Plant material
Fresh, young and tender leaves of five different species of the order Zingiberales (Kaempferia galanga, Kaempferia marginata, Zingiber officinale, Zingiber zerumbet, Musa sp.) which contain high amounts of polysaccharides and polyphenols were collected and wiped with 70% ethanol. For PCR based detection of virus, leaves of banana plant infected with two viruses associated with Musa sp. i.e. Banana bunchy top virus (BBTV) and Banana streak virus (BSV) were collected. A minimum of ten replicates were taken for each species.
Reagents
100 mM Tris–HCl (pH 8),
1.4 M Sodium Chloride (NaCl),
20 mM-Ethylenediaminetetraacetic acid (EDTA) (pH 8),
2% (w/v) Cetyl trimethylammonium bromide (CTAB)
Chloroform-isoamyl alcohol (24:1)
Isopropanol, 70% ethanol
TE buffer (pH 8): 10 mM Tris–HCl, 1 mM EDTA
0.5× Tris/Borate/EDTA (TBE) (10× stock contained 1 M Tris, 0.8 M boric acid, 0.5 M EDTA)
Agarose (molecular grade)
DNA extraction protocol
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1.
Preheat the extraction buffer containing 100 mM Tris–HCl (pH 8), 1.4 M NaCl, 20 mM
EDTA (pH 8), 2% (w/v) CTAB in water bath at 60°C for about 15 minutes.
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2.
Submerge 1 g of plant tissue in 5 ml of absolute alcohol for 5 minutes and allow alcohol to evaporate.
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3.
Grind the tissue in presence of 1% PVP (Polyvinylpyrrolidone) and pre-warmed extraction buffer by using a pre-chilled mortar and pestle (-40°C/-80°C) at room temperature.
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4.
Transfer the ground material into 2 ml centrifuge tubes and incubate in water bath at 60°C for 1 hour.
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5.
Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant in 1.5 ml centrifuge tube using wide bored tip.
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6.
To the supernatant add equal volume of chloroform: isoamyl alcohol (24:1) and mix by inversion for 15 minutes.
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7.
Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant in 1.5 ml centrifuge tube.
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8.
Again add equal volume of chloroform: isoamyl alcohol (24:1) to the supernatant and mix by inversion for 15 minutes.
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9.
Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the supernatant.
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10.
To the supernatant add twice the volume of chilled isopropanol to precipitate the DNA and incubate it at -20°C for 30 minutes.
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11.
Centrifuge the tubes at 10,000 rpm for 10 minutes at 4°C and collect the pellet.
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12.
Wash the pellet 2–3 times with 70% ethanol and air dry the pellet in room temperature.
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13.
Add 50–100 μl of TE buffer to dissolve the DNA.
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14.
Store at -20°C for further use.
Quantification and visualization of DNA
DNA was quantified by measuring optical density (O.D.) at A260 and A280 with a Nanodrop Spectrophotometer (ND2000). Samples were subjected to electrophoresis in 1× TBE buffer for 1 hour at 80 V. 5 μl of the isolated genomic DNA was loaded on 0.8% agarose gel stained with ethidium bromide to check DNA quality. The gels were photographed under a Gel Documentation system (Perkin Elmer Geliance 200).
RAPD and ISSR study
PCR amplification of ten replicates of genomic DNA of Zingiber zerumbet and Zingiber officinale was carried out using RAPD and ISSR primers respectively which were synthesized by Sigma Aldrich Chemicals Pvt. Ltd., India as per the sequence of Operon Technologies, Inc., USA. PCR amplifications were performed routinely using the following PCR reaction mixture: 25 μl contained 50 ng of template DNA, 1× PCR buffer, 1.5 mM of magnesium chloride (MgCl2) 200 μM of deoxynucleotide triphosphates (dNTPs), 10 picomol of each primer, and 1 U of Taq polymerase. PCR amplification was carried out in a thermal Cycler (Eppendorf Mastercycler Pro S). Thermal cycling conditions were as follows: initial denaturation step for 5 min at 94°C, followed by 35 cycles each of 1 min at 94°C (denaturation), 1 min at 37°C (annealing), 2 min at 72°C (extension) followed by one final extension of 7 min at 72°C. The annealing temperatures of ISSR primers were different for each primer depending upon the melting temperature. The amplification products were electrophoresed in 1.8% agarose gels in 0.5× TBE (10× stock contained 1 M Tris, 0.8 M boric acid, 0.5 M EDTA) and stained with ethidium bromide (0.5 μg/ml). The gels were photographed under a Gel Documentation system (Perkin Elmer Geliance 200).
ITS and trnL-F gene amplification
Genomic DNA of Kaempferia galanga and Kaempferia marginata isolated by the present method was used as template for nuclear and chloroplast gene amplification. Ten replicates of Kaempferia galanga DNA was amplified with primers ITS1 (5′-TCCGATGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Ten replicates of using Kaempferia marginata DNA was amplified with primers trnL-Fc (5′-GAAATCGGTAGACGCTACG-3′) and trnL-Ff (5′-ATTTGAACTGGTGACACGAG-3′). The PCR reaction mixture composition was same as that used for RAPD analysis. The reaction mixture of 25 μl contained 50 ng of template DNA, 1× PCR buffer, 1.5 mM of MgCl2, 200 μM of dNTPs, 0.25 μM of each primer, and 1 U of Taq polymerase. PCR amplification was carried out in a thermal Cycler (Eppendorf Mastercycler pro S). Thermal cycling conditions were as follows: initial denaturation step for 5 min at 94°C, followed by 35 cycles each of 1 min at 94°C (denaturation), 1 min at 59.3°C for ITS1 and ITS4 and 61.8°C for trnL-Fc and trnL-Ff (annealing), 2 min at 72°C (extension) followed by one final extension of 7 min at 72°C. The amplification products were electrophoresed in 1.8% agarose gels in 0.5× TBE (10× stock contained 1 M Tris, 0.8 M boric acid, 0.5 M EDTA) and stained with ethidium bromide (0.5 μg/ml). The gels were photographed under a Gel Documentation system (Perkin Elmer Geliance 200).
Virus DNA detection
BBTV and BSV were detected by PCR using specific primers of BBTV: BTVCPF (forward primer) 5′- GCTAGGTATCCGAAGAAATC-3′, BTVCPR (reverse primer) 5′- TCAAACATGATATGTAATTC-3′ (Burns et al. 1995) and BSV: Mys-F1 5′- TAAAAGCACAGCTCAGAACAAACC-3′, Mys R1 5′-CTCCGTGATTTCTTCGTGGTC-3′ (Geering et al. 2000). Virus DNA amplifications were performed routinely using PCR reaction mixture of 25 μl containing 100 ng of template DNA, 1× PCR buffer, 1.5 m MgCl2, 0.2 mM of each dNTPs, 50 ng forward primer, 50 ng reverse primer and 1 U of Taq polymerase. PCR amplification was carried out in a thermal cycler (Eppendorf Mastercycler Pro S). Thermal cycling conditions were as follows: BBTVCPF- 94°C for 3 min, then subjected to 35 cycles of 94°C for 45 s, 50°C for 45 s, and 72°C for 1 min; and finally 1 cycle of 72°C for 10 min and for BSV Mys F1and R1- 94° 4 min, 50° 1 min; 72° 2 min for 1 cycle, then subjected to 30 cycles 94° 1 min; 50° 1 min; 72° 2 min and finally 1 cycle of 72° 10 min. Amplification products were electrophoresed in 1.8% agarose gels in 0.5× TBE (10× stock contained 1 M Tris, 0.8 M boric acid, 0.5 M EDTA) and stained with ethidium bromide(0.5 μg/ml). The gels were photographed under a Gel Documentation system (Perkin Elmer Geliance 200).