2.2 DNA Extraction, PCR Amplification, and Sequencing
Total genomic DNA was extracted from dried leaf tissue using a modified
cetyltrimethylammonium bromide (CTAB) protocol(E. & C., 1980). Raw data
of the 10 newly sequenced whole plastid genomes from Notholirionspecies were generated by Illumina platform, generating 150 bp
paired-end reads at Novogene (Beijing, China). The reads were assembled
using the program NOVOPlasty v2.6.2 (Dierckxsens, Mardulyn, & Smits,
2017) and the genome annotation was performed using Plastid Genome
Annotator (PGA)(Qu, Moore, Li, & Yi, 2019). Manual adjustment compared
with related species’ plastomes was conducted in Geneious v9.0.2(Kearse
et al., 2012). For the plastid DNA (cpDNA) fragment, we designed 15
primer pairs using the whole plastid genome of Notholirion(GenBank accessions NC046464, MH011354) and finally got five chloroplast
fragments (matK , ndhA , ndhG-I , petB-D andpetL-G ) for analysis. We amplified and sequenced ITS(ITS1–5.8sRNA–ITS2 )(White, Bruns, Lee, & Taylor, 1990) and the
five chloroplast fragments above
at Sangon Biotech Co. Ltd. (Chengdu, China) and all DNA fragments were
aligned and manually adjusted using the software MEGA X(Kumar, Stecher,
Li, Knyaz, & Tamura, 2018).