Non-coding RNA Identification And Single-cell Transcriptome Atlas Of Angus Bull Testis

Angus cattle is one of the main breeds of beef cattle.It has the advantages of early sexual maturity,easy production,low birth weight,fast growth,good meat quality,and strong adaptability.As a representative breed of good meat quality,Angus cattle have been introduced in large numbers to improve local cattle breeds and has achieved remarkable results in the production of high-quality and high-grade beef.Bull is an important part of the breeding herd and plays an important role in the improvement of the genetic performance of beef cattle populations.Breeding bulls provide a large amount of high-quality semen for the production of beef cattle so that excellent genetic traits can be stably inherited.Thus,ideal reproductive performance is very important for breeding bulls.The testis is an important organ for spermatogenesis and normal testicular development is the basis for the excellent reproductive performance of bulls.Therefore,exploring the genetic and molecular regulation mechanisms of testicular development and spermatogenesis in bulls is of great significance to the breeding of high-yield and high-quality bulls.The normal progress of testicular development and spermatogenesis depends on the regulation of transcription and post-transcriptional levels of related genes.Non-codingRNA has been shown to play an extremely important role in the regulation of transcription or post-transcriptional levels.Therefore,this study performed identification analysis and functional verification of ncRNA（lncRNA,miRNA,circRNA）at the transcriptome level by selecting Angus cattle as the research object.This study aimed to screen the ncRNA related to testicular development and spermatogenesis during sexual maturation of Angus cattle to analyze the genetic basis of bovine testicular development from the perspective of ncRNA.At the same time,single-cell transcriptome technology was used to analyze the regulation mechanism of bull testis development,explore the molecular regulation mechanism of bull spermatogenesis and testicular somatic cell development,and provide valuable resources for enriching the transcription regulation mechanism of bull testis development.The main results of this study are as follows:（1）Through the identification and analysis of lncRNAs of Angus cattle testis at different developmental stages,23,735 lncRNAs were identified,of which,540 lncRNA（P<0.05）and3,525 mRNA（P-adj<0.05）were differentially expressed;GO and KEGG enrichment exhibited that the target genes of differentially expressed lncRNA and mRNA were enriched in a large number of pathways related to spermatogenesis;through the lncRNA-mRNA interaction analysis,an interaction network of 15 DE lncRNAs and 12 cis target genes related to testicular development was constructed,among them,lncRNA target genes（SPATA16,TCF21,ZPBP,PACRG,ATP8B3,COMP,ACE and OSBP2）are related to bovine testicular development and sexual maturation.（2）Through the identification and analysis of miRNAs of Angus cattle testis at different developmental stages,566 known and 86 new miRNAs were identified;differential expression analysis showed that 223 miRNAs（202 known and 21 novel miRNAs）were differentially expressed in neonatal and sexual maturity stage;there were 112 miRNAs（92 known and 20novel miRNAs）up-regulated and 111 miRNAs（110 known and 20 novel miRNAs）down-regulated in the sexually mature testis（P-adj<0.05,|log₂fold-change|>1）;according to KEGG enrichment analysis,differentially expressed miRNA target genes are enriched in the pathways related to spermatogenesis in testicular development.（3）Through the identification and analysis of circRNAs of Angus cattle testis at different developmental stages,28,065 candidate circRNAs were identified,of which,7,458circRNAs were co-expressed in the bovine testes at the neonatal and sexual maturity stages;differential expression analysis showed that 987 circRNAs differentially expressed（P<0.05）;taking the neonatal stage as a control,710 circRNAs were up-expressed and 277 circRNAs were down-expressed in sexual maturity stage;the GO and KEGG functional enrichment analysis were found that the host genes of differential circRNA were significantly enriched in36 GO terms and 8 pathways.（4）According to the circRNA sequencing data,the differentially expressed circ SMC1B was screened out,and it was verified that circ SMC1B was highly expressed in the sexually mature stage and was highly expressed in bovine m GSCs.Therefore,overexpression of circ SMC1B demonstrated that circ SMC1B promotes the proliferation and apoptosis of bovine m GSCs by using CCK-8,Ed U,flow cytometry,RT-q PCR and other experiments;RNAhybrid software and the dual-luciferase reporting system showed that circ SMC1B competitively binds to let-7i;CCK-8,Ed U,flow cytometry,RT-q PCR and other experiments showed that let-7i targets HMGA1 and NR6A1 to inhibit the proliferation and apoptosis of bovine m GSCs;and found that circ SMC1B can regulate the expression of target genes HMGA1 and NR6A1by binding let-7i to promote the proliferation and apoptosis of m GSCs.（5）The testicular cells were divided into 12 groups,including 9 types of somatic cells and 3 types of germ cells,through cluster analysis by using scRNA-seq technology in bovine testicular cells at sexual immature and mature stages;specific markers of different types of bovine testicular cells were successfully identified,such as ARSE,CLEC12B,GAS1,KRT8,LOC101908015,HIGD1B,CCL21,ESX1,MEIOB,SPATA19,etc.;the testicular germ cells were grouped into 13 groups,in which specifically expressed genes were identified,which comprehensively explains the molecular mechanism of bovine testicular development and sperm production.In this study,the expression of coding genes and ncRNA in cattle testis tissue were comprehensively analysed by usingRNA-seq and scRNA-seq.There were a large number of differentially expressed ncRNA and mRNA related to testicular development and spermatogenesis were found in cattle testis tissue,and the cattle testis were successfully identified the specific marker genes of different cell groups in the tissue.These results provide valuable resources for enriching the transcriptional regulatory mechanism of bull testis development and spermatogenesis,and provide reliable theoretical guidance and technical support for the use of molecular-assisted breeding technology to screen excellent bulls and establish a bull breeding system.