Abstract

Because DNA degradation is mediated by secondary plant products such as phenolic terpenoids, the isolation of high quality DNA from plants containing a high content of polyphenolics has been a difficult problem. We demonstrate an easy extraction process by modifying several existing ones. Using this process we have found it possible to isolate DNAs from four fruit trees, grape ( Vitis spp.), apple ( Malus spp.), pear ( Pyrus spp.) and persimmon ( Diospyros spp.) and four species of conifer, Pinus densiflora, Pinus koraiensis, Taxus cuspidata and Juniperus chinensis within a few hours. Compared with the existing method, we have isolated high quality intact DNAs (260/280 = 1.8–2.0) routinely yielding 250–500 ng/µl (total 7.5–15 µg DNA from four to five tissue discs).

Many studies require isolation of genomic DNA from various kinds of plant species. Because DNA degradation is mediated by secondary plant products such as phenolic terpenoids which may bind to DNA after cell lysis (1), the isolation of high quality DNA from plants containing a high content of polyphenolics such as grape ( Vitis spp.), apple ( Malus spp.), pear ( Pyrus spp.) and conifers was a difficult problem. Although several rapid methods exist for genomic DNA extraction from various plant tissues, there is no simple method for obtaining large quantities of DNA from plants containing a high content of polyphenolics. We demonstrate an easy extraction process by modifying several existing ones (1–4). The process minimizes manipulations of the samples, and requires no ultracentrifugation to purify DNA separated from the polyphenolics, but optimizises the yield of DNA extracted from a sample. Micrograms to milligrams of DNA from each tissue sample can be easily isolated and used directly for both PCR amplification and restriction analyses.

Our rapid extraction process consists of three parts: sample preparation for extraction, precipitation using both polyvinylpyrrolidone (PVP) and salt, and chloroform purification. In this method, fresh young leaf tissues are collected using the lid of a sterile 1.5 ml microcentrifuge tube to punch out four to five discs of material into the tube. This ensures uniform sample size and also reduces possible contamination arising from mishandling of the tissues. Leaf material is ground in a 1.5 ml tube with a pestle in 5 µl (one drop) of 1% (v/v) 2-mercaptoethanol. After grinding, 300 µl of extraction buffer (250 mM NaCl, 25 mM EDTA, 0.5% SDS, 200 mM Tris-HCl pH 8.0) is added to the homogenate, and the tube is flicked at the bottom occasionally to keep the extract mixed. The homogenate is incubated at room temperature for 1 h. Freshly prepared PVP (soluble PVP, Sigma, MW 10 000) (6% of final volume) and one half volume of 7.5 M ammonium acetate are added separately. The mixture is incubated on ice for 30 min and centrifuged for 10 min in a microcentrifuge (10 000 g at 4°C). The supernatant is transferred to a fresh tube to which is added 1 vol isopropanol, and left at−20°C for 30 min to precipitate the DNA. After centrifugation at 10 000 g for 10 min, the supernatant is discarded and the DNA pellet is vacuum-dried. The DNA pellet is resuspended in 500 µl TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA pH 8.0) or distilled water. Two microliters of RNase (1 mg/ml) was added to the solution and incubated at 37°C for 15 min. One vol chloroform-isoamyl alcohol (24:1) is added and emulsified by inverted shaking to remove both RNase and plant pigments. The procedure is repeated once again. After centrifugation (10 000 g at 4°C) for 5 min, the supernatant is transferred to a fresh tube to which 1 vol of isopropanol is added and left at −20°C for 10 min. After centrifugation at 10 000 g for 10 min, the pellet is washed with 1 ml 80% ethanol and vacuum-dried. The DNA pellet is redissolved in 30 µl TE or distilled water. DNA obtained with this technique constantly gives a 260/280 absorbance ratio of 1.8–2.0 indicating high quality intact DNA. This DNA can be stored for months at 4°C. Using this process we have found it possible to isolate DNAs from four different kinds of fruit tree samples, grape ( Vitis spp.), apple ( Malus spp.), pear ( Pyrus spp.) and persimmon ( Diospyros spp.) ( Fig. 1 ), and four species of conifers, Japanese red pine ( Pinus densiflora ), Korean pine ( Pinus koraiensis ), Japanese yew ( Taxus cuspidata ) and Chinese juniper ( Juniperus chinensis ) ( Fig. 2 ) within a few hours.

Initially, we isolated DNA using a modification of the widely used procedure (2,3) or rapid genomic DNA extraction process using PVPP (1,4), but found that these methods could not remove polyphenolics from the tree tissues, and that very little DNA was extracted. Compared with the existing method, we have isolated DNAs from grape, pear, apple and persimmon routinely yielding 250–500 ng/µl (total 7.5–15 µg DNA from four to five tissue discs).

All of these DNAs were used for RAPD-PCR analysis using the method previously published by us (5). Arbitrary decamer primers successfully amplified DNA fragments from both those four fruit tree DNAs ( Fig. 3 ) and four different conifer DNAs ( Fig. 4 ). The DNA samples of 32 individual grape cultivars were extracted using this procedure and assayed for rApD-PCR using 15 primers. We found that 40 reliable RAPD markers selected could be used to calculate both the dissimilarity value and the marker difference that were used to reconstruct the genetic relationships (manuscript in preparation). Figure 5 shows the results of PCR amplification using UBC primer #389 of 13 grape cultivars. This DNA preparation method yielded a predominance of high molecular weight DNA reliably produced PCR amplification products >500 bp in length. Considering all factors involved, this method appears to be the most efficient, reliable and labor-effective DNA isolation procedure from plants containing a high content of polyphenolics such as fruit trees and conifers.

Figure 1

Total DNA prepared from apple, grape, pear and persimmon plants. M, DNA size marker (1 kb DNA ladder, GIBCO-BRL). Lanes 1–4: 1, apple (cv. Fuji); 2, grape (cv. Campbell Early); 3, pear (cv. Nijisseiki); 4, persimmon (cv. Fuju).

Figure 1

Total DNA prepared from apple, grape, pear and persimmon plants. M, DNA size marker (1 kb DNA ladder, GIBCO-BRL). Lanes 1–4: 1, apple (cv. Fuji); 2, grape (cv. Campbell Early); 3, pear (cv. Nijisseiki); 4, persimmon (cv. Fuju).

Figure 2

Total DNA prepared from four different species of conifers. M, DNA size marker (1 kb DNA ladder, GIBCO-BRL). Lanes 1–4: 1, Japanese red pine apple ( P. densiflora ); 2, Korean pine ( P.koraiensis ); 3, Japanese yew ( T.cuspida-ta ); 4, Chinese juniper ( J.chinensis ).

Figure 2

Total DNA prepared from four different species of conifers. M, DNA size marker (1 kb DNA ladder, GIBCO-BRL). Lanes 1–4: 1, Japanese red pine apple ( P. densiflora ); 2, Korean pine ( P.koraiensis ); 3, Japanese yew ( T.cuspida-ta ); 4, Chinese juniper ( J.chinensis ).

Figure 3

RAPD profiles of apple, grape, pear and persimmon DNAs. All reactions had a final volume of 14 µl and contained 1 ng template DNA; 200 µM each of dATP, dCTP, dGTP and dTTP; 0.27 µM primer; 0.028 U Taq DNA polymerase (Promega); 1.5 mM MgCl 2 and 1× reaction buffer (10 mM Tris-HCl pH 8.8, 50 mM KCl and 0.1% Triton X-100). Each reaction mix was overlaid with 14 µl mineral oil. Samples for enzymatic amplification were subjected to 45 cycles of the following thermal profile: 1 min at 94°C, 2 min at 37°C and 3 min at 72 Samples were predenatured for 4 min at 94°C and final extension was for 7 min at 72°C. Amplification fragments generated by PCR in a water-bath thermal cycler (FINEPCR, Korea) were separated according to size on 1.2% agarose gels, stained with ethidium bromide. The oligonucleotide primer was 5′-TCCCGAACCG-3′ (UBC #348). M, 1 kb DNA ladder (GIBCO-BRL). Lanes 1–4: 1, apple (cv. Fuji); 2, grape (cv. Campbell Early); 3, pear (cv. Nijisseiki); 4, persimmon (cv. Fuju).

Figure 3

RAPD profiles of apple, grape, pear and persimmon DNAs. All reactions had a final volume of 14 µl and contained 1 ng template DNA; 200 µM each of dATP, dCTP, dGTP and dTTP; 0.27 µM primer; 0.028 U Taq DNA polymerase (Promega); 1.5 mM MgCl 2 and 1× reaction buffer (10 mM Tris-HCl pH 8.8, 50 mM KCl and 0.1% Triton X-100). Each reaction mix was overlaid with 14 µl mineral oil. Samples for enzymatic amplification were subjected to 45 cycles of the following thermal profile: 1 min at 94°C, 2 min at 37°C and 3 min at 72 Samples were predenatured for 4 min at 94°C and final extension was for 7 min at 72°C. Amplification fragments generated by PCR in a water-bath thermal cycler (FINEPCR, Korea) were separated according to size on 1.2% agarose gels, stained with ethidium bromide. The oligonucleotide primer was 5′-TCCCGAACCG-3′ (UBC #348). M, 1 kb DNA ladder (GIBCO-BRL). Lanes 1–4: 1, apple (cv. Fuji); 2, grape (cv. Campbell Early); 3, pear (cv. Nijisseiki); 4, persimmon (cv. Fuju).

Figure 4

RAPD profiles of four different conifers. PCR reaction is performed using the described protocol in Figure 2. DNAs were amplified using primer 5′-CGCCCGCAGT-3′ (UBC #389). M, 1 kb DNA ladder (GIBCO-BRL). Lanes 1–4: 1, Japanese red pine apple ( P.densiflora ); 2, Korean pine ( P.koraiensis ); 3, Japanese yew ( T.cuspidata ); 4, Chinese juniper ( J.chinensis ).

Figure 4

RAPD profiles of four different conifers. PCR reaction is performed using the described protocol in Figure 2. DNAs were amplified using primer 5′-CGCCCGCAGT-3′ (UBC #389). M, 1 kb DNA ladder (GIBCO-BRL). Lanes 1–4: 1, Japanese red pine apple ( P.densiflora ); 2, Korean pine ( P.koraiensis ); 3, Japanese yew ( T.cuspidata ); 4, Chinese juniper ( J.chinensis ).

Figure 5

Amplification of 13 grape DNAs using the descibed protocol in Figure 2. DNAs were amplified using primer 5′-CGCCCGCAGT-3′ (UBC #389). M, 1 kb DNA ladder (GIBCO-BRL); lanes 1–13, 13 different grape cultivars.

Figure 5

Amplification of 13 grape DNAs using the descibed protocol in Figure 2. DNAs were amplified using primer 5′-CGCCCGCAGT-3′ (UBC #389). M, 1 kb DNA ladder (GIBCO-BRL); lanes 1–13, 13 different grape cultivars.

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