Identification Of Cell Diversity And Specific Expressed Genes In Chicken Breast Muscle Using Single-cell RNA Sequencing

As the main component of skeletal muscle,the development of breast muscle has a significant impact on the meat-type chicken industry efficiency.Intramuscular fat(IMF),which is the key factor affecting meat quality.Chicken IMF cannot be directly isolated from muscle tissue,the expression of IMF genes obtained by traditional transcriptome is easily interfered by muscle cells.Therefore,this study aimed to analyze the heterogeneity and cell composition,to screen and identify IMF-specific expressed genes of breast muscle at two different development stages using single-cell RNA sequencing technology.The results could be used for the development of related molecular markers and lay a foundation for the molecular basic research of meat quality traits.Experiment 1 Differentiation of breast muscle cell sub-clusters and IMF-specific genes in chickens at different developmental stages.The Jingxing-Huang chickens which selected for high IMF were used as materials in the early growth period(5-day-old,D5)and the rapid growth period(100-day-old,D100).Primary muscle cells and IMF cells of breast muscle were extracted for 10×Genomics single-cell RNA sequencing.The results were clustered based on the similarity of gene expression.Ten and seven cell clusters were identified in D5 and D100,respectively.At D5,the cluster 0,1,3,5,6 were defined as myoblasts and 2 clusters(cluster 2 and 8)were adipocytes,cluster4 was erythrocytes.However,only one myoblast cluster3 and adipocytes cluster4 were identified in D100.In addition,cluster0 in D100 was erythrocytes,cluster2 was endothelial cells,cluster5 was satellite cells.A comparative analysis of the myoblast and adipocyte at the two developmental stages reveals that Myf5,NRXN1,RASD1,and FGFR4,etc.were related to biological processes such as muscle tissue development,can be used as marker candidate genes for myoblasts 19 genes such as APOA1 and ENSGALG00000036073 in IMF cells were related to biological processes such as metabolism and transport of IMF,and can be used as marker candidate genes for IMF cells.The heterogeneity of chicken skeletal muscle was enriched by studying the cell composition of muscle cells and IMF cells at different developmental stages.Experiment 2 Identification of chicken breast muscle IMF candidate genes.The Jingxing-Huang chickens(98-day-old)and Cobb broiler(42-day-old)at the marketing age were used as materials.According to the content of triglyceride in breast muscle,the high and low phenotype groups were distinguished.The expression differences of 15 candidate genes related to IMF deposition were detected by qPCR between high-and low-TG groups.Between the high and low groups,there were 11 and 8 significant differentially expressed genes in Jingxing-Huang chickens and Cobb broilers(P<0.05).There were 7 genes,the ADIPOQ,APOA1,FKTN,HAT1,HS3ST5,MED4,and TNIP1 showed the same differential expression trend in the two breeds,which can be used as candidate genes related to IMF deposition for subsequent molecular marker detection.Combined analysis with the results of single-cell sequencing,a total of 3 IMF cells candidate marker genes were screened,including known marker gene for adipocyte ADIPOQ,and new candidate genes APOA1 and ENSGALG00000036073.The Jingxing-Huang chickens at 5-and 66-day-old were used as materials.RNA in situ hybridization technology was used to verify its expression level and spatial heterogeneity.The results show that those 3 genes were expressed in the extracellular matrix and cell membrane of myocytes and adipocyte in breast muscle and had a large number of colocalizations.It indicated that APOA1 and ENSGALG00000036073 could be used as molecular markers for chicken IMF cells.