*4.5. RNA Extraction and Quantitative Real-Time RT-PCR*

For analysis of the expression of *YL* in various soybean tissues, total RNA was isolated from root, nodule, stem, cotyledon, expanded leaf, trifoliate leaf, meristem, flower, young seed and pod collected from three individuals. For the analysis of chloroplast RNA editing in the *yl* mutant and wild type, total RNA was isolated from trifoliate leaves of five-week-old soybean plants. Fresh tissue was frozen immediately in liquid nitrogen and ground to powder using a mortar and pestle. Total RNA was extracted using 1 mL TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. After ethanol precipitation, the RNA was dissolved in RNase-free water.

Approximately 2 μg of total RNA were reverse transcribed using the Prime Script RT reagent Kit with gDNA Eraser (TaKaRa, Beijing, China), according to the manufacturer's instructions. Quantitative real-time RT-PCR was performed in a 20 μL reaction mix containing 50 ng of cDNA, 1 × Light Cycler 480 SYBR Green I Master (Roche, Mannheim, Germany), 0.5 μM of each primer on a Lightcycler 480 (Roche, Mannheim, Germany) machine using the following PCR profile: 2 min at 94 ◦C, followed by 40 cycles of 15 s at 94 ◦C, 15 s at 60 ◦C, and 30 s at 72 ◦C. The dissociation curve analysis was conducted to verify the PCR specificity. Three biological replicates with three technical replicates were analyzed to quantify the levels of gene expression. The soybean actin and ATP synthase genes were used as internal standards to normalize the expression of *YL* using the 2-ΔCt method. The primers used for quantitative real-time RT-PCR were designed using the Primer Premier 5 software (Premier, San Francisco, CA, USA) and listed in Table S3.
