Plant genome dna kit

DNA was extracted from the samples using a Tiangen Plant Genome DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The Illumina NovaSeq6000 platform was used for sequencing. The complete chloroplast genomes of P. arisanensis and T. blechnoides were assembled using GetOrganelle [78 (link)]. However, the complete chloroplast genome of P. ensiformis failed to assemble into a circle using GetOrganelle and was instead assembled using Novoplasty [79 (link)]. The sequences were submitted to the National Center for Biotechnology Information (NCBI) under GenBank accession numbers OP441371 (P. arisanensis), OP743918 (P. ensiformis), and OP743919 (T. blechnoides).

The surface of all herbal materials was cleaned with 75% ethanol to avoid fungal DNA contamination. About 60 mg of the materials were cut into pieces, added with 10% polyvinylpyrrolidone (PVP), and then ground with a FastPrep bead mill (Retsch MM400, Germany). The total DNA was extracted with a Plant Genome DNA Kit (Tiangen Biotech Co., China) in accordance with the manufacturer's instructions. The ITS2 sequences were amplified using universal primers ITS-S2F (5′-ATGCGATACTTGGTGTGAAT-3′) and ITS-S3R (5′-GACGCTTCTCCAGACTACAAT-3′) as previously described (Chen et al., 2010 (link)). Polymerase chain reaction (PCR) amplification was performed in a 25 μL reaction mixture containing 12.5 μL of 2 × PCR Master Mix (Aidlab Biotechnologies Co., Ltd.), 1.0 μL of each primer (2.5 μM), 2 μL (about 30 ng) of DNA templates, and filled with double-distilled water. The reactions were performed with the following thermal program: 94°C for 5 min and 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s, followed by 72°C for 10 min. The PCR products were sequenced by the Major Engineering laboratory of the Chinese Academy of Agricultural Sciences (Beijing, China).

Obtaining a more complete chloroplast genome helps to understand their structural evolution. This study added cpDNAs of species Pteris ensiformis, P. arisanensis, Taenitis blechnoides, Adiantum flabellulatum and A. malesianum. Fresh leaves of the first three were sampled from the campus of Shenzhen Fairy Lake Botanical Garden [72 (link)]. Fresh leaves of the latter two were sampled from the campus of South China Agricultural University (SCAU) [48 (link)]. The plant materials used in the study were identified by Ting Wang and deposited in the Herbarium of SCAU with specimen numbers GXL20210901 (A. flabellulatum), GXL20210902 (A. malesianum), GXL20210903 (P. ensiformis), GXL20210904 (P. arisanensis), and GXL20210905 (T. blechnoides). DNA was extracted from the samples using a Tiangen Plant Genome DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The Illumina NovaSeq6000 platform was used for sequencing.