Identification Of The Candidate Genes And Regulatory Network Related To Testicular Development In Hu Sheep Based On RNA-seq And Re-sequencing Data

Reproduction is one of most important economic traits in sheep industry,thus the selection of elite rams at early stage is a huge benefit for improving population reproduction level and economic gains.Testis,as a reproductive organ for male mammals,because of easily for measure,high heritability,significant association with semen quality,has been received more and more attention from breeders.It is recognized that selection rams with larger testis size is the best way to improve male reproduction.Since the development of testis is a sophetic and complex process,it is essential to understand genetic mechanism underlying it.Therefore,the current study histology of testes in different size using hematoxylin-eosin staining?HE?and transmission electron microscopy?TEM?methods;then RNA-seq of testes with different size or at different developmental stages was investigated to identify differentially expressed mRNA,miRNA and piRNA.Meanwhile,Genome-wide association analysis?GWAS?was conducted to select SNPs associated with testis size and derived traits in 207 sheep.Taken together,the aim of this thesis was to find genes and regulatory network controlling testicular development.The main findings were as follow:1.The histomorphological observation of different size testes of 6-month-old Hu sheep was performed.HE staining showed that the testicular tissue of the large-scale testicular tissue had finely defined spermatogenic cells,spermatozoa in the lumen,and small testis spermatogenesis.There were few epithelial germ cells and no obvious sperm production in the lumen.Transmission electron microscopy?TEM?showed that the cytoplasmic bridge between germ cells and germ cells in the large testis group was significantly larger than that in the small testis group.2.The transcriptomic analyese of Hu testes at birth?M0?,three months?M3?,six months?M6?and 12 months?M12?revealed that 4606?2381 up-regulated and 2225 down-regulated?,7500?4368 up-regulated and 3132 down-regulated?and 15?8 up-regulated and 7 down-regulated?differentially expressed gene?DEGs?were identified in M3vsM0,M6vsM3,M12vsM6 comparision groups,respectively.GO and KEGG analyses found that the DEGs above were mainly enriched in spermiogenesis,fertilization,meiosis process and focal adhesion,ECM-receptor interaction,rapI signaling pathway.Subsequently,10 hub genes related to testicular development were determined using weighted gene co-expression network analysis?WGCNA?method.Two hub genes,including vimentin?VIM?and spermatogenesis associated 6?SPATA6?,were selected to analyze expression at protein level using Western-blot method.It was found that the expression level of VIM protein was peak at M0,and the expression level decreased gradually with the ages.The expression level of SPATA6 protein increased gradually with the ages,and the expression level was peak at M6,which was significantly higher than M0 and M3.It was indicated that these two genes may play important roles in testicular development.3.Each three individuals with the largest?LT group,testis weight=480.91+25.07 g?,middle?MT,testis weight=220.86+1.50 g?and smallest?ST,testis weight=62.70+28.38 g?testis size were selected from a large population of 137 sheep reared from weaned time to 165 d old under the same condition and feed.Their testes were collected to do RNA-seq and then find differentially expressed mRNA,miRNA and piRNA.As a result,7019?4143 up and 2876 down?,7101?4279 up and 2822 down?and 1 DEGs were identified in ST vs MT,ST vs LT and MT vs LT,respectively.For miRNA,445?419 up and 26 down?,127?126 up and 1 down?and 2?both down?DE miRNAs were found in ST vs MT,ST vs LT and MT vs LT,respectively.In addition,293?290 up and 3 down?,456?453 up and 3 down?and 75?59 up and 16 down?DE piRNA were found in ST vs MT,ST vs LT and MT vs LT,respectively.Intergted analysis of DEGs,DE miRNA and DE piRNA identified 154 miRNA-mRNA and one piRNA16763_X1—XG—uncon servative₂6₂286849?uncon servative₁7₁152267?regulatory networks that may control testis size of Hu rams.4.The genome-wide association analysis?GWAS?was applied to investigate association of whole genome sequence information with testis size and derived traits in 207 sheep at 6 months.A total of 194 significant SNPs were found,which located in 183 genes.Of which,the SNPs closely related to left testis weight,length,and width were mainly distributed on chromosomes 4,6,15,and 27,and the SNPs closely related to right testis weight,length,and width were mainly distributed on chromosomes 2,3,6,9,12,and 15.These significant SNPs were mainly annotated to THEG,APOL3,PATZI,POU5F1,indicating these genes are candidate genescontrolling sheep testis size,and can be used as marker—assisted selection?MAS?,thus to guide molecular breeding of sheep.