Whole Transcriptome Difference Analysis Of Ovary In Laying And Egg-ceasing Xingguo Grey Goose

Goose,a typical seasonal breeding animal,has various striking changes in behavior and physiological state with seasonal changes.The distinguishing marks of non-breeding goose are egg ceasing and feather replacement.During non-breeding period,the volume and functional of goose ovary is degradation,and the reproductive related hormone levels undulate sharply,which directly lead to uneven egg production throughout the year.However,the underlying molecular mechanism of reproductive cycle and ovarian function changes remains obscure.This study aims to use high-throughput sequencing technology to explore the candidate factors and interaction networks related to ovarian function of Xingguo gray goose during egg production and ceasing period.The main results are as follows:(1)The ovaries of the laying and egg-ceasing geese were collected to morphological observation.It was found that the vascular network of the egg-ceasing geese was underdeveloped and only a few pre-grade follicles were observed on the surface,whereas,the vessel network on hierarchically developed follicles of the laying geese ovaries were intricate,and several graded follicles of different developmental stages were adhering to the ovaries.(2)In this study,high-throughput sequencing was applied to perform whole transcriptome sequencing of mRNA,lncRNA,circRNA,and miRNA of goose ovaries.After filtering,a total of 95.05 Gb of clean reads were obtained.By comparing with the chicken Galgal4 genome,86.62% of clean reads was blasted to the reference genome.Otherwise,77.70 M clean reads were obtained in the small RNA sequencing library.After length screening,a total of 90.73% clean reads was blasted to reference genome.(3)In this study,a total of 33,089 mNRAs,15,573 new lncRNAs,13,857 new circRNAs,272 known miRNAs,and 49 new miRNAs were identified.The expression levels and the differentially expressed transcripts were analyzed,a total of 901 differentially expressed mRNAs(DEM),80 differentially expressed new lncRNAs(DEL),21 differentially expressed new circRNAs(DEC)and 50 differentially expressed miRNAs(DEMI)were screened in the ovaries of the laying and egg-ceasing groups.(4)The GO and KEGG pathway databases were used to perform functional and pathway enrichment analysis of the DEMs.Down-regulated genes of the laying ovaries,such as AMH,NR2C2 and TPD52 X6,were enriched in the androgen receptor signaling pathway and nuclear hormone receptor binding.Up-regulated genes,such as RPS2,RHOA,ANXA5,MSN,and FSHR,were enriched in biological processes such as female gamete formation and follicular cell development.(5)Functional analysis of lncRNA target genes was performed.The results found that LNC＿005144,LNC＿015230,LNC＿000939,and LNC＿003797,which were up-regulated in the laying ovaries,were enriched in the steroid hormones synthetic biological pathways.In addition,most target genes of LNC＿015573 and LNC＿005873were enriched in the cell cycle pathway.As for LNC＿013695,LNC＿005873,and LNC＿006976,owned several target genes in common,and these target genes were involved in many processes of ovarian growth and development.(6)Biological function of circRNA original genes was analyzed.A total of 13 circRNA original genes were enriched to 328 GO entries,including metal ion binding,cell adhesion,biological adhesion,regulation of cytokinetic process,and regulation of cell cycle process.(7)Functional and pathway enrichment analysis on 50 target genes of DEMIs was conducted.We found several important miRNAs involved in the regulation of ovarian development and follicle formation,such as mi R-205 b,which was significantly up-regulated expressed in egg-ceasing ovaries.Nevertheless,the target genes FSHR,ANXA2,and ANXA5 were significantly down-regulated in the egg-ceasing group.All these genes were involved in hormone signal transduction and cell membrane component formation.Moreover,mi R-101-3p also significantly up-regulated expressed in the egg-ceasing ovaries,which regulated the target genes,FSHR and TPD52 starkly down-regulated expressed in the egg-ceasing ovaries.These two genes were mainly involved in cell proliferation and apoptosis.(8)In this study,differentially expressed lncRNA,circRNA,miRNA,and mRNA were analyzed by correlation analysis,respectively,and related regulatory networks were constructed.A total of 54,940 pairs of lncRNA-miRNA-gene pairs and 22612 pairs of circRNA-miRNA-gene pairs were identified.Among them,we screened multiple interaction networks that may be involved in ovary development,such as LNC＿005847-mi R-101-3p-FSHR,LNC＿013788-gga-mi R-365b-5p-NR2C2,novel＿circ＿CA9-gga-mi R-205b-ANXA2,novel＿circ＿BMPER-gga-mi R-101-3p-TPD52,etc.In summary,we exerted high-throughput sequencing technology to perform whole transcriptome sequencing of goose ovaries during the laying and egg-ceasing periods,and screened a great deal of candidate factors,which may play vital roles in regulating the development of goose ovary in different reproductive cycles,such as TPD52,ANAX2,RPS2,mi R-205 b,mi R-101-3p,mi R-365b-5p,and many new lncRNAs and circRNAs.Moreover,a mass of molecular interaction regulatory networks were also screened.All the above may lay a solid foundation for further study to reveal the underlying molecular mechanisms of avain seasonal reproduction.