A feasible method to extract DNA from the cambium of high-canopy trees: from harvest to assessment

Abstract

Many tropical trees have high canopies and their leaves are not accessible. Thus, the use of tissue from a more accessible organ (cambium) for DNA extraction may be an alternative for molecular studies. We adapted a feasible methodology for extracting genomic DNA from cambium tissue harvested in the field for the assessment with PCR. We tested three storage conditions (two buffers and a silica gel) and four periods of time after harvest. We used previously described protocols and tested them on three species that occur in Amazonian forests and other biomes: Anadenanthera peregrina var. peregrina, Cedrela fissilis, and Ceiba speciosa. Our protocol obtained suitable PCR-grade genomic DNA for DNA sequencing and microsatellite genotyping. We recommend the use of silica for long-term storage and the buffer with ascorbic acid for short-term storage.

Full text

335 VOL. 504 2020: 335  338
http://dx.doi.org/10.1590/1809-4392202001571
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CITE AS: Mangaravite, É.; Terra, V.; Hattori, E.K.O.; Dal’sasso, T.C.S.; Bhering, L.L.; Oliveira, L.O. 2020. A feasible method to extract DNA from the cambium of
high-canopy trees: from harvest to assessment. Acta Amazonica 50: 335-338.
A feasible method to extract DNA from the cambium
of high-canopy trees: from harvest to assessment
Érica MANGARAVITE1,2, Vanessa TERRA3, Eric Koiti Okiyama HATTORI4, aís Carolina da Silva
DAL’SASSO1, Leonardo Lopes BHERING5, Luiz Orlando de OLIVEIRA1,*
1 Universidade Federal de Viçosa, Departamento de Bioquímica, Laboratório de Biologia Molecular e Filogeograa, Av. P. H. Rolfs, S/N, Campus Universitário,
36570-900 Viçosa, Minas Gerais, Brazil
2 Centro Universitário UNIFAMINAS, Colegiado de Biomedicina, Av. Cristiano Ferreira Varella, 655, Bairro Universitário, 36888-233 Muriaé, Minas Gerais, Brazil
3 Universidade Federal de Uberlândia, Instituto de Ciências Agrárias, Campus Monte Carmelo, Rodovia LMG 746, Km 01, s/n, Bloco 1A, sala 309, 38500-000
Monte Carmelo, Minas Gerais, Brazil
4 Universidade Federal dos Vales do Jequitinhonha e Mucuri, Instituto de Ciências Agrárias, Campus de Unaí, Av. Vereador João Narciso, 1380, Bairro Cachoeira,
38610-000 Unaí, Minas Gerais, Brazil
5 Universidade Federal de Viçosa, Departamento de Biologia Geral, Laboratório de Biometria, Av. P. H. Rolfs, s/n, Campus Universitário, 36570-900 Viçosa,
Minas Gerais, Brazil
* Corresponding author: luiz.ufv@hotmail.com; https://orcid.org/0000-0002-5578-2260
ABSTRACT
Many tropical trees have high canopies and their leaves are not accessible. us, the use of tissue from a more accessible organ
(cambium) for DNA extraction may be an alternative for molecular studies. We adapted a feasible methodology for extracting
genomic DNA from cambium tissue harvested in the eld for the assessment with PCR. We tested three storage conditions
(two buers and a silica gel) and four periods of time after harvest. We used previously described protocols and tested them
on three species that occur in Amazonian forests and other biomes: Anadenanthera peregrina var. peregrina, Cedrela ssilis, and
Ceiba speciosa. Our protocol obtained suitable PCR-grade genomic DNA for DNA sequencing and microsatellite genotyping.
We recommend the use of silica for long-term storage and the buer with ascorbic acid for short-term storage.
KEYWORDS: ascorbic acid, dithiothreitol, DNA isolation
Um método viável para extrair DNA do câmbio de árvores de dossel alto:
da coleta à aplicação
RESUMO
Muitas árvores tropicais possuem dossel alto e folhas não facilmente acessíveis. O uso de tecido de um órgão mais acessível
(câmbio) para extração de DNA pode ser uma alternativa para estudos moleculares. Nós adaptamos uma metodologia viável
para extrair DNA genômico de tecido cambial coletado no campo para avaliação com PCR. Testamos três condições de
armazenamento (dois tampões e sílica gel) e quatro períodos após a coleta. Utilizamos protocolos descritos anteriormente e
os testamos em três espécies encontradas em orestas amazônicas e outros biomas: Anadenanthera peregrina var. peregrina,
Cedrela ssilis e Ceiba speciosa. Nosso protocolo foi ecaz na obtenção de DNA adequado para sequenciamento e genotipagem
de microssatélites. Recomendamos o uso de sílica para armazenamento de longo prazo e o tampão com ácido ascórbico para
curto prazo.
PALAVRAS-CHAVE: ácido ascórbico; ditiotrietol; isolamento de DNA
Molecular data has been helpful in revealing hidden
aspects of the evolutionary history of plants (e.g., Hughes
et al. 2013). e number of biodiversity studies using plant
molecular data has increased over the last years (Vinson et al.
2018). Most of the traditional methods for DNA extraction
from plants were designed to use leaves as the source of
genomic DNA (e.g., Doyle 1990; Cota-Sánchez et al. 2006).
However, many tropical trees have high canopies and their
leaves are not readily accessible. Moreover, the vegetation of
seasonally dry forests usually releases its leaves during the
driest period (Oliveira-Filho and Fontes 2000). e use of
commercial kits allows for the quick extraction of genomic
DNA from leaves but adds cost to the analysis.
e use of an eective and inexpensive protocol that uses
cambium tissue may be an alternative when leaves are not
readily available. ere are few studies that have used cambium

MANGARAVITE et al. DNA extraction of tree cambium tissue
336 VOL. 504 2020: 335  338
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tissue as a source of genomic DNA, but most of them adopted
a time-consuming method using a commercial kit to achieve
DNA extraction (e.g., Asif and Cannon 2005; Colpaert et
al. 2005; Novaes et al. 2009; Lanes et al. 2013). Herein, we
aimed to adapt a feasible methodology for extracting genomic
DNA from cambium tissue, from the eld sampling to the
assessment of the DNA with PCR.
We sampled cambium tissue from three tree species that
are native to the Amazon region and other Brazilian biomes:
Anadenanthera peregrina (L.) Speg. var. peregrina (Fabaceae),
Cedrela ssilis Vell. (Meliaceae) and Ceiba speciosa (A. St.-
Hil.) Ravenna (Malvaceae). ree trees of each species were
sampled in Viçosa, Minas Gerais, Brazil (20º45’S, 42º52’W).
To collect a piece of cambium, we used a puncher (diameter
= 1 cm; thickness = 0.2 cm; length = 16 cm) and hammered
it into the tree trunk until it reached the wood brous layer.
e extracted sample was cleaned with a piece of tissue paper
and washed with 100% ethanol.
We collected three samples from each tree (nine samples
per species). e rst sample was kept in a dithiothreitol
transport buer (“DTT” treatment). e second sample was
kept in an ascorbic acid transport buer (“AA” treatment).
Both samples were kept in 15-mL Falcon tubes wrapped in
aluminum foil. e third sample was kept in an air-sealed
plastic bag containing approximately 50 g of silica gel beads
(“SIL” treatment). e DTT and AA buers consisted of
2/3 absolute ethanol and 1/3 1× CTAB buer (Colpaert et
al. 2005). Instead of using DTT (or β-mercaptoethanol) and
AA together in the transport buer, as Colpaert et al. (2005)
did, we tested them separately. For the DTT buer, we added
3 mM dithiothreitol; to the AA buer, we added 0.3% (w
v-1) ascorbic acid. We did not use β-mercaptoethanol in these
buers. e DTT and AA buers were kept at 4 ºC and the
SIL was stored at room temperature (approximately 25 ºC).
Another novelty of our study was to test whether the
storage conditions decreased the overall quality of the genomic
DNA over time. Extractions were performed after four storage
periods: “D0” (extraction performed on the same day of
harvest); and “D7”, “D14”, and “D21” (extraction performed
7, 14, and 21 days after harvest, respectively). Extraction from
the SIL samples was performed only after 14 and 21 days
to allow for tissue dehydration. Prior to DNA extraction,
the buer-stored samples (DTT and AA) were washed with
distilled water, sliced, and dried with tissue paper. e SIL
samples were only sliced. Approximately 40 to 70 mg of each
sample was placed into 2.0 mL microtubes together with two
3.2-mm chrome-steel beads per tube. e samples were then
homogenized with a Bead Beater system (Mini-Beadbeater-24,
BioSpec, Paulínia, SP, Brazil). We performed the genomic
DNA extraction with three replicates following an eight-step
protocol, based on Cota-Sánchez et al. (2006) (modied by
Riahi et al. 2010), which was here applied for the rst time
for cambium tissue. e eight steps were applied as follows:
1. We preheated the CTAB buer (2% CTAB, 100 mM
Tris-HCl, pH 7.5, 1.4 M NaCl, 20 mM EDTA, pH 8.0;
4% PVP; adding 1% β-mercaptoethanol immediately prior
to use) to 65 °C;
2. 800 μL of the hot CTAB buer were added to the 2.0 mL
microtube with the samples and beads;
3. e samples were pulverized with a bead beater: 3 min
at 2,500 oscillations per minute for the silica-dried samples,
and 5 min at 3,000 oscillations per minute for the buer-
stored samples. is step was repeated if the grinding was not
complete after the rst round;
4. e tubes were incubated at 65 °C for 15 min with
occasional swirling;
5. e samples were cooled on ice for 2 min and 750 μL CIA
solution (chloroform:isoamyl alcohol; 24:1) were added to
each tube followed by invertion 50 times. e tubes were
centrifuged for 15 min at 10,000 rpm (Eppendorf centrifuge
5424) and the supernatant was transfered to a new microtube
and the CIA step was repeated;
6. e supernatant was transferred to a new microtube,
0.7-volume ice-cold isopropanol was added and followed
by gentle invertion 10 times and centrifugation for 15 min
at 10,000 rpm. The supernatant was discarded without
disturbing the pellet;
7. e pellet was washed by adding 1 mL 70% ethanol.
e microtube was centrifuged for 10 min at 10,000 rpm
and the ethanol was discarded. is step was repeated with
1 mL absolute ethanol. e pellets were then dried at room
temperature, with care not to overdry them;
8. Each pellet was resuspended in 30 μL TE buer (10 mM
Tris-HCl, pH 8.0, 0.1 mM EDTA, pH 8.0), containing
RNAse A (10 mg mL-1). e DNA sample was incubated at
37 °C for 30 min and then stored at 4 °C.
DNA quantication and quality control were performed
with a NanoDrop Spectrophotometer (ermo Scientic).
We next tested whether the genomic DNA was suitable
for PCR. e genomic DNA was amplied (the internal
transcribed spacer, ITS) with the primer pair ITS4
(5’-TCCTCCGCTTATTGATATGC-3’, White et al. 1990)
and ITS Leu (5’-GTCCACTGAACCTTATCATTTAG-3’,
Baum et al. 1998). For the chloroplast gene, we amplied
the intron of trnL (UAA) (CD) with the primer pair
C (5’-CGAAATCGGTAGACGCTACG-3’) and D
(5’-GTTTACTTTTGGGCATGCTTCG-3’) (trnL intron
region, Taberlet et al. 1991). e PCRs were performed
with three replicates, and the amplication (ITS or CD)
was considered positive when a single, sharp band of the
size expected was visible on a 1% (w v-1) agarose gel. We
calculated the percentage of positive amplications from each

MANGARAVITE et al. DNA extraction of tree cambium tissue
337 VOL. 504 2020: 335  338
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extraction period (D0, D7, D14, and D21) and each storage
condition (DTT, AA, and SIL), for each species. We also used
the microsatellite primers Acol15, Acol16 (Feres et al. 2012),
Ced54, and Ced65 (Hernández et al. 2008) in order to test
the suitability of the DNA. e PCR results were checked on
2% (wv-1) agarose gels. ANOVA and Tukey tests, implemented
in RBio version 119 (Bhering 2017), were used to analyze the
dierences in the DNA concentrations and the percentages of
positive PCRs among treatments within species.
e two storage conditions (DTT and AA) allowed for
PCR amplications that were not possible previously for
frozen bark samples of A. peregrina (Novaes et al. 2009) (Table
1). e SIL storage at room temperature (approximately 25
ºC instead of 4 ºC for the buer samples) exhibited some
DNA degradation, which was likely due to the temperature
dierence. However, several positive PCR amplications
were obtained (Table 2). e SIL storage resulted in a higher
amount of DNA compared to previous studies. For samples
of A. peregrina var. peregrina stored in SIL, our methodology
recovered genomic DNA (496 ng mL-1) with a yield higher
than previously reported (364 ng mL-1, Novaes et al. 2009).
During eld expeditions, silica gel is more amenable to
handling than liquid buers. Additionally, the use of silica gel
requires less storage space in the laboratory when considering
the size of the plastic bags and the tubes in the refrigerator.
Despite some DNA degradation, we recommend SIL for
long-term storage.
DNA extraction after different storage periods of
cambium tissue has not been previously reported. ere
was no signicant dierence in the genomic DNA yield or
quality among the four storage periods and none signicantly
impaired the use of the samples for PCR. is suggests that
cambium tissue can be harvested and kept for some weeks
prior to DNA extraction, which is frequently convenient in
eldwork schedules. We showed that buers with either DTT
or AA were suitable for extracting the DNA right after sample
harvest. Additionally, the AA buer was easier to prepare and
carry into the eld, and then be added to the remaining buer
ingredients as needed. e SIL-stored tissues were dicult
to process during the rst weeks. Nonetheless, the cambium
tissues preserved in SIL storage were amenable to PCR four
weeks after harvest (data not shown).
Anadenanthera peregrina exhibited higher genomic
DNA yield than C. speciosa (Table 1). e high standard
deviations might be due to the dierent sizes of the source
tissue samples. Genomic DNA storage in DTT or AA
exhibited sharp bands of a high-molecular mass, with little
to no smears, suggesting high integrity. e A260/A280 ratio
values presented little variation among species. Most of the
samples exhibited a A260/A280 ratio greater than 1.8 (Figure 1),
suggesting high purity. As a consequence, amplications via
PCR were successful. All tree samples were amplied for the
Table 1. Concentrations of genomic DNA (ng µL-1) extracted from cambium
samples of three tree species under dierent treatments (Treat) of storage time
(0, 7, 14, 21 days) and storage media (DTT = dithiothreitol transport buer, AA
= ascorbic acid transport buer, SIL = silica gel beads) per species . Values are
averages and standard deviations of three replicates.
Treat Anadenanthera peregrina Cedrela ssilis Ceiba speciosa
D0 328.1 ± 202.9 379.1 ± 183.8 155.8 ± 76.7
D7 682.1 ± 848.2 353.8 ± 367.3 188.0 ± 79.3
D14 395.8 ± 276.8 294.2 ± 287.6 187.0 ± 180.3
D21 322.5 ± 292.1 213.0 ± 189.5 136.4 ± 130.0
AA 420.9 ± 633.4 269.9 ± 184.8 158.1 ± 120.4
DTT 374.8 ± 226.0 353.3 ± 284.0 182.1 ± 133.6
SIL 496.1 ± 405.6 247.3 ± 352.9 148.6 ± 151.2
Average 431.5 301.5 165.1
Table 2. Percentage of positive amplications per region (CD and ITS) of DNA
extracted from cambium samples of three tree species under dierent treatments
(Treat) of storage time (0, 7, 14, 21 days) and storage media (DTT = dithiothreitol
transport buer, AA = ascorbic acid transport buer, SIL = silica gel beads).
Treat Anadenanthera peregrina Cedrela ssilis Ceiba speciosa
CD
D0 100.0 94.4 55.6
D7 88.9 83.3 61.1
D14 96.3 100.0 85.2
D21 100.0 92.6 100.0
AA 91.7 91.7 75.0
DTT 100.0 94.4 83.3
SIL 100.0 94.4 77.8
ITS
D0 88.9 94.4 33.3
D7 83.3 83.3 27.8
D14 92.6 70.4 81.5
D21 96.3 88.9 81.5
AA 83.3 86.1 61.1
DTT 97.2 86.1 55.6
SIL 94.4 72.2 72.2
Average 93.8 88.0 67.9
Figure 1. NanoDrop absorbances of the average A260/A280 ratios for DNA extracted
from cambium samples of three tree species under dierent treatments of storage
time (0, 7, 14, 21 days) and storage media (DTT = dithiothreitol transport buer, AA
= ascorbic acid transport buer, SIL = silica gel beads). The columns are averages
of three replicates and the bar is the standard deviation. This gure is in color in
the electronic version.

MANGARAVITE et al. DNA extraction of tree cambium tissue
338 VOL. 504 2020: 335  338
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tested regions. Anadenanthera peregrina exhibited the highest
percentage of positive amplications (Table 2) for the CD
and ITS regions, followed by C. ssilis and C. speciosa. Sharp
bands characterized most of the amplications. We checked
the PCR products for microsatellites and the presence of bands
indicated its suitability for genotyping.
To our knowledge, this is the rst report on a multiplexing
PCR system with microsatellites applied to cambium tissue
DNA, with potential for application in studies of the genetic
diversity of tree species. We recommend the use of silica for
long-term storage and the ascorbic acid transport buer for
short-term storage.
ACKNOWLEDGMENTS
EM is grateful to CAPES (Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior) for a fellowship from Oct/2012
to Feb/2014 and for an international fellowship from
Apr/2015 to Mar/2016; and to CNPq (Conselho Nacional
de Desenvolvimento Cientíco e Tecnológico) for a fellowship
from Mar/2014 to Mar/2015 and from Apr/2016 to Jul/2016.
LOO received a fellowship from CNPq (PQ 304153/2012-5)
and grants from FAPEMIG (Fundação de Amparo à Pesquisa
do Estado de Minas Gerais) (PPM-00561-15, 18 BPD-00282-
10) and CNPq (562249/2010-9).
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RECEIVED: 23/04/2020
ACCEPTED: 19/08/2020
ASSOCIATE EDITOR: Izeni P. Farias
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