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Aglaia Stellatopilosa

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Aglaia Stellatopilosa (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 SARAWAK, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, MALAYSIA [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 Borneo 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 AGLAIA 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 plant 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 Meliaceae, which is a medium sized family of woody trees 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 taxonomy 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.
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