DNA extraction of sweetpotato
529
Note
CTAB METHODS FOR DNA EXTRACTION OF
SWEETPOTATO FOR MICROSATELLITE ANALYSIS
Aline Borges1 ; Mariana Silva Rosa2 ; Gustavo Henrique Recchia 1 ; Jurema Rosa de QueirozSilva3 ; Eduardo de Andrade Bressan1 ; Elizabeth Ann Veasey4 *
1
USP/CENA - Programa de Pós-Graduação Biologia na Agricultura e no Ambiente.
UNESP/FCAV - Programa de Pós-Graduação em Produção e Tecnologia de Sementes.
2
UFRB - Programa de Pós-Graduação em Ciências Agrárias
4
USP/ESALQ - Depto. de Genética, C.P. 83 - 13400-970 - Piracicaba, SP - Brasil.
*Corresponding author <eaveasey@esalq.usp.br>
2
ABSTRACT: Microsatellite markers have proved to be useful in genetic diversity assessments of
sweetpotato (Ipomoea batatas) but practical DNA extraction methods to ensure good quality and
quantity DNA for these studies are yet to be established. This study compares the efficiency of three
modified methodologies for DNA extraction of six sweetpotato landraces using the CTAB extraction
buffer in regard to quantity and purity of DNA quantification and microsatellite band patterns. All
methodologies yielded satisfactory results, but the method based in leaf tissue macerated in liquid
nitrogen was deemed more adequate because of its simplicity and lower cost. However, the method
based in dry leaf tissue was considered more advantageous, first because elicits practicability in the
plant acquisition and drying process, especially when the collection is performed in situ, and also
because its simplicity makes possible the cold storage of the dry, ground samples for future DNA
extractions.
Key words: Ipomoea batatas, SSR, DNA isolation, landraces, protocol
MÉTODOS CTAB DE EXTRAÇÃO DE DNA PARA A ANÁLISE DE
MICROSSATÉLITES EM BATATA-DOCE
RESUMO: Os marcadores microssatélites são úteis para a análise da diversidade genética de variedades
tradicionais de batata-doce (Ipomoea batatas). Para estes estudos, métodos práticos de extração de
DNA precisam ser estabelecidos para assegurar uma boa qualidade e quantidade de DNA extraído.
Assim, foi comparada a eficiência de três metodologias para extração de DNA usando o tampão de
extração CTAB, todas com modificações. Para verificar a quantidade e pureza na quantificação de
DNA, bem como o padrão de bandas de microssatélites para as três metodologias utilizaram-se seis
etnovariedades de batata-doce. Os testes mostraram que as três metodologias apresentaram resultados
satisfatórios. Uma das metodologias baseada em tecido foliar macerado em nitrogênio líquido mostrouse a mais adequada devido à simplicidade e menor custo. Entretanto, o método baseado em tecido
foliar seco foi o mais vantajoso devido à praticidade na aquisição da planta e no processo de secagem,
principalmente quando a coleção encontra-se em condições in situ, e pela possibilidade do
armazenamento refrigerado das amostras secas e maceradas para futuras extrações de DNA.
Palavras-chaves: Ipomoea batatas, SSR, isolamento de DNA, etnovariedades, protocolo
INTRODUCTION
Sweetpotato [Ipomoea batatas (L.) Lam.] belongs
to the Convolvulaceae family. The Ipomoea genus encompass 600-700 species, as many as 500 concentrated in the Americas, mostly native with few introduced species. Part of the Brazilian territory is considered a sweetpotato center of diversity. Within the
species there is a high variability, probably because of
its high ploidy level, which needs to be preserved and
studied to contribute with future plant breeding programs (Austin & Huamán, 1996; Austin, 1988).
Several genetic studies have been conducted with
molecular markers in sweetpotato, such as RAPD (random amplified polymorphic DNA) (Ukoskit & Thompson, 1997; Thompson et al., 1997; Sagredo et al.,
1998; Zhang et al., 1998; Gickuki et al., 2003), DAF
(DNA amplification fingerprinting) (He et al., 1995),
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530
Borges et al.
microsatellites or SSR (simple sequence repeats)
(Jarret & Bowen, 1994; Buteler et al., 1999; Hu et al.,
2004), ISSR (inter-simple sequence repeats) (Hu et al.,
2003), AFLP (amplified fragment length polymorphism)
(Zhang et al., 2000; Fajardo et al., 2002; Zhang et al.,
2004; Bruckner, 2004), and SAMPL (selective amplification of microsatellite polymorphic loci) (Tseng et
al., 2002). Among the DNA extraction methodologies
used by these authors, Doyle & Doyle (1990), with
or without modifications and Gawel & Jarret (1991),
both based on CTAB (cetyltrimethylammonium bromide) extraction buffer, or Wilson et al. (1992), based
on the MATAB (mixed alkyltrimethylammonium bromide) extraction buffer, were the preferred ones. Kim
& Hamada (2005) presented a rapid and reliable DNA
extraction method for sweet-potato also based on
CTAB extraction buffer. Other studies used the
DNAzol methodology (Chomczynski et al., 1997;
Buteler et al., 1999). In general, all these studies used
the maceration of young leaves recently collected or
freeze-dried with liquid nitrogen.
In this study, the widely used methodologies of
Doyle & Doyle (1987) and Doyle & Doyle (1990),
based on CTAB extraction buffer and the maceration
of recently collected leaves with liquid nitrogen, were
compared with the methodology used for cassava
(Manihot esculenta Crantz) (Elias et al., 2004), with adaptations, based on 3% CTAB, using young leaves dried
in an oven at 45ºC for 72 h. The use of dried leaves
for DNA isolation, without using liquid nitrogen, has not
yet been described for sweetpotato. This methodology
was reported in the genetic diversity analysis of South
American cassava landraces with SSR (Elias et al.,
2004). Other studies with cassava used silica gel-dried
leaves for DNA extraction ground in liquid nitrogen before adding the extraction buffer (Colombo, 2000a; Colombo, 2000b). However, the use of the dry leaves for
DNA extraction in sweetpotato is original.
This study compares the three methodologies of
Doyle & Doyle (1987), Doyle & Doyle (1990) and
Elias et al. (2004), with modifications, to the original
methods. The extracted DNA was then used in SSR
analysis to compare band standards and to test the efficient of the methodologies for future studies.
MATERIAL AND METHODS
Plant materials
Sweetpotato landraces used in this study were
sampled at Iguape (24º42’S, 47º33’W) and Cananéia
(25º00’S, 47º55’W), São Paulo State, Brazil (Table 1),
from an ex situ germplasm collection of the Genetics
Department of Escola Superior de Agricultura “Luiz de
Queiroz”, University of São Paulo, Brazil.
DNA extraction
DNA extraction methodologies of Doyle & Doyle
(1987), Doyle & Doyle (1990) and Elias et al. (2004)
based in Dellaporta et al. (1983), all with modifications
from the original methods, were compared. Recently
expanded young leaves of sweetpotato were collected
early in the morning from a multiplication field, washed
and dried with paper towels to eliminate excess dirt,
and then submitted to the following DNA extraction
methods:
A) Modified Doyle & Doyle (1987) methodology
• 50-mg samples of young leaf tissues were ground
to a fine powder in liquid nitrogen;
• powder was then placed in 1.5-mL microtubes containing 700 µL 2% CTAB extraction buffer [20 mM
EDTA, 0.1 M Tris-HCl pH 8.0, 1.4 M NaCl, 2%
CTAB, plus 0.4% β-mercaptoethanol added just before use];
• the solution was incubated at 65ºC for 45 min, gently mixing by inversion every 15 min; 500 µL of chloroform-isoamylalcohol (24:1) was added to the tubes
and gently mixed for 1 min;
• samples were centrifuged for 10 min. at 12,000 rpm;
600 µL of the supernatant was then transferred to a
fresh tube following the addition of 500 µL chloroform-isoamylalcohol (24:1); this procedure was repeated twice;
• 500 µL of the supernatant was then transferred to a
fresh tube with 700 µL of cold isopropanol (-20ºC);
samples were gently mixed by inversion and centri-
Table 1 - List of the sweetpotato landraces assessed in this study and their origin.
Landrace code
Municipality
Community
Local names
Latitude
Longitude
1
Landrace
DGB 5.0
Iguape
Pontal de Icapara
Native/Sambaqui
24º39'S
47º26'W
2
DGB 10.0
Iguape
Praia do Leste
Purple potato
24º40'S
47º25'W
3
DGB 23.0
Iguape
Cavalcanti
White potato
24º42'S
47º41'W
4
5
DGB 32.0
DGB 40.0
Cananéia
Cananéia
Agrossolar
S.P. Bagre
Rio Grande potato
White potato
24º58'S
24º57'S
47º54'W
47º53'W
6
DGB 42.0
Cananéia
Aroeira
White potato
24º52'S
47º52'W
Sci. Agric. (Piracicaba, Braz.), v.66, n.4, p.529-534, July/August 2009
DNA extraction of sweetpotato
fuged at 12,000 rpm for 10 min, and so it was possible to visualize the DNA adhered to the bottom of
the tube;
• the liquid solution was then released and the DNA
pellet washed with 700 µL of 70% ethanol to eliminate salt residues adhered to the DNA, and set to dry
for approximately 12 h, or until the next day, with the
tubes inverted over a filter paper, at room temperature;
• the pellet was then ressuspended in 100 µL TE buffer
(10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) plus
5 µL ribonuclease (RNAse 10 mg mL–1) in each tube;
this solution was incubated at 37ºC for 1h, and after
stored at -20ºC.
B) Modified Doyle & Doyle (1990) methodology
• young leaves were ground to a fine powder in liquid
nitrogen;
• the powder (50 mg) was placed in 1.5-mL
microtubes containing 400 µL of the 2% CTAB extraction buffer with modifications [20mM EDTA, 0.1 M
Tris-HCl pH 8.0, 1.4 M NaCl, 2% CTAB, plus 1% βmercaptoethanol added just before use]; microtubes
were then vortexed for 10 s and incubated at 60ºC for
30 min;
• next, 60 µL of chloroform-isoamylalcohol (24:1) was
added to the solution, which was vortexed for 10 s
and centrifuged at 10,000 rpm for 3 min; the supernatant was transferred to a fresh tube and this stage
was repeated once;
• cold isopropanol (-20ºC) was added to the supernatant (0.7 of the total volume of supernatant collected);
samples were gently mixed by inversion and centrifuged at 10,000 rpm for 3 min; the DNA pellet adhered to the tube was then visualized;
• the liquid phase was then released and DNA washed
twice with 500 µL 70% ethanol; the pellet was set to
dry for approximately 12 h with the tubes inverted
upon filter paper at room temperature;
• the pellet was ressuspended in 100 µL TE buffer solution plus 5 µL RNAse (10 mg mL–1); the solution was
then incubated at 37ºC for 1 h, and after stored at –
20ºC.
C) Modified Elias et al. (2004), based on CTAB extraction buffer, Dellaporta et al. (1983) methodology
• recently expanded young leaves were dehydrated in
the oven at 45ºC for 72h, and then ground to fine powder in porcelain crucibles; the powder was stored in
1.5 mL microtubes at -20ºC until use;
531
• a 50 mg sample was then transferred to a fresh, 1.5mL microtube containing 800 µL of 3% CTAB extraction buffer [30mM EDTA, 0.1 M Tris-HCl pH 8.0, 1.2
M NaCl, 3% CTAB, plus 3% β-mercaptoethanol added
just before use].
• tubes were then incubated at 65ºC for 1 h, gently
mixed every 15 min for adequate homogenization;
• 500 µL of chloroform-isoamylalcohol (24:1) was then
added, mixed gently for 1 min, and centrifuged at
8,000 rpm for 10 min;
• after centrifugation, 500 µL of the supernatant was
transferred to a fresh tube with an equal volume of
chloroform-isoamylalcohol (24:1) plus 200 µL CTAB
3% (without β-mercaptoethanol); this solution was
mixed gently and centrifuged again at 8,000 rpm for
10 min and 500-µL sample of the supernatant was
transferred to a fresh tube with 350 µL cold isopropanol at –20ºC, and gently mixed by inversion;
• the solution was centrifuged at 8,000 rpm for 10 min,
and the resulting pellet was set to dry for approximately
12 h with the tubes inverted over a filter paper at room
temperature;
• the pellet was ressuspended in TE buffer solution,
adding 200 µL TE and 4 µL de RNAse (10 mg mL–1);
the tubes were incubated at 37ºC for 30 min, and then
stored at -20ºC.
DNA quantification
DNA was quantified in 4% polyacrylamide gels.
Electrophoresis was conducted in a 1X TBE buffer
[100 mL 10X TBE (0.89M Tris base, 0.89M Boric
acid, 20 mM EDTA pH 8.0) and 900 mL distilled water] at 60 V for 30 min and then at 120 V for 1.5 h.
The gel was then stained in silver nitrate (Bassam et
al., 1991), using 125 mL fixation buffer (10% absolute ethanol, 5% absolute acetic acid, 895 mL distilled
water) and 0.2% silver nitrate for 5 min. The gel then
was washed twice with 150 mL distilled water, and
stained with 125 mL revelation buffer (30% sodium
hydroxide in 1000 mL distilled water), adding 0.4%
phormaldeid.
PCR amplification and electrophoresis
Eight primers (Table 2) were used for each DNA
extraction method and assessed landraces. PCR reactions were conducted at a final volume of 10.2 µL,
containing 0.2 µL Taq polymerase (5 units µL–1), 1.0
µL 10X Amplification Buffer (Mg+ Free), 1.0 µL MgCl2
(50 mM), 0.5 µL Forward Primer (5 pmoles µL–1), 0.5
µL Reverse Primer (5 pmoles µL–1), 1.0 µL dNTP’s
(2.5 mM each), 3 µL de Milli-Q H2O and 3 µL DNA
(5 ng µL–1). The PCR reaction conditions, conducted
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Borges et al.
Table 2 - List of the eight primers 1 used for the DNA amplification of sweetpotato varieties, showing their expected fragment
sizes and annealing temperatures used in this study.
Locus
Primer pairs 5' ® 3'
F: CAAACGCACAACGCTGTC
R: CGCGTCCCGCTTATTTAAC
F: AGAACGCATGGGCATTGA
R: CCCACCGTGTAAGGAAATCA
F: CGTCCATGCTAAAGGTGTCAA
R: ATAGGGGATTGTGCGTAATTTG
F: AGCCACTCCAACAGCACATA
R: GGTTTCCCAATCAGCAATTC
F: TGGGCATTCTCATATTTTGCT
R: GCCACTCCAACAGCACATAA
F: GCGGAACGGACGAGAAAA
R: ATGGCAGAGTGAAAATGGAACA
F: GAGAGGCCATTGAAGAGGAA
R: AAGGACCACCGTAAATCCAA
F: CAATTTCACACACAAACACG
R: CCCTTCTTCCACCACTTTCA
Ib - 316
Ib - 318
Ib - 255F1
Ib - 286
Ib - 255
Ib-242
Ib-248
Ib - 297
1
Expected size (bp)
Annealing temperature
140-155
60ºC
125-135
53ºC
210-245
53ºC
90-122
51ºC
165-170
62ºC
105-142
52ºC
155-170
62ºC
130-200
51ºC
Buteler et al. (1999).
Table 3 - DNA concentration of the sweetpotato 50 mg
tissue sampled for three methodologies tested:
(A) Elias et al. (2004), (B) Doyle & Doyle (1990),
and (C) Doyle & Doyle (1987).
Landrace
Landrace code
DNA concentration
A
B
C
-------- ng mL -------–1
1
DGB 5.0
30
80
50
2
3
DGB 10.0
10
60
20
DGB 23.0
40
30
30
4
DGB 32.0
30
30
30
5
DGB 40.0
40
40
30
6
DGB 42.0
30
20
30
at a MyCycler BioRad thermocycler, were the following: 3 min at 95ºC; 5 cycles of 45 s at 94ºC, 15 s for
the primer annealing temperature (Table 2), 45 s at 72ºC;
and 20 cycles of 1 min at 90ºC, 1 min for the annealing temperature, 1 min at 72ºC; and 7 min at 72ºC for
the final extension (Buteler et al., 1999).
The products of amplification were separated in 6%
polyacrilamyde gels, at 60 V for 30 min and 120 V
for 3 h. Ladders of 10bp (Invitrogen) and 100bp
(AMRESCO) were used as molecular weight patterns.
The gels were stained in silver nitrate, as described for
the DNA quantification and photodocumented with a
digital camera.
RESULTS AND DISCUSSION
The three methodologies yielded satisfactory results
for DNA extraction, quantification and microsatellites
band resolution for all sweetpotato landraces (Figures
1 and 2). Although the DNA concentration was higher
for both Doyle & Doyle methods in landraces 1 and 2
when compared to the Elias et al. (2004) method,
which is composed of an aggressive process for DNA
extraction of dried material, for the other landraces the
DNA concentration was equivalent for the three methodologies (Table 3). However, the Elias et al. (2004)
methodology showed better quality DNA because of
the lower incidence of impurities compared to the other
methods. This could be related with the modifications
in the methodology based on dry leaves.
Being an hexaploid species (Ozias-Akins & Jarret,
1994), sweetpotato shows complex microsatellite band
patterns (Figure 2). Considering the band patterns
shown by the three methodologies, although a few
landraces did not present good band resolution, in general, the three methodologies showed the same band
patterns. For example, two bands were considered for
landraces 5 and 6 for primers Ib-297 and Ib-286, respectively, while three bands were considered for
landrace 2 for primer Ib-242.
Few modifications were made from the original
CTAB method used by Elias et al. (2004), based on
Dellaporta et al. (1983), such as temperature and drying time in the oven, performed in this study at 45ºC
for 72 h. The lower temperature was important to obtain higher DNA quantity in each sample. The 1% βmercaptoethanol concentration was increased to 3%.
Although this increase contributed to a lower DNA quantity, it was important to obtain a more purified DNA.
This concentration can be increased up to 5% (Pereira
et al., 2007), depending on the quality of the material.
Another modification was the addition of 200 µL CTAB
3% (without β-mercaptoethanol) to the DNA purification stage with chloroform-isoamylalcohol (24:1),
which was important to increase DNA purification.
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DNA extraction of sweetpotato
533
Figure 1 - Quantification polyacrilamyde gel showing the three methodologies: (A) Elias et al. (2004), (B) Doyle & Doyle (1990) and (C)
Doyle & Doyle (1987), all with modifications, plus eight DNA markers, varying from 5 to 60 ng µL-1 Lambda DNA.
Figure 2 - Microsatellite band patterns obtained for three primers (A: Ib-297; B: Ib-286; C: Ib-242) and six sweetpotato landraces (1,
2, 3, 4, 5, 6, from left to right), for the three DNA extraction methodologies: (1) Elias et al. (2004), (2) Doyle & Doyle (1990)
and (3) Doyle & Doyle (1987).
Doyle & Doyle (1990) method was considered
more practicable because required fewer centrifugations with less reagents. Also, a modification to one
of the stages of Doyle & Doyle (1990) included the
substitution of a refrigerated microcentrifuge for a
non-refrigerated microcentrifuge. However, the Elias
et al. (2004) methodology, used for cassava with modifications, presents an advantage over the other two
methodologies in using dried leaf material compared
to grinding of recently collected or freeze-dried leaves
in liquid nitrogen. During plant collection, one of the
difficulties is the storing of the material before analysis, especially when laboratory facilities are distant.
Thus, this method is useful, for example, when in situ
and on farm sweetpotato collections are made. When
using freeze-dried leaves, they usually need to be immediately immersed into liquid nitrogen and transported
to a -20ºC or -80°C freezer until its use. With the Elias
et al. (2004) modified methodology, even sun dried
leaves may be used, or young leaves mounted within
sheets of folded newspaper which are then left to dry
in plant presses at room temperature (Pereira et al.,
2007) or in the sun. Moreover, the young leaves can
be packed within two sheets of filter paper together
with silica gel to be left at the laboratory for dehydration. The fine powder obtained after grinding can be
easily stored in 1.5 mL microtubes at -20ºC for further analysis.
Most genetic studies using molecular markers in
sweetpotato have used methods based on the 2%
CTAB buffer extraction, such as those of Doyle &
Doyle (1990), with or without modification (Tseng et
al., 2002; Zhang et al., 1998, 2000, 2004; Hu et al.,
2003, 2004), or the Gawel & Jarret (1991) method
(Ukoskit & Thompson, 1997), or the Rogers &
Bendich (1988) modified method (Sagredo et al.,
1998). He et al. (1995) and Prakash et al. (1996) used
methods based on the MATAB 2% extraction buffer,
such as Wilson et al. (1992). Other studies used the
DNAzol reagent method (Chomczynski et al., 1997;
Buteler et al., 1999), or the ‘Dneasy plant minikit’
(QIAGEN) (Gichuki et al., 2003), or also the Promega
Wizard® Magnetic 96 DNA Plant System extraction
method (Madison, WI) (Bruckner, 2004). All methodologies cited above used recently collected young leaves
ground in liquid nitrogen (He et al., 1995; Prakash et
al., 1996; Thompson et al., 1997; Zhang et al., 1998;
Tseng et al., 2002; Hu et al., 2003, 2004) or freezedried leaves stored at –20ºC or -80ºC (Fajardo et al.,
2002; Gichuki et al., 2003; Bruckner, 2004). Until now,
apparently no studies with sweetpotato molecular markers have used young leaves dehydrated in the oven for
DNA extraction.
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Borges et al.
Summing up, all three methodologies tested in this
study were shown to be adequate for microsatellites
studies in sweetpotato. However, the cassava Elias et al.
(2004) modified methodology is recommended for
sweetpotato based on 3% CTAB and dehydrated young
leaves, due to lesser costs and the practicability of the
method, especially considering an in situ plant collection.
ACKNOWLEDGEMENTS
To the program Biota/FAPESP and CNPq for providing financial support and scholarships.
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