Chloroplast Genome Sequencing from Plant Leaves

Total genomic DNA was isolated from fresh leaves (~100 mg) using the modified CTAB method (Doyle and Doyle 1987). Subsequently, the cp genomes were amplified using long-range PCR with fifteen primers [27 ]. The PCR products were fragmented for constructing short-insert (500 bp) libraries following the Illumina Nextera XT DNA library preparation instructions. Paired-end sequencing (250 bp) was performed on the Illumina MiSeq 2000 at the Laboratory of Molecular Biology of Germplasm Bank of Wild Species in Southwest China. Quality control of the raw sequence reads was performed using the NGS QC Tool Kit [28 (link)], with a cut-off value for percentage of read length and PHRED quality score as 80 and 30 following Yang et al. [5 (link)]. High-quality reads were assembled into contigs using the de novo assembler in CLC Genomics Workbench v6.5 (CLC Bio), using a k-mer of 64 and a minimum contig length of 500 base pairs (bp). The de novo contigs were assembled into complete chloroplast genomes followed the procedure of Yang et al. [5 (link)].