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).