Gel mini purification kit

PCR products were evaluated using electrophoresis on agarose gels and purified using a Gel Mini purification Kit (ZomanBio Inc., Beijing, China). All of the purified DNA products were sequenced using an ABI 3730 DNA sequencer. Sequence assemblies were performed using CAP3 [39 (link)] and manually confirmed against the reference CPG of Pinus taeda (KC427273.1).
The genome annotation was performed using a Dual Organellar GenoMe Annotator with default parameters [40 (link)], coupled with manual corrections of the start and stop codon positions. All of the tRNAs were identified using tRNAscan-SE v2.0 program (http://lowelab.ucsc.edu/tRNAscan-SE/) [41 (link)]. The boundaries of the exons and introns were verified using the BLASTn algorithm (2.6.0, National Center for Biotechnology Information, Bethesda, MD, USA, 2017) against other closely related pine species. The annotation map of the CPG was generated using Organellar Genome DRAW v1.2 program (http://ogdraw.mpimp-golm.mpg.de/) [42 (link)].

Bacterial cells obtained through functional selection were utilized in colony PCR reactions to amplify the inserts. The PCR reactions employed the M13 primer pair, with 4 μL of 10 μM primers, 25 μL of Green Taq Mix (Vazyme biotech, P131), 4 μL of bacterial suspension, and 17 μL of ddH2O, resulting in a final volume of 50 μL. DNA amplification was conducted in a thermocycler at 95°C for 10 min, followed by 30 cycles of 95°C for 15 s, 55°C for 15 s, and 72°C for 4 min. The final extension was conducted at 72°C for 7 min. Gel recovery of PCR samples was carried out using the Gel Mini Purification Kit (Zomanbio, ZP202), adhering to the manufacturer’s instructions.