title: An improved protocol for intact chloroplasts and cpDNA isolation in conifers.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Departamento de Fitotecnia, Programa de Pós Graduação em Recursos Genéticos Vegetais, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors.forename: Leila do Nascimento
authors.initials: Ldo N
authors.lastname: Vieira
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Núcleo de Fixação Biológica de Nitrogênio, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.
authors.forename: Helisson
authors.initials: H
authors.lastname: Faoro
authors.affiliation: Departamento de Fitotecnia, Programa de Pós Graduação em Recursos Genéticos Vegetais, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors.forename: Hugo Pacheco de Freitas
authors.initials: HP
authors.lastname: Fraga
authors.affiliation: Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Rogalski
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Núcleo de Fixação Biológica de Nitrogênio, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.
authors.forename: Emanuel Maltempi
authors.initials: EM
authors.lastname: de Souza
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Núcleo de Fixação Biológica de Nitrogênio, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.
authors.forename: Fábio
authors.initials: F
authors.lastname: de Oliveira Pedrosa
authors.affiliation: Departamento de Fitotecnia, Programa de Pós Graduação em Recursos Genéticos Vegetais, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors.forename: Rubens Onofre
authors.initials: RO
authors.lastname: Nodari
authors.affiliation: Departamento de Fitotecnia, Programa de Pós Graduação em Recursos Genéticos Vegetais, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil.
authors.forename: Miguel Pedro
authors.initials: MP
authors.lastname: Guerra
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D002736:Chloroplasts / Q000737:chemistry*; D018742:DNA, Chloroplast / Q000302:isolation & purification*; D054628:Genome, Chloroplast; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions; D064028:Tracheophyta / Q000737:chemistry*
chemical_list: D018742:DNA, Chloroplast