(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date -» - n 8 March 2012 (08.03.2012) 2U12/U3U2U6 Al

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every CI2Q 1/68 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/MY20 11/000061 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, 31 May 201 1 (3 1.05.201 1) KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (25) Filing Language: English NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (26) Publication Language: English SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: PI 2010004090 30 August 2010 (30.08.2010) (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant (for all designated States except US): THE GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, GOVERNMENT OF THE STATE OF , ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, [MY/MY]; Level 17, Wisma Bapa TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Malaysia, Petra Jaya, Kuching, Sarawak 93502 (MY). EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventors; and SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (75) Inventors/ Applicants (for US only): YEO, Tiong Chia GW, ML, MR, NE, SN, TD, TG). [MY/MY]; c/o Sarawak Biodiversity Centre, KM 20, an Heights, Semengoh, Locked Bag No. 3032, Declarations under Rule 4.17 : 93990, Kuching, Sarawak, (MY). NG LING NAH, Be¬ — as to the identity of the inventor (Rule 4.17 (i)) linda [MY/MY]; c/o Sarawak Biodiversity Centre, KM 20, an Borneo Heights, Semengoh, Locked Bag No. — as to applicant's entitlement to apply for and be granted 3032, 93990, Kuching, Sarawak (MY). MARIANI BIN- a patent (Rule 4.1 7(H)) TI, Omarzuki@Marzuki [MY/MY]; c/o Sarawak Biodi — of inventorship (Rule 4.1 7(iv)) versity Centre, KM 20, Man Borneo Heights, Semengoh, Locked Bag No. 3032, 93990, Kuching, Sarawak (MY). Published: (74) Agent: CHUAH Jern Ern; ADVANZ FIDELIS Sdn. — with international search report (Art. 21(3)) Bhd., Suite 609, Block D, Phileo Damansara 1, No. 9, — with sequence listing part of description (Rule 5.2(a)) Man 16/1 1, 46350 Petaling Jaya, Selangor (MY).

(54) Title: METHOD FOR IDENTIFICATION OF STELLATOPILOSA

o o Figure 2 ©

(57) Abstract: A method for identification of Aglaia stellatopilosa is by identifying a set of nucleotides in the internal transcribed o spacer (ITS) regions of Aglaia stellatopilosa ribosomal DNA (rDNA), which only exists and belongs to said Aglaia stellatopilosa. This set of nucleotides, which serves as an identity of Aglaia stellatopilosa, is then used to identify and distinguish Aglaia s tel o latopilosa from other Aglaia species, by checking the presence of said set of nucleotides in the ITS regions of sample to be identified. METHOD FOR IDENTIFICATION OF AGLAIA STELLATOPILOSA

TECHNICAL FIELD OF THE INVENTION This invention is related to a method for identification of Aglaia stellatopilosa, and more particularly, to a method for molecular identification of Aglaia stellatopilosa by verifying the presence of a specific set of nucleotides in plant sample to be identified.

BACKGROUND OF THE INVENTION Aglaia stellatopilosa belongs to the tropical and subtropical angiosperm family of , which is a medium sized family of woody or shrubs of both hemispheres, comprising of 5 1 genera. Among those 5 1 genera, Aglaia is the largest genus of the Meliaceae family and it is estimated that there are about 100 known species of Aglaia in the world. As the morphologies of Aglaia species are very similar, there is a need to produce a method to identify and distinguish Aglaia stellatopilosa from other Aglaia species for the purposes of raw material collection, propagation and selection, and extraction and isolation of compound from said Aglaia stellatopilosa. Aglaia species has been known for its medicinal properties and are being used by indigenous people as traditional treatment for fever, fractures, parturition, and inflammation.

The conventional method for identifying and distinguishing between Aglaia species via classification based on morphology has sometimes led to controversial opinions, misidentifications and/or reclassifications, etc. These limitations could jeopardize the selection of the targeted species.. For one instance to a non-expert, it is sometimes difficult to distinguish the leaves of Aglaia glabriflora from those of Aglaia stellatopilosa as their morphologies are very similar. Hence, the conventional taxonomic identification approach is less reliable, of limited application by a small group of experts, resulting in need of an alternative method to identify the species. As a result, it is prudent to solve the aforesaid problems by providing a method for molecular identification of Aglaia stellatopilosa by verifying the presence of a specific set of nucleotides in plant sample to be identified.

SUMMARY OF THE PRESENT INVENTION It is an aim of this present invention to address the aforementioned problems by providing a method to identify and distinguish Aglaia stellatopilosa from other Aglaia species by identifying a specific set of nucleotides within the internal transcribed spacer (ITS) regions of Aglaia stellatopilosa ribosomal DNA (rDNA) sequence, which only exists and belongs to Aglaia stellatopilosa. This specific set of nucleotides, which serves as an identity of Aglaia stellatopilosa, is then used to identify and distinguish Aglaia stellatopilosa from other Aglaia species, by checking the presence of said set of nucleotides in the ITS regions of plant sample to be identified. In another words, said set of nucleotides enables species-level discrimination of Aglaia stellatopilosa from other Aglaia species. This method is developed for the purposes of facilitating raw material collection, propagation, selection and verification, and extraction and isolation of compound with medicinal properties from Aglaia stellatopilosa.

In an embodiment of the present invention, the specific set of nucleotides are T, T, A , T, T, T, T, A , T at respective specific positions of 185, 217, 310, 360, 376, 6 15, 714, 786, and 790 Aglaia stellatopilosa rDNA sequence, which is set forth as SEQ ID N0.1 . Note that the numerical positions of these 9 nucleotides are calculated from the first base of the forward primer, which is set forth as SEQ ID NO.2. Also note that the nucleotides at the positions of 185, 217, 310, 360, and 376 of SEQ ID N0.1 are located at the ITS 1 region o Aglaia stellatopilosa rDNA, whereas the nucleotides at the positions of 615, 714, 786, and 790 of SEQ ID N0.1 are located at the ITS 2 region f Aglaia stellatopilosa rDNA.

Briefly, the specific set of nucleotides as described above serving as the identity of Aglaia stellatopilosa is developed through experimental procedures, which comprise steps of extracting nucleic acids from Aglaia stellatopilosa; amplifying the ITS regions from the rDNA of Aglaia stellatopilosa via polymerase chain reaction with a pair of primers capable of flanking part of 18S rDNA and part of 26S rDNA of Aglaia stellatopilosa; purifying and sequencing the amplified product; blasting the ITS sequence of Aglaia stellatopilosa against National Center for Biotechnology Information (NCBI) database to identify species that scored high sequence similarity; and aligning the ITS sequence of Aglaia stellatopilosa against the ITS sequences of identified species to identify the nucleotides within the ITS 1 and ITS 2 regions that only exist and belong to Aglaia stellatopilosa.

In another embodiment of the present invention, the method for determining the exact identity of plant sample to be identified as Aglaia stellatopilosa or non-Aglaia stellatopilosa is firstly by extracting nucleic acids from said plant sample, wherein the nucleic acids are DNAs. Secondly, a pair of primers is added to the extracted nucleic acids, wherein said primers are capable of flanking part of 18S rDNA and part of 26S rDNA of said plant sample, which include the ITS regions. In a preferred embodiment, the primers are set forth as SEQ ID NO.2 and SEQ ID NO.3, or equivalents thereof, provided said equivalents are also capable of flanking part of 18S rDNA and part of 26S rDNA of said plant sample, which include the ITS regions. Subsequently, the flanked region is amplified by way of polymerase chain reaction. The amplified region is then purified and sequenced prior to verification. During the verification process, the ITS sequence of said plant sample is checked for the presence of the specific set of nucleotides in accordance with the present invention at the specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790. The identity of said plant sample is verified as Aglaia stellatopilosa when the nucleotides of said plant sample at the specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 are T, T, A , T, T, T, T, A , and T, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A, 1B, 1C, and 1D illustrate sequence alignments of ITS sequence of Aglaia stellatopilosa with ITS sequences of other Aglaia plant species; and Figure 2 illustrates the set of nucleotides and positions of each nucleotide in rDNA of Aglaia stellapilosa in accordance with the present invention.

SEQUENCE LISTING The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotides bases. Only one strand of nucleic acid sequence is shown, but the complementary strand is understood as included by reference to the displayed strand.

SEQ ID NO.1 shows the nucleic acid sequence of rDNA of Aglaia stellatopilosa, wherein the first 28 nucleotides are forward primer and the last 28 nucleotides are reverse complement sequence of reverse primer;

SEQ ID NO.2 shows the nucleic acid sequence of forward primer; and

SEQ ID NO. 3 shows the nucleic acid sequence of reverse primer.

DETAILED DESCRIPTION OF THE PRESENT INVENTION The above mentioned and other features and objects of this invention will become more apparent and better understood by reference to the following detailed description. It should be understood that the detailed description made known below is not intended to be exhaustive or limit the invention to the precise form disclosed as the invention may assume various alternative forms. On the contrary, the detailed description covers all the relevant modifications and alterations made to the present invention, unless the claims expressly state otherwise.

In accordance with the present invention, there is disclosed a method for identification of Aglaia stellatopilosa. In particular, the method is employed to identify and distinguish Aglaia stellatopilosa from other >Ag/a/'a species at molecular level. More particularly, the employed method is to identify and distinguish Aglaia stellatopilosa from other Aglaia species by identifying a specific set of nucleotides that only exists and belongs to Aglaia stellatopilosa. This specific set of nucleotides, which serves as an identity of Aglaia stellatopilosa, is then used to identify and distinguish Aglaia stellatopilosa from other Aglaia species, by checking the presence of said set of nucleotides in plant sample to be identified. The identity of the plant sample is confirmed as

Aglaia stellatopilosa when the presence of the specific set of nucleotides is confirmed in said plant sample. This method is developed for the purposes of facilitating raw material collection, propagation and selection, and extraction and isolation of compound with medicinal properties from Aglaia stellatopilosa.

The method of the present invention involves the usage of ITS sequences of rDNA as said sequences are highly conserved among species. Basically, ITS region has been widely employed in taxonomy and molecular phytogeny especially for fungi, bacteria, and . The ITS region of rDNA codes for a piece of non-functional RNA situated between structural ribosomal RNA

(rRNA). In general, the rRNA is made up of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2 , and 26S rRNA. For Aglaia species, the length of ITS 1, 5.8S rRNA, and ITS 2 regions varies from 650 to 662 base pair (bp). Some of the advantages of using ITS are: 1) easy to amplify due to high copy number (500 - 40,000) of rDNA genes; 2) high degree of interspecific variation but low degree of intraspecific variation which is useful for comparisons among closely related species; and 3) readily available universal primers designed from the highly conserved regions flanking the ITS region.

The specific set of nucleotides in accordance with the present invention that serves as the identity of Aglaia stellatopilosa comprises T , T , A, T , T , T , T , A , and T at the respective specific positions of 185, 217, 310, 360, 376, 615,

714, 786, and 790 o Aglaia stellatopilosa rDNA sequence, which is set forth as SEQ ID N0.1 . Note that the nucleotides at the positions of 185, 217, 310, 360, and 376 of SEQ ID N0.1 are located at the ITS 1 region of Aglaia stellatopilosa rDNA, whereas the nucleotides at the positions of 615, 714, 786, and 790 of SEQ ID N0.1 are located at the ITS 2 region of Aglaia stellatopilosa rDNA. Experimental procedures for obtaining the specific set of nucleotides serving as Aglaia stellatopilosa identity

The specific set of nucleotides in accordance with the present invention is obtained through experimental procedures, which comprise steps of extracting nucleic acids from Aglaia stellatopilosa; amplifying the ITS regions from the rDNA of Aglaia stellatopilosa via polymerase chain reaction with a pair of primers capable of flanking part of 18S rDNA and part of 26S rDNA of Aglaia stellatopilosa; purifying and sequencing the amplified product; blasting the ITS sequence of Aglaia stellatopilosa against NCBI database to identify species that scored high sequence similarity; and aligning the ITS sequence of Aglaia stellatopilosa against the ITS sequences of identified species to identify the nucleotides within the ITS 1 and ITS 2 regions that only exist and belong to Aglaia stellatopilosa.

Initially, nucleic acids are extracted from freshly collected leaves of Aglaia stellatopilosa following the procedures of Edwards et al. (1991). A disc of the leaf is pinched out using the lid of sterile tube and macerated for 15 seconds at room temperature using disposable grinders. Thereafter, 400 µ Ι of extraction buffer, which comprises of 200 mM Tris HCI pH 7.5, 250 mM NaCI, 25 mM EDTA, and 0.5% SDS, is added into the macerated sample and vortexed for 5 seconds. The mixture is left to stand at room temperature for 1 hour prior to centrifugation at 13000 revolutions per minute (rpm) for 1 minute. Subsequently, 300 µ Ι of the supernatant is transferred into a fresh tube containing 300 µ Ι isopropanol and incubated at room temperature for 2 minutes. Consequently, the mixture is subjected to centrifugation at 13000 rpm for 5 minutes. The nucleic acids, which are in the form of pellet at this point of time, are vacuum dried and dissolved in 100 µ Ι 1X Tris-EDTA (TE) buffer.

Next, the extracted nucleic acids are subjected to amplification process by way of polymerase chain reaction (PCR), specifically to amplify the ITS regions of Aglaia stellatopilosa. Prior to that, the extracted nucleic acids, which is dissolved in 100 µ Ι 1X TE buffer, has to be mixed with several components and reagents to form a PCR reaction mix. The complete PCR reaction mix is presented in the table below:

Table 1: PCR reaction mix

A pair of primers is added into the PCR reaction mix, wherein one of the primers is being a forward primer and the other being a reverse primer (as indicated in Table 1). The forward primer is set forth as SEQ ID NO.2 and binds to part of the 18S of Aglaia stellatopilosa rDNA, whereas the reverse primer is set forth as SEQ ID NO.3 and binds to part of the 26S of Aglaia stellatopilosa rDNA. However, the uses of other primers are possible, provided that the other primers are capable of flanking the aforesaid region.

Thereafter, the PCR reaction mix undergoes the amplification process using BioRad MyCycler™ thermal cycler under the cycling condition which is presented in the table below: Table 2 : PCR cycling condition

The product of amplification process is then purified using the lllustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). Briefly, the purification process consists of four main steps, which are sample capture, sample binding, wash and dry, and elution. During the sample capture step, for up to 100 µ Ι sample, 500 µ Ι capture buffer is added and mixed thoroughly, wherein said capture buffer contains a pH indicator that changes colour at various pH levels to identify whether the capture buffer plus sample mix is at the optimal pH for DNA to bind to the silica membrane. If the pH indicator is a yellow or pale orange colour, capture buffer-sample mix is at optimal pH for efficient DNA binding to the silica membrane, whereas if the pH indicator is a dark pink or red colour, the pH of the capture buffer-sample mix is too high to achieve efficient DNA adsorption to the silica membrane. During this stage, proteins are denatured and/or agarose is dissolved. Subsequently, the capture buffer-sample mix is introduced into the assembled GFX MicroSpin™ column and collection tube and spun at 16000 x g for 30 seconds. The collected flow through at the collection tube is discarded. During this stage, the DNA binds to the membrane. Thereafter, 500 µ Ι wash buffer is added into the assembled GFX MicroSpin™ column and collection tube and spun again at 16000 x g for 30 seconds. This time around, the collection tube is discarded and the GFX MicroSpin™ column is transferred to a clean 1.5 ml DNase-free microcentrifuge tube. During this stage, the combined washing-drying step removes salts and other contaminants from the membrane bound DNA. Consequently, 30 µ Ι elution buffer is added onto the membrane and left to stand for 60 seconds at room temperature. Next, the DNA is eluted by centrifuging at 16000 x g for 60 seconds. The flow through, which is the purified DNA sample, is retained and stored at -20°C.

Prior to sequencing, the purified product is subjected to 2 main processes, namely, cycle sequencing and purification of the product. During the first stage, the reaction mixtures for the forward and reverse sequencing reactions are prepared by adding all the reagents as shown in Table 3 and Table 4. Thereafter, each prepared reaction mixture is added into each well of optical 96 wells reaction plate. The cycle sequencing is performed by a thermal cycler such as GeneAmp ® PCR System 9700 with the cycling conditions stated in Table 5.

Table 3: Composition of the forward sequencing reaction Reagents Volume (µΙ) Final concentration Autoclaved ultra pure water 5.43 - BigDye® Terminator v3.1 Ready Reaction 0.5 0.1 25X Premix (2.5X) BigDye® Terminator v3.1 Sequencing 1.75 0.875X Buffer (5X) Forward primer (10 pmol/ µ Ι) 0.32 3.2 pmol DNA template 2.00 - Final volume 10 - Table 4: Composition of the reverse sequencing reaction

Table 5: The programme setting of GeneAmp PCR System 9700 for cycle sequencing

The second stage of sequencing process involves purification of the cycle sequencing product using ethanol/EDTA precipitation. Initially, the optical 96 wells reaction plate is removed from the thermal cycler and briefly spun. 2.5 µ Ι of 125 mM EDTA is added into each well containing sample and allowed to settle to the bottom of the well to ensure it is well mixing. Later, 25 µ Ι of absolute ethanol is added to each well. The contents of each well are then pipetted up and down three times to ensure the ethanol is well mixed with the sample. Thereafter, the plate is incubated at room temperature for 15 minutes prior to centrifugation at 3220 x g for 20 minutes at 4°C. After that, the plate is inverted and spun up to 190 x g for 1 minute at 4°C. Subsequently, 30 µ Ι of 70% ethanol is added to each well and proper mixing is done by pipetting up and down three times the contents of each well, before subjecting the plate to another spinning process at 1650 x g for 15 minutes at 4°C. The plate is then inverted once again and spun up to 190 x g for 1 minute at 4°C. Next, the plate is dried by using Eppendorf Concentrator 5301 for 10 minutes and further heated at 95°C for 5 minutes by using the thermal cycler with the lid open. Then, 10 µ Ι Hi-Di™ Formamide is added into each well. The plate is then fed into Applied Biosystem 3130x/ Genetic Analyzer for running the sequencing process.

From then on, the sequenced region, which is set forth as SEQ ID N0.1 , is then blasted against NCBI database to identify the top 5 species that scored high sequence similarity to Aglaia stellatopilosa. From the blast results, the top 5 species are (97% match), Aglaia silvesths (97% match), Aglaia forbesii (96% match), (95% match), and Aglaia korthalsii (95% match). The detailed blast results are tabulated below:

Table 6: Blast results showing other Algaia species with high sequence similarity to Aglaia stellatopilosa Accession Description Max Total Query E Max Score Score coverage value ident AY695556.1 Aglaia perviridis 1265 1265 0.82 0 0.97 voucher HG13 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1 and 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence Table 6 : continued Accession Description Max Total Query E Max Score Score coverage value ident AY695562.1 1251 1251 0.82 0 0.97 voucher HG718 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 26S ribosomal RNA gene, partial sequence AY695546.1 Aglaia forbesii 1418 1418 0.93 0 0.96 voucher HG538 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 26S ribosomal RNA gene, partial sequence Table 6: continued Accession Description Max Total Query E Max Score Score coverage value ident AY695561.1 Aglaia silvestris 1249 1249 0.82 0 0.96 voucher HG679 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 26S ribosomal RNA gene, partial sequence AY695553.1 Aglaia odoratissima 1368 1368 0.93 0 0.95 voucher HG623 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 26S ribosomal RNA gene, partial sequence Table 6 : continued

Referring now to Figure 1A, Figure 1B, Figure 1C, Figure 1D, and Figure 2 . The ITS sequences of these species are aligned against the ITS sequence of Aglaia stellatopilosa and analyzed using BioEdit Sequence Alignment

software. From the alignment analysis, nucleotides that are specific to Aglaia stellatopilosa are identified. The analysis reveals a specific set of nucleotides

consisting of 9 nucleotides having a unique pattern that are specific to Aglaia stellatopilosa, wherein the nucleotides are T , T , A, T , T , T , T , A, and T at respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 of SEQ ID N0.1 . The numerical positions of these 9 nucleotides are calculated from the first base of the forward primer, which is set forth as SEQ ID NO.2. However, these numberings may be affected by alignment gaps due to extra nucleotides present in other species. For instance, referring now to Figure 1A, note that there are two alignment gaps at the positions of 237, and 238. This alignment gaps are created by extra nucleotides in Aglaia forbesii, which in turn causes a shift in numbering of Aglaia stellatopilosa. Therefore, the actual nucleotides of Aglaia stellatopilosa at the positions of 360 and 376 are shifted 2 positions to 362 and 378, respectively, with reference to Figure 1B. Referring now to Figure 1C, also note that there is one alignment gap at the position of 595. This alignment gap is due to an extra nucleotide in Aglaia pervindis, Aglaia odoratissima, and Aglaia korthalsii, which in turn causes a further shift in numbering of Aglaia stellatopilosa. Therefore the actual nucleotides of Aglaia stellatopilosa at the positions of 615, 714, 786, and 790 are shifted 3 positions (taking in the account of the previous alignment gaps at the positions of 237 and 238) to 618, 717, 789, and 793, respectively, with reference to Figure 1C and Figure 1D.

Screening for the specific set of nucleotides in accordance with the present invention in Aglaia and non-Aglaia plant species

The identified specific set of nucleotides is tested for validation to ensure that said set of nucleotides is only present in Aglaia stellatopilosa, therefore enabling said set of nucleotides to serve as the unique identity for Aglaia stellatopilosa. The validation process involves the step of checking the presence of said set of nucleotides at the specific positions within the ITS regions of other Aglaia and non-Aglaia species.

The ITS sequence of Aglaia stellatopilosa is compared with ITS sequences of 37 Aglaia plant species and 50 non-Aglaia plant species, which are available in the GenBank database. The comparison is made specifically at the positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 of Aglaia stellatopilosa. The sequences obtained from said database are truncated so as to contain only the ITS 1, 5.8S rDNA, and ITS 2 for ease of comparison. Notably, none of these species obtained from the database possesses the specific set of nucleotides of T , T , A , T, T , T , T , A, and T at the respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790. The comparison results for 37 Aglaia plant species and 50 o -Ag aia plant

species are shown in Table 7 and Table 8. Additionally, the alignment of these

sequences shows length variation and little similarity across the species,

suggesting that said set of nucleotides according to the present invention are species-specific to Aglaia stellatopilosa.

Table 7 : Presence of specific nucleotides in 38 Aglaia species

Species 185 217 310 360 376 615 714 786 790 / 9

name (T) (T) (A) (T) (T) (T) (T) (A) (T) Aglaia 9 stellatopilosa Aglaia 0 forbesii Aglaia 1 odoratissima Aglaia 0 korthalsii Aglaia 0 perviridis Aglaia 0 silvestris Aglaia 1 archboldiana Aglaia 1 argentea Aglaia 1 australiensis Table 7 : continued No Species 185 217 310 360 376 615 714 786 790 / 9 name (T) (T) (A) (T) (T) (T) (T) (A) (T) 10 Aglaia 1 basiphylla

11 Aglaia 1 chittagonga 12 Aglaia 2 coriacea 13 Aglaia 2 crassinervia 14 Aglaia 2 cucullata 15 ------3 16 Aglaia ✓ 1 elaeagnoldea 17 Aglaia 1 elliptica 18 Aglaia 0 eximia 19 Aglaia 2 exstipulata 20 Aglaia ✓ 1 glabrata 2 1 Aglaia 1 grandis 22 ------0 23 Aglaia 1 leucophylla 24 Aglaia 1 macrocarpa 25 Aglaia 2 meridionalis 26 Aglaia 1 odorata Table 7 : continued No Species 185 217 310 360 376 615 714 786 790 / 9 name (T) (T) (A) (T) (T) (T) (T) (A) (T) 27 Aglaia 2 oligophylla 28 Aglaia 1 pachyphylla 29 Aglaia ✓ 2 rugulosa 30 Aglaia 1 samoensis 31 Aglaia 2 sapindina 32 Aglaia 2 sexipetala 33 Aglaia 2 simplicifolia 34 Aglaia 0 spectabilis 35 Aglaia 2 tenuicaulis 36 Aglaia 0 teysmanniana 37 Aglaia 3 tomentosa 38 Aglaia 1 vitiensis Table 8 : Presence of specific nucleotides in 50 other plant species o Species 185 217 310 360 376 615 714 786 790 / 9 name (T) (T) (A) (T) (T) (T) (T) (A) (T) Alhagi 6 maurorum Ardisia 6 crenata Avena 2 atlantica Avena 2 murphyi Brintonia 5 discoidea Ebenus 4 laguroides Eversmannia 4 subspinosa Faguetia 5 falcata Glechoma ✓ 2 hederacea Halimodendron ✓ 5 halodendron Hedysarum ✓ 4 wrightianum Helichrysum 4 lanceolatum Heracleum 3 austriacum Heracleum 2 inexpectatum Table 8: continued No Species 185 217 310 360 376 615 714 786 790 / 9 name (T) (T) (A) (T) (T) (T) (T) (A) (T) 15 Heracleum 3 lanatum 16 Heracleum 3 persicum 17 Heracleum 2 transcaucasi cum 18 Lamium 0 amplexicaule 19 Leucogenes 4 tarahaoa 20 Liparis ✓ 5 auriculata 21 Liparis 3 cordifolia 22 Liparis ✓ 3 krameri 23 Liparis 2 nervosa 24 Malabaila ✓ 2 aurea 25 Onobrychis ✓ 5 altissima 26 Onobrychis 5 arnacantha 27 Onobrychis ✓ 5 laxiflora 28 Operculicarya V 2 decaryi 29 Orobanche 3 minor Table 8 : continued No Species 185 217 310 360 376 615 714 786 790 / 9 name (T) (T) (A) (T) (T) (T) (T) (A) (T) 3 0 Pinguicula 0 vulgaris

3 1 Plagiochila 2 arbuscula 3 2 Platanus 3 occidentalis

3 3 Potamogeton 0 distinctus 34 Poupartia 1 minor

3 5 Poupartiopsis 1 spondiocarpus 3 6 Protorhus 3 longifolia

3 7 Rhus 2 pendulina

3 8 Rhus 4 taratana

39 Rhus 3 thouarsii

4 0 Seseli 1 diffusum 4 1 Solidago ✓ ✓ 5 arguta 4 2 Solidago ✓ 5 caesia

4 3 Solidago ✓ 5 gigantea

4 4 Solidago 5 riddellii Table 8: continued

Validation for the specific set of nucleotides in accordance with the present invention in randomly collected Aglaia plant species

Over 5 1 Aglaia samples are randomly collected. The ITS sequence of each sample is obtained through the experimental procedures as described above, which include extracting nucleic acids from each sample to be identified; adding a pair of primers to the extracted nucleic acids; amplifying the flanked region; purifying and sequencing the amplified product.

The ITS sequences of collected Aglaia samples are then used to distinguish whether they are Aglaia stellatopilosa or -Aglaia stellatopilosa, by checking the presence of the specific set of nucleotides according to the present invention in said ITS sequences of collected Aglaia samples. The identity of the Aglaia sample is confirmed as Aglaia stellatopilosa when the nucleotides at the positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 of ITS sequence of said Aglaia sample are T, T, A , T, T, T, T, A , and T, respectively. Out of the 5 1 Aglaia samples, 36 Aglaia samples comprise the nucleotides T, T, A , T , T, T , T , A, and T at respective specific positions of 185, 217, 310, 360,

376, 615, 714, 786, and 790. The results of the identification of 5 1 Aglaia

samples are presented in Table 9.

Additionally, the identification results are also compared against taxonomic

identification results to determine the degree of concordance. There is a 100% rate of concordance between the presence of the set of nucleotides and the taxonomic identification of Aglaia stellatopilosa i.e. 36/36. The results are

merged in Table 9 and summarized in Table 10.

Table 9 : Identification of 5 1 Aglaia samples collected from various areas in the State of Sarawak, Malaysia

Specific positions (nucleotides) Identification method

Sample 185 217 310 360 376 615 714 786 790 Set of Taxonomic No. (T) (T) (A) (T) (T) (T) (T) (A) (T) nucleotides 1 ✓ Aglaia Aglaia stellatopilosa stellatopilosa

2 ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa

3 ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa

4 ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa

5 ✓ V ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa

6 ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa

7 ✓ Aglaia Aglaia stellatopilosa stellatopilosa

8 ✓ Aglaia Aglaia stellatopilosa stellatopilosa

9 ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa

10 ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa Table 9 : continued Specific positions (nucleotides) Identification method Sam pi 185 217 310 360 376 615 714 786 790 Set of Taxonomic e No. (T) (T) (A) (T) (T) (T) (T) (A) (T) nucleotides 1 1 ✓ Aglaia Aglaia stellatopilosa stellatopilosa- 12 Aglaia sp. Aglaia glabriflora 13 Aglaia sp. Aglaia glabriflora 14 ✓ ✓ Aglaia sp. Aglaia glabriflora 15 ✓ ✓ ✓ Aglaia sp. Aglaia glabriflora 16 ✓ Aglaia sp. Aglaia glabriflora 17 Aglaia sp. Aglaia glabriflora 18 ✓ /a /a sp. Aglaia glabriflora 19 ✓ ✓ Aglaia sp. Aglaia glabriflora 2 0 ✓ ✓ ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 2 1 ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 2 2 ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa

2 3 ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 2 4 Aglaia Aglaia stellatopilosa stellatopilosa

2 5 ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa

2 6 ✓ Aglaia Aglaia stellatopilosa stellatopilosa 2 7 ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa Table 9 : continued

Specific positions (nucleotides) Identification method Sample 185 217 310 360 376 615 714 786 790 Set of Taxonomic No. (T) (T) (A) (T) (T) (T) (T) (A) (T) nucleotides 28 ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 29 ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 30 V ✓ V Aglaia Aglaia stellatopilosa stellatopilosa 3 1 ✓ ✓ V ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 32 Aglaia sp. Aglaia silvestris 33 ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 34 ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 35 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 36 ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 37 ✓ ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 38 ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 39 ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 40 ✓ V Aglaia Aglaia stellatopilosa stellatopilosa 4 1 ✓ ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 42 ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 43 ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa 44 ✓ ✓ ✓ ✓ ✓ Aglaia Aglaia stellatopilosa stellatopilosa Table 9: continued

Table 10: Comparison between the results of samples identified based on the set of nucleotides in accordance with the present invention and results of samples identified based on taxonomy Method for identification of Aglaia stellatopilosa by utilizing the specific set of nucleotides in accordance with the present invention

Aglaia plant sample with vague identity is initially collected. The nucleic acids of said plant sample, which are DNAs, are then extracted, following the extraction procedures as described above. Thereafter, a pair of primers is added to the extracted nucleic acids, wherein the primers are capable of flanking part of 18S rDNA and part of 26S rDNA of said plant sample, which include the ITS regions. In an embodiment of the present invention, one of the primers is being the forward primer which is set forth as SEQ ID NO.2, whereas the other primer is being the reverse primer which is set forth as SEQ ID NO.3. In another embodiment of the present invention, other equivalent pair of primers can be employed for this purpose, provided that said equivalent pair of primers is also capable of flanking the aforesaid region. Subsequently, the flanked region of said plant sample is amplified, following the amplification procedures as described above. Consequently, the amplified region of said plant sample is purified and sequenced, following the purification and sequencing procedures as described above, in order to obtain the sequence of the rDNA of said plant sample, specifically the ITS sequence.

The exact identity of said plant sample can be determined as Aglaia stellatopilosa or non-Aglaia stellatopilosa by checking the presence of the specific set of nucleotides in accordance with the present invention in the ITS sequence of said plant sample, wherein the specific set of nucleotides, which serves as the identity of Aglaia stellatopilosa, is T, T, A , T, T , T , T, A , and T at respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 of Aglaia stellatopilosa rDNA sequence as set forth as SEQ ID NO. . The identity of the said plant sample is confirmed and verified as Aglaia stellatopilosa when the nucleotides of the ITS sequence of said plant sample are T, T, A , T, T, T, T, A, and T at respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790. l/WE CLAIM . A method for identification of Aglaia stellatopilosa, by utilizing a set of nucleotides, characterized in that the set of nucleotides is T, T, A , T, T, T, T, A , and T at respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 of Aglaia stellatopilosa rDNA sequence, which is set forth as SEQ D N0.1 .

2. A method in accordance with claim 1, wherein the method comprises at least the step of checking the presence of the set of nucleotides in plant sample to be identified, wherein the set of nucleotides is T, T, A , T, T, T, T, A, and T at respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 of Aglaia stellatopilosa rDNA sequence, which is set forth as SEQ ID NO.1 .

3. A method in accordance with claim 1, wherein the method further comprises the steps of: (i) extracting nucleic acids from plant sample to be identified; (ii) adding a pair of primers to the extracted nucleic acids, said primers flanking part of 8S rDNA and part of 26S rDNA of said plant sample; (iii) amplifying the flanked region of said plant sample; (iv) purifying the amplified region of said plant sample; (v) sequencing the purified region of said plant sample; and (vi) checking the presence of the set of nucleotides in the ITS sequence of said plant sample, wherein the set of nucleotides is T, T, A , T, T, T, T, A , and T at respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 of Aglaia stellatopilosa rDNA sequence as set forth as SEQ ID NO.1 ; wherein the plant sample is Aglaia stellatopilosa when the nucleotides of the ITS sequence of said plant sample are T, T, A , T, T , T, T, A , and T at the respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790. 4 . A method in accordance with claim 3, wherein the nucleic acids are DNAs.

5. A method in accordance with claim 3, wherein the amplifying procedure is polymerase chain reaction.

6. A method in accordance with claim 3 , wherein the pair of primers is set forth as SEQ ID NO.2 and SEQ ID NO.3, or equivalents thereof, provided said equivalents specifically flank part of the 18S rDNA and part of the 26S rDNA of said plant sample.

7. A method in accordance with any one of claims 1 and 3, wherein the nucleotides of SEQ ID N0.1 at the positions of 185, 217, 310, 360, and 376 are in the ITS 1 region of rDNA of Aglaia stellatopilosa.

8. A method in accordance with any one of claims 1 and 3, wherein the nucleotides of SEQ ID N0.1 at the positions of 615, 714, 786, and 790 are in the ITS 2 region of rDNA o Aglaia stellatopilosa.

9. A method for identification of Aglaia stellatopilosa for the purposes of facilitating raw material collection, propagation, selection and verification, and extraction and isolation of compound from said Aglaia stellatopilosa, by utilizing a set of nucleotides, characterized in that the set of nucleotides is T , T , A, T , T , T , T , A, and T at respective specific positions of 185, 217, 310, 360,

376, 615, 714, 786, and 790 of Aglaia stellatopilosa rDNA sequence, which is set forth as SEQ ID N0.1 .

10. A method in accordance with claim 9, wherein the method comprises at least the step of checking the presence of the set of nucleotides in plant sample to be identified, wherein the set of nucleotides is T , T , A, T , T , T , T , A , and T at respective specific positions of 185, 217, 310, 360, 376, 615, 714,

786, and 790 of Aglaia stellatopilosa rDNA sequence, which is set forth as SEQ ID NO.1 . 11. A method in accordance with claim 9, wherein the method further comprises the steps of: (i) extracting nucleic acids from plant sample to be identified; (ii) adding a pair of primers to the extracted nucleic acids, said primers flanking part of 18S rDNA and part of 26S rDNA of said plant sample; (iii) amplifying the flanked region of said plant sample; (iv) purifying the amplified region of said plant sample; (v) sequencing the purified region of said plant sample; and (vi) checking the presence of the set of nucleotides in the ITS sequence of said plant sample, wherein the set of nucleotides is T, T, A, T, T, T, T, A , and T at respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790 of Aglaia stellatopilosa rDNA sequence as set forth as SEQ ID NO.1 ; wherein the plant sample is Aglaia stellatopilosa when the nucleotides of the ITS sequence of said plant sample are T, T, A , T, T, T, T, A , and T at the respective specific positions of 185, 217, 310, 360, 376, 615, 714, 786, and 790.

12. A method in accordance with claim 11, wherein the nucleic acids are DNAs.

13. A method in accordance with claim 11, wherein the amplifying procedure is polymerase chain reaction.

14. A method in accordance with claim 11, wherein the pair of primers is set forth as SEQ ID NO.2 and SEQ ID NO.3, or equivalents thereof, provided said equivalents specifically flank part of 18S rDNA and part of 26S rDNA of said plant sample.

15. A method in accordance with any one of claims 9 and 11, wherein the nucleotides of SEQ ID N0.1 at the positions of 185, 217, 310, 360, and 376 are in the ITS 1 region of rDNA of Aglaia stellatopilosa. 16. A method in accordance with any one of claims 9 and 11, wherein the nucleotides of SEQ ID N0.1 at the positions of 615, 714, 786, and 790 are in the ITS 2 region of rDNA of Aglaia stellatopilosa.

AL H REPOR Internat ona app cation No. PCT/MY20 11/000061

A. CLASSIFICATION OF SUBJECT MATTER Int. Cl. C12Q 1/68 (2006.01) ·

According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) MEDLINE, CAPLUS, BIOSIS, AGRICOLA, EPODOC, WPI (Aglaia, mahogany, Meliaceae, phylogeny, taxonomy, systematics, Bayesian, parsimony, internal transcribed spacer, and like terms); GENOMEQUEST (SEQ ID NOs: 1-3)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

MUELLNER, A.N. et al., "Aglaia (Meliaceae): An evaluation of taxonomic concepts based on DNA data and secondary metabolites" American Journal of Botany, 2005, vol. 92, no. 3, pages 534-543 See abstract

MUELLNER, A.N. et al:, "An evaluation of tribes and generic relationships in Melioideae (Meliaceae) based on nuclear ITS ribosomal DNA" Taxon, 2008, vol. 57, no. 1, pages 98-108 · See abstract

GRIMM, G.W. et al., "A Nuclear Ribosomal DNA Phylogeny of Acer Inferred with Maximum Likelihood, Splits Graphs, and Motif Analysis of 606 Sequences" Evolutionary Bioinformatics Online, 2006, vol. 2, pages 7-22 See abstract

X Further documents are listed in the continuation of Box C See patent family annex

* Special categories of cited documents: "A" document defining the general state of the art which is not "T" later document published after the international filing date or priority date and not in considered to be of particular relevance conflict with the application but cited to understand the principle'or theory underlying the invention "E" earlier application or patent but published on or after the "X" document of particular relevance; the claimed invention cannot be considered novel international filing date or cannot be considered to involve an inventive step when the document is taken alone "L" document which may throw doubts on priority claim(s) or "Y" document of particular relevance; the claimed invention cannot be considered to which is cited to establish the publication date of another involve an inventive step when the document is combined with one or more other citation or other special reason (as specified) such documents, such combination being obvious to a person skilled in the art "O" document referring to an oral disclosure, use, exhibition "&" document member of the same patent family or other means "P" document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 29 July 2011 02 August2011 Name and mailing address of the ISA AU Authorized officer JOHN SHAW AUSTRALIAN PATENT OFFICE PO BOX 200, WODEN ACT 2606, AUSTRALIA AUSTRALIAN PATENT OFFICE E-mail address: [email protected] (ISO 9001 Quality. Certified Service) Facsimile No. +61 2 6283 7999 Telephone No : +61 2 6283 2545

Form PCT/ISA/210 (second sheet) (July 2009) nternat ona app cat on o. PCT /MY20 11/00006 1 C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

US 5876977 A (WANG et al.) 02 March 1999 See claim 1

Form PCT/ISA/210 (continuation of second sheet) (July 2009) INTERNATIONAL SEARCH REP RT International application No. PCT /MY20 11/000061

Box No. I Nucleotide and/or amino acid sequence(s) (Continuation of item l.c of the first sheet)

1. With regard to any nucleotide and/or amino acid sequence disclosed in the international application, the international search was carried out on the basis of a sequence listing filed or furnished:

a. (means)

on paper

I in electronic form

b. (time)

I I in the international application as filed

together with the international application in electronic form

I I subsequently to this Authority for the purposes of search

. [ ] In addition, in the case that more than one version or copy of a sequence listing has beeii filed or furnished, the required statements that the information in the subsequent or additional copies is identical to that in the application as filed or does not go beyond the application as filed, as appropriate, were furnished.

3. Additional comments:

Form PCT/ISA/210 (continuation of first sheet (1)) (July 2009) Information on patent family members PCT/MY201 1/000061

This Annex lists the known "A" publication level patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.

Patent Document Cited in Patent Family Member Search Report

US 5876977 CN 11971 16 US 6309840 US 2002146705

Due to data integration issues this family listing may not include 10 digit Australian applications filed since May 2001.

END OF ANNEX

Form PCT ISA 2 0 (patent family annex) (July 2009)