An improved protocol for intact chloroplasts and cpDNA isolation in conifers.
<h4>Background</h4>Performing 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 ge...
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2014
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oai:doaj.org-article:e69c8779d2774cc599b40704ccc3c7e42021-11-18T08:39:03ZAn improved protocol for intact chloroplasts and cpDNA isolation in conifers.1932-620310.1371/journal.pone.0084792https://doaj.org/article/e69c8779d2774cc599b40704ccc3c7e42014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24392157/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Performing 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.<h4>Methodology/principal findings</h4>We 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.<h4>Conclusion</h4>Results 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.Leila do Nascimento VieiraHelisson FaoroHugo Pacheco de Freitas FragaMarcelo RogalskiEmanuel Maltempi de SouzaFábio de Oliveira PedrosaRubens Onofre NodariMiguel Pedro GuerraPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 1, p e84792 (2014) |
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Medicine R Science Q Leila do Nascimento Vieira Helisson Faoro Hugo Pacheco de Freitas Fraga Marcelo Rogalski Emanuel Maltempi de Souza Fábio de Oliveira Pedrosa Rubens Onofre Nodari Miguel Pedro Guerra An improved protocol for intact chloroplasts and cpDNA isolation in conifers. |
| description |
<h4>Background</h4>Performing 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.<h4>Methodology/principal findings</h4>We 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.<h4>Conclusion</h4>Results 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. |
| format |
article |
| author |
Leila do Nascimento Vieira Helisson Faoro Hugo Pacheco de Freitas Fraga Marcelo Rogalski Emanuel Maltempi de Souza Fábio de Oliveira Pedrosa Rubens Onofre Nodari Miguel Pedro Guerra |
| author_facet |
Leila do Nascimento Vieira Helisson Faoro Hugo Pacheco de Freitas Fraga Marcelo Rogalski Emanuel Maltempi de Souza Fábio de Oliveira Pedrosa Rubens Onofre Nodari Miguel Pedro Guerra |
| author_sort |
Leila do Nascimento Vieira |
| title |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers. |
| title_short |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers. |
| title_full |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers. |
| title_fullStr |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers. |
| title_full_unstemmed |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers. |
| title_sort |
improved protocol for intact chloroplasts and cpdna isolation in conifers. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2014 |
| url |
https://doaj.org/article/e69c8779d2774cc599b40704ccc3c7e4 |
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