An improved protocol for intact chloroplasts and cpDNA isolation in conifers
Autor(a) principal: | |
---|---|
Data de Publicação: | 2014 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | http://dx.doi.org/10.1371/journal.pone.0084792 http://www.locus.ufv.br/handle/123456789/12957 |
Resumo: | 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. 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 (26250 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. 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. |
id |
UFV_18e078652600f5d198dbe1f2b0790aec |
---|---|
oai_identifier_str |
oai:locus.ufv.br:123456789/12957 |
network_acronym_str |
UFV |
network_name_str |
LOCUS Repositório Institucional da UFV |
repository_id_str |
2145 |
spelling |
Vieira, Leila do NascimentoFaoro, HelissonFraga, Hugo Pacheco de FreitasRogalski, MarceloSouza, Emanuel Maltempi dePedrosa, Fábio de OliveiraNodari, Rubens OnofreGuerra, Miguel Pedro2017-11-09T13:16:46Z2017-11-09T13:16:46Z2014-01-021932-6203http://dx.doi.org/10.1371/journal.pone.0084792http://www.locus.ufv.br/handle/123456789/12957Performing 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. 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 (26250 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. 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.engPlos One9(1), e84792, Jan 2014Intact chloroplastsIsolation in conifersAn improved protocol for intact chloroplasts and cpDNA isolation in conifersinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALpone.0084792.pdfpone.0084792.pdftexto completoapplication/pdf1168849https://locus.ufv.br//bitstream/123456789/12957/1/pone.0084792.pdffe187bf2e42ddc7224724e79f1107137MD51LICENSElicense.txtlicense.txttext/plain; charset=utf-81748https://locus.ufv.br//bitstream/123456789/12957/2/license.txt8a4605be74aa9ea9d79846c1fba20a33MD52THUMBNAILpone.0084792.pdf.jpgpone.0084792.pdf.jpgIM Thumbnailimage/jpeg6468https://locus.ufv.br//bitstream/123456789/12957/3/pone.0084792.pdf.jpgd15a6ca72ae233150f1a5594b3e8aeeaMD53123456789/129572017-11-09 22:00:34.897oai:locus.ufv.br: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Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452017-11-10T01:00:34LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.en.fl_str_mv |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers |
title |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers |
spellingShingle |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers Vieira, Leila do Nascimento Intact chloroplasts 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 |
An improved protocol for intact chloroplasts and cpDNA isolation in conifers |
author |
Vieira, Leila do Nascimento |
author_facet |
Vieira, Leila do Nascimento Faoro, Helisson Fraga, Hugo Pacheco de Freitas Rogalski, Marcelo Souza, Emanuel Maltempi de Pedrosa, Fábio de Oliveira Nodari, Rubens Onofre Guerra, Miguel Pedro |
author_role |
author |
author2 |
Faoro, Helisson Fraga, Hugo Pacheco de Freitas Rogalski, Marcelo Souza, Emanuel Maltempi de Pedrosa, Fábio de Oliveira Nodari, Rubens Onofre Guerra, Miguel Pedro |
author2_role |
author author author author author author author |
dc.contributor.author.fl_str_mv |
Vieira, Leila do Nascimento Faoro, Helisson Fraga, Hugo Pacheco de Freitas Rogalski, Marcelo Souza, Emanuel Maltempi de Pedrosa, Fábio de Oliveira Nodari, Rubens Onofre Guerra, Miguel Pedro |
dc.subject.pt-BR.fl_str_mv |
Intact chloroplasts Isolation in conifers |
topic |
Intact chloroplasts Isolation in conifers |
description |
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. 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 (26250 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. 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. |
publishDate |
2014 |
dc.date.issued.fl_str_mv |
2014-01-02 |
dc.date.accessioned.fl_str_mv |
2017-11-09T13:16:46Z |
dc.date.available.fl_str_mv |
2017-11-09T13:16:46Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1371/journal.pone.0084792 http://www.locus.ufv.br/handle/123456789/12957 |
dc.identifier.issn.none.fl_str_mv |
1932-6203 |
identifier_str_mv |
1932-6203 |
url |
http://dx.doi.org/10.1371/journal.pone.0084792 http://www.locus.ufv.br/handle/123456789/12957 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartofseries.pt-BR.fl_str_mv |
9(1), e84792, Jan 2014 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Plos One |
publisher.none.fl_str_mv |
Plos One |
dc.source.none.fl_str_mv |
reponame:LOCUS Repositório Institucional da UFV instname:Universidade Federal de Viçosa (UFV) instacron:UFV |
instname_str |
Universidade Federal de Viçosa (UFV) |
instacron_str |
UFV |
institution |
UFV |
reponame_str |
LOCUS Repositório Institucional da UFV |
collection |
LOCUS Repositório Institucional da UFV |
bitstream.url.fl_str_mv |
https://locus.ufv.br//bitstream/123456789/12957/1/pone.0084792.pdf https://locus.ufv.br//bitstream/123456789/12957/2/license.txt https://locus.ufv.br//bitstream/123456789/12957/3/pone.0084792.pdf.jpg |
bitstream.checksum.fl_str_mv |
fe187bf2e42ddc7224724e79f1107137 8a4605be74aa9ea9d79846c1fba20a33 d15a6ca72ae233150f1a5594b3e8aeea |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 |
repository.name.fl_str_mv |
LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV) |
repository.mail.fl_str_mv |
fabiojreis@ufv.br |
_version_ |
1801212974095925248 |