Chinese Journal of

Natural Chinese Journal of Natural Medicines 2015, 13(9): 06530659 Medicines

doi: 10.3724/SP.J.1009.2015.00653

·Research articles·

Identification of maca ( meyenii Walp.) and its adulterants by a DNA-barcoding approach based on the ITS sequence

CHEN Jin-Jin1, 2, ZHAO Qing-Sheng1, LIU Yi-Lan1, 2, ZHA Sheng-Hua1, 2, 3, ZHAO Bing1*

1 National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China; 2 University of Chinese Academy of Sciences, Beijing 100049, China; 3 Beijing Tong Ren Tang Health Pharmaceutical Co., Ltd., Beijing 100062, China

Available online 20 Sep., 2015

[ABSTRACT] Maca (Lepidium meyenii) is an herbaceous that grows in high plateaus and has been used as both food and folk medicine for centuries because of its benefits to human health. In the present study, ITS (internal transcribed spacer) sequences of forty-three maca samples, collected from different regions or vendors, were amplified and analyzed. The ITS sequences of nineteen potential adulterants of maca were also collected and analyzed. The results indicated that the ITS sequence of maca was consistent in all samples and unique when compared with its adulterants. Therefore, this DNA-barcoding approach based on the ITS sequence can be used for the molecular identification of maca and its adulterants.

[KEY WORDS] Lepidium meyenii; Maca; ITS; DNA-barcoding; Molecular identification [CLC Number] R282.5 [Document code] A [Article ID] 2095-6975(2015)09-0653-07

 even corn have been found to be used to adulterate into maca Introduction products [13], which would have a adverse effect on the Maca (Lepidium meyenii Walp.), a Peruvian plant of the healthy development of the maca industry. Therefore, a rapid, family, has been used as both food and specific, and accurate method is needed to identify maca and traditional medicine in the for a long time [1-2]. its adulterants in commercial samples. [13-15] Biochemical and pharmacological studies have shown that There are a number of reports mentioning the maca has various health-related properties, including beneficial identification of maca by phenotypic or phytochemical effects on fertility [3], sexual behavior [4], energy [5], memory [6], characteristics. However, phenotypic characteristics might be osteoporosis [7], prostate [8], and skin [9]. Therefore, maca has totally destroyed during post-harvest processing, and chemical been introduced to Asia, Europe and North America during components are highly affected by the environment, [16-17] the last decade, with an increasing consumption worldwide post-harvest processing, and storage . Therefore, it is [10-11]. Since 2002, maca has been transplanted successfully in necessary to develop a more accurate, reliable method for maca Yunnan, Shanxi, and Xinjiang provinces of China, and has identification. formed large-scale plantings [12]. With the rapid development DNA barcoding is a new molecular diagnostic technique of the commercialization of maca, , , potato, and for identifying species. ITS (internal transcribed spacer) sequences had been proven to be a very useful DNA barcode for identifying species in [18-19]. However, until

now, there has been no report on the identification of maca [Received on] 05-Jun-2014 based on its ITS sequences. As considerable intra-specific [Research funding] This work was supported by the High-Tech Research Program–863 Program in China (No. 2012AA021702-4). variations in ITS sequences have been reported in many plant [20-21] [*Corresponding author] Tel/Fax: 86-10-62574372, E-mail: bzhao@ species , there also are different species sharing the same [22] ipe.ac.cn ITS . In the present study, the ITS of forty-three maca These authors have no conflict of interest to declare. samples from different locations were amplified and

– 653 – CHEN Jin-Jin, et al. / Chin J Nat Med, 2015, 13(9): 653659 sequenced, and the intra-species variations in ITS sequences originating from different locations in China(Table 1). All the were analyzed.. The inter-species variation of ITS sequences specimens were authenticated by Prof. WANG Yun-Chun between maca and its adulterants were also assessed. The from Institute of Process Engineering, Chinese Academy of results from the present study provided an accurate method Sciences, Beijing, China. Meanwhile, macamides, the marker for molecular identification of maca. compounds of maca, were detected using HPLC (Shimadzu LC-20AT, Kyoto, Japan) to authenticate these specimens Materials and Methods further [23]. The sample of ‘yuangen’ (local name of turnip) Materials was obtained from Qinghai Province, China. In addition, the Forty-three maca samples were examined in the present ITS sequences of fifteen other Lepidium species and four study, including four powders imported from , eight fresh common adulterants were collected from GenBank roots, sixteen dried slices or fruits, and fifteen essential tablets (http://www.ncbi.nlm.nih.gov/genbank/) as shown in Table 2.

Table 1 Maca samples used in the present study Sample Sample Type Source Type Source No. No. 1 Fresh root Shangri-La County, Yunnan Province 25 Essential tablet Tiantian Biotech Co., Ltd. Taiyuan Biological Technology Development Co.,, 2 Fresh root Yongshan County, Yunnan Province 26 Essential tablet Ltd. 3 Fresh root Dongchuan City, Yunnan Province 27 Essential tablet Lijiang Green Enhancer Biological Plant Co., Ltd. 4 Fresh root Dahai Village, Huize County, Yunnan Province 28 Essential tablet Yunnan Tiangen Maca Industry Co., Ltd. Yunping Village, Dongchuan City, Yunnan Lijiang Baisuifang Biotechnology Development 5 Fresh root 29 Essential tablet Province (3 600 m) Co., Ltd. 6 Fresh root Dongchuan City, Yunnan Province 30 Essential tablet Yunnan Guzhiji Nourishment Co., Ltd. Yunping Village, Dongchuan City, Yunnan 7 Fresh root 31 Essential tablet Lijiang Longjian Biotechnology Co., Ltd. Province (3 400 m) Hongqi Village, Shangri-La County, Yunnan Shantou Shuangjun Biological Engineering Co., 8 Fresh root 32 Essential tablet Province Ltd. Hongqi Village, Shangri-La County, Yunnan Yunnan Tianyuan Huama Bio-technology Co., Ltd. 9 Dried fruit 33 Essential tablet Province (pure maca) Yunnan Tianyuan Huama Bio-technology Co., Ltd. 10 Dried fruit Pamirs, Xinjiang Uygur Autonomous Region 34 Essential tablet ( extract added) Yunnan Tianyuan Huama Bio-technology Co., Ltd. 11 Dried fruit Nakhi Garden, Taobao online shop 35 Essential tablet (Almond extract added) Lijiang Evergreen Maca Biotechnology R&D Co., 12 Dried fruit Shanxi Guhua Ecological Technology Co., Ltd. 36 Essential tablet Ltd. Yunnan Maca Manor Agricultural Science and 13 Dried fruit 37 Essential tablet Yunnan MaiKa Biotechnology Co., Ltd. Technology Co., Ltd. 14 Dried slice Lijiang Longjian Biotechnology Co., Ltd. 38 Essential tablet Yunnan MaiKa Biotechnology Co., Ltd. 15 Dried slice Yunnan Guzhiji Nourishment Co., Ltd. 39 Essential tablet La Molina, Taobao online shop 16 Dried slice Yunnan Tiangen Maca Industry Co., Ltd. 40 Powder Life-flo health 17 Dried slice Tiantian Biotech Co., Ltd. 41 Powder Amax International Co., Ltd. Lijiang Baisuifang Biotechnology 18 Dried slice 42 Powder 2 Dragon Trading Co., Ltd. Development Co., Ltd. Lijiang Green Enhancer Biological Plant Co., 19 Dried slice 43 Powder Kindly provided by Prof. LI Yu-Lai Ltd. 20 Dried slice Taiyuan Biol. Technol. Development Co., Ltd. Lijiang Evergreen Maca Biotechnol. R&D Co., 21 Dried slice Ltd. 22 Dried slice Yunnan Tianyuan Huama Biotechnol. Co., Ltd. 23 Dried slice Kunming Hongyi Biotechnology Co., Ltd. 24 Dried slice Yunnan MaiKa Biotechnology Co., Ltd.

DNA extraction, amplification, and sequencing concentration on 0.8% agarose gels stained with Goldview The materials were ground into powders for total DNA (SBS Genetech Co., Ltd., Beijing, China). Then the DNA extraction. Total DNA was extracted from maca samples (0.2 samples were diluted to approximately 20 ng·μL−1 as working g) with the GenStar Plant DNA Kit according to the solution. manufacturer’s instructions (GenStar Biosolutions Co., Ltd., The PCR amplification was performed in a volume of Beijing, China). The DNA concentrations were estimated by 25 μL containing 2.5 μL 10 × buffer (with Mg2+), 0.5 mmol·L−1 comparing band intensity with the DNA marker of known dNTP, 1.5 U Taq DNA polymerase, 10 pmol·L−1 each primer, and

– 654 – CHEN Jin-Jin, et al. / Chin J Nat Med, 2015, 13(9): 653659

Table 2 Overview of the studied species and information of their ITS sequences GenBank a Length (bp) Variations GC content No. Name accession ID b c (%) No. of ITS ITS ITS1 ITS2 5.8S Sites Rate 1 L. meyenii 153348 JX908826 621 267 190 164 - - 55.7 2 L. africanum 153324 AY254529 621 267 190 164 25 4.0 56.2 3 L. apetalum 153459 JF976770 621 267 190 164 16 2.6 55.6 4 L. bonariense 153333 HM134831 620 267 189 164 7 1.1 55.6 5 L. campestre 65351 AF055197 620 267 189 164 46 7.4 55.3 6 L. draba 153317 EF367913 620 267 189 164 45 7.2 55.0 7 L. lacerum 743983 FN821676 621 267 190 164 24 3.9 55.4 8 L. montanum 153349 EF367869 622 267 191 164 16 2.6 55.0 9 L. papilliferum 454201 EF368005 622 267 191 164 16 2.6 55.0 10 L. perfoliatum 153358 EF368007 619 266 189 164 51 8.2 55.1 11 L. phlebopetalum 153359 AY254528 617 267 186 164 103 16.6 51.9 12 L. ruderale 153365 JF976776 621 267 190 164 16 2.6 55.6 13 L. sisymbrioides 106949 DQ997561 622 268 190 164 13 2.1 55.5 14 L. solandri 413682 DQ997557 622 268 190 164 13 2.1 55.5 15 L. squamatum 228869 AY254533 623 273 186 164 110 17.7 49.6 16 L. virginicum 59292 HM134830 621 267 190 164 5 0.8 55.1 17 Brassica rapa 51350 GQ268060 608 257 187 164 126 20.3 51.8 18 Raphanus sativus 41679 AY746462 620 267 189 164 120 19.3 55.5 19 Zea mays 4579 AF019817 597 213 220 164 332 53.5 67.7 20 Solanum tuberosum 4113 KF022369 642 235 254 153 240 38.6 63.2 a Variations between maca and the other species; b No. of different sites; c Rates of different sites approximately 50 ng DNA template. The PCR reactions model [27]. The resultant distance matrix was then computed consisted of an initial denaturation at 94 °C for 5 min, 35 to generate a phylogenetic tree by the neighbor-joining cycles of 30 s at 94 °C, 30 s at 52 °C, 1 min at 72 °C, and a method [28]. The 1 000 bootstrap replicates were used to final elongation step of 5 min at 72 °C. The primers for evaluate the strength of resulting branches. amplification and sequencing were ITS4 (5'-TCCTCCGCT Results TATTGATATGC-3') and ITS5 (5'-GGAAGTAAAA [24] GTCGTAACAAGG-3') . Analysis of ITS sequences from maca samples The amplification products were separated on 1.5% Genomic DNA was extracted and used as templates for agarose TAE gels, stained with Goldview, and visualized ITS sequence amplification. In the present study, the ITS under UV light. Successful amplification was signed by the sequences of all maca samples were successfully amplified appearance of a single DNA band of approximately 700 bp. and displayed as a single DNA band of approximately 700 PCR products were directly sequenced bi-directionally on an bp. After sequencing, assembling and aligning, all 43 ITS ABI 3730XL DNA sequencer. sequences were exactly the same. A BLAST search was Data analysis performed to confirm the identity of the ITS sequences, and The sequences were assembled using the ContigExpress the alignment was used to determine the boundaries. The program. Boundaries of ITS1-5.8S-ITS2 of maca were results showed that the complete ITS sequence of maca was determined by comparisons with published sequences of other 621 bp (ITS1-267 bp, 5.8S-164 bp, and ITS2-190 bp) (Fig. Lepidium species in GenBank. All ITS sequences were 1), and it exhibited mostly significant alignments with [25] aligned with Clustal X version 2.0 and manually checked. available ITS sequences from other Lepidium species. The [26] MEGA v.5 was used to estimate the GC content and the sequence data are available in GenBank under the reference: genetic distance matrix according to Kimura’s two-parameter JX908826.

– 655 – CHEN Jin-Jin, et al. / Chin J Nat Med, 2015, 13(9): 653659

Fig. 1 ITS sequence of Maca (621bp); 5.8S region was underlined Analysis of ITS sequences from maca and its adulterants fifteen Lepidium species varied from 617 bp in L. In the subsequent experiments, the variations in the ITS phlebopetalum to 623 bp in L. squamatum, and the variation sequences among maca and its potential adulterants were sites ranged from 5 (0.8%) in L. virginicum to 110 (17.7%) in L. analyzed (Table 2). The largest variation was found between squamatum. The smallest variation was found between maca the ITS sequences of maca and corn, as the ITS length of the and L. virginicum, as the ITS length of the latter was 621 bp latter was 597 bp and the variation sites were 332 (53.5%). and the variation sites were 5 (0.8%). The GC contents varied Besides, the other three common adulterants, turnip, radish, and from 49.6% to 56.2% for all of the studied Lepidium species. potato, also showed large variations with maca, as the ITS Furthermore, the genetic distance matrix based on the lengths were 608 bp, 620 bp, and 642 bp, respectively, and the ITS sequences was calculated according to the Kimura-2 variation sites were 126 (20.3%), 120 (19.3%), and 240 model (Table 3). And the derived neighbor-joining tree is (38.6%), respectively. The GC contents of the four common illustrated in Fig. 2; the tree is highly supported by bootstrap adulterants ranged from 51.8% to 67.7%. The variations were values. Maca showed minimum distances with L. bonariense smaller between maca and the other fifteen Lepidium species, (0.006) and L. virginicum (0.006), which also originated from as they belonged to the same genus. The ITS lengths of the America, but large distances from its adulterants (≥ 0.189).

Table 3 Genetic distance matrix among studied species based on ITS sequences data calculated using the Kimura-2 model. Bold values correspond to the smallest and the largest genetic distances, respectively 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2a 0.036 3 0.028 0.032 4 0.006 0.038 0.030 5 0.082 0.088 0.084 0.084 6 0.078 0.082 0.080 0.080 0.015 7 0.038 0.021 0.036 0.040 0.090 0.086 8 0.023 0.048 0.044 0.023 0.101 0.097 0.054 9 0.023 0.048 0.044 0.023 0.101 0.097 0.054 0.002 10 0.082 0.086 0.082 0.084 0.044 0.040 0.084 0.101 0.101 11 0.166 0.166 0.166 0.166 0.147 0.143 0.171 0.161 0.159 0.156 12 0.028 0.032 0.000 0.030 0.084 0.080 0.036 0.044 0.044 0.082 0.166 13 0.019 0.040 0.032 0.021 0.090 0.086 0.042 0.032 0.032 0.088 0.168 0.032 14 0.019 0.040 0.032 0.021 0.090 0.086 0.042 0.032 0.032 0.088 0.168 0.032 0.000 15 0.173 0.161 0.173 0.176 0.187 0.182 0.166 0.175 0.175 0.175 0.164 0.173 0.173 0.173 16 0.006 0.042 0.030 0.007 0.084 0.080 0.044 0.026 0.026 0.084 0.168 0.030 0.024 0.024 0.175 17 0.199 0.182 0.196 0.204 0.179 0.170 0.189 0.201 0.201 0.168 0.182 0.196 0.196 0.196 0.127 0.199 18 0.189 0.179 0.191 0.194 0.177 0.167 0.182 0.194 0.194 0.163 0.177 0.191 0.191 0.191 0.155 0.189 0.066 19 0.581 0.576 0.592 0.580 0.586 0.582 0.584 0.592 0.593 0.599 0.607 0.592 0.589 0.589 0.658 0.581 0.610 0.601 20 0.588 0.578 0.597 0.592 0.575 0.575 0.589 0.589 0.593 0.595 0.573 0.597 0.606 0.606 0.599 0.593 0.563 0.591 0.421 a The numbers assigned to samples correspond to those used in Table 2

– 656 – CHEN Jin-Jin, et al. / Chin J Nat Med, 2015, 13(9): 653659

Fig. 2 Neighbor-joining phenogram of Maca and its adulterants based on ITS sequences. Numbers at nodes are bootstrap values

Verification test After sequencing and alignment, it was found that A powder of maca adulterated with 10% of ‘yuangen’ the ITS sequences from all of the maca samples were was used to evaluate the applicability of this method. The identical. The highly conserved ITS sequence of maca sequencing results showed that there was obvious impurity might be due to the harsh environment and long-term interference (Fig. 3), demonstrating that this method is domestication. Meanwhile, considerable variations in ITS effective to identify maca when it is adulterated with other sequences were found between maca and its adulterants, plants. especially the common adulterants turnip, radish, potato, Discussion and corn, as they belonged to different genera. Furthermore, the NJ tree showed the obvious divergence highly supported In the present study, forty-three maca samples were by bootstrap values between maca and its adulterants. collected from different places and in different forms in order Therefore, the ITS is suitable as a DNA barcode for maca to make the samples representative. The genomic DNAs were identification. isolated successfully for all the samples, indicating that the Morphological and sensory inspection is a simple and simple processing did not degrade the genomic DNA. direct method to identify maca. However, accurate Furthermore, the ITS sequences from all the samples were authentication is heavily dependent on personal experience, amplified and sequenced successfully, due to several and will also be more difficult or impossible when the advantages of the ITS region, such as easy PCR amplification, products are processed into powders. Physicochemical the multiple sets with thousands of copies, and the moderate methods, such as FTIR, TLC, HPLC, GC and MS [13, 32], size for easy sequencing [30]. have been used to identify maca. However, these methods Baraket et al. have reported that there are big required expensive instruments. Moreover, the chemical variations in ITS sequences among Ficus carica L. composition and contents in maca may be affected by the cultivars [16]. In traditional Chinese medicines, such as growing environment, post-harvest processing, and storage Corni Fructus, Hou et al. have also found some intra-specific variations [30]. However, there also exist conditions. Analysis becomes more difficult when the different species sharing the same ITS [22]. Moreover, we samples are maca powder adulterated with a few other plant found that, in the present study, L. apetalum and L. materials. In the present study, the accuracy of the ITS ruderale, L. sisymbrioides and L. solandri had the same method developed was tested using a powder of maca mixed ITS sequence. Hence, it is important to ensure that the ITS with 10% of ‘yuangen’. Our results confirmed the accuracy sequence is conserved in Maca samples, and unique when of this molecular identification method for maca based on compared with its adulterants. the ITS sequence.

– 657 – CHEN Jin-Jin, et al. / Chin J Nat Med, 2015, 13(9): 653659

Fig. 3 Validation of the method developed in the present study in Maca powder adulterated with 10% ‘yuangen’ (an alias name of turnip)

meyenii) increases swimming endurance capacity in rats [J]. J Conclusion Funct Foods, 2012, 4(2): 568-573. The ITS method developed and validated here can be used [6] Cordova-Ruiz ME. Effect of Maca (Lepidium meyenii Walp.) to assure the authenticity of maca raw material, including fresh, in cognitive functions in an experimental animal model [J]. Neuroimmunomodulat, 2011, 18(6): 369. dried, slices, and powders. This DNA-barcoding method, [7] Zhang YZ, Yu LJ, Ao MZ, et al. Effect of ethanol extract of together with other morphological and sensory judgment and Lepidium meyenii Walp. on osteoporosis in ovariectomized rat physicochemical method, would improve the quality control of [J]. J Ethnopharmacol, 2006, 105(1-2): 274-279. the products of this medicinal plant. [8] Gasco M, Villegas L, Yucra S, et al. Dose-response effect of Red Maca (Lepidium meyenii) on benign prostatic hyperplasia References induced by testosterone enanthate [J]. Phytomedicine, 2007, [1] Wang YL, Wang YC, McNeil B, et al. Maca: An Andean crop 14(7-8): 460-464. with multi-pharmacological functions [J]. Food Res Int, 2007, [9] Gonzales-Castañeda C, Gonzales GF. Hypocotyls of Lepidium 40(7): 783-792. meyenii (maca), a plant of the Peruvian highlands, prevent [2] Bai NS, He K, Roller M, et al. Flavonolignans and other ultraviolet A-, B-, and C-induced skin damage in rats [J]. constituents from Lepidium meyenii with activities in Photodermatol Photo, 2008, 24(1): 24-31. anti-inflammatory and human cancer cell lines [J]. J Agr Food [10] Lee MS, Shin BC, Yang EJ, et al. Maca (Lepidium meyenii) for Chem, 2015, 63(9): 2458-2463. treatment of menopausal symptoms: A systematic review [J]. [3] Ruiz-Luna AC, Salazar S, Aspajo NJ, et al. Lepidium meyenii Maturitas, 2011, 70(3): 227-233. (Maca) increases litter size in normal adult female mice [J]. [11] Esparza E, Hadzich A, Kofer W, et al. Bioactive maca Reprod Biol Endocrin, 2005, 3: 16. (Lepidium meyenii) alkamides are a result of traditional [4] Zheng BL, He K, Kim CH, et al. Effect of a lipidic extract Andean postharvest drying practices [J]. Phytochemistry, from Lepidium meyenii on sexual behavior in mice and rats [J]. 2015, 116: 138-148 Urology, 2000, 55(4): 598-602. [12] Jin WW, Wang QF, Li S, et al. GC-MS analysis of chemical [5] Choi EH, Kang JI, Cho JY, et al. Supplementation of components of essential oil from Lepidium meyenii grown in standardized lipid-soluble extract from maca (Lepidium Xinjiang area [J]. Food Sci, 2009, 30(12): 241-245.

– 658 – CHEN Jin-Jin, et al. / Chin J Nat Med, 2015, 13(9): 653659

[13] Jin WW, Zhang YZ, Mei S, et al. Identification of Lepidium molecular markers and ITS sequences [J]. J Zhejiang Univ meyenii (Walp.) based on spectra and chromatographic (Agr Life Sci), 2008, 34: 473-481. characteristics of its principal functional ingredients [J]. J Sci [22] Jordon-Thaden I, Hase I, Al-Shehbaz I, et al. Molecular Food Agr, 2007, 87(12): 2251-2258. phylogeny and systematics of the genus Draba (Brassicaceae) [14] Muhammad I, Zhao JP, Dunbar DC, et al. Constituents of and identification of its most closely related genera [J]. Mol Lepidium meyenii ‘maca’ [J]. Phytochemistry, 2002, 59(1): Phylogenet Evol, 2010, 55(2): 524-540. 105- 110. [23] Chain FE, Grau A, Marti JC, et al. Macamides from wild [15] McCollom MM, Villinski JR, McPhail KL, et al. Analysis of ‘Maca’, Lepidium meyenii Walpers (Brassicaceae) [J]. macamides in samples of Maca (Lepidium meyenii) by Phytochem Lett, 2014, 8: 145-148. HPLC-UV-MS/MS [J]. Phytochem Analysis, 2005, 16(6): 463- [24] Amplification and direct sequencing of fungal ribosomal RNA 469. genes for phylogenetics. PCR Protocols, a Guide to Methods [16] Zhao JP, Avula B, Chan M, et al. Metabolomic differentiation and Applications [M]. Beijing (China): Academic Press, 1990: of maca (Lepidium meyenii) accessions cultivated under 315-321. different conditions using NMR and chemometric analysis [J]. [25] Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Planta Med, 2012, 78(1): 90-101. Clustal X version 2.0 [J]. Bioinformatics, 2007, 23(21): 2947- [17] Yabar E, Pedreschi R, Chirinos R, et al. 2948. content and myrosinase activity evolution in three maca [26] Kumar S, Nei M, Dudley J, et al. MEGA: a biologist-centric (Lepidium meyenii Walp.) ecotypes during preharvest, software for evolutionary analysis of DNA and protein harvest and postharvest drying [J]. Food Chem, 2011, sequences [J]. Brief Bioinform, 2008, 9(4): 299-306. 127(4): 1576-1583. [27] Kimura M. A simple method for estimating evolutionary rates [18] Li DZ, Gao LM, Li HT, et al. Comparative analysis of a large of base substitutions through comparative studies of nucleotide dataset indicates that internal transcribed spacer (ITS) should sequences [J]. J Mol Evol, 1980, 16(2): 111-120. be incorporated into the core barcode for seed plants [J]. P Natl [28] Saitou N, Nei M. The neighbor-joining method: a new method Acad Sci USA, 2011, 108(49): 19641-19646. for reconstructing phylogenetic trees [J]. Mol Biol Evol, 1987, [19] Xin TY, Yao H, Gao HH, et al. Super food Lycium barbarum 4(4): 406-425. (Solanaceae) traceability via an internal transcribed spacer 2 [29] Liu B, Lowes F. Multiple divergent ITS1 copies were barcode [J]. Food Res Int, 2013, 54(2): 1699-1704. identified in single tomato genome using DGGE analysis [J]. [20] Song JY, Shi LC, Li DZ, et al. Extensive pyrosequencing Plant Mol Biol Rep, 2012, 31(2): 272-279. reveals rrequent intra-genomic variations of internal [30] Hou DY, Song JY, Yao H, et al. Molecular identification of transcribed spacer regions of nuclear ribosomal DNA [J]. PLoS Corni Fructus and its adulterants by ITS/ITS2 sequences [J]. ONE, 2012, 7(8): e43971. Chin J Nat Med, 2013, 11(2): 121-127. [21] Tao XY, Gui XQ, Fu CX, et al. Analysis of genetic [31] Uchiyama F, Jikyo T, Takeda R, et al. Lepidium meyenii (Maca) differentiation and phylogenetic relationship between enhances the serum levels of luteinising hormone in female Changium smyrnioides and Chuanminshen violaceum using rats [J]. J Ethnopharmacol, 2014, 151(2): 897-902.

Cite this article as: CHEN Jin-Jin, ZHAO Qing-Sheng, LIU Yi-Lan, ZHA Sheng-Hua, ZHAO Bing. Identification of maca (Lepidium meyenii Walp.) and its adulterants by a DNA-barcoding approach based on the ITS sequence [J]. Chinese Journal of Natural Medicines, 2015, 13(9): 653-659.

– 659 –