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207Total 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 µL 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 The amplification was performed in an Eppendorf Master cycler Germany with reaction conditions programmed as initial predenaturation 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 Amplification products were separated by electrophoresis on 1 2 agarose gels stained with ethidium bromide at constant voltage 3V cm of gel till bromophenol blue loading dye migrated to other end of the gel The gel was visualized on a UV transilluminator and photographed using gel documentation system Alpha DigiDoc Germany 2 3 2 ISSR PCR amplification A total of 20 primers identified by the University of British Columbia UBC were procured from Bangalore Genei Pvt Ltd Bangalore India and were used for ISSR PCR optimization trials Fifteen primers which gave the best amplification results with the sample DNA were selected for final ISSR PCR analysis PCR amplification was carried out in a 20 µL reaction volume containing 200 µM of dNTP mix 1U of Taq polymerase 1 5 mM MgCl2 1X of reaction buffer 0 5 µM of primer and double distilled water and 20 ng genomic DNA
Using the Mantel test Mantel 1967 a comparison between both methods was performed for morphological data of the ashwagandha genotypes for which both data sets were available by calculating the correlation between the two data sets in NTSYS pc Using the same software PCA was also carried out to identify any genetic association among the genotypes 2 5 Polymorphism Information Content PIC Effective multiple ratio EMR Resolving Power Rp and Marker index MI To measure the polymorphism information of RAPD and ISSR marker systems the PIC was calculated according to following formula Smith et al 1997 PIC Where N total number of allele detected for a locus of a marker Pi frequency of the 1st allele Effective multiple ratio EMR np β which is the product of number of polymorphic loci np in the genotype analysed and the fraction of markers that were polymorphic β The resolving power Rp of each primer was calculated as Rp Σ Ib where Ib band informativeness takes the value of 1 2 0 5 p p being the proportion of genotypes of different Withania species containing that band Prevost and Wilkinson 1999 Marker index MI defined as the product of the polymorphism percentage and PIC is used to estimate the overall utility of each marker system and was calculated using the following equation Marker Index MI PIC EMR Milbourne et al 1997