An Improved Protocol for Intact Chloroplasts and cpDNA

Isolation in Conifers
Leila do Nascimento Vieira1, Helisson Faoro2, Hugo Pacheco de Freitas Fraga1, Marcelo Rogalski3,
Emanuel Maltempi de Souza2, Fábio de Oliveira Pedrosa2, Rubens Onofre Nodari1, Miguel
Pedro Guerra1*
1 Departamento de Fitotecnia, Programa de Pós Graduação em Recursos Genéticos Vegetais, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil,
2 Departamento de Bioquı́mica e Biologia Molecular, Núcleo de Fixação Biológica de Nitrogênio, Universidade Federal do Paraná, Curitiba, Paraná, Brazil, 3 Departamento
de Biologia Vegetal, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

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.

Methodology/Principal Findings: 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.

Conclusion: 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.

Citation: Vieira LdN, Faoro H, Fraga HPdF, Rogalski M, de Souza EM, et al. (2014) An Improved Protocol for Intact Chloroplasts and cpDNA Isolation in
Conifers. PLoS ONE 9(1): e84792. doi:10.1371/journal.pone.0084792
Editor: Steven M. Theg, University of California - Davis, United States of America
Received September 17, 2013; Accepted November 27, 2013; Published January 2, 2014
Copyright: ß 2014 Vieira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES) and Conselho Nacional de desenvolvimento
Cientı́fico e Tecnológico (CNPq) with fellowships, and by Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC) under project number
14848/2011-2, 3770/2012, and 2780/2012-4. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]

Introduction more, chloroplast genome sequences have been used to investigate

gene function [12,13], and they have been targeted for biotech-
The chloroplast genome of land plants usually harbors a nological applications [14,15,16,17]. Based on the importance of
conserved set of approximately 120 genes in a 120–160 kb pair land plant chloroplast DNA (cpDNA) in plant genetics, evolution
genome, out of a genome of some 3,200 genes present in their and biotechnology, it has been a target in many plant genome
cyanobacterial ancestor [1]. Land plant plastomes are mostly sequencing projects [18,19,20]. To date, complete cpDNAs of
conserved and present little variation in size and gene content, more than 300 plants have been sequenced (ncbi.nlm.nih.gov/
ranging from 70,028 nucleotides and 25 protein coding genes in genomes/GenomesGroup.cgi?taxid=2759&opt=plastid).
the nonphotosynthetic parasitic plant, Epifagus virginiana [2], to
With rapid progress in sequencing technologies, chloroplast
217,942 nucleotides and 131 protein coding genes in Pelargonium6
genome sequencing can be realized quickly as a result of small size
Hortorum [3]. Although chloroplast genomes contain highly
and structural simplicity when compared to nuclear genomes.
conserved essential genes for plant growth and development, they
However, chloroplast genome sequences have been determined
also contain variable regions, i.e., intergenic regions and structural
for only a very few families belonging to gymnosperms [18,20,21].
variations. In addition, they contain one of the few sets of
Especially for conifers, chloroplast genome sequences are available
characters that can transcend the life history of green plants and,
for families that include Cephalotaxaceae [11], Cupressaceae [19],
hence, generate important evolutionary information. Therefore,
Pinaceae [20,22,23], Podocarpaceae (database accession no.
chloroplast genome sequences can be used for comparative
evolutionary studies within and between different groups of plants NC_020361.1) and Taxaceae (database accession no.
[4,5,6], as demonstrated by several works [7,8,9,10,11]. Further- NC_020321.1), but not the Araucariaceae family.

PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e84792

 Efficient Protocol for cpDNA Isolation in Conifers

Chloroplast DNA isolation has been a major challenge,

hindering widespread applications in different plant groups.
Chloroplast isolation in conifers by such conventional methods
as sucrose gradient [24] and high salt [25] has, thus far, not been
successful. This most likely results from the high volume of
contaminants, including polyphenols, oleoresins, terpenoids and
polysaccharides, present in conifer needles, making difficult the
acquisition of intact isolated chloroplasts and high quality cpDNA
[26]. For conifers, whole cpDNA sequencing protocols have been
based on total DNA isolation, followed by cpDNA fragments
amplification by use of polymerase chain reaction (PCR) with
degenerate primers [10,11,20,23]. However, this strategy is known
to be time-consuming and difficult to implement because of
differences in gene organization among different plant species [27]
and ‘‘promiscuous’’ cpDNA present in the nucleus and mitochon-
drial genome [28,29,30,31].
Therefore, the overall aim of the present work was to develop
an efficient protocol for chloroplast isolation and subsequent high
quality cpDNA extraction in conifers, using three different conifer
families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae),
Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae).

Materials and Methods

Plant Material
Local A. angustifolia and P. patula seeds were purchased and
germinated in the greenhouse of Federal University of Santa
Catarina, Brazil. Needles were collected from 6 months plants; this
procedure does not require authorization. P. lambertii young plants
(n = 10) were collected at a private area, located at Lages, Santa
Catarina, Brazil (27u 489 5799 S, 50u 199 3399 W), where the species
is abundant, with the previous owner permission (José Antônio
Ribas Ribeiro). This species are not considered under threat.
After, the young plants were transplanted to greenhouse and
maintained under this condition until the collection of needles. A.
bidwilli young needles were collected at Botanical Garden,
authorized by Federal University of Santa Catarina, Brazil. For
each plant species, 25 g of fresh young needles were collected and
stored in 4uC refrigerator for further chloroplast extractions.

Protocols
The three chloroplast DNA isolation methods used here are
described as follows:
A) High salt plus saline Percoll gradient method
(Figure 1). All the following steps were carried out at 0uC, if
not otherwise stated.

1. Prior to extraction, 25 g (fresh weight) of young needles were

collected and kept in dark for 10 days at 4uC to decrease starch
and resin level. Fresh needles were cleaned with 0.5% sarkosyl
(Fluka, Ronkonkoma, NY) for 5 min to reduce microbial
contamination and then washed 4 times with distilled water.
2. Needles were homogenized in 400 ml ice-cold isolation buffer
(Table 1) for 30 s in a pre-chilled blender. Homogenate was
filtered primarily into two layers of gauze bandage and then
filtered again using two layers of Miracloth by softly squeezing
the cloth.
3. Homogenate was centrifuged at 200 g for 15 min at 4uC. The
nucleus pellet and cell-wall debris were discarded. The
supernatant included chloroplasts suspended in it.
4. The supernatant was centrifuged at the higher centrifugal force Figure 1. Flowchart showing the major steps for chloroplast
isolation according to high salt plus saline Percoll.
of 3000 g for 20 min at 4uC, resulting in a chloroplast pellet
doi:10.1371/journal.pone.0084792.g001
with some contamination.

PLOS ONE | www.plosone.org 2 January 2014 | Volume 9 | Issue 1 | e84792

 Efficient Protocol for cpDNA Isolation in Conifers

5. The pellet was gently resuspended in 12 ml of wash buffer attached to the chloroplast membrane, followed by centrifu-
(Table 1) using a paintbrush. gation at 3500 g for 20 min at 4uC. The supernatant was
6. Homogenate was divided into 6 tubes (50 ml), each containing discarded.
20 ml Percoll (GE Healthcare, Uppsala, Sweden) gradient 6. The pellet was resuspended again with 250 ml wash buffer and
(70%–30%) and then centrifuged at 5000 g for 25 min at 4uC. centrifuged at 3500 g for 20 min at 4uC to obtain the final
The interface 70%–30% containing chloroplasts was collected. chloroplast pellet.
7. Collected interface containing chloroplasts was washed twice
C) Sucrose gradient method [33].
with 100 ml of wash buffer and centrifuged at 3000 g for
20 min at 4uC to obtain the purified chloroplast pellet. 1. Prior to extraction, about 25 g (fresh weight) of young needles
were collected and kept in dark for 72 h at 4uC in order to
B) Modified high salt method [32]. All the following steps
decrease the starch level stored in the leaves. Fresh needles
were carried out at 0uC, if not otherwise stated.
were cleaned with distilled water.
1. Prior to extraction, 25 g (fresh weight) of young needles were 2. Needles were homogenized in 400 ml of ice-cold isolation
collected and kept in dark for 72 h at 4uC to decrease starch buffer (Table 1) for 30 s. The homogenate was filtered into
level stored in the needles. Fresh needles were cleaned with centrifuge bottles using two layers of Miracloth by softly
distilled water. squeezing the cloth.
2. Needles were homogenized in 400 ml of isolation buffer 3. The homogenate was centrifuged at 200 g for 15 min at 4uC.
(Table 1) for 30 s. Homogenate was filtered into centrifuge The nucleus pellet and cell-wall debris were discarded. The
bottles, using two layers of Miracloth (Calbiochem, San Diego, supernatant included chloroplasts suspended in it.
CA) by softly squeezing the cloth. 4. The supernatant was centrifuged at a higher centrifugal force
3. The homogenate was centrifuged twice at 200 g for 20 min at (2000 g) for 20 min at 4uC, and the resulting chloroplast pellet
4uC. The nucleus pellet and cell-wall debris were discarded. showed some contamination.
Supernatant included chloroplasts suspended in it. 5. The pellet was resuspended in 7 ml of ice-cold wash buffer
4. The supernatant was submitted to a higher centrifugal force (Table 1), using a soft paintbrush.
(3500 g) for 20 min at 4uC, resulting in a chloroplast pellet 6. The homogenate was gently loaded into 6 tubes (50 ml)
contaminated with some nuclear DNA. containing sucrose step gradient consisting of 18 ml of 52%
5. The pellet was gently resuspended in 250 ml of wash buffer sucrose and overlaid with 7 ml of 30% sucrose.
(Table 1), using a paintbrush to wash the nuclear DNA 7. Step gradients were centrifuged at 3500 g for 60 min at 4uC.

Table 1. Composition of chloroplast isolation buffers and wash buffers for modified high salt method, high salt plus saline Percoll
method and sucrose gradient method.

High salt plus saline Percoll method Modified high salt method [32] Sucrose gradient [33]

Isolation Buffer (pH 3.8) Isolation Buffer (pH 3.8) Isolation Buffer

1.25 M NaCl 1.25 M NaCl 50 mM Tris-HCl (pH 8.0)

0.25 M ascorbic acid 0.25 M ascorbic acid 0.35 M sorbitol
10 mM sodium metabisulfite 10 mM sodium metabisulfite 7 mM EDTA
0.0125 M Borax 0.0125 M Borax 0.1% 2-mercaptoethanol
50 mM Tris-HCl (pH 8.0) 50 mM Tris-HCl (pH 8.0) 0.1% BSA
7 mM EDTA 7 mM EDTA
1% PVP-40 (w/v) 1% PVP-40 (w/v)
0.1% BSA (w/v) 0.1% BSA (w/v)
1 mM DTT

Wash Buffer (pH 8.0) Wash Buffer (pH 8.0) Wash Buffer

1.25 M NaCl 1.25 M NaCl 50 mM Tris-HCl (pH 8.0)

0.0125 M Borax 0.0125 M Borax 0.35 M sorbitol
50 mM Tris-HCl (pH 8.0) 50 mM Tris-HCl (pH 8.0) 25 mM EDTA
25 mM EDTA 25 mM EDTA
1% PVP-40 (w/v) 1% PVP-40 (w/v)
0.1% BSA (w/v) 0.1% BSA (w/v)
1 mM DTT

Both BSA and DTT were added just before the start of the experiment.
Percoll gradient solutions consisted of wash buffer with Percoll at a final concentration of 70% (v/v) and 30% (v/v).
Sucrose gradient solutions consisted of 50 mM Tris-HCl (pH 8.0), 25 mM EDTA and sucrose addition for a final concentration of 52% sucrose (w/v) and 30% (w/v)
sucrose.
doi:10.1371/journal.pone.0084792.t001

PLOS ONE | www.plosone.org 3 January 2014 | Volume 9 | Issue 1 | e84792

 Efficient Protocol for cpDNA Isolation in Conifers

Figure 2. Chloroplast visualization of Araucaria angustifolia in phase contrast microscopy. (A) Chloroplasts isolated with improved high salt
method; (B) Chloroplasts isolated with sucrose method; (C) Chloroplasts isolated with high salt plus Percoll method; (D–F) Micrographs during
chloroplast isolation with high salt plus saline Percoll method; (D) Broken and intact chloroplasts before Percoll gradient centrifugation; (E) Intact
isolated chloroplasts in interface 70/30% after Percoll gradient centrifugation; (F) Broken chloroplasts in upper 30% phase after Percoll gradient
centrifugation. Dotted arrows indicate broken chloroplasts. Solid arrows indicate intact chloroplasts. Bar –50 mM.
doi:10.1371/journal.pone.0084792.g002

8. The band from the 30–52% interface containing chloroplasts Chloroplast DNA Isolation
was collected, diluted twice with 200 ml of wash buffer, and Chloroplast DNA isolation was the same for all chloroplast
centrifuged at 1500 g for 15 min at 4uC to gain the purified pellets obtained using the three different isolation methods. DNA
chloroplast pellet. isolation buffer consisted of 100 mM NaCl, 100 mM Tris-HCl
(pH 8.0), 50 mM EDTA, and 1 mM DTT.

1. The chloroplast lyse was obtained by incubating the chloroplast

pellet with 8 ml of DNA isolation buffer, 1.5 ml 20% SDS,
20 ml 2-Mercaptoethanol and 30 ml Proteinase K (10 mg/ml)
into a centrifuge tube at 55uC for 4 h.
2. The centrifuge tube was incubated on ice for 5 min, and then
1.5 ml 5 M KAc (pH 5.2) was added to the lyse mixture and
chilled for more than 30 min. After that, the tube was
centrifuged at 10000 g for 15 min at 4uC, and the pellet was
discarded.
3. The supernatant was extracted with an equal volume of
saturated phenol and chloroform:isoamyl-alcohol (24:1) and
centrifuged twice at 10000 g for 20 min.
4. An equal volume of isopropyl alcohol (about 10 ml) was added
to the upper aqueous phase and incubated at 220uC
overnight.
5. To obtain the DNA pellet, the tube was centrifuged at 10000 g
for 20 min at 4uC. The cpDNA pellet was washed with 70%
and 96% ethanol, air dried, and redissolved in 50 ml TE buffer.
6. The cpDNA samples were treated with RNAse, and the DNA
band was visualized on a 0.7% agarose gel.
Figure 3. Chloroplast DNA visualization of Araucaria angustifolia 7. DNA purity and concentration were evaluated with Nano-
in 0.7% agarose gel stained with ethidium bromide. (A) Ladder dropH, based on 260/280 and 260/230 ratios.
1 kb and cpDNA isolated with modified high salt method; (B) Ladder
1 kb and cpDNA isolated with sucrose method; (C) Ladder 1 kb and
cpDNA isolated with high salt plus saline Percoll method.
doi:10.1371/journal.pone.0084792.g003

PLOS ONE | www.plosone.org 4 January 2014 | Volume 9 | Issue 1 | e84792

 Efficient Protocol for cpDNA Isolation in Conifers

Microscopy Analysis Table 3. cpDNA ratios of selected conifers evaluated using

The integrity of isolated chloroplasts was assessed with a phase- NanodropH.
contrast light microscopy using an inverted Olympus IX81
microscope [34]. Intact chloroplasts were considered those with
pale yellow-green color and refractive, with a bright halo DNA concentration
appearance around each plastid, whereas broken chloroplasts Plant species (ng/ml) 260/280 260/230
were those with a dark green, granular, and non-refractive Araucaria angustifolia 3438.3 2.05 1.99
appearance [34].
Araucaria bidwilli 3038.0 1.95 1.74
Podocarpus lambertii 430.6 2.01 1.89
Chloroplast Genome Sequencing
Pinus patula 1799.5 2.05 2.10
A. angustifolia, A. bidwilli, P. patula and P. lambertii cpDNAs were
isolated using the high salt plus Percoll method. For each species, Samples were isolated with the high salt plus Percoll method in different conifer
approximately 50 ng of DNA were prepared with the Nextera species. Final volume of 40 ml.
DNA Sample Prep Kit (Illumina, San Diego, USA) according to doi:10.1371/journal.pone.0084792.t003
the manufacturer’s instructions. Chloroplast DNAs were se-
quenced using Illumina MiSeq (26250 read length) at the Federal decrease in viscosity of the solution. The viscosity normally found
University of Paraná - Brazil. The obtained paired-end reads were in the homogenate (conifer needles and isolation buffer) is related
assembled to reference genome sequence and estimate genome to the high amount of resins and polysaccharides present in conifer
coverage, using the CLC Genomics Workbench 5.5 software. The needles, and its reduction enables faster and more efficient
reference chloroplast genome sequences of Podocarpus totara filtration, with lower material loss. In a previous paper [39], it was
(NC_020361.1) and Pinus thunbergii (NC_001631.1) were down- observed that the use of high salt buffers for DNA extraction
loaded from GenBank. increased the quality and yield of DNA extracted from plant
tissues rich in polysaccharides.
Results and Discussion Similarly, the initial centrifugation step, performed to pellet
cellular debris was reduced to 200 g. We also reduced the initial
Chloroplast Isolation centrifugation step at 200 g to only one centrifugation, while in the
Chloroplast isolation protocols are generally based on methods modified high salt method [32], it was performed twice. This
that employ high salt concentration buffers [32], sucrose density reduction increased chloroplast yield and did not entail any
gradient [33], high salt buffers followed by sucrose gradient [35] reduction in the quality of isolated chloroplast as a result of the
and high sorbitol concentration buffers followed by Percoll Percoll gradient step. The major steps for chloroplast isolation
gradient [36]. As the purity of intact chloroplasts is one of the using the high salt plus Percoll method, including original, new
critical steps of whole sequencing, a previous paper [19] and modified steps, are summarized in Figure 1.
considered the use of sucrose density gradients as the best method Microscopy images showed the presence of some intact
for separating nuclear DNA contamination from cpDNA. The chloroplasts and a large amount of broken chloroplasts and other
present protocol is based on a high salt isolation buffer followed by cellular debris in extraction when using the modified high salt and
saline Percoll gradient. The combination of these two strategies sucrose methods (Fig. 2A, B). On the other hand, the high salt plus
provided two advantages: better isolation of chloroplasts by use of saline Percoll method resulted in the presence of abundant intact
the Percoll gradient and decreased contamination by polysaccha- chloroplasts (Fig. 2C).
rides, polyphenols, and proteins. Aiming to better characterize the efficiency of Percoll gradient,
The first significant change in isolation protocol was the microscopy analysis was performed prior to the Percoll gradient
increase in storage time to 10 days at 4uC prior to extraction. This centrifugation step, and the presence of both intact and ruptured
change led to a significant reduction in the viscosity of extraction chloroplasts could be observed (Fig. 2D). However, after
buffer, possibly caused by a decrease in the polysaccharides and centrifugation in Percoll gradient, the 70%/30% interface
oleoresin concentrations. A similar strategy has been used in other (Fig. 2E) contains abundant intact chloroplasts and only a few
protocols, in which 48–72 h at 4uC was enough to reduce the broken chloroplasts, while the upper phase contains many broken
stored polysaccharides [32,33]. However, because of the high chloroplasts (Fig. 2F). In addition, below the 70% gradient, the
amount of oleoresins and thick outer periclinal walls in conifers formation of a white colored pellet composed of polysaccharides
[37,38], a longer time is required to reduce the concentration of and other contaminants could be seen. Despite the high purity of
these compounds. chloroplasts observed immediately after Percoll gradient centrifu-
Subsequently, needles were washed with sarkosyl, reducing gation, two subsequent centrifugations are essential to remove any
material contamination. At the time of homogenization, it was residue of Percoll. The isolation of cpDNA without performing
observed that the high salt buffer was also responsible for the these two washes would be greatly affected by its presence. It is

Table 2. cpDNA from different isolation methods in Araucaria angustifolia sample.

Isolation Method DNA concentration (ng/ml) 260/280 260/230

Modified high salt method 975.6 1.66 0.92

High Salt plus saline Percoll method 3438.3 2.05 1.99
Sucrose Gradient Method 472.4 1.52 0.47

Ratios evaluated with NanodropH, in a final volume of 40 ml.

doi:10.1371/journal.pone.0084792.t002

PLOS ONE | www.plosone.org 5 January 2014 | Volume 9 | Issue 1 | e84792

 Efficient Protocol for cpDNA Isolation in Conifers

Figure 4. Reference graph track showing observed coverage values. Different colors show the minimum (light blue), mean (blue), and
maximum (dark blue) observed coverage values for all genomic regions (data aggregation above 100 bp). Araucaria angustifolia, Araucaria bidwilli,
and Podocarpus lambertii sequence reads were mapped on Podocarpus totara; Pinus patula sequence reads were mapped on Pinus thunbergii.
doi:10.1371/journal.pone.0084792.g004

noteworthy that changes in the protocol enabled the isolation of

the best quality chloroplasts, without the need of ultracentrifuga-
Table 4. Average coverage of cpDNA evaluated from tion, which could be a limiting point in the procedure.
selected conifers with CLC Genomics Workbench 5.5 software. In addition to facilitating whole cpDNA sequencing, isolation of
intact chloroplasts can also be applied in plastid proteome
characterization studies. Comparative proteomics in Triticum
Plant Species Average Coverage Reference Genome aestivum and Arabidopsis thaliana chloroplasts have been recently
Araucaria angustifolia 24.63 Podocarpus totara developed using intact isolated chloroplasts. The results demon-
Araucaria bidwilli 135.97 Podocarpus totara strated that the quality of chloroplast isolation is a fundamental
step of complete proteome characterization [40,41].
Podocarpus lambertii 1196.10 Podocarpus totara
Pinus patula 64.68 Pinus thunbergii
cpDNA Isolation
cpDNA reads were mapped to reference genomes. We also obtained isolated cpDNA with better quality and yield
doi:10.1371/journal.pone.0084792.t004 using this high salt plus saline Percoll method. Using the modified

PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e84792

 Efficient Protocol for cpDNA Isolation in Conifers

high salt and sucrose methods, bands in agarose gel revealed the the use of the technique. A reference-guided chloroplast genome
presence of degraded DNA, indicating contamination with nuclear mapping was performed to estimate the genome average coverage
DNA and polysaccharides (Fig. 3A, B, respectively), while isolated (Fig. 4). The cpDNA sequencing generated a high average
cpDNA formed a well-defined band, which is indicative of high coverage for all species evaluated: 24.63 for A. angustifolia, 135.97
purity and polysaccharide-free cpDNA (Fig. 3C). for A. bidwilli, 1,196.10 for P. lambertii, and 64.68 for P. patula
In addition, Nanodrop evaluation indicated higher cpDNA (Table 4). Thus, in this study, all of the reference genomes were
yield with the high salt plus saline Percoll method, about 3 times sufficiently covered for assembly.
higher when compared to high salt methods and almost 9 times This protocol presents higher genome coverage when compared
higher when compared to the sucrose method. Enhanced 260/280 to protocols recently applied to conifers chloroplast genome
and 260/230 ratios were observed in the high salt plus saline sequencing, as those using total DNA followed by PCR
Percoll method, 2.05 and 1.99, respectively (Table 2). These ratios amplification with degenerated primers that resulted in genome
indicate a high purity of isolated cpDNA, which is a prerequisite coverage only about 8-fold [11,20]. Furthermore, this strategy is
for whole chloroplast sequencing. In the two other methods time-consuming and difficult to implement because of differences
evaluated, contamination was observed with polyphenols and in gene organization among different plant species. Cryptomeria
polysaccharides (Table 2). Moreover, when we used the sucrose japonica cp genome was sequenced using sucrose gradient method,
method, a highly contaminated and oxidized DNA pellet was followed by DNA isolation with phenol/chloroform, DNA
obtained. Taken together, we considered the high salt plus saline purification with DNeasy Plant Mini Kit (QIAGEN) and ATP-
Percoll protocol as having the best yield and quality for cpDNA dependent DNase (TOYOBO) [19]. As shown in the present
isolation from A. angustifolia. Thus, this method was applied to work, the protocol based on saline buffer followed by Percoll
cpDNA isolation of A. bidwilli, P. patula and P. lambertii. As gradient results in higher quality DNA than sucrose gradient.
expected, a high quality in cpDNA isolated from all evaluated Moreover, all these purification steps applied to the isolated DNA,
species was realized at 260/280.1.95 and 260/230.1.74 ratios
such as the utilization of ATP-dependent DNase, led to a lower
(Table 3). All species showed cpDNA yield similar to A. angustifolia,
DNA yield [32].
with the exception of P. lambertii. However, even its cpDNA yield
In summary, the results obtained in the present work show that
was sufficient for sequencing (Table 3).
these improvements in the general protocol for chloroplasts and
cpDNA isolation in conifers enhance the overall quality and yield
Chloroplast Genome Sequencing of chloroplasts and cpDNA isolation, providing a useful tool for
Improving technologies have made DNA sequencing faster,
studies that require isolated chloroplasts and/or plastid genome
more accurate and far cheaper, creating opportunities to sequence
sequence. Facilitating chloroplast sequencing of this species group
the whole chloroplast genome in order to perform evolutionary
and, hence, increasing the amount of information about the plastid
and phylogenomic studies. To test the quality of cpDNA isolated
genome of conifers may, in turn, lead to greater understanding
by our new method, we sequenced the plastid genome of four
about plant evolution, as well as the structural and functional
conifer species (A. angustifolia, A. bidwilli, P. patula and P. lambertii)
genomics in plants other than conifers.
using the Illumina sequencing technology.
To estimate the efficiency of chloroplast genome sequence
assembly with our cpDNA isolation protocol, we sequenced these Author Contributions
four chloroplast genomes by using MiSeq Illumina sequencing Conceived and designed the experiments: LNV MR MPG RON EMS
with only 50 ng of cpDNA. In other sequencing protocols, about FOP. Performed the experiments: LNV HPFF HF MR. Analyzed the data:
5–10 mg [32,33] were used for sequencing, thereby increasing the LNV HF HPFF. Contributed reagents/materials/analysis tools: EMS FOP
amount of plant material required for isolation and often limiting RON MPG. Wrote the paper: LNV MR MPG.

References
1. Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, et al. (1996) Sequence 10. Wu CS, Wang YN, Hsu CY, Lin CP, Chaw SM (2011) Loss of different inverted
analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain repeat copies from the chloroplast genomes of Pinaceae and Cupressophytes and
PCC6803: II. Sequence determination of the entire genome and assignment of influence of heterotachy on the evaluation of gymnosperm phylogeny. Genome
potential protein-coding regions. DNA Res 3: 109–136. Biol Evol 3: 1284–1295.
2. Wolfe KH, Morden CW, Palmer JD (1992) Function and evolution of a minimal 11. Yi X, Gao L, Wang B, Su YJ, Wang T (2013) The complete chloroplast genome
plastid genome from a non-photosynthetic parasitic plant. Proc Natl Acad Sci sequence of Cephalotaxus oliveri (Cephalotaxaceae): evolutionary comparison of
USA 89: 10648–10652. Cephalotaxus chloroplast DNAs and insights into the loss of inverted repeat
3. Chumley TW, Palmer JD, Mower JP, Boore JL, Fourcade HM, et al. (2006) The copies in Gymnosperms. Genome Biol Evol 5: 688–698.
complete chloroplast genome sequence of Pelargonium6hortorum: organization and 12. Rogalski M, Schoettler MA, Thiele W, Schulze WX, Bock R (2008) Rpl33, a
evolution of the largest and most highly rearranged chloroplast genome of land nonessential plastid encoded ribosomal protein in tobacco, is required under
plants. Mol Biol Evol 23: 1–16. cold stress conditions. Plant Cell 20: 2221–2237.
4. Timmis JN, Ayliffe MA, Huang CY, Martin W (2004) Endosymbiotic gene 13. Alkatib S, Scharff LB, Rogalski M, Fleischmann TT, Matthes A, et al. (2012)
transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet 5: The contributions of wobbling and superwobbling to the reading of the genetic
123–135. code. PLoS Genet. 8(11): e1003076.
5. Wolf PG, Roper JM, Duffy AM (2010) The evolution of chloroplast genome 14. Clarke JL, Daniell H, Nugent JM (2011) Chloroplast biotechnology, genomics
structure in ferns. Genome 53: 731–738. and evolution: current status, challenges and future directions. Plant Mol Biol.
6. Greiner S, Bock R (2013) Tuning a ménage à trois: Co-evolution and co- 76: 207–209.
adaptation of nuclear and organellar genomes in plants. Bioessays 35: 354–365. 15. Maliga P, Bock R (2011) Plastid biotechnology: food, fuel and medicine for the
7. Moore MJ, Bell CD, Soltis PS, Soltis DE (2007) Using plastid genomic-scale data 21st century. Plant Physiol 155: 1501–1510.
to resolve enigmatic relationships among basal angiosperms. Proc Natl Acad Sci 16. Rogalski M, Carrer H (2011) Engineering plastid fatty acid biosynthesis to
USA 104: 19363–19368. improve food quality and biofuel production in higher plants. Plant Biotechnol J
8. Jansen RK, Cai Z, Raubeson LA, Daniell H, dePamphilis CW, et al. (2007) 9: 554–564.
Analysis of 81 genes from 64 plastid genomes resolves relationships in 17. Bock R (2013) Strategies for metabolic pathway engineering with multiple
angiosperms and identifies genome-scale evolutionary patterns. Proc Natl Acad transgenes. Plant Mol Biol doi: 10.1007/s11103-013-0045-0.
Sci USA 104: 19369–19374. 18. Wu CS, Wang YN, Liu SM, Chaw SM (2007) Chloroplast genome (cpDNA) of
9. Moore MJ, Soltis PS, Bell CD, Burleigh JG, Soltis DE (2010) Phylogenetic Cycas taitungensis and 56 cp protein-coding genes of Gnetum parvifolium: insights
analysis of 83 plastid genes further resolves the early diversification of eudicots. into cpDNA evolution and phylogeny of extant seed plants. Mol Biol Evol 24:
Proc Natl Acad Sci USA 107: 4623–4628. 1366–1379.

PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e84792

 Efficient Protocol for cpDNA Isolation in Conifers

19. Hirao T, Watanabe A, Kurita M, Kondo T, Takata K (2008) Complete 31. Rousseau-Gueutin M, Ayliffe MA, Timmis JN (2011) Conservation of plastid
nucleotide sequence of the Cryptomeria japonica D. Don. chloroplast genome and sequences in the plant nuclear genome for millions of years facilitates
comparative chloroplast genomics: diversified genomic structure of coniferous endosymbiotic evolution. Plant Physiol 157: 2181–2193.
species. BMC Plant Biol 8: 70–78. 32. Shi C, Hu N, Huang H, Gao J, Zhao Y, et al. (2012) An improved chloroplast
20. Lin CP, Huang JP, Wu CS, Hsu CY, Chaw SM (2010) Comparative chloroplast DNA extraction procedure for whole plastid genome sequencing. PLoS ONE 7:
genomics reveals the evolution of Pinaceae genera and subfamilies. Genome Biol e31468.
Evol 2: 504–517. 33. Jansen RK, Raubeson LA, Boore JL, dePamphilis CW, Chumley TW et al.
21. Werner O, Patino J, Gonzáles-Mancebo JM, Gabriel RMD, Ros RM (2009) (2005) Methods for obtaining and analyzing whole chloroplast genome
The taxonomic status and the geographical relationships of the Macaronesian sequences. Methods Enzymol 395: 348–384.
endemic moss Fissidens luisieri (Fissidentaceae) based on DNA sequence data. 34. Walker DA, Cerovic ZEG, Robinson SP (1987) Isolation of intact chloroplasts:
Bryologist 112: 315–324. general principles and criteria of integrity. Methods Enzymol 148: 145–157.
22. Wakasugi T, Tsudzuki J, Ito S, Nakashima K, Tsudzuki T, et al. (1994) Loss of 35. Diekmann K, Hodkinson TR, Fricke E, Barth S (2008) An optimized chloroplast
all ndh genes as determined by sequencing the entire chloroplast genome of the DNA extraction protocol for grasses (Poaceae) prove suitable for whole plastid
black pine Pinus thunbergii. Proc Natl Acad Sci USA 91: 9794–9798.
genome sequencing and SNP detection. PLoS ONE 3: e2813.
23. Cronn R, Liston A, Parks M, Gernandt GS, Shen R, et al. (2008) Multiplex
36. Kubis SE, Lilley KS, Jarvis P (2008) Isolation and preparation of chloroplast
sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis
from Arabidopsis thaliana plants. In: Posch A, editor. 2D-PAGE: sample
technology. Nucleic Acids Res 36: e122.
preparation and fractionation: Humana Press. 171–183.
24. Palmer JD, Sein DB (1986) Conservation of chloroplast genome structure among
vascular plants. Curr Genet 11: 823–834. 37. Mastroberti AA, Mariath JEA (2003) Leaf anatomy of Araucaria angustifolia
25. Bookjans G, Stummann BM, Henningsen KW (1984) Preparation of chloroplast (Bertol.) Kuntze (Araucariacea). Rev Bras Bot 26: 343–353.
DNA from pea plastids isolated in a medium of high ionic strength. Anal 38. Yamamoto ES, Otto A, Simoneit BRT (2004) Lignans and resin of Araucaria
Biochem 141: 244–247. angustifolia by gas chromatography/mass spectrometry. J Mass Spectrom 39:
26. Keeling CI, Bohlmann J (2006) Diterpene resin acids in conifers. Phytochem 67: 1337–1347.
2415–2423. 39. Arif IA, Bakir MA, Khan HA, Ahamed A, Farhan AH, et al. (2010) A simple
27. Atherton RA, McComish BJ, Shepherd LD, Berry LA, Albert NW, et al. (2010) method for DNA extraction from mature date palm leaves: impact of sand
Whole genome sequencing of enriched chloroplast DNA using the Illumina grinding and composition of lysis buffer. Int J Mol Sci 11: 3149–3157.
GAII platform. Plant Methods 6: 22. 40. Gargano D, Maple-Grødem J, Reisinger V, Eichacker LA, Mølle SG (2013)
28. Ayliffe MA, Timmis JN (1992) Tobacco nuclear DNA contains long tracts of Analysis of the chloroplast proteome in arc mutants and identification of novel
homology to chloroplast DNA. Theor Appl Genet 85: 229–238. protein components associated with FtsZ2. Plant Mol Biol 81: 235–244.
29. Ayliffe MA, Scott NS, Timmis JN (1998) Analysis of plastid DNA like sequences 41. He ZH, Li HL, Shen Y, Li ZS, Mi H (2013) Comparative analysis of the
within the nuclear genomes of higher plants. Mol Biol Evol 15: 738–745. chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line
30. Goremykin VV, Salamini F, Velasco R, Viola R (2009) Mitochondrial DNA of and its parents. J Plant Physiol doi:10.1016/j.jplph.2013.03.016.
Vitis vinifera and the issue of rampant horizontal gene transfer. Mol Biol Evol 26:
99–110.

PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e84792