*4.2. DNA Sequencing, Genome Assembly, and Validation*

The total DNA of fresh young leaves was extracted using a plant DNA extraction kit (Tiangen, Beijing, China). Agarose gel electrophoresis was used to detect DNA integrity, and purity and concentration were ascertained. The Illumina HiSeq platform was used to sequence the total DNA. After sequencing, the raw data was initially screened to remove low quality regions affecting the data quality and subsequent analysis needed to obtain the expected clean data. The SOAPdenovo2.01 [43] oligonucleotide analysis package was used to assemble the contig sequence. BLAT36 [44] was used to locate the assembled long sequence on the chloroplast reference genome of the relative species and to obtain the relative position of the contig sequence to enable splicing of the contig according to its relative position, and to correct assembly errors. A full-length frame map of the chloroplast genome was obtained. GapCloser software was used to fill gaps on the frame map sequence with high-quality short sequences. Any remaining gaps and suspected regions were supplemented and confirmed by generation sequencing, and the small single copy (SSC) and inverted repeat (IR) region junctions were verified. Finally, a complete ring chloroplast genome sequence was obtained.
