Plastid DNA Isolation and Sequencing

Plastid DNA isolation was performed in October 2012 following the method of Shi et al.^{34 (link)} and optimized using the cp DNA extraction protocol developed by Diekmann et al.^{35 (link)}. All the procedures were carried out on ice or at 4 °C with buffers prechilled to 4 °C. The enriched cp pellet was allowed to thaw at room temperature, and DNA was extracted using the Qiagen DNeasy Plant Mini kit standard method on a QIAcube robot (Qiagen, Mississauga, Canada) and eluted in 1/3 × Qiagen AE buffer (3.33 mM Tris–Cl, 0.17 mM EDTA, pH9.0). DNA samples were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Burlington, Canada). Final DNA yields ranged from 0.2 to 4.3 ng/µl and were diluted to 0.2 ng/µl with 10 mM Tris–HCl, with a pH of 8. The acquired cp DNAs were subjected to genomic DNA library preparation with a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, USA) which uses a tagmentation step to produce DNA fragments of length ranging from 250 to 1000 bp and averaging roughly 500 bp. Four MiSeq runs, each with four libraries and 2 × 250 bp paired-end reads, were performed in January-March 2013 to generate 16 forward and 16 reverse FASTQ files. All raw reads were deposited into NCBI under the BioProject PRJNA433726 (Table 1, Table S2).