Abstract
The complexity of the leaf constitution of foxtail millet (Setaria italica (L.) P. Beauv.) makes it difficult to obtain high-purity cpDNA. Here, we developed a protocol to isolate high-quality cpDNA from foxtail millet and other crops. The new protocol replaces previous tissue grinding and homogenization by enzyme digestion of tiny leaf strips to separate protoplasts from leaf tissue and protects chloroplasts from damage by undue grinding and homogenization and from contamination of cell debris and nuclear DNA. Using the new protocol, we successfully isolated high-quality cpDNAs for whole-genome sequencing from four foxtail millet cultivars, and comparative analysis revealed that they were approximately 27‰ longer than their reference genome. In addition, six cpDNAs of four other species with narrow and thin leaf blades, including wheat (Triticum aestivum L.), maize (Zea may L.), rice (Oryza sativa L.) and sorghum (Sorghum bicolor (L.) Moench), were also isolated by our new protocol, and they all exhibited high sequence identities to their corresponding reference genomes. A maximum-likelihood tree based on the chloroplast genomes we sequenced here was constructed, and the result was in agreement with previous reports, confirming that these cpDNA sequences were available for well-supported phylogenetic analysis and could provide valuable resources for future research.
Highlights
Chloroplasts serve as important cytoplasmic organelles in higher plants
The tissue grinding procedure was replaced by cutting leaves into tiny pieces using a surgical blade, which minimized the contamination of cell debris and chloroplast damage
The protoplasts were re-suspended in Buffer 2 followed by two additional centrifugation steps at 500 g for 10 min and 3000 g for 15 min to separate www.nature.com/scientificreports chloroplasts from cell debris and mitochondrial DNA and collect crude chloroplast precipitate based on the difference in sedimentation rates between mitochondria and chloroplast
Summary
Chloroplasts serve as important cytoplasmic organelles in higher plants. Multiple biochemical processes take place in chloroplasts, including photosynthesis, nitrogen metabolism, sulfate reduction, and synthesis of starch, amino acid and lipid. The new protocol significantly increases chloroplast and cpDNA purity and eliminates nuclear and mitochondria DNA contamination Using this protocol, intact cpDNAs with sequencing quality were isolated from four cultivars of foxtail millet and six cultivars of wheat (Triticum aestivum L.), maize (Zea may L.), rice and sorghum (Sorghum bicolor (L.) Moench). Intact cpDNAs with sequencing quality were isolated from four cultivars of foxtail millet and six cultivars of wheat (Triticum aestivum L.), maize (Zea may L.), rice and sorghum (Sorghum bicolor (L.) Moench) The availability of these chloroplast genomes was proven by comparative analysis and phylogenetic analysis, and they will provide valuable information for future research
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