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

Abstract

Premise of the Study: High-yield pure chloroplast DNA (cpDNA) is necessary for whole genome sequencing. Chloroplast extraction with traditional high-salt methods causes damage to nuclei and destroys the integrity of organelles, which leads to high genomic contamination from the nucleus and mitochondria. To overcome this issue, we modified a traditional high-salt method to obtain a new approach called the NaOH low-salt method (NLS). Methods and Results: The NLS method is based on the mild alkaline lysis of plant cells, followed by homogenization with ultrasonic waves and fractionation under reduced osmotic pressure. Results showed that this modified protocol worked efficiently to extract the intact chloroplast from Aeluropus littoralis and other grasses to obtain high-quality pure cpDNA, which was confirmed by fluorescent microscopy, qPCR, and Illumina paired-end sequencing analysis. Conclusions: Compared with high-salt methods, the NLS method has proven robust for extraction of intact chloroplasts and preparation of high-yield pure cpDNA from grasses.