Identification Of CaRNA Related To Skeletal Muscle Development Difference Between Duroc Pig And Enshi Black Pig Based On GRID-seq Technology

Skeletal muscle is the largest tissue in the body.The development of skeletal muscle is closely related to growth rate,feed efficiency,carcass traits and meat quality traits.Chromatin-Associated RNA(caNA)is a kind of RNA that interacts with chromatin in space and regulates gene expression by interacting with chromatin.Recent studies have shown that caRNA has a potential regulatory effect on skeletal muscle development,but its mechanism remains unclear,and there are few reports on the identification of caRNA and its regulation of skeletal muscle development in pigs.In this study,2-weeks-old lean Duroc pigs(DU)and local meat-lard Enshi black pigs(ES)were used as research,through Global RNA interactions with DNA by deep sequencing(GRID-seq)identified caRNAs associated with porcine skeletal muscle development difference;ATAC-seq and WGS datas were used to analyze the potential regulatory mechanisms and related target genes of caRNA on the development difference of porcine skeletal muscle.The main results of this study are as follows:(1)The experimental system of pig muscle tissue GRID-seq library construction was established independently in our laboratory:After exonuclease treatment,the free linker was removed,the linker connection efficiency was improved,the background noise was reduced,and the data quality met the high-throughput sequencing standard after quality control;the results of high-throughput sequencing showed that 38.7 million and 39.5 million read pairs were obtained in DU and ES,respectively.The results showed that the GRID-seq library of porcine muscle tissue was constructed successfully.(2)Based on porcine GRID-seq data,caRNAs was identified in the whole genome:1070 and 1308 caRNAs were identified in the whole genomes of DU and ES,respectively,and the whole genomic RNA-chromatin interaction maps of DU and ES were obtained.(3)The characteristics of RNA-chromatin interaction between DU and ES genomes were analyzed:DNA reads was mainly distributed in the gene region,and RNA reads was mainly distributed in the gene region,indicating that the main source of caRNA was the newborn pre-mRNA;the analysis of the interaction characteristics showed that the protein coding RNA was mainly involved in the cis interaction,while the non-coding RNA was mainly involved in the trans interaction.(4)Eight predominantly expressed caRNAs were identified in DU:The numbers of caRNAs binding peaks in DU and ES were compared,compared with ES,eight caRNAs up-regulated by peaks were identified in DU as RBFOX1,ENSSSCG00000010359,MBNL1,SVIL,TPM1,ENSSSCG00000024481,ACTN2 and MYBPC1,respectively;binding site annotation analysis showed that differential caRNAs was mainly enriched in super enhancers and typical enhancers,indicating that 8 differential caRNAs had potential regulatory activity.(5)The potential regulatory effects of caRNA on skeletal muscle development were analyzed by integrating ATAC-seq data of DU and ES:H3K27ac and H3K4me3 signals in differential caRNAs enrichment regions in DU were significantly higher than those in ES;the Motif analysis results showed that the chromatin open regions enriched by differential caRNAs significantly enriched transcription factors related to skeletal muscle development,such as MyoG,E2A and Ascl2.ATAC-seq and H3K27ac signal analysis showed that enhancers were the target functional elements of differential caRNAs.GO functional annotation showed that the target genes of enhancers regulated by differential caRNAs were significantly enriched in the process related to skeletal muscle development.It is suggested that caRNA has a potential regulatory effect on the development of skeletal muscle.(6)MYBPC1 and RBFOX1 was identified as a candidate gene related to porcine skeletal muscle development by integrating WGS and GRID-seq data of DU and ES:Based on selective sweep,371 and 431 selected regions were identified in DU and ES,respectively,and it was found that MYBPC1 and other genes were selected genes.GO functional annotation showed that the selected genes in DU were significantly enriched in the process related to muscle growth and development,while the selected genes in ES were significantly enriched in immune related processes.The integration of GRID-seq data showed that FST signal and RNA interaction signal are obviously enriched near MYBPC1 and RBFOX1 gene,and the enrichment intensity of DU was significantly higher than that of ES,indicating that MYBPC1 and RBFOX1 gene are a key candidate gene for skeletal muscle development in DU.To sum up,this study identified the caRNAs and candidate genes related to the difference in the development of porcine skeletal muscle,and found that differential caRNAs can regulate the expression of target genes related to muscle development through interaction with enhancers,thus playing a potential role in regulating the development of porcine skeletal muscle.This study provides a new strategy for analyzing the mechanism of differences in skeletal muscle development between Duroc and Enshi black pigs and mining candidate genes related to skeletal muscle development.