Optimized Method of Extracting Rice Chloroplast DNA for High-Quality Plastome Resequencing and de Novo Assembly

Abstract

Chloroplasts, which perform photosynthesis, are one of the most important organelles in green plants and algae. Chloroplasts maintain an independent genome that includes important genes encoding their photosynthetic machinery and various housekeeping functions. Owing to its non-recombinant nature, low mutation rates, and uniparental inheritance, the chloroplast genome (plastome) can give insights into plant evolution and ecology and in the development of biotechnological and breeding applications. However, efficient methods to obtain high-quality chloroplast DNA (cpDNA) are currently not available, impeding powerful sequencing and further functional genomics research. To investigate effects on rice chloroplast genome quality, we compared cpDNA extraction by three extraction protocols: liquid nitrogen coupled with sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation. The liquid nitrogen–sucrose gradient method gave a high yield of high-quality cpDNA with reliable purity. The cpDNA isolated by this technique was evaluated, resequenced, and assembled de novo to build a robust framework for genomic and genetic studies. Comparison of this high-purity cpDNA with total DNAs revealed the read coverage of the sequenced regions; next-generation sequencing data showed that the high-quality cpDNA eliminated noise derived from contamination by nuclear and mitochondrial DNA, which frequently occurs in total DNA. The assembly process produced highly accurate, long contigs. We summarize the extent to which this improved method of isolating cpDNA from rice can provide practical progress in overcoming challenges related to chloroplast genomes and in further exploring the development of new sequencing technologies.

Proteomics Analysis Reveals Non-Controlled Activation of Photosynthesis and Protein Synthesis in a Rice npp1 Mutant under High Temperature and Elevated CO2 Conditions

“…The procedure of chloroplast isolation was essentially identical to the method described earlier [ 16 , 71 ]. Rice seeds were germinated and grown for 7 days under normal (28 °C, 14 h day/23 °C, 10 h night, 40 Pa CO 2 ) and elevated temperature and CO 2 (33 °C, 14 h day/28 °C, 10 h night, 160 Pa CO 2 ) conditions.…”

Functional Analysis of Rice Long-Chain Acyl-CoA Synthetase 9 (OsLACS9) in the Chloroplast Envelope Membrane

“…All these procedures were performed under aseptic conditions. The TEM analysis of the WT and transgenic rice cells expressing ST-GFP without expression of OsLACS9 (lacs9-1 line) was carried out as described previously [45]. WT and lacs9 mutant cells were immediately placed on a flat specimen carrier and frozen in a high-pressure freezer (EM-PACT; Leica Microsystems).…”

“…Intact chloroplasts were purified from seedlings grown in a light condition using the Percoll (GE Healthcare) density-gradient centrifugation method, as described earlier [45,46] and analyzed by SDS-PAGE and immunoblotting. Proteins were extracted from brown rice seed (harvested in the field), calli (cultured in a dedifferentiation medium for four weeks), shoot (germinated at 30 • C for seven days in the dark and moved to a growth chamber (28:23 • C, 12:12 h, light:dark; 20,000 lux) for three days), and mature leaves (germinated at 30 • C for seven days in the dark and moved to a growth chamber (28:23 • C, 12:12 h, light:dark; 20,000 lux) for eight days).…”

Isolation of Intact Chloroplast for Sequencing Plastid Genomes of Five Festuca Species

“…Intact chloroplast isolation is a critical procedure used to help purify cpDNA for its application in genetic research. In the previous studies, plant-specific chloroplast isolation methods were optimized to extract cpDNA based on a high salt buffer followed by a saline Percoll gradient [8], high salt concentration buffer [16], sucrose density gradient [6,17], liquid nitrogen-sucrose gradient method [18], and high sorbitol concentration buffer, followed by a Percoll gradient [19]. Since polysaccharide and oleoresin contamination is a major problem when isolating cpDNA from many plant species [20] including Festuca grass species, purifying intact chloroplasts is required to extract quality cpDNA for genome sequencing.…”