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https://locus.ufv.br//handle/123456789/12957
Tipo: | Artigo |
Título: | An improved protocol for intact chloroplasts and cpDNA isolation in conifers |
Autor(es): | 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 |
Abstract: | 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. |
Palavras-chave: | Intact chloroplasts Isolation in conifers |
Editor: | Plos One |
Tipo de Acesso: | Open Access |
URI: | http://dx.doi.org/10.1371/journal.pone.0084792 http://www.locus.ufv.br/handle/123456789/12957 |
Data do documento: | 2-Jan-2014 |
Aparece nas coleções: | Artigos |
Arquivos associados a este item:
Arquivo | Descrição | Tamanho | Formato | |
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pone.0084792.pdf | texto completo | 1,14 MB | Adobe PDF | Visualizar/Abrir |
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