High-Quality DNA Extraction and Sequencing

We used a modified cetyltrimethylammonium bromide (CTAB) method to extract high-quality DNA (Doyle and Doyle, 1987 ), which was then purified using the Wizard^® DNA cleanup system (Promega, Madison, WI, USA). DNA quality was assessed using a NanoDrop spectrophotometer (Thermo Scientific, Carlsbad, CA, USA), and DNA integrity was evaluated by electrophoresis on a 1% (w/v) agarose gel. A DNA library was prepared using the NEB Next Ultra DNA Library Prep Kit for Illumina (San Diego, CA, USA). Libraries for paired-end 150-bp sequencing were analyzed on an Illumina NovaSeq 6000 platform (Novogene Co., Ltd., Tianjin, China) to generate approximately 10 GB of data for each sample. Raw reads were filtered using SOAPnuke to remove sequencing adaptors and low-quality bases (Chen et al., 2018 (link)). The filtered reads were assembled using GetOrganelle (Jin et al., 2020 (link)) with a range of 21, 45, 65, 85, and 105 k-mers for plastomes and 35, 85, and 115 k-mers for nrDNA. Subsequently, ITS and ETS sequences were uploaded to the NCBI GenBank database (accession numbers are listed in Table 2).