| In broiler sheep production,shedding in spring can save the tedious operation of hair cutting and facilitate production management,which is one of the breeding directions of modern breeding.In this study,the skin samples of Dorper ewes with extreme phenotypes of shedding(group S,n=5)and nonshedding(group N,n=3)were sampled in September 27,2019,January 3,2020 and March 17,2020,and the differentially expressed genes were analyzed by high-throughput sequencing and Bioinformatics,GO analysis,KEGG signaling pathway analysis,expression pattern analysis,WGCNA analysis,and other bioinformatics methods were used to screen genes associated with shedding traits and qRT-PCR validation was performed.The results were as follows:1.Through RNA-seq technology,there were 2533 differentially expressed genes(DEGs),among which 2120 DEGs were screened out in the s group,389 DEGs were screened in the N group,and 1011 DEGs were screened in the S-vs-N group.GO and KEGG analysis of all DEGs showed that more DEGs were enriched in functional entries such as cellular process,biological regulation,metabolic process,cell,cell part,binding,catalyst activity and were enriched in disease-related,immune and endocrine system,signal transduction,signal molecule and interaction,lipid metabolism and other related pathways.2.Through expression pattern analysis of Wnt pathway DEGs and keratins,gene expression patterns associated with traits were determined,which were A pattern(highly expressed in anagen)and T pattern(highly expressed in telogen).Five genes of A pattern in the Wnt pathway,including FZD3,FZD10,LEF1,WNT10B and WNT6,were selected as candidate genes to play a role in the anagen.Meanwhile the stages of hair follicle cycle in shedding Doper sheep were determined as:September for anagen,January for telogen and March for early anagen.The stages of hair follicle cycling in non-shedding Dorper sheep are:September anagen,January early catagen,March mid catagen.3.Expression pattern analysis of all DEGs,a total of 970 DEGs were in accordance with the A or T pattern,of which 394 DEGs were in T pattern and 576 DEGs were in A pattern.Through KEGG pathway analysis of 970 DEGs,a total of 302 pathways were enriched.It was found that pathways such as Wnt、Cell cycle,Estrogen signaling pathway,Melanogenesis,Gap junction,and RNA transport may play a role in the anagen of hair follicles.PI3K-Akt,Ras,NF-kappa B,Jak-STAT,Antigen processing and presentation,Fc gamma R-mediated phagocytosis,Endocytosis,Ubiquitin mediated proteolysis,and calcium signaling,may play a role in the telogen of hair follicles.According to the KEGG map of PI3KAkt,Ras,NF-kappa B and Jak-STAT pathways,the hair follicle telogen regulatory network was constructed.Ten genes were screened:PDGFD,VEGFA,PDGFRB,JAK1,BCL-2,MMP14,ZAP70,GNB3,GAB2 and IRS-1 that may influence shedding traits in sheep.4.By performing WGCNA analysis on all genes,the firebrick3 and grey60 modules were found to be the highest associated with shedding traits.Expression pattern analysis was performed on the top ten hub genes in the connectivity of the two modules,and it was found that the genes in the modules were in line with the A and T patterns,and these genes will be used as candidate genes for further research.5.The expression levels of the 20 genes were quantified by real-time PCR,and the results showed that the qRT-PCR quantification levels were generally consistent with the changing trends of the transcriptome sequencing results.Several candidate genes associated with hair removal traits in sheep were screened in this test,which may help to dissect the molecular regulatory mechanisms associated with hair removal traits in sheep,and to establish a theoretical basis for the eventual breeding of new breeds of broiler sheep capable of shedding in spring. |
