TECHNICAL FIELD

[0001] A method for extracting plant nucleic acids from lignified plant tissue, and methods for using the extracted nucleic acids to determine the source of the lignified plant tissue.

BACKGROUND ART

[0002] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[0003] Extraction of high quality DNA or RNA from fresh plant leaf and bud tissue has become routine, rapid and can now be semi- or completely automated with robotic workstations and specialised extraction 'kits' {e.g. from Qiagen, or other manufacturers). DNA may also be routinely extracted from fungal cells using techniques such as that described in Wolfe et al (New Phytologist; 20 0)

[0004] DNA extracted from freshly harvested wood, particularly if cambium tissue is incorporated, can also yield high quality DNA, which can be comparable to leaf sources (Colpaert et al. 2005). However, extraction of DNA from dried wood is more problematic, with lower yields farther from the cambium (Lowe and Cross 201 1 ).

[0005] Overall, the challenges with using dried wood for genetic work are twofold: both the quantity and quality of the nucleic acids extracted are low. The number of cells in wood that can be expected to contain nucleic acids is low compared to leaves or buds, thus the quantity of nucleic acids is low. Nucleic acid degradation in plants is characterized by lengthwise fractures, with the result that only smaller fragments can be amplified and sequenced and, for example, DNA in wood is typically degraded (Lowe and Cross 201 1). Some of the standard extraction kits available are not designed to recover these small fragments, and therefore these methods often will not work for extremely degraded tissues. [0006] Other aspects of wood anatomy will affect the quality of the nucleic acids that are isolated from wood. In addition to the difficulties posed by macromolecules like lignin and cellulose, there are a wide-range of co-extracted compounds that can also inhibit the polymerase chain reaction (PCR) and other laboratory analyses. In one study, wood DNA extracts added to a PCR reaction with fresh leaf DNA showed significant inhibitory effects, which decreased with increasing dilution of the wood DNA sample (Rachmayanti et al. 2009). These experiments showed that the inhibitory effects of wood components on the nucleic acids that can be extracted were worse in the outer sapwood and not as bad in the inner heartwood. A range of approaches have improved the success rate for nucleic acid extraction from wood and limited the effects of inhibitors. Several additives can also be incorporated at the PCR step to improve amplification in the presence of some potentially inhibiting contaminants.

[0007] However, there does not seem to be any agreement as to the best approach, and results seem to vary widely. One factor may be that some woods are more amenable to nucleic acid extraction than others. Other studies have shown a great deal more success with extraction of nucleic acids from tropical woody species.

[0008] In general, these studies show that it is possible to extract nucleic acids such as DNA from wood using specifically designed laboratories, where contamination issues are minimized and a traceable process is implemented (Gugerli et al. 2005), and that success has been higher for non-tropical plants than for tropical species, but that the process is still not reliable. There is also still work to be done on how far down the timber treatment process (fresh wood, field-dried wood, sawn timber, kiln dried, or other processing) nucleic acids can be extracted.

[0009] It is against this background that the present description has been developed. It seeks to overcome, or at least ameliorate, one or more of the deficiencies of the prior art mentioned above to provide a nucleic acid extraction method that reliably works with a wide variety of lignified plant tissue from a range of plant species and on a range of timber products from along the processing chain (fresh timber, kiln dried timber and veneers), or to at least provide the consumer with a useful or commercial choice. SUMMARY OF INVENTION

[0010] The present invention provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[001 1] The lignified plant tissue is preferably subjected to the extraction solution for between 0.5 and 16 hours, more preferably between 0.5 and 6 hours, between 1 h and 6h, between 2h and 6h, between 3h and 6h, between 4h and 6h, or between 4.5 and 5.5 hours. Preferably, the lignified plant tissue is exposed to the solution for about 4h, 4.25h, 4.5h, 5h, 5.25 or 5.5 hours to enable extraction of the nucleic acids.

[0012] The exposure of the lignified plant tissue to the extraction solution should preferably occur at a temperature of between 40° and 65°, between 45° and 65° between 47° and 60°, between 49.5°C and 60.5°C, between 50°C and 57°C, or between 52°C and 56°C. Most preferably, the temperature of the solution during exposure of the lignified plant tissue to extract nucleic acids is about 52°C, 53°C, 54°C, 55°C, 56°C or 57°C.

[0013] The present invention also provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a chelating agent and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0014] The present invention also provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a salt and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a). [0015] The present invention also provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, an excipient and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0016] The present invention also provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a biological antioxidant and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0017] The present invention also provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a serine protease and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0018] The present invention also provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a chelating agent, a salt, an excipient, a biological antioxidant, a serine protease and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0019] The present invention also provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: CTAB, Tris, NaCI, EDTA, Boric Acid, PVP K30, DTT, and Proteinase K; b) purifying the nucleic acids extracted in (a).

[0020] The present invention also provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: 55m CTAB, 100mM Tris, 1.4M NaCI, 20mM EDTA, 1% (w/v) Boric Acid, 2% (w/v) PVP K30, 47m DTT, and 1.86% Proteinase K; b) purifying the nucleic acids extracted in (a).

[0021] The present invention also provides a method of determining the genus and/or species a sample of lignified plant tissue is from, comprising the steps of: a) extracting plant nucleic acids from the lignified plant tissue using a method comprising the following steps: i. exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid and buffer to extract the nucleic acids; ii. purifying the nucleic acids extracted in (i); b) using the nucleic acids from (a) to develop a genetic profile for the lignified plant tissue sample; c) comparing the genetic profile from (b) with a library of genetic reference profiles.

[0022] The present invention also provides a method of determining the plant source of a sample of lignified plant tissue, comprising the following steps: a) extracting plant nucleic acids from the lignified plant tissue using a method comprising the following steps: i. exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid and buffer to extract the nucleic acids; ii. purifying the plant nucleic acids extracted in (i); b) using the plant nucleic acids from (a) to develop a genetic profile for the lignified plant tissue sample; c) comparing the genetic profile from (b) with a library of genetic reference profiles.

[0023] The present invention also provides a method for developing a genetic profile for a sample of lignified plant tissue comprising the steps of: a) extracting plant nucleic acids from lignified plant tissue using a method comprising the following steps: i. exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid and buffer to extract the nucleic acids; ii. purifying the nucleic acids extracted in (i); b) subjecting the isolated plant nucleic acids of step (a) to a procedure chosen from the list comprising: PCR, RAPD analysis, AFLP analysis, CAPS analysis, RFLP analysis, SSR analysis, SNP analysis, SSCP analysis, sequencing.

[0024] The present invention also provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, boric acid and buffer; b) instructions for use.

[0025] Preferably, the instructions for use of the kit designate that the extraction is carried out by exposing the lignified plant tissue to the extraction solution for about 5 hours. Preferably, the instructions for use of the kit designate that the extraction is carried out by exposing the lignified plant tissue to the extraction solution at a temperature of about 55°C.

[0026] The present invention also provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, a chelating agent, boric acid and buffer; b) instructions for use.

[0027] The present invention also provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, a salt, boric acid and buffer; b) instructions for use.

[0028] The present invention also provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising; a) a cationic surfactant, an excipient, boric acid and buffer; b) instructions for use.

[0029] The present invention also provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, a biological antioxidant, boric acid and buffer; b) instructions for use.

[0030] The present invention also provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, a serine protease, boric acid and buffer; b) instructions for use.

[0031] [The present invention also provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, boric acid, a buffer a chelating agent, a salt, an excipient, a biological antioxidant and a serine protease; b) instructions for use. [0032] The present invention also provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) CTAB, Tris, NaCI, EDTA, Boric Acid, PVP K30, DTT, Proteinase K; b) instructions for use.

DETAILED DESCRIPTION

Detailed Description

[0033] It is often important to determine the source of lignified tissue such as timber, to answer questions including:

• Was this timber harvested from the declared country or region?

• Does this timber come from a conservation area?

• Does this timber come from natural forest or a plantation?

• Does this product come from the declared log or stump (is the chain-of- custody intact)?

• Has this log or lot been swapped with other plants of the same species (log laundering)?

• Is this timber of the declared species?

• is this timber a Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) listed species or not?

Method of Extracting Nucleic Acids

[0034] In order to assist with determining the answer to question such as these, the present description provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0035] The lignified plant tissue is preferably subjected to the extraction solution for between 0.5 and 16 hours, more preferably between 0.5 and 6 hours, between h and 6h, between 2h and 6h, between 3h and 6h, between 4h and 6h, or between 4.5 and 5.5 hours. Preferably, the lignified plant tissue is exposed to the solution for about 4h, 4.25h, 4.5h, 5h, 5.25 or 5.5 hours to enable extraction of the nucleic acids.

[0036] The exposure of the lignified plant tissue to the extraction solution should preferably occur at a temperature of between 40° and 65°, between 45° and 65° between 47° and 60°, between 49.5°C and 60.5°C, between 50°C and 57°C, or between 52°C and 56°C. Most preferably, the temperature of the solution during exposure of the lignified plant tissue to extract nucleic acids is about 52°C, 53°C, 54°C, 55°C, 56°C or 57°C.

[0037] Without wishing to be bound by any particular theory, it is believed that the boric acid assists with the removal of carbohydrate impurities in the extraction solution.

[0038] The boric acid is preferably provided in the solution at between 0.1% (w/v) and 5% (w/v). Preferably, the boric acid is provided at between 0.25% and 4%, between 0.5% and 3%, between 0.5% (w/v) and 2% (w/v), or between 0.9% (w/v) and 1 .1 % (w/v). Most preferably, the boric acid is provided in the solution at 0.5%, 0.75%, 1 %, 1 .25% or 1 .5% (w/v).

[0039] The cationic surfactant used in the method of the present invention functions to at least collate and emulsify the lipids in the reaction mixture. Any cationic surfactant that can perform at least this function may be used. For example, the cationic surfactant may be chosen from the list comprising: cetyitrimethylammonium bromide (CTAB), centrimonium chloride, benzalkonium chloride, and benzethonium chloride.

[0040] The cationic surfactant is preferably provided in the solution at between 10mM and 100mM. Preferably, the cationic surfactant is provided at between 20mM and 80mM, between 30mM and 70mM, between 45mM and 65mM, or between 49.5mM and 60.5mM. Most preferably, the cationic surfactant is provided in the solution at 45mM, 50mM, 55mM, 60mM or 65mM.

[0041 ] As referred to herein, the term "cationic surfactant" refers to any surfactant molecule comprising a hydrophobic "tail" group and a hydrophilic "head" group, wherein the hydrophilic head group comprises a positive charge.

[0042] In some embodiments, the cationic surfactant comprises a quaternary ammonium cation as the head group. "Quaternary ammonium cations" are positively charged ions of the structure:

wherein each of R ¹ , R ² , R ³ and R ⁴ are independently selected from H, alkyl or aryl groups, with the proviso that at least one of R ¹ , R ² , R ³ and R ⁴ comprises a hydrophobic alkyl or aryl group.

[0043] "Alkyl" as a group or part of a group denotes an optionally substituted straight or branched aliphatic hydrocarbon group. The group may be a terminal group or a bridging group.

[0044] "Aryl" as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 18 atoms per ring. Examples of aryl groups include optionally substituted phenyl, optionally substituted naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a Cs-7 cycloalkyl or Cs _-7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.

[0045] The term "optionally substituted" as used throughout the specification denotes that the group may or may not be further substituted or fused with one or more non- hydrogen substituent groups. In certain embodiments the substituent groups are one or more groups independently selected from the group consisting of halogen, =0, =S, -CN, -N0 ₂₎ -CF ₃ , -OCF _3l alkyl, alkenyl, alkynyl, haloalkyi, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycioalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, aryl heteroalkyl, heteroarylheteraalkyl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxycycloalkyl, alkyloxyheterocycloalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkyloxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, amino, alkylamino, acyiamino, aminoalkyl, arylamino, sulfonylamino, sulfmylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, C(0)OH, - C(=0)R ^a , -C(=0)OR ^a , C(=0)NR ^a R ^b , C(=NOH)R ^a , C(=NR ^a )NR ^b R°, NR ^a R ^b , NR ^a C(=0)R ^b , NR ^a C(=0)OR ^b , NR ^a C(=0)NR ^b R°, NR ^a C(=NR ^b )NR°R ^d , NR ^a S0 ₂ R ^b ,-SR ^a , S0 ₂ NR ^a R ^b , -OR ^a , OC(=0)NR ^a R ^b , OC(=0)R ^a and acy!, wherein R ^a , R ^b , R° and R ^d are each independently selected from the group consisting of H, Ci-C<i2alky], Ci- C^haloalkyl, C2-Ci2alkenyl, C2-Ci ₂ alkynyl, C2-Cioheteroaikyl, C3-Ci2cycloalkyl, C3- Ci2cycloalkenyl, C2-Ci ₂ heterocycloalkyl, C2-Ci2heterocycloalkenyl, C ₆ -Ci ₈ aryl, Ci- Ci ₈ heteroaryl, and acyl, or any two or more of R ^a , R ^b , R ^c and R ^d , when taken together with the atoms to which they are attached form a heterocyclic ring system with 3 to 12 ring atoms.

[0046] In some embodiments each optional substituent is independently selected from the group consisting of: halogen, =0, =S, -CN, -N0 _2l -CF ₃ , -OCF3, alkyl, alkenyl, alkynyl, haloalkyi, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, -COOH, -SH, and acyl.

[0047] In some embodiments, at least one, at least two, at least three or each of R , R ² , R ³ and R ⁴ is alkyl.

[0048] Quaternary ammonium cations may be permanently charged, independent of the pH of their solution. Quaternary ammonium salts or quaternary ammonium compounds as referred to herein may include salts of quaternary ammonium cations with an anion.

[0049] As set out above, cationic surfactants, including those comprising a quaternary ammonium cation, include one or more hydrocarbon tail groups. Typically, the hydrocarbon tail is of sufficient length to be lipophilic or hydrophobic. In some embodiments, the one or more hydrocarbon tail groups of the cationic surfactant, ie. one or more of R ¹ , R ² , R ³ and R ⁴ in structure (I), may comprise an alkyl chain at least 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons in length.

[0050] The buffer used in the method of the present invention functions to at least maintain the pH of the solution during extraction of the nucleic acids. Any buffer that can perform at least this function may be used. For example, the buffer may be chosen from the list comprising: tris(hydroxymethyl)aminomethane (Tris), piperazine- N,N'-bis(2-ethanesulfonic acid) (PIPES), (M)ethanosulfonic acid and succonic acid.

[0051] The buffer is preferably provided in the solution at between 50mM and 150mM. Preferably, the buffer is provided at between 60mM and 140mM, between 70mM and 130mM, between 80mM and 120mM or between 90mM and 110mM. Most preferably, the buffer is provided in the solution at 70mM, 80mM, 90mM, 100mM, 110mM, 120mM or 130mM.

[0052] The solution that the lignified plant tissue is exposed to in the method of the present invention may additionally comprise a chelating agent for binding metal ions such as magnesium and/or calcium ions. Preferably, the chelating agent chelates magnesium and/or calcium ions to reduce their ability to act as a co-factor to endogenous nucleases that may cause damage to the extracted nucleic acids. Any chelating agent that can perform at least this function may be used. For example, the chelating agent may be chosen from the list comprising: ethylene diamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), ethylenediamine- Ν,Ν'-disuccinic acid (EDDS) and diethylene triamine pentaacetic acid (DTPA).

[0053] The chelating agent is preferably provided in the solution at between 1 mM and 50mM. Preferably, the chelating agent is provided at between 5mM and 40mM, between 10mM and 30mM, between 15mM and 25mM or between 18mM and 22mM. Most preferably, the chelating agent is provided in the solution at 10mM, 12mM, 15mM, 17mM, 20mM, 22mM, 25mM or 27mM.

[0054] The invention thus provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a chelating agent and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0055] The solution that the lignified plant tissue is exposed to in the method of the present invention may additionally comprise a salt for maintaining the osmolarity of the solution and assist in nucleic acid stabilisation. Any salt that can perform at least this function may be used. For example, the salt may be chosen from the list comprising: sodium chloride, sodium citrate.

[0056] The salt is preferably provided in the solution at between 0.5M and 3mM. Preferably, the salt is provided at between 0.5M and 2.0M, between 1 M and 2M, between 1.26M and 1.54M or between 1 M and 1.4M. Most preferably, the salt is provided in the solution at 1.4M.

[0057] The invention thus provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a salt and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0058] The solution that the lignified plant tissue is exposed to in the method of the present invention may additionally comprise an excipient to allow the components of the solution to react with the liginifed tissue and enhance precipitation of components other than the nucleic acids such as phenolic compounds. The excipient used in the present method should preferably form complex hydrogen bonds with phenolics and co-precipitates with cell debris upon cell lysis. Any excipient that can perform at least this function may be used. For example, the excipient may be chosen from the list comprising: polyvinylpyrrolidone (PVP including PVP 360, PVP K30, PVP-40); polyvinylpyrrolidone (PVPP) and polyethylene glycol (PEG 200).

[0059] The excipient is preferably provided in the solution at between 0.5% and 4% (w/v). Preferably, the excipient is provided at between 1% (w/v) and 3% (w/v), or between 1.8% (w/v) and 2.2% (w/v). Most preferably, the excipient is provided in the solution at 1%, 1.5%, 1.75%, 2%, 2.25%, 2.5% or 3.0% (w/v).

[0060] The invention thus provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, an excipient and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0061] The solution that the lignified plant tissue is exposed to in the method of the present invention may additionally comprise a biological antioxidant which will serve to reduce the disulfide bonds of at least the nucleic acids in the solution and/or to reduce the tannins and other polyphenols present in the extraction mixture. Any biological antioxidant that can perform at least this function may be used. For example, the biological antioxidant may be chosen from the list comprising: β- mercaptoethanol, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), ascorbic acid, and sodium ascorbate. [0062] The biological antioxidant is preferably provided in the solution at between 5 and 100mM, or between 5 and 75mM. Preferably, the biological antioxidant is provided at between 20mM and 70mM, between 30mM and 60mM, 30mM and 65mM, or between 42.3mM and 51 .7mM. Most preferably, the biological antioxidant is provided in the solution at 35mM, 37mM, 40mM, 45mM, 47mM, 50mM, 52mM or 55mM.

[0063] The invention thus provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a biological antioxidant and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0064] The solution that the lignified plant tissue is exposed to in the method of the present invention may additionally comprise a protease to assist in the breakdown of cellular wall material in the lignified plant tissue and inactivate nucleases.

[0065] Preferably, the protease in the solution that the lignified plant tissue is exposed to in the method of the present invention is a serine protease. Any serine protease that can perform to assist in the breakdown of cellular wall material in the lignified plant tissue and inactivate nucleases may be used. For example, the serine protease may be chosen from the list comprising: proteinase K, OB protease.

[0066] Alternatively, the solution that the lignified plant tissue is exposed to in the method of the present invention may additionally comprise a cysteine protease to assist in the breakdown of cellular wall material in the lignified plant tissue and inactivate nucleases. Any cysteine protease that can perform at least this function may be used. For example, the cysteine protease may be chosen from the list comprising: brofasin, cysteine proteinase. In another aspect of the invention, the solution that the lignified plant tissue is exposed to in the method of the present invention may additionally comprise a broad specificity protease such as nagarse, [0067] The serine protease is preferably provided in the solution at between 0.1 % and 3.5%. Preferably, the serine protease is provided at between 0.5% and 3.0%, between 1 .0% and 2.4%, or between 1 .67% and 2.05%. Most preferably, the biological antioxidant is provided in the solution at 1.25%, 1.5%, 1.75%, 1 .86%, 1.95%, 2.0%, 2.15%, 2.25% or 2.5%.

[0068] The invention thus provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a serine protease and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0069] Alternatively, the solution that the lignified plant tissue is exposed to in the method of the present invention may additionally comprise two or more of the additional agents listed above. Therefore, the solution that the lignified plant tissue is exposed to may comprise (in addition to a cationic surfactant, boric acid and buffer) two or more of the following: a chelating agent, a salt, an excipient, a biological antioxidant, and a serine protease.

[0070] In one embodiment of the invention, the invention provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid, a chelating agent, a salt, an excipient, a biological antioxidant, a serine protease and buffer to extract the nucleic acids; b) purifying the nucleic acids extracted in (a).

[0071] The solution may comprise additional reagents such as RNAse, DNAse, or other reagents depending on the nature of the nucleic acids it is desired to extract.

[0072] Once the nucleic acids have been extracted from the lignified plant tissue, the technique of purification of those nucleic acids is dependent on the preference of the investigator. Any technique, from caesium chloride gradients to proprietary kits may be used in this step of the method. For example, the extracted nucleic acids may be purified with chloroform or a phenol-chloroform mixture, followed by precipitation an alcohol such as ethanol or isopropanol. Alternatively, the nucleic acids may be precipitated with sodium or ammonium acetate.

[0073] In a preferred embodiment, the extracted nucleic acids are purified using more than one technique sequentially. For example, the extracted nucleic acids may be purified using chloroform/isoamyl alcohol, followed by precipitation using isopropanol (preferably in the presence of glycogen), followed by purification using a proprietary kit. If precipitation is carried out with isopropanol, this step is preferably carried out in the presence of glycogen either overnight at about -20°C, or at -80° for between 0.5h and 2h, preferably between 0.5 and 1 h, most preferably for 1 h. Alternatively, the isopropanol precipitation in the presence of glycogen could be carried out overnight at -80°C, or for 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h,12h etc. The isopropanol precipitation could also be carried out at -20°C for 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 12h etc.

[0074] Preferably the nucleic acids are purified using the proprietary kit MO BIO Ultra Clean® 15 DNA Purification Kit [catalogue #:12100; MO B!O Laboratories, Inc., Carlsbad, USA]. However, other DNA extraction kits from a variety of sources can be used in the present method.

[0075] The nucleic acids extracted using the method of the present description may be DNA or RNA, from either the genome of the cells of the lignified plant tissue, or from chloroplasts or mitochondria.

[0076] In one aspect of the invention, the lignified plant tissue is exposed to a solution comprising: CTAB, Tris, NaCI, EDTA, Boric Acid, PVP K30, DTT.and Proteinase K.

[0077] In one embodiment of the invention, the invention provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: CTAB, Tris, NaCI, EDTA, Boric Acid, PVP K30, DTT, and Proteinase K; b) purifying the nucleic acids extracted in (a).

[0078] In one example of the invention, the lignified plant tissue is exposed to a solution comprising 55mM CTAB, 100mM Tris, 1.4M NaCI, 20mM EDTA, 1% (w/v) Boric Acid, 2% (w/v) PVP K30, 47mM DTT; and 1.86% Proteinase K.

[0079] The invention therefore provides a method for extracting plant nucleic acids from lignified plant tissue comprising the steps of: a) exposing the lignified plant tissue to a solution comprising: 55m CTAB, 100mM Tris, 1.4M NaCI, 20mM EDTA, 1 % (w/v) Boric Acid, 2% (w/v) PVP K30,47mM DTT, and 1.86% Proteinase K; b) purifying the nucleic acids extracted in (a).

[0080] Before the method of nucleic extraction is performed on the lignified plant tissue, the tissue may be exposed to a sample preparation method that breaks down at least part of the structure of the lignified plant tissue. For example, the tissue may be flaked, diced, cut or grated, and/or ground with a mortar and pestle, or exposed to a ball mill. The sample preparation method may be carried out at room temperatures, or may be performed at low temperatures such as in the presence of dry ice or liquid nitrogen. It is believed that iow temperatures such as those provided by exposing the tissue to liquid nitrogen may help preserve the nucleic acids during extraction.

[0081] In the present specification, the term "lignified plant tissue" is understood to encompass plant tissue comprising lignin. For example, the lignified plant tissue may be chosen from tissue such as stems, roots, seeds, seed coats, flower buds, leaf buds, wood and timber.

[0082] In the present specification, the term "trees" is understood to encompass bushes, shrubs and other sources of lignified plant tissue.

[0083] The terms "wood" and "timber" encompasses any hard, fibrous structural tissue containing lignin found in the stems and roots of trees and other woody plants. This includes heartwood and sapwood. The wood or timber maybe from hardwood trees or softwood trees. The terms "wood" and "timber" also encompasses manufactured or engineered wood including glued laminated timber (giulam), wood structural panels (including plywood, oriented strand board and composite panels), laminated veneer lumber (LVL) and other structural composite lumber (SCL) products, parallel strand lumber, l-joists, chipboard, hardboard, and medium-density fiberboard (MDF).

[0084] Preferably, the lignified plant tissue is wood or hard tissue such as stems, roots, seeds, seed coats, flower buds and leaf buds from sources such as trees, shrubs and bushes. Alternatively, the lignified plant tissue may be wood from dried sources such as dried wood, furniture, wooden barrels, wood pulp, plywood, laminated wood, lumber or logs.

Use of Extracted Nucleic Acids

[0085] It is known to use nucleic acid-based techniques to investigate natural variation within populations of plants to identify genus and species (DNA barcoding). However, difficulties in extracting suitable nucleic acid material from lignified plant tissue renders these processes less effective when applied to timber products, particularly dried timber products. Therefore, the present method of nucleic acid extraction may be used to assist in the determination of the genus and/or species of the source tree that the lignified plant tissue comes from.

[0086] There is therefore provide a method of determining the genus and/or species a sample of lignified plant tissue is from, comprising the steps of: a) extracting plant nucleic acids from the lignified plant tissue using a method comprising the following steps: i) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid and buffer to extract the nucleic acids; ii) purifying the nucleic acids extracted in (i); b) using the nucleic acids from (a) to develop a genetic profile for the lignified plant tissue sample; c) comparing the genetic profile from (b) with a library of genetic reference profiles.

[0087] It is known to use the natural variation within populations of plants to determine whether that tree comes from a specific geographical location or population of plants using nucleic acid-based techniques. The nucleic acid based techniques allow the determination of whether the lignified plant tissue comes from sustainable, or at least legal, sources and/or whether the lignified plant tissue comes from the geographical source the provider has declared it originates from. It also allows the determination of whether the lignified plant tissue comes from the declared log or stump that the provider has been certified the tissue to come from. Thus, there are a number of different genetic methods to be used for verification of source, both at the regional (phylogeography) and concession scales (population genetic assignment), and for tracking individual logs or timber products (DNA fingerprinting). However, difficulties in extracting suitable nucleic acid material from lignified plant tissue renders these processes less effective when applied to timber products, particularly dried timber products. Therefore, the present method of nucleic acid extraction may be used to assist in the determination of the geographical location of the source tree that the lignified plant tissue comes from.

[0088] There is therefore provide a method of determining the plant source of a sample of lignified plant tissue, comprising the following steps: a) extracting plant nucleic acids from the lignified plant tissue using a method comprising the following steps: i) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid and buffer to extract the nucleic acids; ii) purifying the nucleic acids extracted in (i); b) using the nucleic acids from (a) to develop a genetic profile for the lignified plant tissue sample; c) comparing the genetic profile from (b) with a library of genetic reference profiles. [0089] The genetic profile may be developed for a sample of lignified plant tissue by: a) extracting plant nucleic acids from lignified plant tissue using the method described above; and b) subjecting the isolated nucleic acids of step (a) to a procedure that allows determination of genomic, mitochondrial or chloroplast molecular markers.

[0090] The molecular markers may be chosen from the list comprising: randomly amplified polymorphic DNA (RAPD); amplified fragment length polymorphisms (AFLP); restriction fragment length polymorphisms (RFLP); cleaved amplified polymorphic sequences (CAPS), simple sequence repeats/microsatellites (SSR), single nucleotide repeats (SNP), single strand conformational polymorphisms (SSCP). Alternatively, other molecular markers known to the skilled reader may be used in the present method. The nucleic acids may be subject to sequencing, and/or PCR in its various forms (such as RT-PCR, Allele-specific PCR, Dial-out PCR, Asymmetric PCR, Intersequence-specific PCR, RACE LaNe RAGE etc).

[0091 ] There is therefore provided a method for developing a genetic profile for a sample of lignified plant tissue comprising the steps of: a) extracting plant nucleic acids from lignified plant tissue using a method comprising the following steps: i) exposing the lignified plant tissue to a solution comprising: a cationic surfactant, boric acid and buffer to extract the nucleic acids; ii) purifying the nucleic acids extracted in (i); b) subjecting the isolated nucleic acids of step (a) to a procedure chosen from the list comprising: PCR, RAPD analysis, AFLP analysis, CAPS analysis, RFLP analysis, SSR analysis, SNP analysis, SSCP analysis, sequencing.

[0092] When carrying out the method of: determining the genus and/or species a sample of lignified plant tissue is from; the plant source of a sample of lignified plant tissue; or developing a genetic profile for a sample of lignified plant tissue, the lignified plant tissue may be exposed to one or more of the following (in addition to a cation ic surfactant, boric acid and buffer): a chelating agent, a salt, an excipient, a biological antioxidant, or a serine protease.

[0093] In one aspect of the invention, the lignified plant tissue is exposed to a solution comprising: CTAB, Tris, NaCI, EDTA, Boric Acid, PVP K30, DTT, and Proteinase K.

[0094] In one example of the invention, the lignified plant tissue is exposed to a solution comprising: 55mM CTAB, 100mM Tris, 1 .4M NaCI, 20mM EDTA, 1 % (w/v) Boric Acid, 2% (w/v) PVP K30, 47mM DTT, and 1.86% Proteinase K.

Kits

[0095] The present description further provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, boric acid and buffer; b) instructions for use.

[0096] Preferably, the instructions for use designate that the extraction is carried out by exposing the lignified plant tissue to the extraction solution for between 0.5 and 16 hours, more preferably between 0.5 and 6 hours, between 1 h and 6h, between 2h and 6h, between 3h and 6h, between 4h and 6h, or between 4.5 and 5.5 hours. Preferably, the lignified plant tissue is exposed to the solution for about 4h, 4.25h, 4.5h, 5h, 5.25 or 5.5 hours to enable extraction of the nucleic acids.

[0097] Preferably, the instructions for use designate that the extraction is carried out by exposing the lignified plant tissue to the extraction solution at a temperature of between 40° and 65°, between 45° and 65° between 47° and 60°, between 49.5°C and 60.5°C, between 50°C and 57°C, or between 52°C and 56°C. Most preferably, the temperature of the solution during exposure of the lignified plant tissue to extract nucleic acids is about 52°C, 53°C, 54°C, 55°C, 56°C or 57°C.

[0098] The present description further provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, a chelating agent, boric acid and buffer; b) instructions for use.

[0099] The present description further provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, a salt, boric acid and buffer; b) instructions for use.

[00100] The present description further provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, an excipient, boric acid and buffer; b) instructions for use.

[00101] The present description further provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, a biological antioxidant, boric acid and buffer; b) instructions for use.

[00102] The present description further provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, a serine protease, boric acid and buffer; b) instructions for use.

[00103] The present description further provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) a cationic surfactant, boric acid, a buffer a chelating agent, a salt, an excipient, a biological antioxidant and a serine protease; b) instructions for use.

[00104] The present description further provides a kit for extracting plant nucleic acids from lignified plant tissue, said kit comprising: a) CTAB, Tris, NaCI, EDTA, Boric Acid, PVP K30, DTT, Proteinase K; b) instructions for use. General

[00105] Those skilled in the art will appreciate that the description herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the description includes all such variations and modifications. The description also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[00106] The present description is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the description as described herein.

[00107] The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.

[00108] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[00109] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. [00110] As used herein the term "derived" and "derived from" shall be taken to indicate that a specific integer may be obtained from a particular source albeit not necessarily directly from that source.

[001 1 1] As used herein, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a terpene synthase that catalyzes the formation of a terpene includes synthases that catalyze the productions of one or a plurality of terpenes.

[001 2] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[00113] Other than in the operating example, or where otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Hence "about 80 %" means "about 80 %" and also "80 %". At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[001 14] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the description are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value; however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements

[001 15] Other definitions for selected terms used herein may be found within the detailed description and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the description belongs. [00116] The following examples serve to more fully describe the manner of using the invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these methods in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

EXAMPLES

Example 1

Method

Sample preparation

1. Remove the outer part of the timber sample (removal of contaminated tissue)

2. Make small (curly) flakes with a clean scalpel

3. Fill a 2ml_ microcentrifuge tube about 1/2 to 2/3 of the volume with flakes

4. Add 2 clean steel balls

5. Freeze the sample in liquid nitrogen

6. Homogenate the sample with a Bead Beater (Retsch ^® mixer mill) at 17.5s ^"1 for 100 Seconds

7. Repeat steps 5 and 6 once (or twice) more until fine sawdust can be seen

8. Remove the steel balls

Extraction

1. Add 900μΙ_ BoTab buffer, 45μΙ_ DTT" (1 M) and 18μ[_ Proteinase K and mix well (vortex)

2. Incubate for 5 hours at 55°C (mixing occasionally)

Purification

1. Add 450μΙ_ Chioroform/lsoamyl alcohol (24:1 v/v) and mix (vortex) the sample

2. Incubate for 10 minutes at room temperature, mixing regularly

3. Centrifuge for 15 minutes at 16 200g (4°C)

4. Prepare a 1.5mL microcentrifuge tube with 225μΙ_ 3M NaOAc (pH 5.2) 5. Add 600μΙ_ of the aqueous phase to the NaOAc

6. Add 600μΙ_ Isopropanol and 0.5-4μΙ_ Glycogen

7. Incubate at -20°C overnight

8. Centrifuge for 5 minutes at 16 200g (4°C)

9. Discard the supernatant

10. Add 300μ[_ 70% EtOH

1 1. Centrifuge for 10 minutes at 16 200g (4°C)

12. Discard the supernatant

13. Add 25μΙ_ H ₂ O to the pellet (do not dry the sample)

^Λ DTT was substituted for β-Mercapto-ethanol because of its lower toxicity

Cleaning DNA using MO BIO UltraClean® 15 DNA Purification Kit

1. Add 90-100μ[_ ULTRA SALT (3 times the volume of the sample)

2. Add 5.8μί ULTRA BIND (homogenated) and mix well

3. Incubate for 5-10 minutes at room temperature

4. Centrifuge for 5 seconds at 10 OOOg (room temperature)

5. Discard the supernatant

6. Add 900μΙ_ ULTRA WASH and mix well

7. Centrifuge for 5 seconds at 10 OOOg (room temperature)

8. Discard the supernatant

9. Centrifuge for 5 seconds at 10 OOOg (room temperature)

10. Remove the supernatant

11.Add 23μΙ_ of H ₂ O and mix well

12. Incubate for ~5 minutes at room temperature

13. Centrifuge for 1 minute at 10 OOOg (room temperature) - the supernatant contains the DNA and can be diluted approximately 1 :1 0 NB: The pellet from step 13 is unstable, use supernatant quickly. Rerun centrifuge every two samples if extracting multiple samples of supernatant.

[MO BIO Ultra Clean ^® 15 DNA Purification Kit (catalogue #:12100); MO Laboratories, Inc. , Carlsbad, USA].

BoTab buffer (for 250mL):

Results

[00117] To evaluate the success of the mBO extraction method it has been compared with the most commonly used plant DNA extraction protocol (CTAB) as well as five selected commercial kits:

• Nucleon Phytopure Genomic DNA Extraction kit (NPh)

• DNeasy ^® Plant mini Kit (DNP)

• GenElute™ Plant Genomic DNA Miniprep Kit (GEP)

• innuPREP Plant DNA Kit (iPP)

• sbeadex mini plant kit (smp)

[00118] These kits were chosen because of their successful application by other groups working with plant material:

[00119] Samples of saw timber of four individuals for each of the four tree species Swietenia sp. (Sw1 , Sw2, Sw3, Sw4), Intsia sp. (In1 , In2, In3, In4), Quercus sp. (Qui , Qu2, Qu3, Qu4) and Larix sp. (La1 , La2, La3, La4) were used in the present test. [00120] For each sample, chloroplast fragments of a size between 120-300bp were amplified.

[00121] The success of the PCR and thus the DNA extraction was checked by agarose-gel electrophoresis. A strong PCR band was classified as 1 point, a weak PCR band by a 0.5 and no PCR product by 0 points. For each extraction method the sum of values was calculated as an overall indicator of the quality of the DNA extraction protocol. Three tests could not been done because of lack of chemicals

(*) ^■

[00122] The mBo extraction method was the most successful, with a success rate of 75%. The commercial kits, as well as the common CTAB protocol, all showed clearly lower success rates. The innuPREP Plant DNA Kit is the best commercial kit, but it only had a success rate of 56 %, all the others are below the 50 % rate.