Comparative Analysis of two DNA Extraction Protocols from Fresh and Dried wood of Cunninghamia

IAWA Journal, V ol. 33 (4), 2012: 441–456

COMPARATIVE ANALYSIS OF TWO DNA EXTRACTION

PROTOCOLS FROM FRESH AND DRIED WOOD OF

CUNNINGHAMIA LANCEOLATA (TAXODIACEAE) Lichao Jiao1, Yafang Yin1,*, Fuming Xiao2, Qingpeng Sun3, Kunlin Song1 and

Xiaomei Jiang1

SUMMARY

DNA was isolated from the sapwood, transition wood and heartwood of

fresh and dried Cunninghamia lanceolata wood using two DNA extrac-

tion protocols: the modified CTAB method and the modified Qiagen

kit. Our major objective was to (i) determine an optimized method for

retrieving good quality and sufficient quantity of DNA from wood, and

to (ii) investigate the effect of different radial positions of fresh and dried

wood for DNA extraction. In comparison with the modified CTAB

method, a greater quantity of higher quality DNA – both chloroplast and

nuclear ribosomal DNA – was retrieved using the Qiagen kit protocol. The

chloroplast DNA regions retrieved from both fresh and dried wood were

successfully amplified using both protocols, but the PCR amplification for

the rDNA-ITS region from the heartwood failed using both protocols. The

quantity and purity of the DNA from the sapwood and transition wood

(derived from nuclei and plastids in the parenchyma cells) was greater

than that from the heartwood (derived mainly from amyloplasts). Due

to the influence of the drying treatment, the quantity of DNA decreased

by more than 50%. The optimized radial position for DNA extraction in

the stem was demonstrated based on anatomical observation.

Key words: Wood identification, DNA extraction protocols, comparison,

dried wood, chloroplasts, nuclei, amyloplasts, Cunninghamia lanceo-

lata.

INTRODUCTION

Illegal logging, excessive logging and trading in illegal timber are causing deforestation in natural forests, along with a great deal of ecological and economic damage. Wood identification is therefore so crucial and significant that it is a technical prerequisite for monitoring the chain of wood certification (Jiang et al. 2010; Jiao et al. 2012). It is

1) Wood Anatomy and Utilization Department, Chinese Research Institute of Wood Industry, Chinese Academy of Forestry, No. 1 Dongxiaofu, Beijing, CN 100091, China.

2) Jiangxi Academy of Forestry, No. 1629 Fenglin Street, Nanchang, Jiangxi Province, CN 330032, China.

3) College of Biotechnology, Beijing University of Agriculture, No.7 Beinong Road, Beijing, CN 102206, China.

*) Corresponding author [E-mail: yafang@https://www.wendangku.net/doc/7f18131686.html,].

Associate Editor: Frederic Lens

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often difficult or even impossible for traditional wood identification methods based on anatomical sectioning to identify timbers on a species level alone. To overcome such limitations, DNA methods may significantly improve the identification process of an unknown wood sample (Deguilloux et al. 2002; Lowe & Cross 2011).

DNA extraction from fresh materials (leaves or buds) is a matter of routine in mo-lecular biology. However, DNA extraction from harvested wood tissues becomes more problematic and complicated, especially when it comes to dried wood. Firstly, only small amounts of DNA are present in wood tissues, even in living trees (Abe et al. 2011). The cross-section of a tree consists of the bark, cambium, xylem and pith. Xylem is the most valuable part of a tree in the wood industry; it is generally composed of sapwood, transition wood and heartwood. In the sapwood, only parenchyma cells are living, while all the cells in the heartwood are dead (Bamber 1976; Deguilloux et al. 2002). Furthermore, previous reports state that the degeneration of DNA starts after the death of a cell, which results in the splitting of intact DNA into small fragments (Rachmayanti et al. 2009; Finkeldey et al. 2010). With the influence of storage time and drying treatment, wood DNA becomes more seriously degraded.

The usual methods of DNA extraction from wood are the Hexadecyl trimethyl am-monium Bromide (CTAB) protocol (White et al. 2000; Ohyama et al. 2001; Reynolds & Williams 2004; Fladung et al. 2004) and the DNeasy Plant Mini Kit protocol (Qiagen) (Dumolin-Lapègue et al. 1999; Deguilloux et al. 2002; Rachmayanti et al. 2006, 2009; Yoshida et al. 2007; Abe et al. 2011; Hanssen et al. 2011). However, there are few studies comparing the two different protocols for DNA extraction from wood (Asif & Cannon 2005; Tnah et al. 2012). Moreover, an effective DNA extraction protocol from wood has not yet been determined. Tnah et al. (2012) reported that a greater quantity of DNA extracts could be retrieved from wood using the Qiagen kit protocol, which yielded a higher PCR amplification success rate. Another study demonstrated that the CTAB and the Qiagen kit protocol retrieved only a small quantity of low quality DNA from wood, while the DNA extracts could not be amplified (Asif & Cannon 2005). Con-sequently, it is very important to determine which is the most efficient DNA extraction protocol from wood. Additionally, the factors influencing DNA extraction success rate from wood, including the radial position of wood in the stem and the drying treatment, have not been fully explored.

DNA extracts from wood are usually highly degraded, so choosing multiple copies of the target gene sequence in each cell is conducive to increasing the chances of a successful PCR amplification (Cano 1996; Deguilloux et al. 2002). Thus, compared to single-copied nuclear genes, multiple copies of chloroplast genes are advantageous for detecting degraded DNA in wood.

The chloroplast genome has a highly conserved sequence, a uniparental inheritance (usually maternal in angiosperms and paternal in gymnosperms) and no recombination (Tang et al. 2011). Moreover, the chloroplast gene for DNA barcoding is appropriate for the taxonomic identification of a species (Kress & Erickson 2007; Taberlet et al. 2007; Lahaye et al. 2008; H?ltken et al. 2012). In higher plants, plastids originate from proplastids in the cells of the meristem and they take on different forms, such as chloroplasts, chromoplasts, leucoplasts and amyloplasts, according to their cellular

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function, biochemistry and storage capacity. For trees, proplastids differentiate to chlo-roplasts in leaves and to amyloplasts or chromoplasts in other tissues (Pizzolato 1978; Deguilloux et al. 2002). Meanwhile, they almost retain the same genetic information, even after differentiation (Ohyama et al. 2001). In addition, the sequence divergence between nuclear ribosomal DNA internal transcribed spacer (rDNA-ITS) copies within individuals of one species is very low, whereas higher levels of divergence sequence are found between species (Hanssen et al. 2011). Thus rDNA-ITS region is often con-sidered as an excellent tool for taxonomic purposes (Szymanski et al. 1999). Recently, the rDNA-ITS region has been proposed to incorporate into the core DNA barcoding for seed plants identification (China Plant BOL Group 2011). Therefore, we have chosen chloroplast DNA (cpDNA) and rDNA-ITS gene regions for the PCR amplification of wood DNA in Cunninghamia lanceolata, an important timber crop.

The aims of this study are (i) to determine the optimized chloroplast and nuclear DNA extraction protocol by means of a comparison between the modified CTAB method and the Qiagen kit protocol and (ii) to explore how the radial positions in wood and the drying procedure affect the efficiency of DNA extraction. Chinese fir [Cunninghamia lanceolata (Lamb.) Hook.], one of the major commercial plantation species distributed within 17 provinces with the largest planting area in China, was selected for this study. In addition, the distribution of nuclei and amyloplasts containing DNA in the different radial positions of fresh and dried wood were investigated, using microscopy to provide a better understanding of the quantity of DNA extraction from wood materials.

MATERIALS AND METHODS

Sample preparation

Two 36-year-old trees of Chinese fir [Cunninghamia lanceolata (Lamb.) Hook.] (average diameter at breast height: 24.2 cm) were collected randomly from the Chenshan Forestry Centre in Ji’an, in the Jiangxi Province, China. Wood discs with a thickness of 50 mm were cut at a level of 2 m from the ground. Two discs were then immediately placed into an ice storage box. The other two discs were instantly put into a plastic bag in situ. Meanwhile, small wood blocks were cut using a razor blade and immersed immediately in a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M of phosphate buffer (pH 7.2).

After returning to the laboratory, the discs that had been placed in the ice storage box were transferred at once to a -20°C cryogenic freezer to maintain their freshness. The other two discs were preserved at room temperature for about 3 months after drying in an oven for 5 h at 70°C, in order to simulate wood-drying condition.

Before DNA extraction, the surface of the wood tissues and the cutter were washed in diluted bleach to avoid contamination. Wood specimens were divided into 3 sectors according to the different radial positions of the wood, e.g. sapwood, transition wood and heartwood. Wood sections were then sawn into dimensions of 10 (L) × 10 (R) × 20 (T) mm from the wood sectors, in accordance with the annual ring (Table 1). Slices of an approx. 3–5 μm thickness were prepared from each block using a sliding microtome (TU-213, Japan) to produce small wood shavings. The slices or wood shavings were

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444then rapidly ground into fine powder using a mortar and pestle in liquid nitrogen. These wood powders were placed in a 1.5 ml microcentrifuge tube and stored in a -80°C cryogenic freezer before DNA analysis took place.

DNA isolation

All the DNA isolations were carried out under sterile conditions in order to prevent external environmental contamination. Two DNA extraction protocols, viz. the modi -fied CTAB method (Murray & Thompson 1980) and the modified Qiagen kit protocol (Rachmayanti et al . 2006), respectively, were carried out in the study. For both fresh and dried wood samples, five DNA extractions were performed at each radial position. In addition, DNA extracts from leaves were used as positive control. And a negative control was also tested in each extraction, which was carried out using the same treat-ment as for the normal samples except that no wood powders were added.

Modified CTAB method Solutions

CTAB extraction buffer: 2% (w/v) CTAB, 5% (w/v) PVP, 1.4 mM NaCl, 20 mM EDTA, 100 mM Tris-HCl (PH 8.0), 2% (v/v) β-mercaptoethanol.Protocol

Incubate CTAB extraction buffer for 10 min at 65°C. And add 750 μl of CTAB ex -traction buffer to the 1.5 ml microcentrifuge tube with approximately 100 mg of wood powder and mix them thoroughly. The mixture is incubated for more than 5 h under occasional swirling in order to release the DNA as much as possible. The wood powder has a high absorption capacity, just like a sponge, so the step of mixing thoroughly is of great significance. After mixing cool for 2 min, add equal volume of chloroform-isoamylalcohol (24:1) and homogenize with a Vertical mixer (HS-3) at 60 r/min for 10 min, then centrifuge at 10,000 rpm for 10 min and transfer the aqueous phase carefully to a new 1.5 ml microcentrifuge tube. Repeat the step again. Add 4 μl of RNase A (10 mg/mL DNase free) to the supernatant and incubate for 30 min at 37°C. Subsequently, add 1.5 volumes of cold isopropanol to precipitate DNA, mix well and incubate for 1 h at -20°C. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. The pellet is washed by adding cold 75% ethanol. Repeat 2 to 3 times. Discard the washing buffer and dry the pellet at room temperature. Dissolve the pellet with 50 μl of double-distilled water and conserve DNA samples at -20°C in a freezer.

Modified DNeasy Plant Mini Kit protocol (Qiagen)

Genomic DNA of Cunninghamia lanceolata wood was extracted using the modified Qiagen kit protocol (Rachmayanti et al . 2006).

Table 1. Radial positions in a wood disc.

Sample position Rings of sapwood

Rings of transition wood

Rings of heartwood

Annual ring

28–32

19–20

13–15

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DNA quantity and quality assessment

The DNA extracts were loaded on 1% agarose gels, followed by ethidium bromide staining. Moreover, the quantity of the DNA was determined by means of measuring the absorbance at the wavelength of 260 nm in a UV spectrophotometer (TU-1901). The DNA purity was determined by calculating the ratio of the A260/A280 (Asif & Cannon 2005; Rachmayanti et al. 2006; Abe et al. 2011). In general, the ratio A260/ A280 of pure DNA is indicated as 1.8 (Sambrook & Russell 2001).

PCR amplification

PCR primers specific to chloroplast and nuclear genomes were used to examine the quality of DNA extracted from leaves and wood tissues. Two universal primer pairs that had been selected from the highly conserved regions of 21 tree chloroplast genomes (Tang et al. 2011) were tested on the DNA extracts. Their chloroplast DNA sequence regions were as follows: Intron in the ycf3 gene (forward primer: 5’-AGAAC-CGTACTTGAGAGTTTCC-3’; reverse primer: 5’-CTGTCATTACGTGCG(A/G) CTATCT-3’); non-coding region between the psb C gene and the trn S gene (for-ward primer: 5’-GCAGCTGCAGCAGGATTTG-3’; reverse primer: 5’-GGAGA-GATGGCCGAGTGGTT-3’). Moreover, the nuclear ribosomal DNA internal tran-scribed spacer (rDNA-ITS) partial region was PCR amplified with a forward primer (5’-GCCGTTAAGACCAGGGAT-3’, nucleotide positions 337 to 354) and a reverse primer (5’-TGATTCACGGGATTCTGC-3’, nucleotide positions 800 to 817). PCR amplifications were carried out using 20 μl of PCR reactions mixture, consist-ing of 1 unit of Taq DNA polymerase (TaKaRa), 1.5 mM of MgCl2, 200 μM of each dNTP (TaKaRa), 0.4 μM of each primer and approximately 20 ng of template DNA. The amplifications were carried out using the Mastercycler Personal (Eppendorf) for an initial denaturing step at 95°C for 5 min, 35 cycles at 94°C for 1 min, at an annealing temperature of 55°C (50°C for rDNA-ITS region) for 1 min and at 72°C for 1 min. This was followed by a final extension step for 10 min at 72°C. The PCR products were detected afterwards on 1% agarose gels. Meanwhile, the amplification products of leaves that were in the same amplification protocol as the wood samples were used as a positive control.

A total of 180 PCR amplifications on DNA extracted by two DNA extraction methods from three different radial positions in both fresh and dried wood were performed in this study (Table 2).

Light microscopy

Small wood blocks fixed in a mixture of 2% paraformaldehyde and 2.5% glutar-aldehyde in 0.1 M of phosphate buffer (pH 7.2) and dried wood samples were cut separately into small pieces (10 (R) × 10 (T) × 10 (L) mm) according to different radial positions. Thereafter, radial sections were cut into thicknesses of 20 μm on a freezing microtome (LEICA, CM3050S) at -20°C. They were then observed under a light microscope (Olympus, BX61) after being stained with a 1% aqueous solution of acetocarmine for nuclei investigation. Moreover, radial sections were stained with a 1% aqueous solution of iodine-potassium iodide for observing starch grains in order to investigate the distribution of amyloplasts (Nakaba et al. 2006).

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Table 2. The number of PCR amplifications for DNA from different radial positions in fresh and dried wood, using the modified CTAB method and the Qiagen kit protocol (number three indicates three pairs of PCR primers containing two pairs for chloroplast DNA and one pair for nuclear ribosomal DNA, and number five indicates five repeated samples of DNA extracts).

sapwood transition wood heartwood Total

CTAB Fresh wood 3 × 5 3 × 5 3 × 5 45

Dried wood 3 × 5 3 × 5 3 × 5 45

Qiagen kit Fresh wood 3 × 5 3 × 5 3 × 5 45

Dried wood 3 × 5 3 × 5 3 × 5 45

Total 180

Table 3. Analysis of variance in the quantity of DNA (ng/mg) extracted from different radial positions in fresh and dried wood, using the modified CTAB method and the Qiagen kit protocol.

For both fresh and dried wood, the mean values have been averaged from five DNA extractions of each radial position, respectively. The data in parentheses represent the standard deviation.

sapwood transition wood heartwood F-probability

Fresh wood69.95 A 57.08 B 12.70 C <0.0001 CTAB(8.40) (2.78) (3.06)

Dried wood24.67 A 25.43 A 7.90 B 0.0002

(1.29) (3.34) (1.95)

Fresh wood107.02 A 76.10 B 13.83 C <0.0001 Qiagen kit(7.63) (6.80) (4.43)

Dried wood47.68 A 36.17 B 8.38 C 0.0004

(3.61) (9.11) (1.78)

Significant differences among the radial positions are denoted by different letters (P<0.05).

Table 4. Analysis of variance in the purity of DNA (A260/A280) extracted from different radial positions in fresh and dried wood, using the modified CTAB method and the Qiagen kit protocol.

For both fresh and dried wood, the mean values have been averaged from five DNA extractions of each radial position, respectively. The data in parentheses represent the standard deviation.

sapwood transition wood heartwood

Fresh wood 1.32 A 1.38 A 1.14 A CTAB(0.17) (0.13) (0.12)

Dried wood 1.30 A 1.32 A 1.18 A

(1.11) (0.16) (0.06)

Fresh wood 1.47 A 1.35 AB 1.17 B Qiagen kit(0.10) (0.17) (0.05)

Dried wood 1.50 A 1.31 A 1.22 A

(0.06) (0.18) (0.09)

Significant differences among the radial positions are denoted by different letters (P<0.05).

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Jiao et al. — DNA extraction from wood In general, a larger quantity of DNA could be retrieved using the Qiagen kit protocol than that when using the modified CTAB method (Fig. 2). The quantity of DNA extracted from fresh wood using the Qiagen kit protocol was 40.9% greater than that retrieved using the modified CTAB method. In dried wood, the quantity of DNA retrieved using the Qiagen kit protocol was 59.0% greater than that isolated by the modified CTAB method. For fresh wood, there were significant differences in the quantity of extracted DNA between sapwood and heartwood (P<0.05) when using both the modified CTAB method and the Qiagen kit protocol. Similarly, the differences in the quantity of re -trieved DNA were also statistically significant between the sapwood and heartwood of dried wood when using these two DNA extraction protocols (P<0.05) (Table 3). It was indicated that the quantity of DNA was greater in the sapwood and less in the heartwood for both the fresh and the dried wood (Fig. 2, Table 3).

Furthermore, the purity of the DNA extracted from the wood illuminated by the ratio of A 260/A 280 was between 1.1 and 1.6, which was lower than that yielded from the leaves (Sambrook & Russell 2001). The purity of the DNA obtained using the Qiagen kit protocol was generally higher than the DNA isolated using the modified CTAB method (Fig. 3), while the purity of the DNA extracts from the sapwood and transition wood was higher than that from heartwood (Fig. 3, Table 4). For both fresh and dried

Statistical analysis

Statistical analysis was carried out using the one-way analysis of variance (ANOV A , SAS program 9.0) to evaluate differences in the quantity of the DNA from the various radial positions in fresh and dried wood.

RESULTS

DNA extraction

The modified CTAB method and the Qiagen kit protocol were carried out to extract DNA from fresh and dried wood of Cunninghamia lanceolata , respectively. Both of these two techniques could yield a large quantity of genomic DNA from fresh sapwood and transition wood, whereas the DNA extracted from dried sapwood and transition wood was severely degraded. Furthermore, genomic DNA could not be visualized in agarose gel for fresh and dried heartwood (Fig. 1).

bp M FS FT F H D S DT DH L bp M FS FT F H D S DT D H L

10k 10k

Figure 1. The total genomic DNA extracted from fresh and dried wood, using the modified CTAB method and the Qiagen kit protocol. – A: The modified CTAB method. – B: The Qiagen kit protocol. – M = 10 kb marker; FS = fresh sapwood; FT = fresh transition wood; FH = fresh heartwood; DS = dried sapwood; DT = dried transition wood; DH = dried heartwood; L = leaves (The concentration of DNA extracts from leaves was diluted 10 times).

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wood, there was no significant difference in the ratio of A 260/A 280 for the DNA retrieved using the modified CTAB method, when it came to sapwood and heartwood (P >0.05). However, a significant difference in the purity of the DNA obtained using the Qiagen kit protocol was shown between the fresh sapwood and heartwood (P<0.05), although no significant difference was found between the dried sapwood and heartwood (P >0.05) (Table 4).

PCR amplification

For cpDNA, the intron in the ycf 3 gene (668 bp) and the noncoding region between the psb C gene and the trn S gene (401 bp) were successfully amplified in the DNA

120100806040200 CTAB Qiagen kit CTAB Qiagen kit

Fresh wood Dried wood

sapwood transition wood heartwood

n g /m g

Figure 2. The quantity of DNA (ng/mg of wood powder) extracted from different radial positions in fresh and dried wood, using the modified CTAB method and the Qiagen kit protocol.

CTAB

Qiagen kit

FS

FT

FH

DS

DT

DH

L

1.81.61.41.21.00.80.60.40.20A 260/A 280

Figure 3. The purity of the DNA (A 260/A 280) extracted from different radial positions of fresh and dried wood, using the modified CTAB method and the Qiagen kit protocol. – FS = fresh sapwood; FT = fresh transition wood; FH = fresh heartwood; DS = dried sapwood; DT = dried transition wood; DH = dried heartwood; L = leaves.

449

Jiao et al. — DNA extraction from wood extracted using the modified CTAB method and the Qiagen kit protocol, when it came to the sapwood, transition wood and heartwood of both fresh and dried wood. In addi-tion, it was indicated that the efficiency of the PCR amplification of the DNA extracts taken from the heartwood was less than that from sapwood and transition wood (Fig. 4). The PCR amplification success rates of cpDNA regions are shown in Figure 5. The amplification success rate of cpDNA fragments extracted from fresh and dried wood, using the Qiagen kit protocol, was almost 100%, whereas that retrieved from fresh and dried heartwood using the modified CTAB method was slightly less, at 80–90% (Fig. 5).

Figure 4. The PCR amplification products generated with chloroplast DNA (A, B, C & D) and nuclear DNA (E & F), isolated from the different radial positions in the fresh and dried wood. – A & B: PCR product-668 bp. – C & D: PCR product-401 bp. – E & F: PCR product- 481 bp. – A, C & E: DNA fragment retrieved by modified CTAB method. – B, D & F: DNA fragment retrieved by the Qiagen kit protocol. – M = 2000 bp marker; FS = fresh sapwood; FT = fresh transition wood; FH = fresh heartwood; DS = dried sapwood; DT = dried transition wood; DH = dried heartwood; L = leaves.

FS

FT

FH

DS

DT

DH

L

1.00.80.60.40.20.0CTAB Qiagen kit

S u c c e s s r a t e

Figure 5. PCR amplification success rate of chloroplast DNA regions extracted from different radial positions of fresh and dried wood, using the modified CTAB method and the Qiagen kit protocol. – FS = fresh sapwood; FT = fresh transition wood; FH = fresh heartwood; DS = dried sapwood; DT = dried transition wood; DH = dried heartwood; L = leaves.

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For nuclear DNA, the ITS region (481 bp) was amplified in the experiment. This DNA region extracted by the modified CTAB method from fresh and dried sapwood was amplified successfully, but in the transition wood and heartwood of fresh and dried wood, the ITS region failed in subsequent PCR amplifications. For the DNA extracted by the Qiagen kit protocol, it was amplified successfully from the sapwood and transi-tion wood of fresh and dried wood, whereas the DNA from fresh and dried heartwood failed in the PCR amplification. Additionally, the PCR amplification success rate of rDNA-ITS region extracted from fresh and dried sapwood using the modified CTAB method was almost 100%. Similarly, the success rate of rDNA-ITS region yielded by the Qiagen kit protocol from sapwood and transition wood of fresh and dried wood was also 100% in the experiment.

Distribution of nuclei and amyloplasts

In the fresh wood, abundant acetocarmine-stained nuclei were detected in the ray parenchyma cells of sapwood (Fig. 6A). Numerous amyloplasts were observed in the ray parenchyma cells (Fig. 7A) or axial parenchyma cells (Fig. 7B). In the transition wood, the number of nuclei (Fig. 6B) and amyloplasts (Fig. 7C) was less than that in the sapwood. However, in the heartwood, ray parenchyma cells with acetocarmine-stained nuclei were not clearly visible (Fig. 6C) and amyloplasts stained with iodine-potassium iodide were sporadically detected (Fig. 7D).

Generally, less nuclei (Fig. 6) and amyloplasts (Fig. 7) were observed in the dried wood, when compared to the fresh wood. Moreover, the number of nuclei and amy-loplasts decreased from the sapwood to the heartwood. In the dried heartwood, nuclei were not clearly visible (Fig. 6F), while amyloplasts were sometimes detected in the ray parenchyma cells (Fig. 7G).

DISCUSSION

Comparison of the modified CTAB method and the Qiagen kit protocol Compared to the modified CTAB method, the Qiagen kit protocol could yield a greater quantity of better quality DNA extracts. Meanwhile, it was demonstrated that DNA extracted from wood by both protocols could be successfully amplified except for rDNA-ITS region from transition wood and heartwood. Furthermore, the PCR amplification success rate of the cpDNA retrieved from heartwood using the Qiagen kit protocol was higher than that extracted when using the modified CTAB method, although PCR amplification for nuclear DNA of heartwood using both extraction methods failed. The results obtained here agreed with those by Tnah et al. (2012), who reported that a greater quantity of DNA extracts could be isolated using the Qiagen kit protocol and that the Qiagen kit protocol yielded a higher PCR amplification success rate, when compared with the CTAB method. Most previous studies confirmed that the Qiagen kit protocol was more efficient to extract DNA from wood and that the DNA extracts could be amplified (Dumolin-Lapègue et al. 1999; Deguilloux et al. 2002; Rachmayanti et al. 2006, 2009; Yoshida et al. 2007; Abe et al. 2011; Hanssen et al. 2011; Tang et al. 2011). This could be due to the spin columns in the Qiagen kit, which contain silica-gel-based membranes that bind the DNA and so contaminants that

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inhibited the PCR amplification could be eluted from the membranes using a washing buffer. Accordingly, more pure DNA extracts could be obtained using the Qiagen kit than that obtained with the modified CTAB method.

Moreover, the Qiagen kit protocol is more convenient, time-saving and does not contain harmful chemicals such as phenol or chloroform. Nevertheless, in the current experiment, the disadvantage of the Qiagen kit is its cost, which is about 5 to 10 times more expensive per extraction than the modified CTAB method.

The quantity and quality of extracted DNA in different radial positions of fresh and dried wood

The study demonstrates that the quantity of DNA decreases in heartwood compared to sapwood in both fresh and dried Cunninghamia lanceolata wood, which is consistent with the results of previous studies (Yoshida et al. 2007; Rachmayanti et al. 2009; Abe et al. 2011; Tang et al. 2011). The differences in the quantities of DNA extracted from the different radial positions can be explained by the fact that living cells exist in the sapwood but that DNA is gradually degraded during cell death in heartwood formation (Bamber 1976; Deguilloux et al. 2002; Abe et al. 2011).

Lumber is frequently artificially dried before being processed and used (Yoshida et al. 2007). Consequently, we have explored the influence of drying on the efficiency of DNA extraction from wood. It was reported that the quantity of DNA in wood de-creased after high temperature treatment (Yoshida et al. 2007; Tnah et al. 2012). This study demonstrates the same result in relation to the quantity of DNA in fresh wood being greater than that from wood dried at 70°C. The most likely explanation for this is the drying treatment that caused the DNA degradation and diminished the presence of DNA in the wood tissues (Tnah et al. 2012).

Furthermore, several studies on the purity of DNA extracts from fresh wood had been published (De Filippis et al. 1998; Novaes et al. 2009). De Filippis et al. (1998) demonstrated that the A260/A280 ratios of DNA extracts from fresh Robinia wood were between 1.88 and 1.48, and the purity decreased when going from the outer sapwood to the inner heartwood. There was no detailed study on dried wood, although Rachmayanti et al. (2009) reported that the purity of DNA extracts from processed wood was very low. The present study shows that, utilizing UV spectrophotometer detection, the purity of DNA extracts (1.1–1.6) from both fresh and dried Cunninghamia lanceolata wood is less than that of the previous study, which suggests that a higher level of impurities, including proteins, phenols and carbohydrates are present in the DNA extracts of the Cunninghamia lanceolata wood samples.

PCR amplification of DNA taken from different radial positions in fresh and dried wood

In the present study, the cpDNA of all DNA extracts could be successfully ampli-fied (Fig. 4A–D), as well as rDNA-ITS of sapwood (Fig. 4E, F) using two extraction methods and transition wood by Qiagen kit (Fig. 4F), although a large quantity of high quality DNA was not retrieved from the wood, especially the dried Cunninghamia lanceolata wood. Therefore, the application of retrieving DNA information from dried or processed wood has potential and is feasible for future wood identification.

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452Figure 6. Light micrographs of radial sections, stained with 1% acetocarmine, showing nuclei (arrows) in the ray parenchyma cells in different radial positions of the fresh and dried wood. – A, B & C: Radial sections of sapwood, transition wood and heartwood of fresh wood, respec -tively. – D, E & F: Radial sections of sapwood, transition wood and heartwood of dried wood, respectively. — Scale bars of A–F = 50 μm.

In general, the overall trend of the amplification success rate on cpDNA region is consistent with both the fresh and dried wood (Fig. 5). The success rate of the cpDNA region from the heartwood is slightly less than from sapwood and transition wood, which is confirmed by previous research (Rachmayanti et al . 2009; Tnah et al . 2012). On the other hand, PCR amplification failed for the rDNA-ITS region of fresh and dried heartwood. A possible explanation is that DNA is gradually degraded during cell death of heartwood formation. Furthermore, it was demonstrated that greater amounts of essen-

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Jiao et al. — DNA extraction from wood tial oils, whose major chemical component was cedrol, existed in Cunninghamia lanceo- lata wood, especially in its heartwood (Huang et al . 2004; Ye et al . 2005). This could be also an important factor involved in interference to DNA isolation and the inhibition of PCR amplification. On the other hand, chemical contaminations including cedrol are bet- ter removed with Qiagen kit protocol compared with the modified CTAB method, which can explain the somewhat lower PCR success rate with the modified CTAB method.

Figure 7. Light micrographs of radial sections, stained with a 1% aqueous solution of iodine-potassium iodide, showing the starch grains (arrows) in the ray parenchyma cells or axial paren-chyma cells in different radial positions of the fresh and dried wood. – A, C & D: Radial sections of sapwood, transition wood and heartwood of fresh wood, respectively. – B: Axial parenchyma cells in fresh sapwood. – E, F & G: Radial sections of sapwood, transition wood and heartwood of dried wood, respectively. — Scale bars of A–G = 50 μm.

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Distribution of DNA in different radial positions in fresh and dried wood

It has been suggested that ray parenchyma cells remain alive with protoplasm from the sapwood to the transition wood and that the number of organelles declines from the sapwood to the transition wood (Nakaba et al. 2006). Moreover, Abe et al. (2011) reported that, using fluorescence microscopy, DNA stained with 0.2% (4)4’-6-diamid-ino-2-phenylindole (DAPI) could be detected in nuclei and amyloplasts of the fresh sapwood, and amyloplasts treated with I2-KI were sometimes observed in the ray parenchyma of the fresh heartwood using an optical microscope, although Song et al. (2011) indicated that nuclei and protoplasm could not be detected in the ray parenchyma cells of fresh heartwood with transmission electron microscopy (TEM). In the stored samples, DNA could be detected in the nuclei of the sapwood and DNA in amyloplasts was occasionally observed in the heartwood (Abe et al. 2011). Generally, the present research is consistent with the above mentioned studies. Nuclei are observed in the sapwood and the transition wood of fresh and dried wood, while nuclei can not be clearly observed in the heartwood of Cunninghamia lanceolata. Furthermore, numerous amyloplasts are detected in the sapwood and transition wood of fresh wood. In the fresh heartwood and in the dried wood, amyloplasts are observed occasionally. Therefore, it can be inferred that DNA generally comes from nuclei and plastids, such as amyloplasts in the parenchyma cells of sapwood and transition wood and that DNA mainly comes from amyloplasts in heartwood.

CONCLUSION

Our results clearly show that a greater quantity of higher quality DNA could be extracted from wood of Cunninghamia lanceolata using the Qiagen kit protocol compared to the modified CTAB method. For both fresh and dried wood, the DNA content decreased when going from the sapwood to the heartwood. In addition, the purity of the DNA extracts from the sapwood and transition wood was greater than that of the heartwood. Due to the influence of the drying procedure, the quantity of DNA in the dried wood was less than that of the fresh wood. Additionally, it was indicated that the cpDNA regions retrieved from wood using both protocols could be successfully amplified. As for the rDNA-ITS region from heartwood yielded by both protocols, the PCR amplifi-cation failed. Furthermore, the amplification success rate for the DNA extracted from the sapwood and the transition wood was generally higher than that for the heartwood. Anatomical analysis shows that the extracted DNA of sapwood and transition wood gen-erally comes from the nuclei and amyloplasts in the parenchyma cells; in the heartwood, the amyloplasts are likely to be the source of DNA. This also implies that nuclear DNA of heartwood is more difficult to amplify successfully compared with cpDNA. Based on microscopic observation, this study demonstrates the optimized radial position for DNA extraction and subsequent analysis in a stem. In conclusion, the success of DNA extraction from wood and PCR amplification is a necessary first step in the application of DNA methods in wood identification. Further improvement of DNA extraction and amplification techniques in the future will undeniably aid in identification of wood samples and will become a reliable tool in timber certification.

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ACKNOWLEDGEMENTS

This work was supported financially by a project in a Chinese State Forestry Administration Project (201104058) and a Chinese National Natural Science Foundation Project (No. 30972303). We would like to express our gratitude for the technical help given by Professor Mrs. M.Xu and L.Liu at the Research Institute of Wood Industry, the Chinese Academy of Forestry.

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