Screening Of Genes Related To Different Stages Of Hair Follicle Growth In Subo Merino

In present study,we performed transcriptome sequencing on the left scapular skin tissue(three biological replicates were used for each stage)of Subo Merino sheep at multiple embryonic days 65d(G1,formation of primary hair follicles),85d(G2,formation of secondary hair follicles),105d(G3,formation of secondary acquired hair follicles),135d(G4,mature hair follicles),lambs aged one week 7d(G5),and one month 30d(G6)using RNA-seq technology.The bioinformatics method was used to analyze the differentially expressed genes at different hair follicle developmental stages,in order to screen out the candidate genes related to hair follicle development,and to provide a theoretical basis for exploring the mechanism of hair follicle development.At the same time,RT-PCR method was used to verify the sequencing results,and PCR-SSCP technique was used to verify the KAP16 gene in large population.The main results are as follows:(1)RNA-seq analysis was performed on 18 skin samples of Subo Merino sheep for 6 time points by using Illumina Hiseq 2000.With an average of 12 G data were produced for each sample.The raw reads of each sample reached more than 73 million,while clean reads reached more than 68 million,and clean ratios ranged from 90.98% to 94.79%.(2)Analysis of differentially expressed genes revealed that 7879 differentially expressed genes were found during hair follicle development in Subo Merino sheep;in which,1562 differentially expressed genes were found at the G2/G1 stages;2302 differentially expressed genes were found at the G3/G2 stages;1520 differential expressed genes were found at the G4/G3 stages;699 differential expressed genes were found at the G5/G4 stages;166 differential expressed genes were found at the G6/G5 stages;and 6561 differential expressed genes were found at the G6/G1 stages.A total of 12623 novel differentially expressed genes were captured during the hair follicles developmental stages in Subo Merino sheep.Among them,1762 differential genes were found at the G2/G1 stages,2066 differential genes were found at the G3/G2 stages;611 genes differences were found at the G4/G3 stages;842 differential genes were found at the G5/G4 stages;28 differential genes were found at the G6/G5 stages,and 10691 differential genes were found at the G6/G1 stages.(3)GO function analysis revealed that the differentially expressed genes were significantly enriched in cell components,molecular functions and biological processes(P<0.05).Based on the KEGG pathway analysis,some of the differentially expressed genes are enriched into the Wnt,Notch,Hedgehog,TGF-β,MAPK,TNF,and Hippo signaling pathways.K-means clustering analysis revealed that all of the differentially expressed genes at the 6 time points were classified into 10 clusters based on their group mean and changes with respect to other compartments.PPI analysis of differentially expressed genes in different periods was performed by Cytoscape software,and KRT14,FGF7,Notch1,ALB and CFTR were found to be the key proteins.(4)RT-PCR was used to verify the gene expression levels of 10 differentially expressed genes.The relative expression levels of selected genes were consistent with the sequencing results.Collectively,the sequencing results were reliable.(5)In this study,re-sequencing and PCR-SSCP methods were used to validate the genetic polymorphisms of 2 SNPs mutation points of KAP16 gene in Chinese Merino(Xinjiang type)population.At the same time,the SAS 8.1 least-squares analysis technique was used to study correlation with wool traits.χ2 test raveled that the C41006938 T and T41007938 C mutation points were in the Hardy-Weinberg disequilibrium(P>0.05).The polymorphic information content of C41006938 T and T41007938 C mutation points were 0.37 and 0.18,respectively;therefore,Subo Merino sheep population was reached the moderate level at the genetic variation of the gene.The different genotypes of C41006938 T had a significant effect on body size and live weight after sharing(P<0.05);the different genotypes of T41007938 C had a very significant effect on fiber diameter and coefficient of variation(P<0.01),and had a significant effect on greasy weight and number of crimps(P<0.05);the combined genotypes of the C41006938 T and T41007938 C had a very significant effect on greasy weight(P<0.01),and had a significant effect on fiber diameter and coefficient of variation(P<0.05).