ArticlePloS one2014
An improved protocol for intact chloroplasts and cpDNA isolation in conifers.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 44 papers.
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44 citing papers in PubMed, 73 citations in OpenAlex.
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- The plastome sequence of Bactris gasipaes and evolutionary analysis in tribe Cocoseae (Arecaceae).PloS one · 2021Article
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- Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining.Frontiers in plant science · 2021Article
- Plastome sequences of the subgenus Passiflora reveal highly divergent genes and specific evolutionary features.Plant molecular biology · 2020Article
- Phylogenetic and evolutionary features of the plastome of Tropaeolum pentaphyllum Lam. (Tropaeolaceae).Planta · 2020Article
- The complete chloroplast genome ofMitochondrial DNA. Part B, Resources · 2020Article
- Illumina sequencing of complete chloroplast genome ofMitochondrial DNA. Part B, Resources · 2020Article
- Characterization of sequence variability hotspots in Cranichideae plastomes (Orchidaceae, Orchidoideae).PloS one · 2020Article
- Comparative analysis of the complete chloroplast genomes from six Neotropical species of Myrteae (Myrtaceae).Genetics and molecular biology · 2020Article
- Identification of soybean trans-factors associated with plastid RNA editing sites.Genetics and molecular biology · 2020Article
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Authors and funding
8 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundPerforming chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement. METHODOLOGY/PRINCIPAL
findingsWe developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula.
conclusionResults show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.
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