Verification of RT-qPCR Analysis

Total RNA extraction from the longissimus dorsi muscle of two sheep populations was performed using a suitable kit, as per the manufacturer's instructions (Vazyme Biotech Co., Ltd., Nanjing); thereafter, RNA samples were used as a template for reverse transcription into cDNA according to the instructions outlined by the manufacturer of the reverse transcription kit (TransGen Biotech Co., Ltd., Beijing). According to the gene sequence provided by NCBI, Primer Premier 5 software was used to design the primers by themselves and the primers were synthesized by Xi'an Qingke Biology Company (Supplementary Table 1). Using cDNA as a template, RT-qPCR analysis was performed considering the transcription levels of IFRD1, PPARD, MYL2, and MSTN genes. According to the instructions outlined by the manufacturers of the fluorescence quantitative PCR kit (Quanshijin Biotech Co., Ltd., Beijing), the reaction volume used was 20 μL and the following constituted the reaction mixture: 10 mmol/L, 0.5 μL of upstream and downstream primers, 10 μL of qPCR Super Mix, 1 μL of cDNA, and 8 μL of ddH2O. RT-PCR reaction program considered in the present study was as follows: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 5 s, and extension at 60°C for 30 s, 40 cycles. Using the β-actin (β-actin) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes as internal reference genes, the fluorescence quantitative results were calculated using the 2^−ΔΔCt method, and each sample was tested three times.