Association Analysis Of Lef1,Dlx3 And Hoxc13 Gene Expression,Polymorphism Identification And Wool Traits In Gansu Alpine Fine-Wool Sheep

Hair follicles undergo cyclic growth during the growth process.Many signaling pathways play an important role in the development of the hair follicle cycle,and these pathways interact with each other to form a complex regulatory network that regulates the hair follicle cycle.According to previous studies,Lef1,Dlx3 and Hoxc13 play important regulatory roles in hair follicle development and hair growth,and mutations in them can affect hair follicle development.Therefore,we collected skin tissues from Gansu Alpine Fine-wool Sheep at different periods(1,30,60,90,180 and 270 d)and applied conventional HE staining to observe the structural characteristics of skin tissues;Application of RT-q PCR technique to analyze the expression of Lef1,Dlx3 and Hoxc13 in skin at different periods;application of immunohistochemistry and immunofluorescence techniques for protein quantification and observation of distribution localization of Lef1,Dlx3 and Hoxc13;The detected SNPs were typed and analyzed for wool trait association by applying sequencing technology and KASP genotyping.The main results were as follows:1.The results of the histomorphological development of the skin showed that the number of hair follicles was found to show a continuous decrease and then stabilization,which dropped to a minimum at 60 d and remained unchanged in the following periods,significantly lower than in the first and 30 d periods(P < 0.05);Hair follicle area varied with age and was significantly higher at 60 d,90 d,and 180 d than at other periods(P< 0.05);epidermal thickness did not vary significantly across ages(P > 0.05);The epidermal thickness did not change significantly(P > 0.05)with age;the dermal thickness changed with age and was significantly higher(P < 0.05)at the 90 th d than in other periods;the follicle diameter likewise changed with age and was significantly higher(P < 0.05)at the 90 th d.The trend was basically consistent with the follicle area and dermal thickness.It indicates that the changes in the number of hair follicles in this study are in accordance with the pattern of hair follicle development,and the most significant changes in follicle diameter,follicle area and dermal thickness were found to be concentrated in the regression phase.2.The results of differential gene spatiotemporal expression analysis showed that Hoxc13,Lef1 and Dlx3 were expressed in the skin at different stages of hair follicle development.The m RNAs were differentially expressed at different stages and showed a strong spatiotemporal expression profile.The m RNA expression trends of Hoxc13,Lef1 and Dlx3 were consistent throughout the hair follicle cycle development,all showing an increase followed by a decrease,and the m RNA expression of Hoxc13,Lef1 and Dlx3 was significantly higher at 180 d than other periods(P < 0.05).3.The results of protein expression and distribution localization studies showed that the mean optical densities of Lef1 and Dlx3 were significantly higher at 30,60 and90 days of age than at 1,180 and 270 days of age(P < 0.05);the mean optical density of Hoxc13 was significantly lower at 1 day of age than at 30,60,90,180 and 270 days of age(P < 0.05).Immunofluorescence results showed that Lef1 was mainly expressed in the dermal papilla with high-density strong positive expression,Dlx3 and Hoxc13 were mainly expressed in the inner root sheath,and the outer root sheath and dermal papilla with high-density strong positive expression.It was found that Lef1,Dlx3 and Hoxc13 play important roles in the development of hair follicles in the inner root sheath,outer root sheath and dermal papilla.4.The results of Lef1 and Hoxc13 gene polymorphism identification and association analysis showed that two SNPs(g.142108969T>G and g.142109237A>G)of Hoxc13 were detected in Gansu alpine fine wool sheep and significantly associated with wool length(P < 0.05).Six SNPs of Lef1 were detected,while five of them(g.20657218C>T,g.20657450C>T,g.20657494G>A,g.20657554A>T and g.20657538G>T)were significantly associated with wool length(P < 0.05);one SNP(g.20657552T>C)was significantly associated with mean fiber diameter(P < 0.05).haplotype combinations H2H3 and H1H3 of Lef1 were significantly correlated with wool length(P < 0.05).In summary,this study found that Hoxc13,Lef1 and Dlx3 showed high expression during the degenerative phase of hair follicle cyclogenesis in Gansu Alpine Fine-wool Sheep.While Hoxc13 and Dlx3 were mainly expressed in the outer root sheath,inner root sheath and dermal papilla of the hair follicle during the whole hair follicle cycle development,Lef1 was only expressed in the dermal papilla.Two SNPs of Hoxc13 and six SNPs of Lef1 were identified,and the Hoxc13 variant was found to be significantly associated with wool length and the Lef1 variant was found to be significantly associated with wool length and average fiber diameter.Hoxc13 and Lef1 can be used as candidate genes for molecular genetic markers to improve wool traits in Gansu alpine Fine-wool Sheep.