3 Biotech

, 8:220 | Cite as

Development of cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers from the chloroplast genome of Glycyrrhiza species

  • Ick-Hyun Jo
  • Jwakyung Sung
  • Chi-Eun Hong
  • Sebastin Raveendar
  • Kyong-Hwan Bang
  • Jong-Wook Chung
Original Article


Licorice (Glycyrrhiza glabra) is an important medicinal crop often used as health foods or medicine worldwide. The molecular genetics of licorice is under scarce owing to lack of molecular markers. Here, we have developed cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers based on single nucleotide polymorphisms (SNP) by comparing the chloroplast genomes of two Glycyrrhiza species (G. glabra and G. lepidota). The CAPS and HRM markers were tested for diversity analysis with 24 Glycyrrhiza accessions. The restriction profiles generated with CAPS markers classified the accessions (2–4 genotypes) and melting curves (2–3) were obtained from the HRM markers. The number of alleles and major allele frequency were 2−6 and 0.31–0.92, respectively. The genetic distance and polymorphism information content values were 0.16–0.76 and 0.15–0.72, respectively. The phylogenetic relationships among the 24 accessions were estimated using a dendrogram, which classified them into four clades. Except clade III, the remaining three clades included the same species, confirming interspecies genetic correlation. These 18 CAPS and HRM markers might be helpful for genetic diversity assessment and rapid identification of licorice species.


Licorice Genetic diversity SNP CAPS HRM 



Single nucleotide polymorphism


Cleaved amplified polymorphic sequence


High-resolution melting


Insertion and deletion


Genetic distance


Polymorphism information content


Number of alleles


Major allele frequency


Simple sequence repeats


Author contributions

I-HJ and JS participated in the study design, coordinated the experiment, and drafted the manuscript; C-EH, SR, and K-HB performed the experiments; J-WC participated in the study design, collected samples, analyzed genetic diversity, and coordinated the draft of the manuscript. All authors critically read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Arora V, Ghosh MK, Pal S, Gangopadhyay G (2017) Allele specific CAPS marker development and characterization of chalcone synthase gene in Indian mulberry (Morus spp., family Moraceae). PLoS ONE 12(6):e0179189CrossRefGoogle Scholar
  2. Ashurmetov OA (2009) Selection of parental pairs for obtaining hybrids in the genera Glycyrrhiza L. and Meristotropis Fisch. et Mey. Genet Res Crop Evol 43:167–171CrossRefGoogle Scholar
  3. Babu BK, Mathur RK, Kumar PN, Ramajayam D, Ravichandran G, Venu MVB, Babu SS (2017) Development, identification and validation of CAPS marker for SHELL trait which governs dura, pisifera and tenera fruit forms in oil palm (Elaeis guineensis Jacq.). PLoS ONE 12(2):e0171933CrossRefGoogle Scholar
  4. Carmen CD, Adriana K, Cristina D, Codruţa Ş, Dian A (2012) Glycyrrhiza glabra and Glycyrrhiza echinata—sources of low hemotoxic saponins. Acta Med Marisiensis 58(3):150–152Google Scholar
  5. Cavalli-Sforza LL, Edwards AW (1967) Phylogenetic analysis: models and estimation procedures. Evolution 21:550–570CrossRefGoogle Scholar
  6. Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X et al (2010) Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS ONE 5(1):e8613CrossRefGoogle Scholar
  7. Choi GY, Kim YH, Chae SW, Lee HW, Ko BS, Lee MY (2011) Discrimination of Chinese Glycyrrhiza uralensis and Uzbek Glycyrrhiza glabra using taste sensor. Korean J Herbol 26:35–39Google Scholar
  8. Erayman M, Ilhan E, Güzel Y, Eren AH (2014) Transferability of SSR markers from distantly related legumes to Glycyrrhiza species. Turk J Agric For 38(1):32–38CrossRefGoogle Scholar
  9. Fenwick GR, Lutomski J, Nieman C (1990) Liquorice, Glycyrrhiza glabra L.—composition, uses and analysis. Food Chem 38(2):119–143CrossRefGoogle Scholar
  10. Fraunfelder FW (2005) Ocular side effects associated with dietary supplements and herbal medicines. Drugs Today 41:537–546CrossRefGoogle Scholar
  11. Ganopoulos I, Tsaballa A, Xanthopoulou A, Madesis P, Tsaftaris A (2013) Sweet cherry cultivar identification by high-resolution-melting (HRM) analysis using gene-based SNP markers. Plant Mol Biol Rep 31:763–768CrossRefGoogle Scholar
  12. Hayashi H, Hosono N, Kondo M, Hiraoka N, Ikeshiro Y, Shibano M, Kusano G, Yamamoto H, Inoue K (2000) Phylogenetic relationship of six Glycyrrhiza species based on rbcL sequences and chemical constituents. Biol Pharm Bull 23:602–606CrossRefGoogle Scholar
  13. Hebert PD, Cywinska A, Ball SL (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B Biol Sci 270:313–321CrossRefGoogle Scholar
  14. Jeong HJ, Jo YD, Park SW, Kang BC (2010) Identification of Capsicum species using SNP markers based on high resolution melting analysis. Genome 53:1029–1040CrossRefGoogle Scholar
  15. Jo IH, Bang KH, Hong CE, Kim JU, Lee JW, Hyun DY, Ryu H, Kim DH (2016) Analysis of the chloroplast genome and SNP detection in a salt tolerant breeding line in Korean ginseng. J Plant Biotechnol 43:417–421CrossRefGoogle Scholar
  16. Kageyama Y, Suzuki H, Saruta T (1992) Glycyrrhizin induces mineralocorticoid activity through alterations in cortisol metabolism in the human kidney. J Endocrinol 135:147–152CrossRefGoogle Scholar
  17. Kim DW, Kim RN, Choi SH, Kim DW, Nam SH, Choi HS, Koh HD, Kim A, Chae SH, Ahn JC et al (2011) EST analysis predicts putatively causative genes underlying the pharmaceutical application of Glycyrrhiza uralensis Fisch. Plant Mol Biol Rep 29:814–824CrossRefGoogle Scholar
  18. Kim HJ, Bae JY, Jang HN, Park SN (2013) Comparative study on the antimicrobial activity of Glycyrrhiza uralensis and Glycyrrhiza glabra extracts with various countries of origin as natural antiseptics. Microbiol Biotechnol Lett 41:358–366CrossRefGoogle Scholar
  19. Kim K, Lee SC, Lee J, Lee HO, Joh HJ, Kim NH, Park HS, Yang TJ (2015) Comprehensive survey of genetic diversity in chloroplast genomes and 45S nrDNAs within Panax ginseng species. PLoS ONE 10:e0117159CrossRefGoogle Scholar
  20. Kim S, Lee J, Baek S, Ahn J, Hong K (2018) Identification of DNA molecular markers by comparison of Pinus densiflora and Pinus sylvestris chloroplast genomes. PeerJ (preprints) 6:e26506v1Google Scholar
  21. Kondo K, Shiba M, Nakamura R, Morota T, Shoyama Y (2007) Constituent properties of licorices derived from Glycyrrhiza uralensis, G. glabra, or G. inflata identified by genetic information. Biol Pharm Bull 30:1271–1277CrossRefGoogle Scholar
  22. Lee YJ, Jo JH (2004) A study on a morphological identification of Glycyrrhiza radix. Korean J Herbol 19:47–52Google Scholar
  23. Lee OR, Kim MK, Yang DC (2012) Authentication of medicinal plants by SNP-based multiplex PCR. In: Sucher NJ, Hennell JR, Carles MC (eds) Plant DNA fingerprinting and barcoding: methods and protocols. Humana Press, New York, pp 135–147CrossRefGoogle Scholar
  24. Liao WC, Lin YH, Chang TM, Huang WY (2012) Identification of two licorice species, Glycyrrhiza uralensis and Glycyrrhiza glabra, based on separation and identification of their bioactive components. Food Chem 132:2188–2193CrossRefGoogle Scholar
  25. Lim JM, Ahn YS, Park CG, Park CB, Cho JH (2012) Authentication of traded medicinal herb, Glycyrrhiza spp. (Licorice), based on nrDNA-ITS2 sequence analysis. J Korean Soc Int Agric 24:435–443CrossRefGoogle Scholar
  26. Linhart YB, Grant MC (1996) Evolutionary significance of local genetic differentiation in plants. Annu Rev Ecol Syst 27:237–277CrossRefGoogle Scholar
  27. Liu JIE, Moeller M, Gao LM, Zhang DQ, Li DZ (2011) DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species. Mol Ecol Resour 11:89–100CrossRefGoogle Scholar
  28. Mackay JF, Wright CD, Bonfiglioli RG (2008) A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods 4:8CrossRefGoogle Scholar
  29. Montoro P, Maldini M, Russo M, Postorino S, Piacente S, Pizza C (2011) Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC–ESI/MS and LC–ESI/MS/MS. J Pharm Biomed Anal 54:535–544CrossRefGoogle Scholar
  30. Raveendar S, So YS, Lee KJ, Lee DJ, Sung J, Chung JW (2017) The complete chloroplast genome sequence of Glycyrrhiza lepidota (Nutt.) Pursh—an American wild licorice. J Crop Sci Biotechnol 20:295–303CrossRefGoogle Scholar
  31. Um Y, Jin ML, Lee Y, Hur M, Cha SW, Jung CS, Kim SM, Lee JH (2016) Genetic diversity analysis of Glycyrrhiza uralensis using 8 novel polymorphic microsatellite markers. J Plant Biotechnol 43:174–180CrossRefGoogle Scholar
  32. Yamazaki M, Sato A, Shimomura K, Saito K, Murakoshi I (1994) Genetic relationships among Glycyrrhiza plants determined by RAPD and RFLP analyses. Biol Pharm Bull 17:1529–1531CrossRefGoogle Scholar
  33. Yang SO, Hyun SH, Kim SH, Kim HS, Lee JH, Whang WK, Lee MW, Choi HK (2010) Differentiation of roots of Glycyrrhiza species by 1H nuclear magnetic resonance spectroscopy and multivariate statistical analysis. Bull Korean Chem 31:825–828CrossRefGoogle Scholar
  34. Yang HQ, Dong YR, Gu ZJ, Liang N, Yang JB (2012) A preliminary assessment of matK, rbcL and trnH—psbA as DNA barcodes for Calamus (Arecaceae) species in China with a note on ITS. Ann Bot Fenn 49:319–330CrossRefGoogle Scholar
  35. Zhang MY, Fan L, Liu QZ, Song Y, Wei SW, Zhang SL, Wu J (2014) A novel set of EST-derived SSR markers for pear and cross-species transferability in Rosaceae. Plant Mol Biol Rep 32:290–302CrossRefGoogle Scholar
  36. Zhao Y, Yin J, Guo H, Zhang Y, Xiao W, Sun C, Wu J, Qu X, Yu J, Wang X et al (2015) The complete chloroplast genome provides insight into the evolution and polymorphism of Panax ginseng. Front Plant Sci 5:696Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Ick-Hyun Jo
    • 1
  • Jwakyung Sung
    • 2
  • Chi-Eun Hong
    • 1
  • Sebastin Raveendar
    • 3
  • Kyong-Hwan Bang
    • 1
  • Jong-Wook Chung
    • 4
  1. 1.Department of Herbal Crop ResearchNational Institute of Horticultural and Herbal Science (NIHHS), Rural Development Administration (RDA)EumseongRepublic of Korea
  2. 2.Soil and Fertilizer DivisionNational Institute of Agricultural Science (NIAS), Rural Development Administration (RDA)WanjuRepublic of Korea
  3. 3.National Agrobiodiversity Center, National Institute of Agricultural Sciences (NIAS), Rural Development Administration (RDA)JeonjuRepublic of Korea
  4. 4.Department of Industrial Plant Science and TechnologyChungbuk National UniversityCheongjuRepublic of Korea

Personalised recommendations