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Development of genome-wide simple sequence repeat markers in Codonopsis lanceolata using next-generation sequencing


Codonopsis lanceolata is an herbaceous perennial plant predominantly cultivated in East Asia and used for medicinal purposes. However, genetic information of C. lanceolata is lacking. Therefore, we sequenced genomic DNA using next-generation sequencing (NGS) and searched for simple sequence repeats (SSRs) to develop molecular markers in C. lanceolata. A total of 250,455 SSRs were identified, and di-nucleotides and tri-nucleotides accounted for the majority of all the SSRs. Among these SSRs, we designed 26,334 primer sets from di- to octa-nucleotide motifs. We used an in silico approach to investigate 2626 SSRs (tri- to penta-nucleotide motifs) and found 573 SSRs showing polymorphism. Of the 573 SSRs showing polymorphism in silico, we randomly selected 39 SSRs and verified polymorphism in 16 C. lanceolata accessions. The number of alleles ranged from 2 to 13, and the mean polymorphic information content value was 0.54. Therefore, we successfully designed 39 SSR markers for use in breeding and genetic studies of C. lanceolata.

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Data availability

Sequencing reads have been deposited in the National Agricultural Biotechnology Information Center (NABIC) Sequence Read Archive (BioProject ID: NN-7251, NN-7252, NN-7253, NN-7254, NN-7255, and NN-7256). Primer sequences have been deposited in the National Center for Biotechnology Information’s GenBank database; accession numbers are listed in Table 3.


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    CAS  Article  Google Scholar 

  2. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinf 30:2114–2120

    CAS  Article  Google Scholar 

  3. Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314–331

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Celik I, Gultekin V, Allmer J, Doganlar S, Frary A (2014) Development of genomic simple sequence repeat markers in opium poppy by next-generation sequencing. Mol Breed 34:323–334

    CAS  Article  Google Scholar 

  5. Edwards KJ, Barker JH, Daly A, Jones C, Karp A (1996) Microsatellite libraries enriched for several microsatellite sequences in plants. Biotech 20:758–760

    CAS  Article  Google Scholar 

  6. Eujayl I, Sledge MK, Wang L, May GD, Chekhovskiy K, Zwonitzer JC, Mian MAR (2004) Medicago truncatula EST-SSRs reveal cross-species genetic markers for Medicago spp. Theor Appl Genet 108:414–422

    CAS  Article  Google Scholar 

  7. He JY, Ma N, Zhu S, Komatsu K, Li ZY, Fu WM (2015) The genus Codonopsis (Campanulaceae): a review of phytochemistry, bioactivity and quality control. J Nat Med 69:1–21

    Article  Google Scholar 

  8. Holt RA, Jones SJ (2008) The new paradigm of flow cell sequencing. Genome Res 18:839–846

    CAS  Article  Google Scholar 

  9. Hosseinzadeh-Colagar A, Haghighatnia MJ, Amiri Z, Mohadjerani M, Tafrihi M (2016) Microsatellite (SSR) amplification by PCR usually led to polymorphic bands: Evidence which shows replication slippage occurs in extend or nascent DNA strands. Mol Bio Res Commun 5:167–174

    CAS  Google Scholar 

  10. Howlader J, Hong Y, Natarajan S, Sumi KR, Kim HT, Park JI, Nou IS (2020) Development of powdery mildew race 5-specific SNP markers in Cucumis melo L. using whole-genome resequencing. Hortic Environ Biotechnol 61:347–357

    CAS  Article  Google Scholar 

  11. Iniguez-Luy FL, Voort AV, Osborn TC (2008) Development of a set of public SSR markers derived from genomic sequence of a rapid cycling Brassica oleracea L. genotype. Theor Appl Genet 117:977–985

    CAS  Article  Google Scholar 

  12. Ishida S, Okasaka M, Ramos F, Kashiwada Y, Takaishi Y, Kodzhimatov OK, Ashurmetov O (2008) New alkaloid from the aerial parts of Codonopsis clematidea. J Nat Med 62:236–238

    CAS  Article  Google Scholar 

  13. Jun TH, Michel AP, Mian MR (2011) Development of soybean aphid genomic SSR markers using next generation sequencing. Genome 54:360–367

    CAS  Article  Google Scholar 

  14. Jung SW, Han AJ, Hong HJ, Choung MG, Kim KS, Park SH (2006) α-glucosidase inhibitors from the roots of Codonopsis lanceolata Trautv. Agric Chem Biotechnol 49:162–164

    CAS  Google Scholar 

  15. Kim HJ, Lee JN, Cho KS, Won HS, Suh JT (2019) Genetic diversity and population structure analysis of Ever-bearing and June-bearing strawberry cultivars using SSR markers. Hortic Sci Technol 37:108–118

    Google Scholar 

  16. Kim S, Jeong JH, Chung H, Kim JH, Gil J, Yoo J, Um Y, Kim OT, Kim TD, Kim YY, Lee DH et al (2016) Simple sequence repeat marker development from Codonopsis lanceolata and genetic relation analysis. J Plant Biotechnol 43:181–188

    Article  Google Scholar 

  17. Kölliker R, Jones ES, Drayton MC, Dupal MP, Forster JW (2001) Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.). Theor Appl Genet 102:416–424

    Article  Google Scholar 

  18. Komoto N, Ichikawa M, Ohta S, Nakano D, Nishihama T, Ushijima M, Kodera Y, Hayama M, Shirota O, Sekita S et al (2010) Murine metabolism and absorption of lancemaside A, an active compound in the roots of Codonopsis lanceolata. J Nat Med 64:321–329

    CAS  Article  Google Scholar 

  19. Lee KT, Choi J, Jung WT, Nam JH, Jung HJ, Park HJ (2002) Structure of a new echinocystic acid bisdesmoside isolated from Codonopsis lanceolata roots and the cytotoxic activity of prosapogenins. J Agric Food Chem 50:4190–4193

    CAS  Article  Google Scholar 

  20. Li Z, Liu X, Wang X, Fan B, Wang X, Zhao G (2013) Isolation and characterization of novel microsatellite markers in Codonopsis tangshen (Campanulaceae). Conserv Genet Res 5:393–395

    Article  Google Scholar 

  21. Li ZH, Wen HY, Chen J, Wu GL, Wang YJ (2009) Development of 10 polymorphic microsatellite loci primers for Codonopsis pilosula Nannf. (Campanulaceae). Conserv Genet 10:747–749

    CAS  Article  Google Scholar 

  22. Liu K, Muse SV (2005) PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinf 21:2128–2129

    CAS  Article  Google Scholar 

  23. Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y et al (2012) SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaSci 1:18

    Article  Google Scholar 

  24. Nguyen TK, Ha STT, Lim JH (2020) Analysis of chrysanthemum genetic diversity by genotyping-by-sequencing. Hortic Environ Biotechnol 61:903–913

    CAS  Article  Google Scholar 

  25. Park G, Kim J, Jin B, Yang HB, Park SW, Kang SC, Chung SM, Park Y (2018) Genome-wide sequence variation in watermelon inbred lines and its implication for marker-assisted breeding. Hortic Sci Technol 36:280–291

    CAS  Google Scholar 

  26. Park JS, Park JH, Kim SJ, Park YD (2020) Genome analysis of tissue culture-derived variations in regenerated Brassica rapa ssp. pekinensis plants using next-generation sequencing. Hortic Environ Biotechnol 61:549–558

    CAS  Article  Google Scholar 

  27. Rafalski JA, Vogel JM, Morgante M, Powell W, Andre C, Tingey SV (1996) Generating and using DNA markers in plants. In Non-mammalian genomic analysis: a practical guide. Academic Press, San Diego, pp 75-134

  28. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386

    CAS  PubMed  Google Scholar 

  29. Shinde D, Lai YL, Sun FZ, Arnheim N (2003) Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)(n) and (A/T)(n) microsatellites. Nucleic Acids Res 31:974–980

    CAS  Article  Google Scholar 

  30. Squirrell J, Hollingsworth PM, Woodhead M, Russell J, Lowe AJ, Gibby M, Powell W (2003) How much effort is required to isolate nuclear microsatellites from plants? Mol Ecol 12:1339–1348

    CAS  Article  Google Scholar 

  31. Stanke M, Steinkamp R, Waack S, Morgenstern B (2004) AUGUSTUS: a web server for gene finding in eukaryotes. Nucleic Acids Res 32(Web server):W309–W312

    CAS  Article  Google Scholar 

  32. Taheri S, Lee Abdullah T, Yusop MR, Hanafi MM, Sahebi M, Azizi P, Shamshiri RR (2018) Mining and development of novel SSR markers using next generation sequencing (NGS) data in plants. Mol 23:399

    Article  Google Scholar 

  33. Ushijima M, Komoto N, Sugizono Y, Mizuno I, Sumihiro M, Ichikawa M, Sekita S (2008) Triterpene glycosides from the roots of Codonopsis lanceolata. Chem Pharm Bull 56:308–314

    CAS  Article  Google Scholar 

  34. Van Oosterhout C, Hutchinson WF, Wills DP, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538

    Article  Google Scholar 

  35. Yang T, Fang L, Zhang X, Hu J, Bao S, Hao J, Li L, He Y, Jiang J, Wang F et al (2015) High-throughput development of SSR markers from pea (Pisum sativum L) based on next generation sequencing of a purified chinese commercial variety. PLoS ONE 10:e0139775

    Article  Google Scholar 

  36. Yoo GE, Lee U (1989) A taxonomic study of the genus Codonopsis in Korea. Kor J Plant Tax 19:81–102

    Article  Google Scholar 

  37. Zhu H, Senalik D, McCown BH, Zeldin EL, Speers J, Hyman J, Bassil N, Hummer K, Simon PW, Zalapa JE (2012) Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (Vaccinium macrocarpon Ait.). Theor Appl Genet 124:87–96

    CAS  Article  Google Scholar 

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This work was carried out with the support of "Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01588301)" Rural Development Administration, Republic of Korea.

Author information




SCK, OTK, S-CK, HBK, YL: conceived and designed the experiments, SK, JG, YU: performed the experiments, NJ, CPH, S-GP, YL: analyzed the data, DHL, B-HJ: contributed reagents/materials/analysis tools, SK, NJ, YL: wrote the paper.

Corresponding author

Correspondence to Yi Lee.

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The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Communicated by Inhwa Yeam.

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Kim, S., Jo, N., Gil, J. et al. Development of genome-wide simple sequence repeat markers in Codonopsis lanceolata using next-generation sequencing. Hortic. Environ. Biotechnol. 62, 985–993 (2021).

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  • Codonopsis lanceolata
  • Marker
  • Medicinal plant
  • Next-generation sequencing
  • Simple sequence repeat