Medicinal plants have been given greatsignificance in recent years due to its demand in industry for human and animal welfare and alluring market prices(Lubbe and Verpoorte 2011).
India is called as the “Botanical Garden” of theworld due to variegated climatic ecosystem which is suitable for cultivationfor medicinal plants. India being one of theworld’s 12 mega biodiversity countries needs to conserve its resources wherethey are being exploited and should be grown commercially to avoid theirsusceptibility to extinction because of indiscriminate use.Among the various medicinal plants, Withaniasomnifera (L.) Dunal(Winter cherry, Ashwagandha or Asgandh of family Solanaceae is animportant medicinal plant that finds extensive use as a potential herb in thetraditional system of medicine as a ‘rasayana’ and ‘medhya rasayana’.
The similarities betweenroots of Ashwagandha and ginseng roots have led to it being called as Indianginseng (Tripathi et al. 1996).W.somnifera is a geneticallysimple species (2n = 48; n = 24; largely self-pollinated) most suited todevelop cultivars for commercial production of novel sterols and alkaloids(Singh and Kumar 1998). It grows in dry and sub-tropical regions. The major Ashwagandha cultivating states areMadhya Pradesh, Rajasthan, Punjab, Uttar Pradesh, Haryana, Gujarat andMaharashtra among which Madhya Pradesh alone is having more than 4000 ha areafor cultivation. Due to presence of alkaloids in roots, leaves and seeds, thesesare used in preparation of Ayurvedic and Unani medicines, to combat a widerange of diseases from tuberculosis to arthritis. Important part of ashwagandhais its roots, followed by leaves and berries due to presence of “Withanolides” (Gupta et al.
1996). Themajor biochemical constituents of W. somnifera are steroidal alkaloids andlactones, a class of constituents together known as withanolides (steroidallactones with ergostane skeleton).
Ongoing trials and research on animal support therole of ashwagandha’s root and leaf extracts in different disorders anddiseases and possess properties like anticancer, antioxidant etc. (Chopra etal. 2004; Cooley et al. 2007; Murthy et al. 2010; Rasool et al. 2000;Padmavathi et al. 2005; Bhattacharya et al. 2006) and act as source of arestorative drug (Asthana and Raina 1989).
Molecularmarkers remain unaffected by physiological condition and environmental factors thatis the reason for their wide application in genetic diversity assessment among W.somnifera (L.) Dunal genotypesand to identify duplicated accessions within the germplasm collections. Due tosame reason, molecular markers are reliable for informative polymorphisms sincegenetic composition is unique for each species. Most important development hasoccurred in the field of molecular genetics with the emergence of molecularmarker since for breeders it is effective tool for investigating novel sourcesof variations and genetic factors controlling quantitativelyinherited traits. These markers areused for the detection and exploitation of DNA polymorphism (Semagn et al. 2010).
For differentiating plants at inter- and/orintra-specific level genetic polymorphism plays significant role, not only inmedicinal plants but also in cereals, cash, plantation and horticulture crops. Themost important role of conservation is to preserve the process of geneticdiversity and development in the viable population of ecology and commerciallyviable varieties / genotypes to avoid possible extinction (Rout et al.2010). Different types of marker systemshave been used for biodiversity analysis. These include RFLP, SSR, RAPD and theAFLP. RAPD and ISSR markers are two molecular approaches that havebeen used to detect variation among plants.
Systematic evaluation and quantification of the variability from the presentstudy will serve as one step towards providing accurate genetic information forfurther breeding programmes for Withaniaimprovement. The assessment of variation would provide us acorrect picture of the extent of variation, further helping us to improve thegenotypes for biotic and abiotic stresses. The main objective of this study was to characterizethe Withania genotypes using morphologicaland molecular markers in order to evaluate the genetic diversity andrelationships among genotypes lines. 2.Materials and methodsThepresent field investigation were carried out during late kharifof 2013 and 2014 InstructionalFarm, Rajasthan College of Agriculture, MPUAT, Udaipur(24035’N, 70042’E), Rajasthan (India).
2.1.Plant materialsPlantsof 25 genotypes lines which include native and foreign plants collected from differentparts of India, were maintained and considered for the present study (Table 1). Newly emerged leaf samples of the cultivarswere used for DNA extraction.2.2.
Morphological analysisSevenmorphometric characters were evaluated from 25 genotypes lines of plantspecimens. Standardization of data on morphological characters was done usingthe YBAR option of the Stand program from the NTSYS-pc 2.1 software (Rohlf 2004).Duplicate measurements for each line were averaged and were used to design adata matrix of pairwise similarities between genotypes lines. The simplematching coefficient (SMC) was used to measure the similarity, as it was thecoefficient with the best results following a cophenetic test.
Principalcomponent analysis (PCA) was also used for non-hierarchical relationships amongthe genotypes. Eigenvalues and eigenvectors were calculated by the Eigenprogram using a correlation matrix as input (calculated using standardizedmorphological data), and a 2-D and 3-D plot was used to generate thetwo-dimensional PCA plot from NTSYS-pc 2.1 (Rohlf 2004).2.
3.Genomic DNA extraction and quantificationTotalgenomic DNA was isolated from 25 genotypes lines using a cetyltrimethylammoniumbromide (CTAB) extraction protocol (Doyle and Doyle 1990) and was then quantifiedspectrophotometrically on Nanospectrophotometer,Implen (Germany).2.3.1.RAPD-PCR amplificationTwenty decamer primers (Operon TechnologiesInc.) were screened in the ashwagandha genotypes, of which 15 primers generatedpolymorphic and reproducible banding patterns and were selected for finalanalysis.
PCR amplification was carried out in a 20 ?L reaction volumecontaining 200 µM of dNTP mix, 1.5 mM MgCl2, 1U of Taq polymerase, 1X of reaction buffer, 0.5 µM of primer and doubledistilled water and 20 ng genomic DNA.