Identification of Differentially Expressed Genes in Different Types of Broiler Skeletal Muscle Fibers Using the RNA-seq Technique.
ABSTRACT: The difference in muscle fiber types is very important to the muscle development and meat quality of broilers. At present, the molecular regulation mechanisms of skeletal muscle fiber-type transformation in broilers are still unclear. In this study, differentially expressed genes between breast and leg muscles in broilers were analyzed using RNA-seq. A total of 767 DEGs were identified. Compared with leg muscle, there were 429 upregulated genes and 338 downregulated genes in breast muscle. Gene Ontology (GO) enrichment indicated that these DEGs were mainly involved in cellular processes, single organism processes, cells, and cellular components, as well as binding and catalytic activity. KEGG analysis shows that a total of 230 DEGs were mapped to 126 KEGG pathways and significantly enriched in the four pathways of glycolysis/gluconeogenesis, starch and sucrose metabolism, insulin signalling pathways, and the biosynthesis of amino acids. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the differential expression of 7 selected DEGs, and the results were consistent with RNA-seq data. In addition, the expression profile of MyHC isoforms in chicken skeletal muscle cells showed that with the extension of differentiation time, the expression of fast fiber subunits (types IIA and IIB) gradually increased, while slow muscle fiber subunits (type I) showed a downward trend after 4 days of differentiation. The differential genes screened in this study will provide some new ideas for further understanding the molecular mechanism of skeletal muscle fiber transformation in broilers.
Project description:Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected P-value<0.05). In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only improves our understanding of the location-specific and breed-specific genes and pathways but also provides some candidate molecular targets for increasing muscle products and meat quality by genetic control.
Project description:Background:The meat quality of yellow feathered broilers is better than the quality of its production. Growth traits are important in the broiler industry. The exploration of regulation mechanisms for the skeletal muscle would help to increase the growth performance of chickens. At present, some progress has been made by researchers, but the molecular mechanisms of the skeletal muscle still remain unclear and need to be improved. Methods:In this study, the breast muscles of fast- and slow-growing female Jinghai yellow chickens (F4F, F8F, F4S, F8S) and slow-growing male Jinghai yellow chickens (M4S, M8S) aged four and eight weeks were selected for transcriptome sequencing (RNA-seq). All analyses of differentially expressed genes (DEGs) and functional enrichment were performed. Finally, we selected nine DEGs to verify the accuracy of the sequencing by qPCR. Results:The differential gene expression analysis resulted in 364, 219 and 111 DEGs (adjusted P-value ? 0.05) for the three comparison groups, F8FvsF4F, F8SvsF4S, and M8SvsM4S, respectively. Three common DEGs (ADAMTS20, ARHGAP19, and Novel00254) were found, and they were all highly expressed at four weeks of age. In addition, some other genes related to growth and development, such as ANXA1, COL1A1, MYH15, TGFB3 and ACTC1, were obtained. The most common DEGs (n = 58) were found between the two comparison groups F8FvsF4F and F8SvsF4S, and they might play important roles in the growth of female chickens. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway also showed some significant enrichment pathways, for instance, extracellular matrix (ECM)-receptor interaction, focal adhesion, cell cycle, and DNA replication. The two pathways that were significantly enriched in the F8FvsF4F group were all contained in that of F8SvsF4S. The same two pathways were ECM-receptor interaction and focal adhesion, and they had great influence on the growth of chickens. However, many differences existed between male and female chickens in regards to common DEGs and KEGG pathways. The results would help to reveal the regulation mechanism of the growth and development of chickens and serve as a guideline to propose an experimental design on gene function with the DEGs and pathways.
Project description:Intramuscular fat (IMF) plays an important role in meat quality. However, the molecular mechanisms underlying IMF deposition in skeletal muscle have not been addressed for the sex-linked dwarf (SLD) chicken. In this study, potential candidate genes and signaling pathways related to IMF deposition in chicken leg muscle tissue were characterized using gene expression profiling of both 7-week-old SLD and normal chickens. A total of 173 differentially expressed genes (DEGs) were identified between the two breeds. Subsequently, 6 DEGs related to lipid metabolism or muscle development were verified in each breed based on gene ontology (GO) analysis. In addition, KEGG pathway analysis of DEGs indicated that some of them (GHR, SOCS3, and IGF2BP3) participate in adipocytokine and insulin signaling pathways. To investigate the role of the above signaling pathways in IMF deposition, the gene expression of pathway factors and other downstream genes were measured by using qRT-PCR and Western blot analyses. Collectively, the results identified potential candidate genes related to IMF deposition and suggested that IMF deposition in skeletal muscle of SLD chicken is regulated partially by pathways of adipocytokine and insulin and other downstream signaling pathways (TGF- ? /SMAD3 and Wnt/catenin- ? pathway).
Project description:Chicken is widely favored by consumers because of some unique features. The leg muscles occupy an important position in the market. However, the specific mechanism for regulating muscle growth speed is not clear. In this experiment, we used Jinghai yellow chickens with different body weights at 300 days as research subjects. The chickens were divided into fast- and slow-growing groups, and we collected leg muscles after slaughtering for use in RNA-seq. After comparing the two groups, 87 differentially expressed genes (DEGs) were identified (fold change ? 2 and FDR < 0.05). The fast-growing group had 42 up-regulated genes and 45 down-regulated genes among these DEGs compared to the slow-growing group. Six items were significantly enriched in the biological process: embryo development ending in birth or egg hatching, chordate embryonic development, embryonic skeletal system development, and embryo development as well as responses to ketones and the sulfur compound biosynthetic process. Two significantly enriched pathways were found in the KEGG pathway analysis (P-value < 0.05): the insulin signaling pathway and the adipocytokine signaling pathway. This study provides a theoretical basis for the molecular mechanism of chicken growth and for improving the production of Jinghai yellow chicken.
Project description:In China, the production for duck meat is second only to that of chicken, and the demand for duck meat is also increasing. However, there is still unclear on the internal mechanism of regulating skeletal muscle growth and development in duck. This study aimed to identity candidate genes related to growth of duck skeletal muscle and explore the potential regulatory mechanism. RNA-seq technology was used to compare the transcriptome of skeletal muscles in black Muscovy ducks at different developmental stages (day 17, 21, 27, 31, and 34 of embryos and postnatal 6-month-olds). The SNPs and InDels of black Muscovy ducks at different growth stages were mainly in "INTRON", "SYNONYMOUS_CODING", "UTR_3_PRIME", and "DOWNSTREAM". The average number of AS in each sample was 37,267, mainly concentrated in TSS and TTS. Besides, a total of 19 to 5377 DEGs were detected in each pairwise comparison. Functional analysis showed that the DEGs were mainly involved in the processes of cell growth, muscle development, and cellular activities (junction, migration, assembly, differentiation, and proliferation). Many of DEGs were well known to be related to growth of skeletal muscle in black Muscovy duck, such as MyoG, FBXO1, MEF2A, and FoxN2. KEGG pathway analysis identified that the DEGs were significantly enriched in the pathways related to the focal adhesion, MAPK signaling pathway and regulation of the actin cytoskeleton. Some DEGs assigned to these pathways were potential candidate genes inducing the difference in muscle growth among the developmental stages, such as FAF1, RGS8, GRB10, SMYD3, and TNNI2. Our study identified several genes and pathways that may participate in the regulation of skeletal muscle growth in black Muscovy duck. These results should serve as an important resource revealing the molecular basis of muscle growth and development in duck.
Project description:The different skeletal muscle fiber types exhibit distinctively different physiological and metabolic properties, and have been linked to both human metabolic diseases and meat quality traits in livestock. Circular RNAs (circRNAs) are a new class of endogenous RNA regulating gene expression, but regulatory mechanisms of skeletal muscle fibers involved in circRNAs remain poorly understood. Here, we constructed circRNA expression profiles of three fast-twitch biceps femoris (Bf) and three slow-twitch soleus (Sol) muscles in pigs using RNA-seq and identified 16,342 distinct circRNA candidates. Notably, 242 differentially expressed (DE) circRNAs between Bf and Sol muscles were identified, including 105 upregulated and 137 downregulated circRNAs, and are thus potential candidates for the regulation of skeletal muscle fiber conversion. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of host genes of DE circRNAs revealed that host genes were mainly involved in skeletal muscle fiber-related GO terms (e.g., muscle contraction, contractile fiber part, and Z disk) and skeletal muscle fiber-related signaling pathways (e.g., AMPK and cGMP-PKG). We also constructed co-expression networks of DE circRNA-miRNA-mRNA using previously acquired high-throughput sequencing mRNA and miRNA data, from which 112 circRNA-miRNA and 95 miRNA-mRNA interactions were identified. Multiple circRNAs essentially serve as a sponge for miR-499-5p, which is preferentially expressed in slow-twitch muscle and reduces the severity of Duchenne muscular dystrophy (DMD). Taken together, a series of novel candidate circRNAs involved in the growth and development of porcine skeletal muscle was identified. Furthermore, they provide a comprehensive circRNA resource for further in-depth research on the regulatory mechanisms of circRNA in the formation of skeletal muscle fiber, and may provide insights into human skeletal muscle diseases.
Project description:Previous studies of horse RNA-seq were performed by mapping sequence reads to the reference genome during transcriptome analysis. However in this study, we focused on two main ideas. First, differentially expressed genes (DEGs) were identified by de novo-based analysis (DBA) in RNA-seq data from six Thoroughbreds before and after exercise, here-after referred to as "de novo unique differentially expressed genes" (DUDEG). Second, by integrating both conventional DEGs and genes identified as being selected for during domestication of Thoroughbred and Jeju pony from whole genome re-sequencing (WGS) data, we give a new concept to the definition of DEG. We identified 1,034 and 567 DUDEGs in skeletal muscle and blood, respectively. DUDEGs in skeletal muscle were significantly related to exercise-induced stress biological process gene ontology (BP-GO) terms: 'immune system process'; 'response to stimulus'; and, 'death' and a KEGG pathways: 'JAK-STAT signaling pathway'; 'MAPK signaling pathway'; 'regulation of actin cytoskeleton'; and, 'p53 signaling pathway'. In addition, we found TIMELESS, EIF4A3 and ZNF592 in blood and CHMP4C and FOXO3 in skeletal muscle, to be in common between DUDEGs and selected genes identified by evolutionary statistics such as FST and Cross Population Extended Haplotype Homozygosity (XP-EHH). Moreover, in Thoroughbreds, three out of five genes (CHMP4C, EIF4A3 and FOXO3) related to exercise response showed relatively low nucleotide diversity compared to the Jeju pony. DUDEGs are not only conceptually new DEGs that cannot be attained from reference-based analysis (RBA) but also supports previous RBA results related to exercise in Thoroughbred. In summary, three exercise related genes which were selected for during domestication in the evolutionary history of Thoroughbred were identified as conceptually new DEGs in this study.
Project description:Modern fast-growing broilers are susceptible to cardiac dysfunctions because their relatively small hearts cannot adequately meet the increased need of pumping blood through a large body mass. To improve cardiac health in broilers through breeding, we need to identify the genes and pathways that contribute to imbalanced cardiac development and occurrence of heart dysfunction. Two broiler lines-Ross 708 and Illinois-were included in this study as models of modern fast-growing and heritage slow-growing broilers, respectively. The left ventricular transcriptome were compared between the two broiler lines at day 6 and 21 post hatch through RNA-seq analysis to identify genes and pathways regulating compromised cardiac development in modern broilers. Number of differentially expressed genes (DEGs, p<0.05) between the two broiler lines increased from 321 at day 6 to 819 at day 21. As the birds grew, Ross broilers showed more DEGs (n = 1879) than Illinois broilers (n = 1117). Both broilers showed significant change of muscle related genes and immune genes, but Ross broilers showed remarkable change of expression of several lipid transporter genes including APOA4, APOB, APOH, FABP4 and RBP7. Ingenuity pathway analysis (IPA) suggested that increased cell apoptosis and inhibited cell cycle due to increased lipid accumulation, oxidative stress and endoplasmic reticulum stress may be related to the increased cardiac dysfunctions in fast-growing broilers. Cell cycle regulatory pathways like "Mitotic Roles of Polo-like Kinases" are ranked as the top changed pathways related to the cell apoptosis. These findings provide further insight into the cardiac dysfunction in modern broilers and also potential targets for improvement of their cardiac health through breeding.
Project description:Sarcopenia is an age-related disease characterized by the loss of muscle mass and muscle function. A proper understanding of its pathogenesis and mechanisms may lead to new strategies for diagnosis and treatment of the disease. This study aims to discover the underlying genes, proteins, and pathways associated with sarcopenia in both genders. Integrated analysis of microarray datasets has been performed to identify differentially expressed genes (DEGs) between old and young skeletal muscles. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed to uncover the functions of the DEGs. Moreover, a protein-protein interaction (PPI) network was constructed based on the DEGs. We have identified 41,715 DEGs, including 19 downregulated and 41,696 upregulated ones, in men. Among women, 3,015 DEGs have been found, with 2,874 of them being upregulated and 141 downregulated genes. Among the top up-regulated and downregulated genes, the ribosome biogenesis genes and genes involved in lipid storage may be closely related to aging muscles in men and women respectively. Also, the DEGs were enriched in the pathways including those of ribosome and Peroxisome proliferator-activated receptor (PPAR) in men and women, respectively. In the PPI network, Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1), Cullin 3 (CUL3) and P53 have been identified as significant hub proteins in both genders. Using the integrated analysis of multiple gene expression profiles, we propose that the ribosome biogenesis genes and those involved in lipid storage would be promising markers for sarcopenia in men and women, respectively. In the reconstructed PPI network, neurotrophic factors expressed in skeletal muscle are essential for motoneuron survival and muscle fiber innervation during development. Cullin E3 ubiquitin ligase (Cul3) is an important component of the ubiquitin-proteasome system-it regulates the proteolysis. P53 is recognized as a central regulator of the cell cycle and apoptosis. These proteins, which have been identified as the most significant hubs, may be involved in aging muscle and sarcopenia.
Project description:BACKGROUND:Pulmonary arterial hypertension, also known as Ascites syndrome (AS), remains a clinically challenging disease with a large impact on both humans and broiler chickens. Pulmonary arterial remodeling presents a key step in the development of AS. The precise molecular mechanism of pulmonary artery remodeling regulating AS progression remains unclear. METHODOLOGY/PRINCIPAL FINDINGS:We obtained pulmonary arteries from two positive AS and two normal broilers for RNA sequencing (RNA-seq) analysis and pathological observation. RNA-seq analysis revealed a total of 895 significantly differentially expressed genes (DEGs) with 437 up-regulated and 458 down-regulated genes, which were significantly enriched to 12 GO (Gene Ontology) terms and 4 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways (Padj<0.05) regulating pulmonary artery remodeling and consequently occurrence of AS. These GO terms and pathways include ribosome, Jak-STAT and NOD-like receptor signaling pathways which regulate pulmonary artery remodeling through vascular smooth cell proliferation, inflammation and vascular smooth cell proliferation together. Some notable DEGs within these pathways included downregulation of genes like RPL 5, 7, 8, 9, 14; upregulation of genes such as IL-6, K60, STAT3, STAT5 Pim1 and SOCS3; IKK?, IkB, P38, five cytokines IL-6, IL8, IL-1?, IL-18, and MIP-1?. Six important regulators of pulmonary artery vascular remodeling and construction like CYP1B1, ALDH7A1, MYLK, CAMK4, BMP7 and INOS were upregulated in the pulmonary artery of AS broilers. The pathology results showed that the pulmonary artery had remodeled and become thicker in the disease group. CONCLUSIONS/SIGNIFICANCE:Our present data suggested some specific components of the complex molecular circuitry regulating pulmonary arterial remodeling underlying AS progression in broilers. We revealed some valuable candidate genes and pathways that involved in pulmonary artery remodeling further contributing to the AS progression.