Medicinal indiscriminate use. Among the various medicinal plants,

Medicinal plants have been given great
significance 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 the
world due to variegated climatic ecosystem which is suitable for cultivation
for medicinal plants. India being one of the
world’s 12 mega biodiversity countries needs to conserve its resources where
they are being exploited and should be grown commercially to avoid their
susceptibility to extinction because of indiscriminate use.

Among the various medicinal plants, Withania
somnifera (L.) Dunal
(Winter cherry, Ashwagandha or Asgandh of family Solanaceae is an
important medicinal plant that finds extensive use as a potential herb in the
traditional system of medicine as a ‘rasayana’ and ‘medhya rasayana’. The similarities between
roots of Ashwagandha and ginseng roots have led to it being called as Indian
ginseng (Tripathi et al. 1996).

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W.
somnifera is a genetically
simple species (2n = 48; n = 24; largely self-pollinated) most suited to
develop 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 are
Madhya Pradesh, Rajasthan, Punjab, Uttar Pradesh, Haryana, Gujarat and
Maharashtra among which Madhya Pradesh alone is having more than 4000 ha area
for cultivation. Due to presence of alkaloids in roots, leaves and seeds, theses
are used in preparation of Ayurvedic and Unani medicines, to combat a wide
range of diseases from tuberculosis to arthritis. Important part of ashwagandha
is its roots, followed by leaves and berries due to presence of   “Withanolides” (Gupta et al.
1996). The
major biochemical constituents of W. somnifera are steroidal alkaloids and
lactones, a class of constituents together known as withanolides (steroidal
lactones with ergostane skeleton).

Ongoing trials and research on animal support the
role of ashwagandha’s root and leaf extracts in different disorders and
diseases and possess properties like anticancer, antioxidant etc. (Chopra et
al. 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 a
restorative drug (Asthana and Raina 1989).  

Molecular
markers remain unaffected by physiological condition and environmental factors that
is the reason for their wide application in genetic diversity assessment among W.
somnifera (L.) Dunal genotypes
and to identify duplicated accessions within the germplasm collections. Due to
same reason, molecular markers are reliable for informative polymorphisms since
genetic composition is unique for each species. Most important development has
occurred in the field of molecular genetics with the emergence of molecular
marker since for breeders it is effective tool for investigating novel sources
of variations and genetic factors controlling quantitatively
inherited traits. These markers are
used for the detection and exploitation of DNA polymorphism (Semagn et al. 2010).
 For differentiating plants at inter- and/or
intra-specific level genetic polymorphism plays significant role, not only in
medicinal plants but also in cereals, cash, plantation and horticulture crops.
            The
most important role of conservation is to preserve the process of genetic
diversity and development in the viable population of ecology and commercially
viable varieties / genotypes to avoid possible extinction (Rout et al.
2010).  Different types of marker systems
have been used for biodiversity analysis. These include RFLP, SSR, RAPD and the
AFLP. RAPD and ISSR markers are two molecular approaches that have
been used to detect variation among plants.
Systematic evaluation and quantification of the variability from the present
study will serve as one step towards providing accurate genetic information for
further breeding programmes for Withania
improvement. The assessment of variation would provide us a
correct picture of the extent of variation, further helping us to improve the
genotypes for biotic and abiotic stresses. The main objective of this study was to characterize
the Withania genotypes using morphological
and molecular markers in order to evaluate the genetic diversity and
relationships among genotypes lines.      

 

2.
Materials and methods

The
present field investigation were carried out during late kharif
of 2013 and 2014 Instructional
Farm, Rajasthan College of Agriculture, MPUAT, Udaipur
(24035’N, 70042’E), Rajasthan (India).

2.1.
Plant materials

Plants
of 25 genotypes lines which  include  native  and
 foreign plants collected from different
parts of India, were maintained and considered for the present study (Table 1). Newly emerged leaf samples of the cultivars
were used for DNA extraction.

2.2.
Morphological analysis

Seven
morphometric characters were evaluated from 25 genotypes lines of plant
specimens. Standardization of data on morphological characters was done using
the 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 a
data matrix of pairwise similarities between genotypes lines. The simple
matching coefficient (SMC) was used to measure the similarity, as it was the
coefficient with the best results following a cophenetic test. Principal
component analysis (PCA) was also used for non-hierarchical relationships among
the genotypes. Eigenvalues and eigenvectors were calculated by the Eigen
program using a correlation matrix as input (calculated using standardized
morphological data), and a 2-D and 3-D plot was used to generate the
two-dimensional PCA plot from NTSYS-pc 2.1 (Rohlf 2004).

2.3.
Genomic DNA extraction and quantification

Total
genomic DNA was isolated from 25 genotypes lines using a cetyltrimethylammonium
bromide (CTAB) extraction protocol (Doyle and Doyle 1990) and was then quantified
spectrophotometrically on Nanospectrophotometer,
Implen (Germany).

2.3.1.
RAPD-PCR amplification

Twenty decamer primers (Operon Technologies
Inc.) were screened in the ashwagandha genotypes, of which 15 primers generated
polymorphic and reproducible banding patterns and were selected for final
analysis. PCR amplification was carried out in a 20 ?L reaction volume
containing 200 µM of dNTP mix, 1.5 mM MgCl2, 1U of Taq polymerase, 1X of reaction buffer, 0.5 µM of primer and double
distilled water and 20 ng genomic DNA.