ORIGINAL ARTICLE Utility of a multidisciplinary approach for genome diagnostics of cultivated and wild germplasm resources of medicinal Withania somnifera, and the status of new species, W. ashwagandha, in the cultivated taxon Arun Kumar • Bilal A. Mir • Deepmala Sehgal • Tanvir H. Dar • Sushma Koul • Maharaj K. Kaul • Soom N. Raina • Ghulam N. Qazi Received: 4 May 2009 / Accepted: 4 October 2010 / Published online: 17 November 2010 Ó Springer-Verlag 2010 Abstract Realizing the inconsistencies that exist in the extent and nature of differentiation in the Withania somni- fera genetic resources in India, the 21 cultivated and wild accessions, and the two hybrids (cultivated 9 wild acces- sions and vice versa) were investigated for morphological, cytogenetical, chemical profiling, and crossability features. Their nuclear and chloroplast genomes were also assayed at the nucleotide sequence level, and by use of DNA markers. Chloroplast DNA diversity and somatic chromo- some number (2n = 48) were not helpful in identifying the differences. Other approaches, on the other hand, espe- cially restriction endonuclease digests, types and sequence length composition of ITS 1 and ITS 2 of nuclear ribo- somal DNA, AFLP fingerprinting, and crossability barriers unambiguously provided startling discrete differences between the cultivated and wild accessions, indicating a clear division of W. somnifera into two distinct lineages. These data, therefore, are indicative of the fact that because of the unique characteristics of its nuclear genome, and strong crossability barriers vis-a `-vis wild accessions of W. somnifera, the cultivated accessions should be relegated to the rank of the separate species, W. ashwagandha. Keywords AFLP markers Á Crossability Á ITS1 and ITS2 RFLP Á Sequence length composition Á Withania ashwagandha Á Withania somnifera Introduction The genus Withania (Solanaceae) has been estimated to consist of 26 species distributed widely in South Asia and the Eastern Mediterranean area (Kaul 1957; Kumar et al. 2007). Of these, only two species, W. somnifera and W. coagulans, have been reported in India. W. somnifera, commonly known as ‘‘Ashwagandha’’ or ‘‘Asgandh’’ is one of the most valued shrubby bushes in Indian traditional systems of medicine, Ayurveda and Unani. ‘‘Ashwagan- dha’’ in the Sanskrit language means smelling like a horse. The plant’s English name is winter cherry. The roots of W. somnifera contain bioactive compounds with sedative, anti-inflammatory, anti-tumour, anti-bacterial, and anti- spasmodic properties (Bhattacharya et al. 1987; Uma et al. 1993, 1996; Singh and Kumar 1998; Scartezzini et al. 2007). ‘‘Ashwagandha’’ roots are compared with ginseng roots for their restorative properties and have been given the name ‘‘Indian ginseng’’. In India, the plant grows widely in drier areas in the subtropical and semitemperate regions from plains to a height of 1,700 m. Commercial cultivation of W. somnifera in India is carried out on *5,000 ha of land in Manasa, Madhya Pradesh, and in some parts of Rajasthan, Andhra Pradesh and Uttar Pradesh (Anon 1976; Kothari et al. 2003). The estimated annual production of ‘‘Ashwagandha’’ roots in India is approxi- mately 2,000 tons. Significant levels of genotypic and phenotypic vari- ability, more especially between the wild and cultivated morphotypes, do exist in the species (Kaul 1957; Atal and A. Kumar (&) Á B. A. Mir Á S. Koul Á M. K. Kaul Á G. N. Qazi Indian Institute of Integrative Medicine, Jammu 180001, India e-mail: arunsonal2001@gmail.com D. Sehgal Á T. H. Dar Department of Botany, University of Delhi, Delhi 110007, India S. N. Raina Amity Institute of Biotechnology, Amity University, Sector - 125, Noida 201303, UP, India 123 Plant Syst Evol (2011) 291:141–151 DOI 10.1007/s00606-010-0372-4