Abstract
Allium ochotense and Allium microdictyon are commonly known as ‘Mountain garlic’ and are popular, economically important species in many countries such as Korea, China, and Mongolia. Their leaves are used as culinary side dishes and in traditional medicines. In Korea, these two species are at risk of extinction due to damage to their natural habitat and thus, conservation and breeding programs are needed. However, their identification relies mostly on morphological data, which is limited and until recently, led to classifying these two species under A. victorialis. In the present study, a simple and reliable method of molecular identification was developed to distinguish A. ochotense from A. microdictyon that targets four barcoding regions: the internal transcribed spacer (ITS), the maturase K gene (matK), the chloroplast psbA-trnH intergenic region, and the ribulose-bisphosphate carboxylase large subunit gene (rbcL). Single nucleotide polymorphisms (SNPs) were found in ITS and matK regions, and species-specific primers were designed based solely on the SNP at position 680 of the ITS region that could differentiate A. ochotense from A. microdictyon. Using these primers in amplification refractory mutation system (ARMS)-PCR, A. ochotense, and A. microdictyon could be simultaneously and efficiently distinguished. This study is the first to report a simple, rapid, and efficient method for discriminating A. ochotense and A. microdictyon, indicating the utility of species-specific markers in the development of conservation and breeding programs.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arif IA, Khan HA, Bahkali AH, Al Homaidan AA, Al Farhan AH, Al Sadoon M, Shobrak M (2011) DNA marker technology for wildlife conservation. Saudi J Biol Sci 18:219–225. https://doi.org/10.1016/j.sjbs.2011.03.002
Choi HJ, Oh BU (2011) A partial revision of Allium (Amaryllidaceae) in Korea and north-eastern China. Bot J Linn Soc 167:153–211. https://doi.org/10.1111/j.1095-8339.2011.01166.x
Choi H-J, Jang C-G, Ko S-C, Oh B-U (2004) A taxonomic review of Korean Allium (Alliaceae). Korean J Plant Taxon 34:119–152
Choi S, Ramekar RV, Kim YB et al (2017) Molecular authentication of two medicinal plants Ligularia fischeri and Ligularia stenocephala using allele-specific PCR (AS-PCR) strategy. Genes Genom 39:913–920
Cuenoud P, Savolainen V, Chatrou LW, Powell M, Grayer RJ, Chase MW (2002) Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. Am J Bot 89:132–144. https://doi.org/10.3732/ajb.89.1.132
El Beyrouthy M, Alain AbiRizk MEB (2013) DNA fingerprinting: the new trend in fighting the adulteration of commercialized and cultivated medicinal plants. Adv Crop Sci Technol 1:e107. https://doi.org/10.4172/2329-8863.1000e107
Friesen N (1995) The genus Allium L. in the flora of Mongolia. Feddes Repert 106:59–81. https://doi.org/10.1002/fedr.19951060116
Friesen N, Fritsch RM, Pollner S, Blattner FR (2000) Molecular and morphological evidence for an origin of the aberrant genus Milula within himalayan species of Allium (Alliacae). Mol Phylogenet Evol 17:209–218. https://doi.org/10.1006/mpev.2000.0844
Friesen N, Fritsch RM, Blattner FR (2006) Phylogeny and new intrageneric classification of Allium (Alliaceae) based on nuclear ribosomal DNA ITS sequences. Aliso 22:372–395
Fritsch RM, Keusgen M (2006) Occurrence and taxonomic significance of cysteine sulphoxides in the genus Allium L. (Alliaceae). Phytochemistry 67:1127–1135. https://doi.org/10.1016/j.phytochem.2006.03.006
Fritsch RM, Blattner FR, Gurushidze M (2010) New classification of Allium L. subg. Melanocrommyum (Webb & Berthel.) Rouy (Alliaceae) based on molecular and morphological characters. Phyton 49:145–220
Ganie SH, Upadhyay P, Das S, Sharma MP (2015) Authentication of medicinal plants by DNA markers. Plant Gene 4:83–99. https://doi.org/10.1016/j.plgene.2015.10.002
Ganopoulos I, Aravanopoulos F, Madesis P, Pasentsis K, Bosmali I, Ouzounis C, Tsaftaris A (2013) Taxonomic identification of mediterranean pines and their hybrids based on the high resolution melting (HRM) and trnL approaches: from cytoplasmic inheritance to timber tracing. PLoS ONE 8:e60945. https://doi.org/10.1371/journal.pone.0060945
Gao T, Yao H, Song J, Liu C, Zhu Y et al (2010) Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2. J Ethnopharmacol 130:116–121. https://doi.org/10.1016/j.jep.2010.04.026
Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30:1229–1235. https://doi.org/10.1093/molbev/mst012
Han J, Zhu Y, Chen X, Liao B, Yao H et al (2013) The short ITS2 sequence serves as an efficient taxonomic sequence tag in comparison with the full-length ITS. Biomed Res Int 2013:741476. https://doi.org/10.1155/2013/741476
Han BK, Rhee SJ, Jang YJ, Sim TY, Kim YJ, Park TS, Lee GP (2016) Identification of a causal pathogen of watermelon powdery mildew in Korea and development of a genetic linkage marker for resistance in watermelon (Citrullus lanatus). Korean J Hortic Sci Technol 34:912–925
Hebert PD, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B 270:313–321. https://doi.org/10.1098/rspb.2002.2218
Herden T, Hanelt P, Friesen N (2016) Phylogeny of Allium L. subgenus Anguinum (G. Don. ex W.D.J. Koch) N. Friesen (Amaryllidaceae). Mol Phylogenet Evol 95:79–93. https://doi.org/10.1016/j.ympev.2015.11.004
In J-G, Kim M-K, Lee O-R, Kim Y-J et al (2010) Molecular identification of Korean mountain ginseng using an amplification refractory mutation system (ARMS). J Ginseng Res 34:41–46. https://doi.org/10.5142/jgr.2010.34.1.041
Jigden B, Wang H, Samdan N, Yang DC (2010) Molecular identification of oriental medicinal plant Anemarrhena asphodeloides Bunge (‘Jimo’) by multiplex PCR. Mol Biol Rep 37:955–960. https://doi.org/10.1007/s11033-009-9747-4
Jung S-Y, Park S-H, Nam C-H, Lee H-J, Lee Y-M, Chang K-S (2013) The distribution of vascular plants in Ulleungdo and nearby island regions (Gwaneumdo, Jukdo), Korea. J Asia-Pac Biodivers 6:123–156. https://doi.org/10.7229/jkn.2013.6.1.123
Kim C-H, Kim T-J, Seon B-Y (2000) Taxonomic identities of some endemic Korean vascular plants. Korean J Plant Taxon 30:355–361
Kim JS, Jang H-W, Kim J-S, Kim H-J, Kim J-H (2012) Molecular identification of Schisandra chinensis and its allied species using multiplex PCR based on SNPs. Genes Genom 34:283–290. https://doi.org/10.1007/s13258-011-0201-3
Kim J, Kim DS, Lee ES, Ahn YK, Chae WB, Lee SS (2017) The construction of a Chinese cabbage marker-assisted backcrossing system using high-throughput genotyping technology. Hortic Sci Technol 35:232–242
Kress WJ, Erickson DL (2007) A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region. PLoS ONE 2:e508. https://doi.org/10.1371/journal.pone.0000508
Kress WJ, Erickson DL, Jones FA, Swenson NG, Perez R, Sanjur O, Bermingham E (2009) Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proc Natl Acad Sci USA 106:18621–18626. https://doi.org/10.1073/pnas.0909820106
Kwok PY (2001) Methods for genotyping single nucleotide polymorphisms. Annu Rev Genomics Hum Genet 2:235–258. https://doi.org/10.1146/annurev.genom.2.1.235
Lahaye R, van der Bank M, Bogarin D, Warner J et al (2008) DNA barcoding the floras of biodiversity hotspots. Proc Natl Acad Sci USA 105:2923–2928. https://doi.org/10.1073/pnas.0709936105
Li Q-Q, Zhou S-D, He X-J, Yu Y, Zhang Y-C, Wei X-Q (2010) Phylogeny and biogeography of Allium (Amaryllidaceae: Allieae) based on nuclear ribosomal internal transcribed spacer and chloroplast rps16 sequences, focusing on the inclusion of species endemic to China. Ann Bot 106:709–733
Liu J, Huang S, Sun M, Liu S, Liu Y et al (2012) An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods 8:34. https://doi.org/10.1186/1746-4811-8-34
Ma XY, Xie CX, Liu C, Song JY, Yao H et al (2010) Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region. Biol Pharm Bull 33:1919–1924
Mishra P, Kumar A, Nagireddy A, Mani DN, Shukla AK, Tiwari R, Sundaresan V (2016) DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market. Plant Biotechnol J 14:8–21. https://doi.org/10.1111/pbi.12419
Newmaster SG, Fazekas AJ, Steeves RA, Janovec J (2008) Testing candidate plant barcode regions in the Myristicaceae. Mol Ecol Resour 8:480–490. https://doi.org/10.1111/j.1471-8286.2007.02002.x
Nguyen NH, Driscoll HE, Specht CD (2008) A molecular phylogeny of the wild onions (Allium; Alliaceae) with a focus on the western North American center of diversity. Mol Phylogenet Evol 47:1157–1172. https://doi.org/10.1016/j.ympev.2007.12.006
Park M-J, Kim MK, In J-G, Yang D-C (2006) Molecular identification of Korean ginseng by amplification refractory mutation system-PCR. Food Res Int 39:568–574. https://doi.org/10.1016/j.foodres.2005.11.004
Sarker D, Johnson MAT, Reynolds A, Brandham PE (1997) Cytology of the highly polyploid disjunct species, Allium dregeanum (Alliaceae), and of some Eurasian relatives. Bot J Linn Soc 124:361–373. https://doi.org/10.1111/j.1095-8339.1997.tb02002.x
Semagn K, Bjørnstad Å, Ndjiondjop MN (2006) An overview of molecular marker methods for plants. Afr J Biotechnol 5:2540–2568
Sun Y, Skinner DZ, Liang GH, Hulbert SH (1994) Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theor Appl Genet 89:26–32. https://doi.org/10.1007/BF00226978
Tate JA, Simpson BB (2003) Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Syst Bot 28:723–737. https://doi.org/10.1043/02-64.1
Techen N, Parveen I, Pan Z, Khan IA (2014) DNA barcoding of medicinal plant material for identification. Curr Opin Biotechnol 25:103–110. https://doi.org/10.1016/j.copbio.2013.09.010
Wang H, Sun H, Kwon WS, Jin H, Yang DC (2010) A PCR-based SNP marker for specific authentication of Korean ginseng (panax ginseng) cultivar “Chunpoong”. Mol Biol Rep 37:1053–1057. https://doi.org/10.1007/s11033-009-9827-5
Wang H, Kim MK, Kim YJ, Lee HN, Jin H, Chen J, Yang DC (2012) Molecular authentication of the oriental medicines Pericarpium citri reticulatae and Citri unshius pericarpium using SNP markers. Gene 494:92–95. https://doi.org/10.1016/j.gene.2011.11.026
Whatmore AM, Murphy TJ, Shankster S, Young E, Cutler SJ, Macmillan AP (2005) Use of amplified fragment length polymorphism to identify and type Brucella isolates of medical and veterinary interest. J Clin Microbiol 43:761–769. https://doi.org/10.1128/JCM.43.2.761-769.2005
Yang JY, Jang SY, Kim H-K, Park SJ (2012) Development of a molecular marker to discriminate Korean Rubus species medicinal plants based on the nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL-F intergenic region sequences. J Korean Soc Appl Biol Chem 55:281–289. https://doi.org/10.1007/s13765-012-1044-6
Yao H, Song JY, Ma XY, Liu C, Li Y et al (2009) Identification of Dendrobium species by a candidate DNA barcode sequence: the chloroplast psbA-trnH intergenic region. Planta Med 75:667–669. https://doi.org/10.1055/s-0029-1185385
Ye S, Dhillon S, Ke X, Collins AR, Day IN (2001) An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29:E88
Yip PY, Chau CF, Mak CY, Kwan HS (2007) DNA methods for identification of Chinese medicinal materials. J Chin Med 2:9. https://doi.org/10.1186/1749-8546-2-9
Yoo KO, Kim WB, Park HJ, Lim SC, Jang HT (1998) Investigation on the ultrastructure of epidermis, anatomical, palynological, and cytological characteristics of Allium victorialis var. platyphyllum collected from three different habitats. J Korean Soc Hortic Sci 39:260–265 [in Korean]
You FM, Huo N, Gu YQ, Luo MC, Ma Y et al (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:253. https://doi.org/10.1186/1471-2105-9-253
Acknowledgments
This study was supported by a 2014 research Grant from Kangwon National University, and by the Rural Development Administration (RDA) and Cooperative Research Program for Agricultural Science and Technology Development (PJ012011032017).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Fig. 1
Multiple sequence alignments of matK and ITS from Allium ochotense and Allium microdictyon (PDF 208 kb)
Rights and permissions
About this article
Cite this article
Kim, YB., Ramekar, R.V., Choi, SJ. et al. Molecular identification of Allium ochotense and Allium microdictyon using multiplex-PCR based on single nucleotide polymorphisms. Hortic. Environ. Biotechnol. 59, 865–873 (2018). https://doi.org/10.1007/s13580-018-0069-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13580-018-0069-0