Indian Journal of Biotechnology Vol 9, July 2010, pp 325-328 Short Communications Intraspecific variation in the internal transcribed spacer (ITS) regions of rDNA in Withania somnifera (Linn.) Dunal Bilal Ahmad Mir 1 , Sushma Koul 1 *, Arun Kumar 1 , Maharaj K Kaul 1 , Amarjit Singh Soodan 2 and Soom Nath Raina 3 1 Biodiversity and Applied Botany Division, Indian Institute of Integrative Medicine, Jammu 180 001, India 2 Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India 3 Cellular and Molecular Cytogenetics Laboratory, University of Delhi, Delhi 110 007, India Received 2 June 2009; revised 1 December 2009; accepted 5 February 2010 Intraspecific variation in ITS regions of the rDNA among the five wild and five cultivated genotypes of Withania somnifera, were evaluated at nucleotide sequence level using restriction fragment length polymorphism (RFLP). The entire internal tran- scribed spacer (ITS1-5.8S-ITS2) region was first amplified by PCR and then cleaved with four different restriction enzymes (EcoRV, Hinf I, Afa I & Hae III). Restriction endonuclease digests, types, and sequence length composition of ITS 1 and ITS 2 of nuclear ribosomal DNA provided discrete differences between the cultivated and wild genotypes. A 710 bp single amplified product was obtained in all the five wild genotypes whereas, two ITS bands named as ITS type A and B of 709 bp and 552 bp, respectively were obtained in the five cultivated genotypes. A single deletion at 672 position was noted in ITS type A of cultivated genotypes. There was no restriction site in 552 bp ITS band for all the four restriction enzymes used. The variation of ITS at amplification as well as digestion level is in conformity with morphological and phytochemical differences in W. somnifera genotypes. Keywords: Withania somnifera, RFLP, internal transcribed spacer (ITS), intraspecific variation Withania somnifera also known as Ashwagandha or Asgandh is considered a wonder herb with diverse therapeutic values in the Ayurvedic and indigenous medical systems. Ashwagandha roots are compared with ginseng roots for their restorative properties and have been given the name ‘Indian ginseng’. The plant is reported to possess adaptogenic, antiinflammatory, immunosuppressive, antioxidant, immunomodulatory and anticancerous properties 1-5 . In India, the plant grows widely in drier areas in the subtropical and semi-temperate regions from plains up to a height of 1700 m. Commercial cultivation of W. somnifera in India is carried out in ∼ 5000 ha land in Manasa (Madhya Pradesh) and in some parts of Rajasthan, Andhra Pradesh and Uttar Pradesh 6,7 . The estimated annual production of Ashwagandha roots in India is about 2000 tons. An extreme degree of variability exists in W. somnifera with respect to growth habit, chemical profiling and morphological characteristics of plants in different parts of India and in other countries 8,9 . The cultivated morphotypes are reported to be distinct from the wild ones 9-12 . In Arabic and Persian literature, both cultivated and wild plants are separately described, former referred to as Kaknaj-i- bostani and the latter as Kaknaj-i-jaballi 10 . Further, Negi et al 13 grouped the Indian germplasm into Nagori and Kashmiri groups based on AFLP data. Internal transcribed spacer (ITS) regions of the nuclear rDNA genes, ITS1 and ITS2, have proved one of the most informative regions of variable DNA for phylogenetic analysis at the level of species relationships within genera 14,15 . The ITS regions are non-coding DNA that are transcribed to RNA, but spliced out during ribosome assembly. Being non- coding, they accumulate DNA mutations much more rapidly than the 5.8S rDNA gene. Ribosomal genes, although present in high copy number, are usually homogeneous in DNA sequence within individuals through the process of concerted evolution and so are effectively equivalent to the study of variation of a single gene locus 16 . However, in some cases, concerted evolution is incomplete and multiple nuclear rDNA sequence types co-exist in the genome 17 . The nuclear genomic ITS1, 5.8S and ITS2 regions of rDNA are contiguous and, being ~700 bases in total length, can be readily amplified by PCR as a single unit and studied together. Significant levels of genotypic and pheno- typic variability, more especially the wild and culti- vated morphotypes, do exist in the species 9-12 . The present study was aimed to confirm the classification into cultivated and wild genotypes based on ITS region of rDNA in W. somnifera. The seeds of the wild and cultivated genotypes (Fig. 1) were collected from Bikaner (Rajasthan) and —————— *Author for correspondence: Tel : 91-191-2569020; Mobile : 09419131891 E-mail : koul_sushma@yahoo.co.in