NCAPG Regulates Myogenesis in Sheep, and SNPs Located in Its Putative Promoter Region Are Associated with Growth and Development Traits

SIMPLE SUMMARY: Screening for polymorphisms in the promoter region of a functional gene is an effective way to identify useful markers for improving sheep growth and development. Non-SMC condensin I complex subunit G (NCAPG) is a candidate gene linked with sheep growth and development. Its explicit role in muscle development is still unclear, and markers in NCAPG’s promoter region have not been explored yet. The goal of this study was to investigate the direct role of NCAPG in regulating myogenic development and the differentiation of myoblasts and explore potential markers in its promoter region in relation to sheep growth and development traits. To achieve this goal, cell proliferation and differentiation after RNA interference with NCAPG were investigated in embryonic myoblasts. In addition, the genetic markers in the promoter region of NCAPG were scanned, and association analysis between the markers and sheep growth and development traits was carried out. The results suggest that interfering with NCAPG inhibits the proliferation and differentiation of myoblasts. Five variants detected in the promoter region of NCAPG were significantly (p < 0.05) associated with sheep growth and development traits. These results provide direct evidence of NCAPG regulating myogenesis and provide useful genetic markers to increase the efficacy of the selection of sheep growth and development traits. ABSTRACT: Previously, NCAPG was identified as a candidate gene associated with sheep growth traits. This study aimed to investigate the direct role of NCAPG in regulating myogenesis in embryonic myoblast cells and to investigate the association between single-nucleotide polymorphisms (SNPs) in its promoter region and sheep growth traits. The function of NCAPG in myoblast proliferation and differentiation was detected after small interfering RNAs (siRNAs) knocked down the expression of NCAPG. Cell proliferation was detected using CCK-8 assay, EdU proliferation assay, and flow cytometry cell cycle analysis. Cell differentiation was detected via cell immunofluorescence and the quantification of myogenic regulatory factors (MRFs). SNPs in the promoter region were detected using Sanger sequencing and genotyped using the improved multiplex ligation detection reaction (iMLDR(®)) technique. As a result, a notable decrease (p < 0.01) in the percentage of EdU-positive cells in the siRNA-694-treated group was observed. A significant decrease (p < 0.01) in cell viability after treatment with siRNA-694 for 48 h and 72 h was detected using the CCK-8 method. The quantity of S-phase cells in the siRNA-694 treatment group was significantly decreased (p < 0.01). After interfering with NCAPG in myoblasts during induced differentiation, the relative expression levels of MRFs were markedly (p < 0.05 or p < 0.01) reduced compared with the control group on days 5–7. The myoblast differentiation in the siRNA-694 treatment group was obviously suppressed compared with the control group. SNP1, SNP2, SNP3, and SNP4 were significantly (p < 0.05) associated with all traits except body weight measured at birth and one month of age. SNP5 was significantly (p < 0.05) associated with body weight, body height, and body length in six-month-old sheep. In conclusion, interfering with NCAPG can inhibit the proliferation and differentiation of ovine embryonic myoblasts. SNPs in its promoter region can serve as potential useful markers for selecting sheep growth traits.