An Improved Chloroplast DNA Extraction Procedure for Whole Plastid Genome Sequencing

Abstract

BackgroundChloroplast genomes supply valuable genetic information for evolutionary and functional studies in plants. The past five years have witnessed a dramatic increase in the number of completely sequenced chloroplast genomes with the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-effective high-throughput chloroplast DNA (cpDNA) extraction becomes a major bottleneck restricting the application, as conventional methods are difficult to make a balance between the quality and yield of cpDNAs.Methodology/Principal FindingsWe first tested two traditional methods to isolate cpDNA from the three species, Oryza brachyantha, Leersia japonica and Prinsepia utihis. Both of them failed to obtain properly defined cpDNA bands. However, we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked efficiently to isolate the cpDNA from the same three plant species. We sequenced the isolated DNA samples with Illumina (Solexa) sequencing technology to test cpDNA purity according to aligning sequence reads to the reference chloroplast genomes, showing that the reference genome was properly covered. We show that 40–50% cpDNA purity is achieved with our method.ConclusionHere we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing results suggest that the isolated cpDNA has reached enough yield and sufficient purity to perform subsequent genome assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing projects.

NaOH low‐salt method for chloroplast isolation and highly pure cpDNA preparation from Aeluropus littoralis

“…Then, leaf samples were kept under a prolonged dark period of 48 h to decrease the starch content, as heavy starch accumulation has been shown to prevent the isolation of intact plastids during extraction (Pongratz and Beck, 1978). Aeluropus littoralis chloroplasts were isolated according to the classical HS (Bookjans et al., 1984) and mHS (Shi et al., 2012) methods as described previously. For the HS method (Fig.…”

“…Therefore, the main goal of our study was to develop an optimal protocol that simultaneously generates an adequate amount of intact chloroplasts and highly pure cpDNA. Our results demonstrated that the HS and mHS methods (Bookjans et al., 1984; Shi et al., 2012) produced a highly damaged chloroplast fraction (Fig. 4) and yielded a cpDNA that was extremely contaminated with nuclear and mitochondrial DNAs (Figs.…”

“…However, a principal drawback to this approach is the resulting low yield of isolated cpDNA, which is not sufficient for chloroplast genome sequencing when using current next‐generation sequencing platforms. To obtain the desired amount of isolated cpDNA, the starting plant materials must be increased to 25–100 g and extraction must be performed in high volumes, which is not possible for most samples and significantly increases the final time and cost (Shi et al., 2012). …”

“…As mentioned above, cpDNAs obtained using the HS and mHS methods (Bookjans et al., 1984; Shi et al., 2012) are highly contaminated with other genomic DNAs. Therefore, we have modified the original HS method and developed a new protocol to extract intact chloroplasts and prepare pure cpDNA from the halophyte grass Aeluropus littoralis (Gouan) Parl.…”

“…Next, an equal volume of isopropyl alcohol was added to the upper clear aqueous phase, and the bottles were kept in the freezer (−20°C) overnight. Finally, the aqueous phase was centrifuged at 10,000 × g for 20 min, and the cpDNA pellet was washed with 70% ethanol, air‐dried, and redissolved in TE buffer (Shi et al., 2012). …”