Bioscience Methods 2025, Vol.16, No.2, 83-99 http://bioscipublisher.com/index.php/bm 84 Traditional transcriptomics is usually based on the average signal of tissues or cell populations, which makes it difficult to resolve the heterogeneity between different cell types. The rise of single-cell sequencing technology provides unprecedented resolution for in-depth analysis of complex tissues, and can characterize the transcriptional characteristics of cell types, states, and rare cell types at the single-cell level. In recent years, single-cell RNA sequencing (scRNA-seq) has been widely used in developmental biology and disease research, but its application in non-model animals such as livestock is relatively rare. In particular, in the field of muscle biology, single-cell transcriptomics can decompose skeletal muscle tissue into different subpopulations, revealing the transcriptional heterogeneity of muscle stem cells, myoblasts, and stromal cells, which is of great significance for understanding muscle development and regeneration (Qiu et al., 2017; Zhu et al., 2024). In addition, single-cell multi-omics is expected to construct a more comprehensive regulatory network by simultaneously or in parallel measuring multiple molecular levels (such as genome, transcriptome, and epigenome) of a single cell. For example, integrating single-cell transcriptome and epigenetic data can associate changes in gene expression with the dynamics of upstream regulatory elements (such as chromatin open regions or DNA methylation), thereby more directly revealing causal relationships (Ren et al., 2023). With the development of single-cell multi-omics technology and analytical methods, its application potential in muscle biology is huge, and it is expected to break through the limitations of traditional population-level research and provide new perspectives for analyzing the complex regulatory mechanisms of muscle development (Wang et al., 2020). This study aims to systematically summarize the latest progress of single-cell multi-omics technology in the study of goat skeletal muscle development, focusing on the regulatory mechanisms at the transcriptional and epigenetic levels. This study first outlines the general process of goat skeletal muscle development, the main cellular and molecular players, and highlights the uniqueness of goats as a species in muscle development. Next, the insights of single-cell transcriptomics into goat muscle development are discussed, including the identification of different cell subpopulations, muscle lineage gene expression patterns, and important findings obtained from transcriptome data. Then, the epigenetic mechanisms related to muscle development are introduced, including the regulatory role of chromatin accessibility, histone modification, and DNA methylation in muscle cells, and how these mechanisms affect the expression of key muscle genes. After that, the application of multi-omics integrated analysis in discovering regulatory mechanisms was discussed, and the strategies for integrating transcriptome and epigenomic data, commonly used computational tools, and the advantages and limitations of multi-omics methods in muscle research were introduced. Then, by listing recent representative cases of single-cell multi-omics research on muscle development (including livestock muscle research), the understanding of epigenetic and transcriptional regulation in these studies, as well as the implications for future scientific research and livestock breeding practices, were summarized. Finally, the application prospects of single-cell multi-omics in livestock research were prospected, how to apply research results to genetic improvement was discussed, and challenges that need to be considered in terms of technical implementation and ethical practice were proposed. This study hopes to provide researchers with a comprehensive reference on the molecular regulation of goat skeletal muscle development, summarize the current understanding, and point out the direction for future work. 2 Overview of Goat Skeletal Muscle Development 2.1 Key stages of muscle generation The process of goat skeletal muscle development can be divided into two main stages: embryonic muscle generation and postnatal muscle growth. During the embryonic period, the number of muscle fibers in adult skeletal muscle is basically determined: muscle stem cells originate from somites, undergo proliferation, migration and differentiation of myogenic precursor cells (myoblasts) during myometrial development, and primary myoblasts first differentiate and fuse to form primary myotubes, and then secondary myoblasts attach to primary myotubes and differentiate to form more secondary myotubes. These myotubes become muscle fibers when they mature. During myogenesis, a series of myogenesis regulatory factors play a core role, including Pax3/7, MyoD, Myf5, myosin heavy chain (MyHC), etc. Classic studies have shown that MyoD and Myf5 are essential factors for the formation of myogenic precursors, while Myogenin and MRF4 (Myf6) mediate the terminal differentiation of myoblasts (Qiu et al., 2020). For example, in early muscle progenitor cells and satellite stem cells, MyoD1 and
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