Advertisement

Molecular identification of Allium ochotense and Allium microdictyon using multiplex-PCR based on single nucleotide polymorphisms

  • Yong-Bog Kim
  • Rahul Vasudeo Ramekar
  • Seong-Jin Choi
  • Byoung-Gon Choi
  • Se-Won Kim
  • Youn-Ki Moon
  • Hee-Sun Noh
  • Ju-Kyong Lee
  • Jin-Sung Hong
  • Nam-Il Park
  • Ik-Young Choi
  • Seon-Kang Choi
  • Kyong-Cheul Park
Research Report Genetics and Breeding
  • 32 Downloads

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.

Keywords

ITS matK psbA-trnH rbcL Amplification refractory mutation system (ARMS)-PCR 

Notes

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).

Supplementary material

13580_2018_69_MOESM1_ESM.pdf (208 kb)
Supplementary Fig. 1 Multiple sequence alignments of matK and ITS from Allium ochotense and Allium microdictyon (PDF 208 kb)
13580_2018_69_MOESM2_ESM.docx (14 kb)
Supplementary material 2 (DOCX 13 kb)
13580_2018_69_MOESM3_ESM.docx (13 kb)
Supplementary material 3 (DOCX 12 kb)

References

  1. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 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 CrossRefGoogle Scholar
  3. Choi H-J, Jang C-G, Ko S-C, Oh B-U (2004) A taxonomic review of Korean Allium (Alliaceae). Korean J Plant Taxon 34:119–152CrossRefGoogle Scholar
  4. 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 39:913–920CrossRefGoogle Scholar
  5. 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 CrossRefPubMedGoogle Scholar
  6. 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 CrossRefGoogle Scholar
  7. Friesen N (1995) The genus Allium L. in the flora of Mongolia. Feddes Repert 106:59–81.  https://doi.org/10.1002/fedr.19951060116 CrossRefGoogle Scholar
  8. 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 CrossRefPubMedGoogle Scholar
  9. Friesen N, Fritsch RM, Blattner FR (2006) Phylogeny and new intrageneric classification of Allium (Alliaceae) based on nuclear ribosomal DNA ITS sequences. Aliso 22:372–395CrossRefGoogle Scholar
  10. 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 CrossRefPubMedGoogle Scholar
  11. 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 49:145–220Google Scholar
  12. 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 CrossRefGoogle Scholar
  13. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 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 CrossRefPubMedGoogle Scholar
  15. Hall BG (2013) Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 30:1229–1235.  https://doi.org/10.1093/molbev/mst012 CrossRefPubMedGoogle Scholar
  16. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 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 34:912–925Google Scholar
  18. 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 CrossRefGoogle Scholar
  19. 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 CrossRefPubMedGoogle Scholar
  20. 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 CrossRefGoogle Scholar
  21. 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 CrossRefPubMedGoogle Scholar
  22. 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 CrossRefGoogle Scholar
  23. Kim C-H, Kim T-J, Seon B-Y (2000) Taxonomic identities of some endemic Korean vascular plants. Korean J Plant Taxon 30:355–361CrossRefGoogle Scholar
  24. 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 CrossRefGoogle Scholar
  25. 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 35:232–242Google Scholar
  26. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 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 CrossRefPubMedGoogle Scholar
  28. 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 CrossRefPubMedGoogle Scholar
  29. 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 CrossRefPubMedGoogle Scholar
  30. 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 106:709–733CrossRefGoogle Scholar
  31. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 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 33:1919–1924CrossRefGoogle Scholar
  33. 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 CrossRefPubMedGoogle Scholar
  34. 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 CrossRefPubMedGoogle Scholar
  35. 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 CrossRefPubMedGoogle Scholar
  36. 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 CrossRefGoogle Scholar
  37. 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 CrossRefGoogle Scholar
  38. Semagn K, Bjørnstad Å, Ndjiondjop MN (2006) An overview of molecular marker methods for plants. Afr J Biotechnol 5:2540–2568Google Scholar
  39. 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 CrossRefPubMedGoogle Scholar
  40. 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 CrossRefGoogle Scholar
  41. 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 CrossRefPubMedGoogle Scholar
  42. 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 CrossRefPubMedGoogle Scholar
  43. 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 CrossRefPubMedGoogle Scholar
  44. 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 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 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 CrossRefGoogle Scholar
  46. 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 CrossRefPubMedGoogle Scholar
  47. Ye S, Dhillon S, Ke X, Collins AR, Day IN (2001) An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res 29:E88CrossRefGoogle Scholar
  48. 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 CrossRefGoogle Scholar
  49. 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] Google Scholar
  50. 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 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Korean Society for Horticultural Science 2018

Authors and Affiliations

  • Yong-Bog Kim
    • 1
  • Rahul Vasudeo Ramekar
    • 2
  • Seong-Jin Choi
    • 1
  • Byoung-Gon Choi
    • 1
  • Se-Won Kim
    • 1
  • Youn-Ki Moon
    • 1
  • Hee-Sun Noh
    • 1
  • Ju-Kyong Lee
    • 2
  • Jin-Sung Hong
    • 2
  • Nam-Il Park
    • 3
  • Ik-Young Choi
    • 4
  • Seon-Kang Choi
    • 4
  • Kyong-Cheul Park
    • 4
  1. 1.Gangwon-do Agricultural Research and Extension ServicesChuncheonSouth Korea
  2. 2.Department of Applied Plant Sciences, College of Agriculture and Life SciencesKangwon National UniversityChuncheonSouth Korea
  3. 3.Department of Plant ScienceGangneung-Wonju National UniversityGangneungSouth Korea
  4. 4.Department of Agriculture and Life IndustryKangwon National UniversityChuncheonSouth Korea

Personalised recommendations