DNA from processed and unprocessed wood: Factors influencing the isolation success

Abstract

Molecular genetic markers have numerous potential applications in environmental forensics if DNA can be isolated from ‘difficult’ non-human biological material such as hairs, feathers, or wood. The identification of the origin of wood is particularly important in order to identify illegally harvested and traded timber and wood products. We describe success rates of DNA isolation from wood based on a simple, previously published extraction protocol. The protocol was used to isolate DNA from a total of 406 wood samples, mainly of the important tropical tree family Dipterocarpaceae. The reliability of the extraction method was confirmed by comparing fragment sizes and sequences after isolation of DNA from leaves and wood of the same trees. We observed the success of amplification of chloroplast DNA (cpDNA) fragments of different lengths by means of PCR, investigated key factors influencing PCR, and conducted inhibitor tests for a subset of the samples. The average rate of successful PCR amplification was 75.7%. Main factors influencing the success of PCR amplification were the size of the amplified fragment and the processing status of the wood. Short fragments and unprocessed wood resulted in higher success rates. The success rate was also dependent on the age (storage duration) of the wood probe and on the investigated species. Amplification success was higher if DNA was isolated from outer sapwood (without cambium) in comparison to DNA isolated from the transition zone between sapwood and heartwood and the inner heartwood. However, inhibitor tests also indicated more PCR inhibitory substances in the outer sapwood in comparison to transition wood and heartwood. The addition of polyvinylpyrolidone (PVP) to the lysis buffer proved to be highly efficient to improve the amplification success if inhibitory substances were present.

Introduction

Molecular genetic markers are very important tools in (human) forensic analyses and are widely used to identify suspects in criminal cases [1]. In most cases the underlying question is to reliably identify the DNA origin of a given sample. Forensic applications of molecular markers from non-human biological material are rapidly developing and the number of marker types and of reference data is increasing both for animal and plant species [2]. An obvious requirement for the application of genetic fingerprinting techniques is the isolation of DNA from different biological materials such as hairs or feathers in animals [3] or wood in plants.

An important application of molecular markers in environmental forensics is to provide evidence for the illegal trade with both living and dead biological material. Forest destruction and degradation continue to be main threats to global biodiversity and cause severe environmental damage particularly, but not only, in developing countries. Hence, the trade with illegally harvested timber and processed wood is a potentially important field to apply molecular genetic tools in forensics.

Potential applications of such tools are manifold. Custom offices in producer and consumer countries, forest certification schemes such as PEFC [4] and FSC [5] and enterprises producing timber according to the principles of sustainable forest management, as well as the timber industry and end consumers potentially benefit from improved methods to infer the origin of wood which might be illegally harvested. Currently available methods only rarely allow to prove false declarations of the origin of wood in court.

Wood anatomy can be macroscopically and microscopically assessed. While it is often possible to identify the species or at least a particular species group based on wood anatomical characters, it is rarely possible to infer the origin within the distribution range of a species. Methods based on the observation of ratios of stable isotopes are widely used to conclude on the growing region of plants with important applications in the food industry [6], [7] and have also been suggested as suitable to infer the origin of wood [8], [9], but first results based on this method indicate less resolution as initially expected.

Two basic requirements need to be fulfilled in order to use DNA variation for the identification of the origin of wood: (i) it is possible to isolate DNA from wood of different age and processing status, and (ii) markers need to be developed which are informative with regard to the identification of the origin of wood.

The latter requirement is based on knowledge on the distribution of genetic diversity within a particular target species or, in case of species-rich groups with many endemics, on differentiation patterns among species. Thus, genetic inventories are needed to gain insights into variation patterns at different spatial scales. Progress has been made in this regard for several temperate species, in particular European white oaks [10]. Promising variation patterns have also been observed for selected tropical species [11], [12]. The observation of maternally inherited genetic information (e.g. cpDNA markers in angiosperms) often proved to be particularly useful to distinguish between plants from widely separated regions since genetic differentiation is often strong at maternally inherited markers [10], [13].

This paper mainly deals with the first requirement to use DNA for wood identification, i.e. the isolation of DNA from different types of wood. Marker development for a particularly important group of tropical timber (Dipterocarpaceae) will be separately described (Rachmayanti et al., in prep.; Nuroniah et al., in prep.).

Main obstacles for DNA extraction from wood and wood products as compared to other plant tissues are: (1) Physical. Wood is a hard plant tissue. A mechanical treatment such as drilling or slicing is required in order to disrupt woody material containing fibres, vessels and parenchymatic tissue with intact DNA and DNA fragments. Thereby, overheating of tissue must be avoided since it may lead to irreversible degradation of DNA [15], [16]. (2) Chemical compounds. Numerous agents and wood compounds potentially inhibit DNA extraction or result in low-quality DNA not suitable for amplification by PCR. For example, many phenolic compounds of the lignin metabolism which are present in different concentrations in all types of wood and many chemicals used for wood treatment are potential PCR inhibitors [15], [17]. (3) Biological. Decomposition of wood by fungi and microorganisms results in degradation of DNA and provides an alternative source of DNA from wood decaying organisms. Contamination with DNA from other organisms is expected to be particularly severe on the surface of wood after long periods of storage [15] and is detrimental particularly for the application of universal genetic markers. (4) Age. Degeneration of DNA will start after the death of a plant cell and hence in the case of timber before and after tree felling. The size of fragments which can be amplified and investigated is expected to continuously decrease after the death of the respective wood tissue [14], [18], [19].

Studies involving the isolation and amplification of DNA from dry wood are still rare. Some studies were reported working with wood from oaks [20], Robinia spp. [21] and Gonystylus bancanus[22]. A simple, but reliable method for DNA isolation is mandatory for large-scale practical applications in different laboratories. We developed such a simple method for DNA isolation for the tropical tree family Dipterocarpaceae based on a widely used extraction kit [16].

In this paper, we report success rates of amplification after DNA extraction following the method described in Ref. [16]. We compare DNA fragment sizes and sequences obtained from wood and leaves of the same tree, and we assess the following factors potentially influencing the success rate of DNA isolation from wood.

• Species: dipterocarps, tropical trees other than dipterocarps, and selected temperate tree species were investigated.

• Processing status of wood before extraction: both untreated timber and wood processed mainly for window framing was analyzed.

• Wood used for extraction: outer (sapwood), middle (transition zone between sapwood and heartwood), and inner (heartwood).

• Size of fragments: success rates after amplification of different fragment sizes ranging from 150 bp to 1.1 kb.