Biologia 69/2: 133—138, 2014 Section Cellular and Molecular Biology DOI: 10.2478/s11756-013-0311-2
An improved method to extract DNA from mango Mangifera indica* Mohammad S. Uddin1,2, Wenli Sun1, Xinhua He3, Jaime A. Teixeira da Silva4 & Qi Cheng1** 1
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China; e-mail:
[email protected] 2 Bangladesh Agricultural Research Institute, Gazipur – 1701, Bangladesh 3 College of Agriculture, Guangxi University, Nanning 530004, People’s Republic of China 4 P. O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken, 761-079, Japan
Abstract: High quality genomic DNA is the first step in the development of DNA-based markers for fingerprinting and genetic diversity of crops, including mango (Mangifera indica L.), a woody perennial. Poor quality genomic DNA hinders the successful application of analytical DNA-based tools. Standard protocols for DNA extraction are not suitable for mango since the extracted genomic DNA often contains secondary metabolites that interfere with analytical applications. In this study, we employed an additional step to remove polysaccharides, polyphenols and secondary metabolites from genomic DNA extracted from young or mature leaf tissue; then a modified traditional cetyl trimethyl ammonium bromide (CTAB) method was applied. The use of 0.4 M glucose improved DNA quality and avoided contamination and browning by polyphenolics, relative to the traditional CTAB method. This is an easy and efficient method for genomic DNA extraction from both young and mature leaves of mango. The isolated DNA was free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by its clear colour, its viscosity, A260 /A280 ratio and suitability for PCR-based reactions. This modified protocol was also used to extract high quality genomic DNA from other woody perennials, including walnut, guava, lychee, pear, grape and sugarcane. Key words: genomic DNA extraction; Mangifera indica; polyphenols; inter simple sequence repeat; 18S rRNA. Abbreviations: CTAB, cetyl trimethyl ammonium bromide; EDTA, ethylenediamine tetraacetic acid; ISSR, inter simple sequence repeat; PVP, polyvinylpyrrolidone; TAE, Tris acetate-EDTA; TE, Tris-EDTA.
Introduction Mango (Mangifera indica L.; Anacardiaceae), commonly known as the ‘King of fruits’ (Singh 1996), is a popular tropical fruit, especially in Asia. In conjunction with other biotechnological applications, such as tissue culture and somatic embryogenesis (Malabadi et al. 2011), the use of high quality genomic DNA would advance molecular studies on this tropical species. Mango leaves contain high levels of polysaccharides, polyphenolics, proteins, tannins and other secondary metabolites, whose presence can inhibit advanced molecular research from extracted genomic DNA (Pirttila et al. 2001). During the extraction process, cells are disrupted and these cytoplasmic compounds can come into contract with nuclei and other organelles (Loomis 1974). In their oxidized form, polyphenols covalently bind to
DNA giving it a brown colour and making it useless for most research applications (Katterman & Shattuch 1983; Guillemaut & Marechal-Drouard 1992). Polyphenolic compounds interact irreversibly with nucleic acid resulting in the inability of different modifying enzymes to manipulate the DNA (Manoj et al. 2007). Polysaccharides are also problematic as they make DNA unruly during pipetting and hinder the activity of polymerases, ligases and restriction endonucleases (Fang et al. 1992; Sharma et al. 2002). The few protocols for mango genomic DNA extraction mostly used young and fresh leaves (Dellaporta et al. 1983; Doyle & Doyle 1990; Davis et al. 1995), but these are not always available on a mango tree. Moreover, vegetative growth occurs only 3–4 times a year on an individual stem, depending upon the cultivar and growth conditions (Davenport & Nunez-Elisea 1997).
* Electronic supplementary material. The online version of this article (DOI:10.2478/s11756-013-0311-2) contains supplementary material, which is available to authorized users. ** Corresponding author
c 2013 Institute of Molecular Biology, Slovak Academy of Sciences
134 In this study, we established a simple protocol for the extraction of good quality DNA with greater yield from mango leaves of different ages. Material and methods Plant material The young and mature leaves of 14 mango varieties (BARI Aam-1, 2, 3, 4, 5, 6, 7, 8, Gopalbhog, Khirsapat, Langra, Ashwina, Mollica and Fazli) were obtained from the Regional Horticulture Research Station, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur, Bangladesh. Ten leaves of different ages (7, 14, 21 and 50–60 days) were collected from each variety, washed with water, blotted dry with filter paper and immediately placed into small sealed polybags to prevent transpiration loss. Leaf material was flown from Bangladesh to China, and no ice or liquid nitrogen was used during transportation. Upon arrival, leaf material was stored at –70 ◦C until use. Walnut (Juglans regia L.), guava (Psidium guajava), lychee (Litchi chinensis), pear (Pyrus communis L.), grape (Vitis vinifera) and sugarcane (Saccharum officinarum L.) leaves, 10 for each species and also of different ages (7, 14, 21 and 50–60 days), were collected from Nanning, Guangxi province, China. The protocol was standardized in the Biotechnology Research Institute laboratory, Graduate School of the Chinese Academy of Agricultural Sciences, Beijing, China. Reagents Two solutions were used to extract DNA. Solution 1 consisted of 0.4 M glucose (Beijing Chemical Works, Beijing, China), 20 mM ethylenediamine tetraacetic acid (EDTA; Amresco, Solon, USA; pH 8.0), 3% (w/v) polyvinylpyrrolidone (PVP)-40 (molecular weight 40,000) (Amresco) and 0.2% (v/v) β-mercaptoethanol. Solution 2 consisted of 2% cetyl trimethyl ammonium bromide (CTAB) (w/v) (Amresco), 100 mM Tris (pH 8.0) (Amresco), 20 mM EDTA (pH 8.0) (Amresco), 1.4 M NaCl, and 0.15% (v/v) βmercaptoethanol. In both cases, β-mercaptoethanol was added just prior to use. In addition, chloroform:isoamyl alcohol (24:1), 70% alcohol, 100% alcohol, 3 M sodium acetate (pH 5.2) (Sinopharm Chemical Reagents Co., Ltd., Shanghai, China) and Tris-EDTA (TE) buffer consisting of 10 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0) and 0.01 µg/µL RNase A (Promega Corp., Madison, USA) were also used. Genomic DNA extraction using new method About 0.2–0.3 g of frozen mango leaves were weighed after removing midribs and secondary veins and homogenized with a pre-chilled mortar and pestle in liquid nitrogen. Leaves were ground to a fine powder and transferred into a 2-mL tube (Axygen, Union City, USA) to which 800 µL of solution 1 was added followed by 0.2% β-mercaptoethanol. The mixture was vortexed, then centrifuged at 12,000 rpm for 10 min at 4 ◦C. The supernatant was discarded. The same procedure was repeated with 700 µL of solution 1. To the pellet, 700 µL of preheated (65 ◦C) solution 2 was added as extraction buffer, 0.15% (v/v) β-mercaptoethanol was added and the mixture was mixed gently. The tubes, each containing a different leaf sample, were incubated at 65 ◦C in a water bath for 1 h with intermittent shaking. Centrifuge tubes were cooled to room temperature and an equal volume of chloroform:isoamyl alcohol (24:1) was added. The contents were mixed well by vortexing or shaken manually for 5 min, then centrifuged at 12,500 rpm for 10 min at room temperature. The supernatant was transferred to a fresh
M.S. Uddin et al. 1.5-mL tube (Axygen) and this clean-up step was repeated three-times or until a clear supernatant was obtained. The upper aqueous phase was transferred into a new Eppendorf tube (1.5 mL) containing twice the volume of 100% ethanol and 1/10 (v/v) of sodium acetate, mixed gently and kept at –20 ◦C for 1 h. The tubes were centrifuged at 12,000 rpm for 15 min and the supernatant was discarded. The DNA pellet was washed twice with 70% ethanol and dried at room temperature. The dried pellet was re-suspended in 200 µL 0.1X TE and incubated at 37 ◦C for 30 min. An equal volume of chloroform was added, mixed and tubes were centrifuged at 12,000 rpm for 5 min. The upper aqueous phase was carefully collected and transferred into a new sterile 1.5-mL Eppendorf tube containing twice the volume of 100% ethanol, mixed gently and kept at –20 ◦C for 1 h. Tubes were centrifuged at 12,000 rpm for 15 min, the liquid was discarded, and tubes were dried at room temperature. The final pellet was dissolved in 20-40 µL ddH2 O or TE buffer and kept at –20 ◦C indefinitely. The Doyle & Doyle (1987) DNA extraction method was considered as the old, standard or control extraction method against which the new method was compared. ISSR-PCR amplification Inter simple sequence repeat (ISSR) analysis was carried out according to He et al. (2007). Each 25-µL reaction mixture consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 0.25 mM of mixed dNTPs, 0.25 µM primer UBC-841 (University of British Columbia; 5’-GAGAGAGAGAGAGAGAYC-3’), 4 units of Taq DNA polymerase (Beijing Sunbiotech Co., Ltd., Beijing, China) and about 50 ng of genomic DNA isolated using the new method described above. Amplification of the PCR was performed in a Peltier thermal cycler (PTC-200, MJ Research Inc., USA) under the following conditions: a denaturation step for 5 min at 94 ◦C followed by 40 cycles of 92 ◦C for 1 min, 47 ◦C for 1 min and 72 ◦C for 2 min, and then a final extension cycle at 72 ◦C for 10 min. 18S rRNA-PCR amplification 18S rRNA analysis was carried out using 14 cultivars including 8 commercial mango varieties, walnut, pear, sugarcane, grape, lychee and guava according to Roy et al. (2011) with some modifications. Each 25-µL reaction mixture consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 0.25 mM of mixed dNTPs, 0.25 µM primers (forward: 5’-CGGAGAATTAGGGTTCGATTC-3’, reverse: 5’CCAGAACATCTAAGGGCATCA-3’), 4 units of Taq DNA polymerase (Beijing Sunbiotech Co., Ltd., Beijing, China) and about 50 ng of genomic DNA isolated using the new method described above. Amplification was performed in a Peltier thermal cycler (PTC-200, MJ Research Inc., USA) under the following conditions: a denaturation step for 5 min at 94 ◦C followed by 40 cycles of 92 ◦C for 1 min, 55 ◦C for 1 min and 72 ◦C for 2 min, and then a final extension cycle at 72 ◦C for 10 min. Agarose gel electrophoresis The amplified products were separated on a 1.5% agarose gel in 1X Tris acetate-EDTA (TAE) buffer for electrophoresis at 100 V for 48 min and then visualized by staining the gel with 0.5 µg/mL of ethidium bromide. Banding was photographed using a Gel-doc system (Bio-Rad Laboratories, Hercules, USA) and stored as digital pictures at 600 dpi. The size of each fragment was estimated relative to a 100-bp Plus DNA ladder (TransGen Biotech., Beijing, China).
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Table 1. DNA yield and purity from leaf tissue of mango and other fruit tree. Mango variety / fruit tree Doyle & Doyle (1987) BARI Aam-2 BARI Aam-2 BARI Aam-2 BARI Aam-2 New developed method BARI Aam-2 BARI Aam-2 BARI Aam-2 BARI Aam-2 Walnut Pear Sugarcane Grape Lychee Guava
Leaf age; colour following DNA extraction
A260 /A280
µg/mL
7 days; dark brown 14 days; brown 21 days; light green 50–60 days; dark green
1.966 1.841 1.861 2.220
332.5 365.5 328.0 19.5
7 days; dark brown 14 days; brown 21 days; light green 50-60 days; dark green Mature Mature Mature Mature Mature Mature
1.820 1.786 1.850 1.842 1.765 1.725 1.902 1.782 1.830 1.841
968.0 1080.0 1018.0 646.50 1140.0 756.0 942.0. 860.0 1011.0 811.5
Fig. 1. Mango leaves of four different ages. (A) dark brown, soft, young, not fully developed, leaf age = 7 days; (B) brown, soft, young, developed, leaf age = 14 days; (C) light green, fully developed, thick, leaf age = 21 days; (D) dark green, fully developed, leaf age = 50–60 days.
Results In this study, mango leaves of four different ages were used to observe the quantity and quality of isolated DNA (Fig. 1). Older leaves yielded less DNA than younger leaves (Table 1). Clean, white and sometime colourless DNA pellets were obtained using the new DNA extraction protocol (Fig. 2). Yield was estimated by gel electrophoresis: the new method always produced brighter bands than the old method (Doyle & Doyle 1990) (Fig. 3) and was superior to it for a number of reasons (Table S1). The isolated DNA was essentially free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by its clear colour, viscosity, A260 /A280 ratio (consistently between 1.75 and
1.95; Table 1), suitability for ISSR-PCR amplification (Fig. 4), and 18S rRNA amplification (Fig. 5). Discussion Most of the protocols for DNA isolation from plant species containing high levels of phenolic compounds are a modified version of standard protocols (Doyle & Doyle 1990; Teixeira da Silva 2005; Teixeira da Silva & Tanaka 2006). Generally, changes have been made to the extraction buffer composition and the pH, and the isolation of nuclei has been added as a prior step (Couch 1990; Guillemaut 1992; Paterson 1993). Mature plant tissues are not preferred for DNA extraction because of the presence of high concentrations of polysaccharides,
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Fig. 2. (A) Brown-black DNA pellet using Doyle & Doyle (1987) method; (B) white DNA pellet following extraction using the new method developed in this paper.
Fig. 3. Relative yields of genomic DNA from mango and other fruit trees extracted according to Doyle & Doyle (1987) and the new method. One µL of genomic DNA from each sample was loaded on a 1% agarose gel and electrophoresed at 100 V for 40 min. Lanes 1, 2, 3, 4 show yields of DNA by CTAB method extracted from different age leaf (7, 14, 21, 50-60 days) of BARI Aam-2. Bands of lane 5, 6, 7, 8 show DNA isolated by the new method from BARI Aam-2 from the same leaf age as before. Lanes 9, 10, 11, 12, 13, 14 represent yield of genomic DNA extracted by the new method using only mature leaf from walnut, pear, sugarcane, grape, lychee and guava.
polyphenols, and other secondary metabolites (Dabo et al. 1993; Zhang & McDonald 2000). In mango, this problem is exacerbated when using fully expanded mature leaves (Azmat et al. 2012). In our new protocol, in the first step, 0.4 M glucose was used, which improved DNA quality, avoiding contamination and browning by polyphenolics, since PVP binds phenolic compounds and β-mercaptoethanol was used as an antioxidant. The addition of glucose avoids contamination and browning by polyphenolics, improving DNA quality (Permingeat et al. 1998). Mango leaf DNA was previously extracted by Yamanaka et al. (2006) using the Doyle & Doyle (1990) method but a DNA purification kit (Mag-Extractor genome kit) was needed to obtain pure and workable DNA, thus making the protocol costly. On the other hand, the DNA extrac-
Fig. 4. ISSR-PCR assay using DNA extracted by the new method as template for amplification. Five µL of amplification products of each mango variety were loaded on a 1.5% agarose gel, subjected to electrophoresis at 100 V for 48 min and stained with ethidium bromide. Lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 represent BARI Aam-1, 2, 3, 4, 5, 6, 7, 8, walnut, pear, sugarcane, grape, lychee and guava, respectively. Each variety was successfully amplified at different length by ISSR marker.
tion method used for young and fresh mango leaves by Kashkush et al. (2001) and Ukoskit (2007) was not suitable for this study since polyphenolic-free DNA samples could not be obtained. Puchooa (2004) reported that young leaves of lychee tend to have less plant metabolites, while mature leaves tend to contain a higher amount of polyphenolic compounds. In this study, both young and mature leaves were used to obtain good quality DNA for cv. ‘BARI Aam-2’, although the quantity of DNA obtained varied among cultivars (Table S2). Thus, PVP and β-mercaptoethanol were added to the DNA isolation buffer as they were previously reported to effectively remove polyphenols from mature, damaged or improperly stored leaf tissues (Doyle & Doyle 1987; Howland et al. 1991; Dawson & Magee 1995; Clark 1997; Zidani et al. 2005). According to John (1992), PVP forms complex hydrogen bonds with polyphenolic compounds which can be separated from DNA by
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References
Fig. 5. 18SrRNA-PCR assay using DNA isolated by the new method as template. Three µL of amplification products of each mango variety were loaded on a 1% agarose gel and electrophoresed at 100 V for 40 min. Desired bands were at 1100 bp. Lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 represent BARI Aam-1, 2, 3, 4, 5, 6, 7, 8, walnut, pear, sugarcane, grape, lychee and guava, respectively.
centrifugation. The midrib and secondary veins were removed prior to DNA extraction in order to ease leaf pulverization. Polysaccharides inhibit PCR amplifications and can lead to erroneous interpretations (Kotchoni et al. 2003). The co-precipitation of polysaccharides was avoided by adding higher concentrations of selective precipitants of nucleic acid, CTAB (0.04 g/mL) and NaCl (3 M) (Dellaporta et al. 1983); the latter was used at 1.4 M in our new method. Long-tail surfactants such as CTAB produce a conformational change in the DNA from a “random coil” to a “compact globule” making DNA precipitation more effective (Azmat et al. 2012). Phenolic compounds are powerful oxidizing agents and bind covalently to the extracted DNA, making it useless for most molecular manipulations (Porebski et al. 1997; Padmalatha & Prasad 2006). A high concentration (0.02 g/mL) of PVP mixed in the extraction buffer (Fang et al. 1992; Moller et al. 1992; Lodhi et al. 1995) binds to phenolic compounds and helps to remove them. In our protocol, superfluous cellular proteins were removed by a triple extraction with chloroform-isoamyl alcohol, which also helped to remove different colouring substances, such as chlorophyll, pigments and dyes (Azmat et al. 2012). He et al. (2007) used RNaseA in 0.1X TE during DNA extraction from mango leaves. DNA isolated by our new method yielded reproducible and consistent amplification among 14 mango cultivars (Table S2) proving its compatibility for PCR-based reactions for a wide range of mango germplasm (Figs S1, S2, S3).
Acknowledgements The authors gratefully acknowledge the financial support by the National Agricultural Technology Project (NATP), PIU-BARC, Phase-1, IDA Credit 4386 Bangladesh.
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Supplementary material Table S1. Comparison between the old and new method. Items
New method
Old method (Doyle & Doyle 1987)
Leaf Time of application DNA yield Applicable Pellet color Use of glucose During transportation Sealed poly bag Sophisticated box
Both young and mature Year round High Wide range of applications White 0.4 M Ice box, liquid nitrogen not used Needed Not used
Young and fresh When new leaves are available Low Not applicable Brown to black Not used Essential Not used Essential
Table S2. DNA yield and purity from leaf tissue of mango cultivars. Cultivars
Leaf age (days), colour
Old method (Doyle & Doyle 1987)
New method
A260/A280
µg/mL
A260/A280
µg/mL
BARI Aam-1
7, dark brown 14, brown 21, light green 50-60, dark green
1.920 1.864 1.960 2.010
266.8 316.2 288.3 16.90
1.806 1.740 1.842 1.851
930.5 955.0 896.6 671.4
BARI Aam-2
7, dark brown 14, brown 21, light green 50-60, dark green
1.966 1.841 1.861 2.220
332.5 365.5 328.0 19.5
1.820 1.786 1.850 1.842
968.0 1080.0 1018.0 646.0
BARI Aam-3
7, dark brown 14, brown 21, light green 50-60, dark green
1.870 1.893 1.960 2.250
303.2 335.8 346.2 22.5
1.75 1.77 1.80 1.83
1023.0 928.2 888.7 678.5
BARI Aam-4
7, dark brown 14, brown 21, light green 50-60, dark green
1.88 1.96 1.98 2.14
272.8 302.0 291.2 16.5
1.78 1.77 1.81 1.84
913.0 1011.0 972.5 700.3
BARI Aam-5
7, dark brown 14, brown 21, light green 50-60, dark green
1.94 1.91 1.97 2.04
318.5 330.0 295.0 24.6
1.75 1.74 1.76 1.81
1026.2 968.5 912.3 627.2
BARI Aam-6
7, dark brown 14, brown 21, light green 50-60, dark green
1.91 1.85 1.96 1.99
332.6 286.2 276.8 20.5
1.76 1.81 1.85 1.81
842.1 965.3 970.5 725.1
BARI Aam-7
7, dark brown 14, brown
1.85 1.92
311.5 286.0
1.77 1.75
942.2 988.8
21, light green 50-60, dark green
1.96 2.20
291.1 23.9
1.81 1.85
846.2 648.6
BARI Aam-8
7, dark brown 14, brown 21, light green 50-60, dark green
1.87 1.96 1.92 2.08
352.0 316.5 298.1 19.9
1.74 1.78 1.81 1.86
1041.1 912.5 846.6 616.4
Gopalbhog
7, dark brown 14, brown 21, light green 50-60, dark green
1.85 1.91 1.96 1.99
332.8 301.2 210.9 23.8
1.72 1.81 1.75 1.83
1011.2 988.7 970.2 652.9
Khirsapat
7, dark brown 14, brown 21, light green 50-60, dark green
1.90 1.88 1.97 2.30
342.1 300.1 272.7 24.6
1.75 1.81 1.83 1.79
980.2 1032.0 911.8 612.9
Langra
7, dark brown 14, brown 21, light green 50-60, dark green
1.88 1.92 1.86 1.98
347.0 293.8 272.1 16.5
1.78 1.75 1.81 1.83
911.4 972.2 928.6 627.1
Ashwina
7, dark brown 14, brown 21, light green 50-60, dark green
1.92 1.88 1.96 1.99
298.6 275.1 260.8 22.8
1.73 1.78 1.81 1.84
1008.4 960.8 912.1 647.8
Mollica
7, dark brown 14, brown 21, light green 50-60, dark green
1.85 1.91 1.93 2.14
358.0 312.4 286.1 23.1
1.76 1.78 1.81 1.83
991.4 978.1 904.1 601.6
Fazli
7, dark brown 14, brown 21, light green 50-60, dark green
1.92 1.86 1.96 2.22
282.6 270.1 212.0 14.6
1.79 1.75 1.81 1.84
846.6 912.8 888.1 652.0
Fazli
Mollica
Ashwina
Langra
Khirsapat
Gopalbhog
BARI Aam-8
BARI Aam-7
BARI Aam-6
BARI Aam-5
BARI Aam-4
BARI Aam-3
BARI Aam-2
BARI Aam-1
Marker 5000 bp 3000 bp
2000 bp 1500 bp 1000 bp 900 bp 800 bp 700 bp 600 bp 500 bp 400 bp 300 bp 200 bp 100 bp
Fig. S1. ISSR-PCR assay using DNA extracted by the new method as template for amplification. Five µL of amplification products of each mango variety were loaded on a 1.5% agarose gel, subjected to electrophoresis at 100 V for 48 min and stained with ethidium bromide. Lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 represent mango cultivars BARI Aam-1, 2, 3, 4, 5, 6, 7, 8, Gopalbhog, Khirsapat, Langra, Ashwina, Mollica and Fazli, respectively.
Fig. S2. 18SrRNA-PCR amplification using DNA extracted by the new method as template. Five µL of amplification products of each mango variety were loaded on a 1.5% agarose gel, subjected to electrophoresis at 100 V for 48 min and stained with ethidium bromide. Lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 represent mango cultivars BARI Aam-1, 2, 3, 4, 5, 6, 7, 8, Gopalbhog, Khirsapat, Langra, Ashwina, Mollica and Fazli, respectively.
Fig. S3. Recombinase aid-amplification (RAA) profile using DNA extracted by the new method as template. Five µL of amplification products of each species were loaded on a 1.5% agarose gel, subjected to electrophoresis at 100 V for 48 min and stained with ethidium bromide. A, B, C, D, E, F and G represent mango, walnut, pear, lychee, guava, grape and sugarcane, respectively. Lanes 1, 2, 3 and 4 represent, respectively, 28, 24, 20 and 18 base-pairs primer length amplification.
