Molecular Authentication of Indigenous Species Cinnamomum ...

Research ArticleMolecular Authentication of Cinnamomum osmophloeum vs. C. burmannii. 193Molecular Authentication of Indigenous SpeciesCinnamomum osmophloeum vs. Exotic Species C.burmannii by a Multiplex-PCR MethodShr-Wei Shiue 1 , Shih-Chieh Lee 2 and Kuen-Yih Ho 1 *1 Department of Forestry and Natural Resources, National ChiayiUniversity, Chiayi 60004, Taiwan ROC2 Department of BioIndustry Technology, Da-Yeh University, ChanghuaHsien 51591, Taiwan ROCABSTRACTMorphologically the exotic plant speciesCinnamomum burmannii looks like indigenousplant species C. osmophloeum in Taiwan.Discrimination between these two species istherefore should not depend solely on theirmorphological characteristics. In the presentstudy, a set of multiplex-PCR primers wasdeveloped to discriminate one species from theother, based on the single nucleotidepolymorphism in the rDNA internal transcribedspacer 2 (ITS2) region of these two species. An125-bp amplicon specific to C. osmophloeum and a204-bp amplicon specific to C. burmannii weresuccessfully generated, and no intra-speciesdifference between individual plants of eitherspecies was observed.Key words: Molecular authentication, Internaltranscribed spacer, Multiplex-PCR.以 複 合 式 聚 合 脢 連 鎖 反 應 進 行 原 生 土 肉 桂與 陰 香 之 分 子 驗 證薛 士 緯 1 、 李 世 傑 2 、 何 坤 益 1 *1 國 立 嘉 義 大 學 森 林 暨 自 然 資 源 學 系2 大 葉 大 學 生 物 產 業 科 技 學 系* 通 信 作 者 , kyho@mail.ncyu.edu.tw投 稿 日 期 :2011 年 9 月 5 日接 受 日 期 :2011 年 9 月 30 日作 物 、 環 境 與 生 物 資 訊 8:193-200 (2011)Crop, Environment & Bioinformatics 8:193-200 (2011)189 Chung-Cheng Rd., Wufeng, Taichung 41362, TaiwanROC摘 要臺 灣 特 有 種 土 肉 桂 (Cinnamomumosmophloeum) 與 外 來 種 陰 香 (C. burmani) 之 苗木 在 外 觀 上 相 似 , 傳 統 的 形 態 鑑 定 方 法 可 能造 成 誤 認 。 因 此 , 利 用 DNA 分 子 標 記 技 術來 鑑 定 , 藉 由 設 計 引 子 擴 增 出 臺 灣 土 肉 桂rDNA 內 轉 錄 間 隔 區 (internal transcribedspacer; ITS) 後 , 將 片 段 定 序 以 進 行 比 較 , 並試 著 找 出 土 肉 桂 專 屬 的 差 異 片 段 , 用 來 作 為臺 灣 土 肉 桂 的 分 子 快 速 鑑 定 判 斷 依 據 。 本 研究 進 行 ITS 核 酸 序 列 的 差 異 比 較 , 發 現 在 同物 種 的 ITS 核 酸 序 列 並 無 變 異 , 有 助 於 釐 清外 觀 形 態 分 類 上 的 混 亂 。 利 用 臺 灣 土 肉 桂 及陰 香 在 ITS2 序 列 上 之 差 異 , 進 行 專 一 性 引 子之 設 計 , 成 功 設 計 出 可 以 使 用 PCR 技 術 及 電泳 分 析 即 可 讓 兩 者 出 現 條 帶 上 的 差 異 。 在 電泳 分 析 兩 物 種 皆 在 286 bp 處 出 現 條 帶 , 而 125bp 處 的 條 帶 則 只 有 臺 灣 土 肉 桂 顯 示 出 來 。 利用 此 判 讀 可 以 快 速 的 檢 測 出 兩 者 間 的 差 異 ,從 而 達 成 分 子 快 速 鑑 定 的 結 果 。關 鍵 詞 .. 分 子 驗 證 、 內 轉 錄 間 隔 區 、 複 合 式聚 合 脢 連 鎖 反 應 。INTRODUCTIONThe indigenous plant species Cinnamomumosmophloeum has been commonly planted byforest farmers in Taiwan because it is rich incinnamaldehyde, a major constituent of C. cassiaforming the cinnamon of commerce, and has anexcellent natural anti-bacterial activity as well as

194Crop, Environment & Bioinformatics, Vol. 8, September 2011other pharmacological properties (Chang et al.2001, Cheng et al. 2004, Cheng et al. 2008).Whereas, Cinnamomum burmannii originates insouthern China and the East Indian archipelago,and is a new species to adapt themselves inTaiwan (Tseng et al. 2008). As these two speciesare similar in appearance, they are not easily bediscerned and thus often cause economic losses toforest farmers because of value discrepancybetween the two species.The internal transcribed spacer sequence (ITS)of rDNA has been widely used in many molecularauthentication techniques by virtue of thepresence of more than one single nucleotidedifference in this region between similar species.ITSs reside within non-coding regions that aresituated between slowly-evolving structuralribosomal genes including the 5.8S, the smallsubunit (SSU; also called 17S or 18S), and thelarge subunit (LSU; also called 24S or 28S) (Jobesand Thien 1997). However, ITSs evolve quickly,and are often employed to resolve species withinthe same genus (Hillis and Dixon 1991, Baldwin etal. 1995, Wendel and Álvarez 2003, Small et al.2004, Lee et al. 2009), a technique with high andreliable accuracy.After Taiwan joined the World TradeOrganization (WTO), many of its agricultural andforestry products have been affected by free tradeand open market demand. Fast authenticationmethods are needed in order to identify and sortout harmful species during the quarantine period.Such precautionary measures are required toprotect the agricultural and forestry well-beingsof our country. In this study, specific primerswere synthesized after comparing variations inthe ITS2 sequences between the indigenousspecies C. osmophloeum and exotic species C.burmannii in order to differentiate these twospecies at molecular level in a timely fashion.MATERIALS AND METHODS1. MaterialsSamples of fresh leaves of Cinnamomumosmophloeum and C. burmannii were collected andidentified by Dr. Fu-Yuan Lu and his colleaguesof National Chiayi University from three differentregions of Taiwan as the experimental materialsfor the study. The leaves were dehydrated, andaliquot parts were put into air-permeable bagsseparately and stored in dry, sealed containers.The collection sites for samples and their DNAsample codes for C. osmophloeum and C. burmanniiare listed Table 1.2. Specific primer designThe primer pairs specific for C. osmophloeumand C. burmannii were designed based on a singlenucleotide difference found after comparing theirGAG AAT GGC CTC CCG TGC-3′; D4-R: 5′-GCGACG TAC GAT CGG CGA CGA-3′; BI-F: 5′-GTGGGG GTT GTG AGA GGC GAC C-3′; and ITS2-R:5′-CTC GCC GTT ACT AGG GGA AT-3′.Table 1. Collection sites of samples and DNA sample codes for Cinnamomum osmophloeum and C.burmannii.Scientific name Sample collection site GPS coordinate CodeC. osmophloeum Sanjiaonan MountainN23 14.439 E120 35.542Os.s(Tainan County)Defulan MountainN24 11.233 E120 57.741Os.d(Taichung County)PinglinN24 08.838 E120 44.121Os.t(Taichung County)C. burmannii LantanN23 28.526E120 28.881Bu.l(Chiayi City)ShekouN23 26.645 E120 32.474Bu.s(Chiayi County)PuliN23 55.490 E120 53.624Bu.p(Nantou County)Note: The DNA code entails the first two letters of the sample species (to the left of the punctuationmark) and the first Roman letter of the sample collection site (to the right of the punctuationmark).

Molecular Authentication of Cinnamomum osmophloeum vs. C. burmannii. 195Fig. 1. Design scheme for primers ITS2-F, D4-R, BI-F, and ITS2-R in the ITS2 region.3. Genomic DNA extraction and PCRamplificationGenomic DNA was extracted according tothe modified cetyl trimethyl ammonium bromide(CTAB) method (Kobayashi et al. 1998) withminor changes. This DNA was used as a templatefor PCR amplification using a pair of primers anda nucleic acid amplifying device, the PCRthermocycler. The total volume of each PCRreaction was 25 μL, containing 1.5 μL of DNA, 2.0μL of dNTPs (2.5 mM), 2.5 μL of 10X Taq buffer, 2μL of DMSO, 2.1 μL of Mg ++ , 0.2 μL of Taq, 1 μLof primers (contains 0.25 μL of ITS2-F, D4-R, BI-Fand ITS2-R), and 13.7 μL of ddH 2 O. PCR reactionswere carried out under the following conditions:denaturing at 96℃ for 12 min, and then 95℃ for30 s, 58℃ for 50 s (annealing), and 72℃ for 1 min(extension), for 35 cycles, and finally 72℃ for 10min for the final extension.4. Electrophoresis and imagingThe PCR products were subjected to anagarose gel electrophoresis and the gel wasstained with ethidium bromide. After obtainingan image of the gel, the bioimaging analysissoftware ImageJ v1.41 (http://rsb.info.nih.gov/ij/index.html) was employed to translate bandintensity to an intensity plot amenable to analysis.5. Sequencing and sequence analysisThe DNA bands corresponding to sources ofC. osmophloeum and C. burmannii were cut fromthe agarose gel using sterile razor blades, and theextracted PCR products were submitted to theGenomics Incorporation for DNA sequencing.Results obtained from the DNA sequencing werecompared to sequences in the National Center forBiotechnology Information (NCBI) database usingthe BLAST software to determine whether theywere the desired sequences.RESULTS AND DISCUSSION1. Specificity of PCR amplification toDNAs of Cinnamomum osmophloeumand C. burmannii using specific primerpairsAs shown in Fig. 2, these two Cinnamomumspecies shared a common peak at 286 bp.Furthermore, C. osmophloeum gave a specific peakat 125 bp while C. burmannii exhibited a specificpeak at 204 bp. It is worth noting that the specificpeak from C. osmophloeum was more intense thanthat of C. burmannii.Three bands from Fig. 2 were excised fromthe agarose gel and subjected to sequencing toconfirm the specificity of the bands. Sequencing

196Crop, Environment & Bioinformatics, Vol. 8, September 2011results revealed that the bands of 286-bp, the204-bp, and the 125-bp were consistent withtargeted sequences between primer pairsITS2-F/ITS2-R, B1F/ITS2-R, and ITS2-F/D4-R,respectively. Moreover, these three sequenceswere then compared to entries in the NCBIdatabase using the BLAST software to confirmthat they were generated from the targetedsequences (Figs. 3−5).(a)(b)Fig. 2. Electrophoretic analysis of PCR products using primers ITS2-F, D4-R, BI-F, and ITS2-R. (a)Agarose gel image; (b) Bioimaging analysis of the electrophoresis gel image using ImageJsoftware. M: 100-bp DNA Markers; Os: Cinnamomum osmophloeum; s, d, and t: SanJiaonanMountain, DeFulan Mountain, and Pinglin, respectively; Bu: C. Burmannii; l, s, and p:Lantan, Shekou, and Puli, respectively. The 286-bp band is common to both Cinnamomumosmophloeum and C. burmannii and the 125-bp band is specific to C. osmophloeum whilethe 204-bp band is specific for C. burmannii.Fig. 3. Results obtained from Cinnamomum osmophloeum (Query) with DNA of 286 bp sequencingwere compared to ITS2-F − ITS2-R sequences in the NCBI database using BLAST todetermine whether they were the desired sequences.

Molecular Authentication of Cinnamomum osmophloeum vs. C. burmannii. 197Fig. 4. Results obtained from Cinnamomum burmannii (Query) with DNA of 204 bp sequencingwere compared to B1-F − ITS2-R sequences in the NCBI database using BLAST todetermine whether they were the desired sequences.Fig. 5. Results obtained from Cinnamomum osmophloeum (Query) with DNA of 125 bpsequencing were compared to ITS2-F − D4-R sequences in the NCBI database using BLASTto determine whether they were the desired sequences.2. Reproducibility of PCR amplification ofC. osmophloeum and C. burmanniiAs shown in Fig. 6, samples of C.osmophloeum from Lienhuachih Research Centerhad bands of 286 bp and 125 bp. Since thesesamples had been positively identified as C.osmophloeum before collection, the resultsindicated that the specific primer pairs designedin this study for the specific band (125 bp) of theformer could be accurately amplified by PCR. Asshown in Fig. 7, samples of C. burmannii fromShekou in Chiayi County had bands of 286 bp and204 bp. Since the samples have been previouslyidentified as C. burmannii, the results indicatedthat the specific primer pairs designed in thisstudy for the specific band (204 bp) of the latterDNA could be accurately amplified by PCR. Thesame results were obtained using samples of C.burmannii collected from Endemic SpeciesResearch Institute in Nantou County (Table 2).This study describes a Multiplex-PCRmethod to amplify PCR products of differentlengths followed by agarose gel electrophoresis toresolve the bands. After staining and imaging thegel, molecular authentication could be performedbased on specific bands from different species.

198Crop, Environment & Bioinformatics, Vol. 8, September 2011(a)(b)Fig. 6. Reproducibility of PCR amplification of Cinnamomum osmophloeum with specific primerpairs ITS2-F, D4-R, BI-F, and ITS2-R. (a) Agarose gel image; (b) Bioimaging analysis of theagarose gel image using ImageJ software. M: 100-bp DNA markers; L1 − L5: Cinnamomumosmophloeum Kanehira samples collected from Lien Hua-Chin Research Center in NantouCounty. The 286-bp band is common for both C. osmophloeum and C. burmannii while the125-bp band is specific for C. osmophloeum.(a)(b)Fig. 7. Reproducibility of PCR amplification of Cinnamomum Burmannii with specific primerpairs ITS2-F, D4-R, BI-F, and ITS2-R. (a) Agarose gel image; (b) Bioimaging analysis of theagarose gel image using ImageJ software. M: 100-bp DNA markers; S1 − S5: C. burmanniisamples collected from Shekou in Chiayi County. The 286-bp band is common for both C.osmophloeum and C. burmannii while the 204-bp band is specific for C. osmophloeum.Park et al. (2005) have successfully designedspecific primer pairs based on a single nucleotidedifference within the 5.8S region and effectivelycharacterized Korean ginseng and other closelyrelated species using Multiplex-PCR.Sequence of the ITS2 region in C.osmophloeum and C. burmannii is nearly identical,except for a single nucleotide difference at 106bp where a T presents in C. osmophloeum and aC in C. burmannii (Fig. 1). The characteristic125-bp band of C. osmophloeum could bespecifically amplified with the ITS2-F/D4-Rprimer pair based on the single nucleotidedifference. Similarly, the BI-F/ITS2-R primerpair can amplify the 204-bp band specific to C.burmannii. The ITS2-F/ITS2 primer pair wasdesigned based on the common DNA sequencein the ITS2 region of the 2 species; therefore, thepair could amplify the 286-bp band specific toboth.

Molecular Authentication of Cinnamomum osmophloeum vs. C. burmannii. 199Table 2. Collection sites of samples and DNA sample codes for Cinnamomum osmophloeum and C. burmanniiScientific name Sampling place GPS coordinate DNA codeC. osmophloeum Lienhuachin Research Center(Nantou County)C. burmannii Endemic Species ResearchInstitute(Nantou County)Shekou Forest Farm(Chiayi County)N23 55.490 E120 53.624N23 49.718 E120 48.022N23 26.648 E120 32.477L1,L2, L3, L4,L5T1,T2,T3,T4,T5S1,S2,S3,S4,S5The intensity of the DNA bands amplified bythe ITS2-F/D4-R primer pair was higher than thatof the bands amplified with the BI-F/ITS2-Rprimer pair, indicating that the current PCRcondition was optimized for the C. osmophloeumDNA amplification. This high level of detectionmet the quarantine requirement and aid in speciesidentification.In this study, in order to examine whetheraccurate analytical results could be obtainedwhen the primer pairs were used on the samplesof indigenous species C. osmophloeum and exoticspecies C. burmannii collected from differentregions, reproducibility experiments wereperformed as well. The results showed that theprimer pairs could accurately amplify thecharacteristic bands of both species.C. osmophloeum, which considered as avaluable native forest resource, belongs to thesame genus as C. burmannii. In recent years, seedsof C. burannii have been imported and extensivelyused for environmental greening. As these twospecies are not easy to be discerned because ofmorphological similarity, there is a need toestablish an appropriate method to avoid theambiguity and improper identification. Inaddition, when exotic species C. burmannii wasused to extract leaf essential oil, it may mess-upthe products with that of C. osmophloeum andinterfere the market price. The impacts of thisexotic species to local forest ecosystem shouldalso be taken into consideration.CONCLUSIONSThis study established a molecularauthentication technique for fast discrimination ofindigenous species Cinnamomum osmophloeumfrom exotic species C. burmannii. The designedspecific primer pairs were proved fromelectrophoresis to be able to generate thedistinguishing bands without resorting tosequencing, and thus saved labor and money fordiscrimination. The analytical process is facile,need no sophisticated protocols and equipment,and is suitable for general laboratories. By takingadvantage of rDNA, the method is reliable,accurate, and free from ambiguity associated withplant morphology. Thus, the developed molecularmethod provides a robust and reproducible resultand may be used complement to chemicalanalysis and traditional way of morphologicalprocessing.REFERENCESBaldwin BG, MJ Sanderson, MF Wojciechowski,CS Campbell, MJ Donoghue (1995) The ITSregion of nuclear ribosomal DNA: a valuablesource of evidence on angiosperm phylogeny.Ann. Mol. Bot. Gard. 82:247-277.Chang ST, PF Chen, SC Chang (2001)Antibacterial activity of leaf essential oils andtheir constituents from Cinnamomumosmophloeum. J. Ethnopharm. 77:123-127.Cheng SS, JY Liu, KH Tsai, WJ Chen, ST Chang(2004) Chemical composition and mosquitolarvicidal activity of essential oils from leavesof different Cinnamomum osmophloeum clones. J.Agric. Food. Chem. 52:4395-400.Cheng SS, JY Liu, CY Lin, YR Hsui, M C Lu, WJWu, ST Chang (2008) Terminating redimported fire ants using Cinnamomumosmophloeum leaf essential oil. Bioresour.Technol. 99:889-3.Hillis DM, MT Dixon (1991) Ribosomal DNA:molecular evolution and phylogeneticinference. Q. Rev. Biol. 66:411-53.

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