DNA Barcodes Distinguish Withania somnifera and Withania ashwagandha

  • Saloni Malik
  • Bilal A. Mir
  • Hemant K. Singh
  • Manju Chaudhary
  • Soom N. Raina
  • Shashi B. Babbar
Research Article


On the basis of morphology, chemical profiling, crossing ability, amplified fragment length polymorphism and comparison of nucleotide sequences of nuclear ribosomal internal transcribed spacer (ITS), the cultivated form of Withania somnifera, a species of therapeutic value, has been circumscribed as a new species, Withania ashwagandha. The present study was undertaken to ascertain whether the two species can be distinguished on the basis of DNA barcoding. Six barcode loci, ITS, ITS2, matK (maturase K), rbcL (ribulose-bisphosphate carboxylase/oxygenase, large subunit), rpoC1 (RNA polymerase-β′ subunit, main catalytic subunit) and trnH-psbA spacer (transfer RNA for histidine-photosystem II protein D1 spacer) from W. somnifera, W. ashwagandha, their hybrid, and ‘ashwagandha’ market samples were amplified, sequenced, and compared. ITS, ITS2 and matK distinguished two species on the basis of phylogenetic tree method. Likewise, BLAST 1 analysis based on ITS, ITS2, matK, and rbcL individually discriminated two species. However, on the basis of Kimura 2 Parameter distances, two species could not be distinguished as the requirement of a distinct barcode gap—the highest intraspecific distance being lower than the lowest interspecific distance—was not met by any of the loci. If compared by character-based method, ITS, ITS2 and matK sequences of the two species had distinct diagnostic nucleotides (pure character attributes) at nine, four and one positions, respectively. Interestingly, all market samples co-segregated and shared character attributes with W. ashwagandha.


DNA barcoding Withania ITS matK rbcL 



The authors thank Dr. Sushma Koul formerly from the Biodiversity and Applied Botany Division, Institute of Integrative Medicine, Jammu for providing the plant material. They gratefully acknowledge the financial support provided by the Indian Council of Medical Research, New Delhi to S.B.B. in form of a research project. S.M. too thankfully acknowledges the receipt of Junior and Senior Research fellowships from this project. The present work was also partially supported by a Research and Development grant provided to S.B.B. by the University of Delhi. The language editing of the manuscript by Dr. Anupam Raina, a professional copy editor, is gratefully acknowledged.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

40011_2017_879_MOESM1_ESM.docx (12.7 mb)
Supplementary material 1 (DOCX 13001 kb)


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Copyright information

© The National Academy of Sciences, India 2017

Authors and Affiliations

  1. 1.Department of BotanyUniversity of DelhiDelhiIndia
  2. 2.Department of Botany, School of Life Sciences, Satellite Campus KargilUniversity of KashmirKargilIndia
  3. 3.Amity Institute of BiotechnologyAmity UniversityNoidaIndia

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