Mining Important Genes For Sheep Reproductive Traits Based On Reproductive Genomics

There are many genes related to reproduction in the genome,which can regulate the development and function of eggs and sperm,affect the morphology and traits of germ cells,and affect implantation and embryonic development.Reproduction is one of the most important economic traits in animal husbandry production,which directly affects the reproductive efficiency and economic benefits of breeding.Sheep reproductive traits are complex traits regulated by multiple genes.The genetic effects and interactions of multiple genes need to be considered in the breeding of reproductive traits.This greatly increases the difficulty of sheep breeding.In view of these problems,through the study of genomics technology,we can deeply analyze the reproductive mechanism of sheep at the genomic level,explore the genetic law and molecular regulatory network of reproduction,and reveal a group of genes that play a key role in the reproductive process.These genes are directly involved in or regulate the formation of reproductive cells,the binding of gametes,embryonic development and the formation of offspring.They may affect sex determination,reproduction rate,reproductive cycle and fertility.In this study,the reproductive traits of sheep were taken as the starting point,and the whole genome association analysis,transcriptome analysis,genome-wide association study based on gene expression,exon selection signal analysis,cell culture experiment and whole genome CRISPR-Cas9 knockout were used to study the genome,tissue expression profile and hormone regulation of sheep.Analyze the genetic parameters and marker-derived genes of litter size and growth traits in sheep,integrate multi-tissue transcriptome data to characterize the genetic basis and biomarkers of sheep reproduction,and explore the regulation of sex hormone FSH(Follicle Stimulating Hormone)on gene expression in sheep granulosa cells,and establish a data set of sheep reproductive genes,analyze the interaction between genes,improve the efficiency and accuracy of sheep breeding,and explore the physiological function and regulation mechanism of sheep reproduction.It provides important reference data and scientific basis for understanding its genetic mechanism and obtaining effective molecular markers for sheep genetic selection.The specific test results are as follows:1、Evaluate the genetic parameters of lambing number and growth traits in sheep,and identify genetic markers and candidate genes.By evaluating the genetic parameters of the lambing traits of 946 ewes and the growth traits of 2289 lambs,it was found that the lamb size(h~2=0.12)and three growth traits were low and medium heritability.The heritability of growth traits birth weight,weaning weight and daily gain of lambs were 0.22,0.28 and 0.29respectively.The breeding value estimation found that the breeding value of males was much greater than that of females.The sheep 50 K medium-density array was further used to conduct genotyping and whole-gene association analysis on sheep with different phenotypes,and a total of 8 SNPs and 16 candidate genes that affect the number of lambs and growth of sheep were discovered.2、Reveal gene expression patterns in multiple tissues of sheep,screen key genes involved in gonadal hormone synthesis,reproduction and other functions in the hypothalamic-pituitary-ovarian axis,and analyze the gonadal axis regulatory mechanism.On the basis of the genetic evaluation in Study 1,the hypothalamus,pituitary gland,ovary,heart,liver,spleen,kidney,uterine horn,uterine body and longissimus dorsi muscle were collected from three sheep with higher estimated breeding values.Samples of 10 tissues were used to construct RNA-seq gene expression maps.Global gene expression analysis found that the ovary,uterus and hypothalamus expressed the largest number of genes,accounting for 85.5%,87.5%and 86.9%of all expressed genes respectively;the heart and liver expressed genes that are common in multiple tissues.Genes expressed in multiple tissues were significantly enriched in the ATM and BMP signaling pathways,and the most expressed genes in liver,spleen and ovary were enriched in the immune response pathway.UMAP cluster analysis(Uniform manifold approximation and projection)clusters 10 tissue samples into 8 categories.Among them,liver and kidney are grouped into one group.The gene expression of the hypothalamic-pituitary-ovarian axis was analyzed.Focused analysis found that the hypothalamus expressed 15,035 genes,the pituitary gland expressed 11,446genes,the ovary expressed 14,919 genes.GO and KEGG enrichment analysis of the top3,000 genes with the highest expression in the three tissues found that the hypothalamus is mainly involved in brain behavior,synaptic transmission and other processes;the pituitary gland is involved in signal transduction and other processes;and the ovary is involved in development,reproduction and immune system process.It is speculated that the hypothalamus and pituitary activate multiple functional signals and transmit these signals to the ovary,thereby activating gamete production and embryonic organ morphogenesis.3、By comparing the differences in ovarian transcriptome of different fecundity,genetic variation related to the growth and differentiation of ovarian tissue was found.In Study 2,it was found that the hypothalamus and pituitary gland activate multiple functional pathways to act on the ovaries,and most of the ovarian genes are enriched in the reproductive process and immune response pathways.Therefore,the expression patterns and functional differences in the ovaries of sheep with different fecundity were further explored.A comparative transcriptome analysis of the ovary tissues of high-and low-fertility sheep found that 884 genes were DEGs with different fecundity,of which 514 genes were highly expressed in the ovary tissues of high-breeding sheep,mainly involved in the reproductive process,organelle assembly,cell movement;there are 370 highly expressed genes in the ovary tissue of low-breeding sheep,which are mainly involved in amino acid metabolism,negative regulation of biological processes,and apoptosis.Among the 17908 genes,9 co-expression gene modules were constructed in WGCNA.Further,expression Genome-Wide Association Study(e GWAs)and exon wide selection(EWSS)were performed signature)screened 12 highly differentiated genes(FGD4,LHFPL2,PPIL6,MILR1,POU2F3,SH3PXD2B,MXRA5,PPP1R9B,NUP62,PHLDB1,XPC and DAXX)affecting high fertility in sheep.These genes can be used as potential new marker genes for sheep reproduction.4、The gene expression patterns of ovarian granulosa cells(GCs)treated with different concentrations of FSH sex hormones were analyzed,and gene clusters related to cell proliferation,differentiation,and reproductive signal transduction were found,and the mechanism of FSH on granulosa cells was inferred.The results of Study 2 and Study 3showed that ovarian expression genes were involved in signaling pathways such as reproductive process and hormone transduction.FSH,as an important regulatory hormone in the hypothalamic-pituitary-ovarian axis,plays a key role in the development and function of GCs.When different concentrations of FSH were added to sheep GC culture in vitro,the proliferation rate of cells supplemented with 10 ng/m L FSH was significantly higher than that of other concentrations(p-value<0.05),indicating that appropriate amount of FSH can significantly improve the proliferation ability of GCs.The cell proliferation rate of GCs cultured with 10 ng/m L FSH was significantly higher than that of other concentrations(p-value<0.05).Transcriptome analysis of GC model treated with high and low concentrations of FSH showed that 10 ng/m L FSH significantly increased the expression of BCL-2 in GCs,accelerated the process of cell proliferation and reduced its apoptosis.Significantly reduced the expression of Casp3 proapoptotic genes.Compared with the control group and 100ng/m L FSH group,10 ng/m L FSH was the best concentration for cultured granulosa cells,and there were 937 highly expressed genes.There were 634 highly expressed genes in the100 ng/m L FSH group.Among the differentially expressed genes,five genes were up-regulated in the 10 ng/m L FSH treatment group and down-regulated in the 100 ng/m L FSH treatment group(EGR1,STAR,IGFBP4,WNT6 and GLI1).The expression of TP53 and MITF genes increased with the increase of FSH concentration.The expression levels of IGFBP3,WNT5A,INHBA,and ESR2 genes decreased with the increase of FSH concentration.The results showed that the addition of appropriate amount of FSH made GCs express more highly expressed genes,promoted the expression of anti-apoptotic genes.5、The first sheep genome-wide CRISPR-Cas9 knockout library and sheep 2,3 and sex chromosome ovarian granulosa cell knockout cell library were constructed.In order to further explore and identify the underlying mechanism of FSH on the regulation of proliferation and apoptosis of GCs,10 ng/m L FSH was added to the cell knockout library for the first time,and the target genes related to reproductive traits were screened,such as EBF2,SP3,FANCG,NEIL2,CNPPD1,UNC5A,RAB14,DAPL4,PTPRN,SELENON,BMP15,GDF7,WNT6,GLI1,FGD4 and MXRA5.These target genes are mainly involved in mitochondrial membrane regulation,lysosomal protein positive regulation and interleukin3,reproductive structure development,dendritic spine morphology regulation,calcium channel inhibitor activity,integrin-mediated positive regulation and glucose binding.CRISPR-Cas9 whole gene knockout technology was used to reveal the FSH regulation mechanism and verify the function of candidate genes.In addition to the known major genes related to reproduction,new regulatory factors were found,which provided a new research direction for further research.In summary,this thesis focuses on the reproductive traits of sheep,taking the litter size of sheep as the starting point,from individual to tissue,from hypothalamus-pituitary-ovarian axis to ovary,from ovary to granulosa cells,through genomics,transcriptomics,cell culture and CRISPR-Cas9 knockout technology system to establish the gene regulation set and interaction network relationship affecting sheep reproduction.These genes play an important role in ovarian maturation and oogenesis,hormone regulation,cell proliferation,migration,invasion,apoptosis,cell metabolism,signal transduction and steroid biosynthesis.It provides a theoretical basis for optimizing sheep breeding strategies,identifying and selecting individuals or strains with good reproductive performance,accelerating the breeding process,and improving the reproductive performance of sheep.