescence knockouts and nonfluorescence knockouts were obtained.The cocoon shape of knockout silkworm was more regular than that of wild type(WT).The proportion of regular cocoons in knockout silkworms was 73%,whereas that in WT was 40%.indicating that LOC101743693 deletion significantly affected cocoon shape.The aspect rate of the hybrids between the knockout and Japanese J8 silkworms was significantly higher than that of the control group,whereas the aspect rate of the hybrids between the knockouts and Chinese L6J5 silkworms was not significantly different from that of the control group,indicating that the deletion of LOC101743693 gene can increase the aspect ratio.The investigation of spinning rate showed that the knockout silkworms were significantly slower at spinning compared with WT and other controls over an 8 h period,suggesting that LOC101743693 gene affects cocoon shape by altering the spinning behavior.The qPCR results related to GABAergic neurotransmission genes in the larvas’ brain of the early stage of silk spinning show that the expression levels of genes involved in GABA synthesis pathway are higher in the mutant type than in the WT and other control groups suggesting that the loss of LOC101743693 gene may affect silk spinning behavior by affecting GABAergic neurotransmission.