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RAPD Based Genetic Diversity Analysis in 25 Genotypes of Withania somnifera (L.) Dunal

Ashwagandha (Fam = Solanaceae) [(ashwa = horse) and (gandha = smell), “horse like smell”], one of the most important medicinal plant also known as Withania somnifera L. Dunal, poison gooseberry, winter cherry and Indian ginseng (parallel to Panax Ginseng, of Chinese origin) (Tripathi et al., 1996; Singh et al., 1998). Due to its medicinal properties, it had been used since ancient times as “rasayana” and “medha rasayana” in Ayurveda (CSIR, 1976) and now-a-day paving pathway for preparing allopathy drugs. C-28 steroidal lactones known as with anolides are mainly responsible for vast medicinal properties such as antiinflammatory, anticonvulsive, antitumor, immunosuppressive, antioxidant and adaptogenic action. The main part of plant which contributes for these with anolides and alkaloids are roots and leaves.


Introduction
Ashwagandha (Fam = Solanaceae) [(ashwa = horse) and (gandha = smell), "horse like smell"], one of the most important medicinal plant also known as Withania somnifera L. Dunal, poison gooseberry, winter cherry and Indian ginseng (parallel to Panax Ginseng, of Chinese origin) (Tripathi et al., 1996;Singh et al., 1998). Due to its medicinal properties, it had been used since ancient times as "rasayana" and "medha rasayana" in Ayurveda (CSIR, 1976) and now-a-day paving pathway for preparing allopathy drugs. C-28 steroidal lactones known as with anolides are mainly responsible for vast medicinal properties such as anti-inflammatory, anticonvulsive, antitumor, immunosuppressive, antioxidant and adaptogenic action. The main part of plant which contributes for these with anolides and alkaloids are roots and leaves.
Pradesh, Gujarat, Maharashtra, Rajasthan and Punjab extending to the mountainous regions of Himachal Pradesh and Jammu.
Molecular marker technique is a powerful tool for evaluating genetic variation and exploring evolutionary relationships among and within plant species (Hoisington et al., 1998;Stuber, 1995). PCR (Polymerase Chain Reaction)-based markers i.e. RAPD are being used in the analysis of genetic diversity in crop plants because of their relative ease. There is no need of prior knowledge about the genome, which makes RAPD a common method for such studies in different plants. The RAPD technique (Williams et al., 1990) is faster and cheaper for revealing genetic polymorphism. RAPD markers have been widely used for evaluating genetic variation and genetic relationships, genetic mapping, identifying genotypes, etc., with various plants.
The objective of this work was to evaluate the extent and distribution of genetic diversity among 25 accessions of Withania somnifera using RAPD markers and to determine the phylogenetic relationship amongst them with the future goal of assisting the marker assisted crop improvement programme.

Materials and Methods
In the present investigation, seed of 25 diverse genotypes of Withania somnifera Dunal were procured from AICRP on MAP&B (Medicinal and Aromatic Plant and Betelvine). Source details of the genotypes used are given in table 1. Young fresh and healthy leaves were collected and DNA extraction was done following the cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1990).The extracted genomic DNA was analysed on 0.8% agarose gel and was diluted to an optimum concentration using TE for polymerase chain reaction (PCR). A total of 20 arbitrary decamer primers were initially used, out of which 15 primers showed clear, scorable and highly polymorphic bands ( Table 2).
For RAPD analysis, the genomic DNA was amplified in a reaction volume of 20 ml containing 50 ng of template DNA,1x reaction buffer, 1 unit of Taq DNA polymerase, 200 mM each of dNTPs mix and 0.5 μM/reaction of primer using a programmable thermo-cycler DNA Engine (Biorad, Germany) with the following cycling parameters: initial denaturation at 94 °C for 4 min, followed by 44 cycles of denaturation at 94 °C for 45 sec, annealing at 32 °C for 2 min, and extension at 72 °C for 2 min. A final extension was done for 10 min at 72 °C with a hold temperature of 4 °C. The amplified PCR products were electrophoretically separated on 1.2% agarose gel prepared in 1x TAE buffer containing ethidium bromide (EtBr) staining dye. The gel was run for 3h at 50V.The size of the amplified DNA fragments were determined using low base pair and 500bp DNA ladders= (Bangalore Genie, India) as standard molecular size markers. DNA fragments were visualized under UV-trans-illuminator and photographed using gel documentation system. Scoring of amplicons obtained from different RAPD markers was done on the basis of presence (taken as 1) or absence (taken as 0) of bands for each primer. For banding pattern only clear and unambiguous bands were scored for each primer. Comparison of band position was done relative to molecular weight of standard DNA ladders. Accordingly, a rectangular binary data matrix was obtained and statistical cluster analysis was performed using the NTSYS-pc version 2.02e (Rohlf, 1998). A pair wise similarity matrix was generated and the cluster analysis was performed via unweighted pair group method with arithmetic averages (UPGMA) to develop a dendrogram. A two-dimensional and three-dimensional principal component analysis (PCA) was constructed to provide another means of testing the relationship among the genotypes.

Results and Discussion
Among the 20 RAPD primers used for initial screening, 15 markers produced polymorphic, reproducible and scorable bands. A total of 150 bands were generated from 15 RAPD primers, of which 141 bands were polymorphic (94%) with an average of 12 polymorphic bands per primer ( Table 2). The total number of amplified bands varied between 6 (primer OPA-02 and OPA-06) and 18 (primer OPB-05). The overall size of PCR amplified products ranged between 100 bp to 3000 bp. The percent polymorphism ranged from as low as 50 (OPA-02) to as high as 100 (C-19, OPA03, OPA06, OPB4, OPB05, OPB06, OPB07, OPC 05, P-20 and S-34).The average PIC was 0.310 ranging from 0.164 to 0.422. The lowest and the highest PIC value were recorded for primer OPA-05 and OPA-02 respectively. Figure 1 shows the amplification pattern obtained from primer OPA-05 produced 9 polymorphic bands. 7 unique bands (band which is present in a particular genotype but absent in rest of the genotypes) were detected in 6 genotypes viz., AWS2B, UWS 56, UWS 59, UWS 32, UWS 37 and UWS 22 with 5 RAPD primers (OPA-05 OPB-04, OPB-05, OPC-05 and S-34). The genotype UWS 32 gave maximum number of distinct bands i.e., 2. The sizes of these unique bands ranged from 350 to 3000 bp (Table 3).
Based on RAPD similarity matrix data, the values of similarity coefficient ranged from 0.29 to 0.85 i.e., 29-85% or genetic diversity ranged from 15 to 71%. The average similarity across all the genotypes was found out to be 0.57 showing that the genotypes were moderately diverse. Maximum similarity value of 0.85 was observed between genotypes UWS 23 and UWS 28 followed with a similarity coefficient value of 0.78 between UWS 11 and UWS 13. Similarly, minimum similarity value of 0.29 was observed between genotypes UWS 10 and UWS 67 and between UWS 10 and UWS 77 followed by a similarity coefficient of 0.3 between UWS 67 and UWS 15 (Table 4).
The RAPD cluster tree analysis of 25 W. somnifera L. Dunal genotypes showed that they could be mainly divided into 2 clusters at a similarity coefficient of 0.43 and 0.61 respectively (Fig. 2). The Cluster I is a major cluster including 20 genotypes and it can be divided into 2 sub-clusters i.e. sub cluster I and II at 0.54 and 0.49 similarity coefficients respectively. Sub cluster I can be further divided into 2 groups, subgroup A and B at 0.74 and 0.76 similarity coefficients.     Cluster I major cluster including 20 genotypes and minor cluster II included 5 genotypes. Genotypes UWS 10 and UWS 22 showed maximum genetic distance and therefore the most divergent genotypes.