Use este identificador para citar ou linkar para este item: 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 TamanhoFormato 
pone.0084792.pdftexto completo1,14 MBAdobe PDFThumbnail
Visualizar/Abrir


Os itens no repositório estão protegidos por copyright, com todos os direitos reservados, salvo quando é indicado o contrário.