Genetic polymorphism of Japanese cultivated Rheum species in the internal transcribed spacer region of nuclear ribosomal DNA

  • Mai Asanuma
  • Shu Zhu
  • Naoki Okura
  • Shao-Qing Cai
  • Kayo Yoshimatsu
  • Katsuko KomatsuEmail author
Original Paper


In order to develop new domestic production of Rhei Rhizoma (RR) from Rheum specimens cultivated in the Sugadaira Medicinal Plant Cultivation Test Field (SMPCF), the ITS sequences of 12 SMPCF specimens and Chinese Rheum specimens of four species, as well as RR samples produced in North Korea, China and Japan, were determined by subcloning and their sequences were compared. As the ITS sequences of 10 SMPCF specimens showed significant intra-individual polymorphism, identification of pseudogenes was conducted by detecting the three motifs of the 5.8S sequence and the stability of the 5.8S secondary structure. Approximately 46% of sequences obtained from the SMPCF specimens were putative pseudogenes. The maximum likelihood tree based on ITS sequences showed three main groups—the outer group and inner clusters I and II; clones from 10 SMPCF specimens including putative pseudogenes belonged to the outer group. Cluster I was composed of two clades, one including clones from R. officinale specimens and R. palmatum-derived samples with matK genotype Rp9, and another including clones from R. coreanum-derived samples. Cluster II consisted of three clades, one including clones from R. palmatum specimens with genotype Rp5, another including clones mainly from R. tanguticum specimens with genotype Rt4, and the third including clones from R. palmatum or R. tanguticum specimens with various matK genotypes. Clones from SMPCF specimen RC5 showed a close relationship with those from R. tanguticum specimens with matK genotype Rt4, whereas those from specimen RC9 related to R. coreanum-derived samples. As a result, specimens RC5 and RC9 were considered as candidates for the development of domestic RR.


Rheum ITS polymorphism Pseudogene Phylogenetic analysis Rhubarb 



The authors would like to thank Mr. Morikazu Murakami and Mr. Takayuki Tamura of the Medicinal Plants Center, Toyama Prefectural Institute for Pharmaceutical Research, all the staff of the SMPCF and Dr. Shengli Wei from the School of Chinese Pharmacy, Beijing University of Chinese Medicine for helping in sample collection. This work was supported by the Research on Development of New Drugs from Japan Agency for Medical Research and Development, AMED [Grant number JP16ak0101034h0002, JP17ak0101034h0003] and in part by 2017 Director Leadership Expenses, University of Toyama and JSPS Core-to-Core Program, B. Asia-Africa Science Platforms.

Supplementary material

11418_2019_1298_MOESM1_ESM.xlsx (60 kb)
Supplementary material 1 (XLSX 60 kb)


  1. 1.
    Namba T (1993) The Encyclopedia of Wakan-Yaku (Traditional Sino-Japanese Medicines) with color pictures, 2nd edn. Hoikusha Publishing Co. Ltd, Osaka, pp 16–20Google Scholar
  2. 2.
    The Ministry of Health, Labour and Welfare (2016) The Japanese pharmacopoeia, 17th edn. The MHLW Ministerial Notification No. 64, Tokyo, pp 1955–1956 (English version) Google Scholar
  3. 3.
    Komatsu K, Nagayama Y, Tanaka K, Ling Y, Cai SQ, Omote T, Meselhy RM (2006) Comparative study of chemical constituents of rhubarb from different origins. Chem Pharm Bull 54:1491–1499. CrossRefGoogle Scholar
  4. 4.
    Hatta R, Matsuoka T (1999) Cultivation of medicinal plants and creation of the new breed of Daiou, Dahuang. Nat Med 53:37–40Google Scholar
  5. 5.
    Goto M, Nagao Y (1984) The quality of rhubarbs and the cultivation of a rhubarb (Shinshu Daio). Kampo Med 35:77–85CrossRefGoogle Scholar
  6. 6.
    Shibata S, Kobayashi S (1983) Trial cultivation of rhubarb in Japan. J Tradit Sino Jpn Med 4:68–71Google Scholar
  7. 7.
    Yang DY, Fushimi H, Cai SQ, Komatsu K (2004) Molecular analysis of Rheum species used as Rhei Rhizoma based on the chloroplast matK gene sequence and its application for identification. Biol Pharm Bull 27:375–383CrossRefGoogle Scholar
  8. 8.
    Birky CW (1995) Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution. Proc Natl Acad Sci USA 92:11331–11338CrossRefGoogle Scholar
  9. 9.
    Foust CM (1994) Mysteries of rhubarb: Chinese medicinal rhubarb through the ages. Pharm Hist 36:155–159Google Scholar
  10. 10.
    Fujita N (1933) Rhubarb. J Jpn Bot 8:367–372Google Scholar
  11. 11.
    Sone T, Fujisawa M, Takenaka M, Nakagawa S, Yamaoka S, Sakaida M, Nishiyama R, Yamato KT, Ohmido N, Fukui K, Fukuzawa H, Ohyama K (1999) Bryophyte 5S rDNA was inserted into 45S rDNA repeat units after the divergence from higher land plants. Plant Mol Biol 41:679–685CrossRefGoogle Scholar
  12. 12.
    Poczai P, Hyvönen J (2010) Nuclear ribosomal spacer regions in plant phylogenetics: problems and prospects. Mol Biol Rep 37:1897–1912CrossRefGoogle Scholar
  13. 13.
    Ma X, Xie C, Guan M, Xu X, Miki E, Takeda O, Xin T (2014) High levels of genetic diversity within one population of Rheum tanguticum on the Qinghai-Tibet plateau have implications for germplasm conservation—Appendix. Pharm Crop 5:1–8CrossRefGoogle Scholar
  14. 14.
    Jobes DV, Thien LB (1997) A conserved motif in the 5.8S ribosomal RNA (rRNA) gene is a useful diagnostic marker for plant internal transcribed spacer (ITS) sequences. Plant Mol Biol Rep 15:326–334CrossRefGoogle Scholar
  15. 15.
    Harpke D, Peterson A (2006) Non-concerted ITS evolution in Mammillaria (Cactaceae). Mol Phylogenet Evol 41:579–593. CrossRefGoogle Scholar
  16. 16.
    Harpke D, Peterson A (2008) Extensive 5.8S nrDNA polymorphism in Mammillaria (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions. J Plant Res 121:261–270. CrossRefGoogle Scholar
  17. 17.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415. CrossRefGoogle Scholar
  18. 18.
    Xu B, Zeng XM, Gao XF, Jin DP, Zhang LB (2017) ITS non-concerted evolution and rampant hybridization in the legume genus Lespedeza (Fabaceae). Sci Rep 7:1–15. CrossRefGoogle Scholar
  19. 19.
    Hřibová E, Čížková J, Christelová P, Taudien S, de Langhe E, Doležel J (2011) The ITS1-5.8S-ITS2 sequence region in the Musaceae: structure, diversity and use in molecular phylogeny. PLoS One 6:e17863. CrossRefGoogle Scholar
  20. 20.
    Wuyts J, De Rijk P, Van De Peer Y, Winkelmans T, De Wachter R (2001) The European large subunit ribosomal RNA database. Nucleic Acids Res 29:175–177CrossRefGoogle Scholar
  21. 21.
    Harpke D, Peterson A (2008) 5.8S motifs for the identification of pseudogenic ITS regions. Botany 86:300–305. CrossRefGoogle Scholar
  22. 22.
    Bailey C, Carr T, Harris S, Hughes C (2003) Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes. Mol Phylogenet Evol 29:435–455. CrossRefGoogle Scholar
  23. 23.
    Xiao LQ, Möller M, Zhu H (2010) High nrDNA ITS polymorphism in the ancient extant seed plant Cycas: incomplete concerted evolution and the origin of pseudogenes. Mol Phylogenet Evol 55:168–177. CrossRefGoogle Scholar
  24. 24.
    Queiroz CS, Batista FRC, Oliveira LO (2011) Evolution of the 5.8S nrDNA gene and internal transcribed spacers in Carapichea ipecacuanha (Rubiaceae) within a phylogeographic context. Mol Phylogenet Evol 59:293–302. CrossRefGoogle Scholar
  25. 25.
    Yokota Y, Kawata T, Iida Y, Kato A, Tanifuji S (1989) Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA. J Mol Evol 29:294–301CrossRefGoogle Scholar
  26. 26.
    Zheng X, Cai D, Yao L, Teng Y (2008) Non-concerted ITS evolution, early origin and phylogenetic utility of ITS pseudogenes in Pyrus. Mol Phylogenet Evol 48:892–903. CrossRefGoogle Scholar
  27. 27.
    Wei S, Zhu S, Tu P, Cai SQ, Komatsu K (2007) Molecular analysis of genus Rheum (4) Geographical characteristics of matK genotypes. Abstract of The 54the Annual Meeting of the Japanese Society of Pharmacognosy. Nagoya, p 266Google Scholar

Copyright information

© The Japanese Society of Pharmacognosy 2019

Authors and Affiliations

  • Mai Asanuma
    • 1
  • Shu Zhu
    • 1
  • Naoki Okura
    • 2
  • Shao-Qing Cai
    • 3
  • Kayo Yoshimatsu
    • 4
  • Katsuko Komatsu
    • 1
    Email author
  1. 1.Division of Pharmacognosy, Department of Medicinal Resources, Institute of Natural MedicineUniversity of ToyamaToyamaJapan
  2. 2.Pharmaceutical Affairs DivisionHealth and Welfare Department of Nagano PrefectureNaganoJapan
  3. 3.School of Pharmaceutical SciencesPeking UniversityBeijingPeople’s Republic of China
  4. 4.Research Center for Medicinal Plant ResourcesNational Institutes of Biomedical Innovation, Health and NutritionTsukubaJapan

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