Essay Example on Plants of 25 genotypes lines representing native as well as foreign Plants

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2 Materials and methods The present field investigation were carried out during late kharif of 2013 and 2014 Instructional Farm Plant materials Plants of 25 genotypes lines representing native as well as 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 plant specimens from 25 genotypes lines Data on morphological characters were standardized using the YBAR option of the Stand program from the NTSYS pc 2 1 software Rohlf 2004 Duplicate measurements for each specimen 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 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 



The amplification was performed in an Eppendorf Master cycler with reaction conditions programmed as initial predenaturation at 94 C for 5 min followed by 35 cycles of denaturation at 94 C for 1 min annealing at 42 9 C 60 C 45 secs and extension at 72 C for 1 min with a hold temperature of 4 C A final extension was done for 5 min at 72 C Amplification products were separated by electrophoresis on 1 2 agarose gels and photographed using gel documentation system 2 4 Data analysis Amplified bands generated from RAPD and ISSR PCR amplification were scored based on the presence 1 or absence 0 of bands for each primer and were used to calculate a SM similarity matrix using NTSYS pc version 2 1 Rohlf 2004 Cluster analysis was performed on both morphological and molecular data Similarity matrices were compiled for all pairs of accessions using SM similarity coefficients using SIMQUAL then cluster analysis was done using the Unweighted Pair Group Method with Arithmetic Mean UPGMA analysis and dendrograms were constructed using the SAHN program The cophenetic correlation was calculated to find the degree of association between the original similarity matrix and the tree matrix in both morphological and molecular analyses

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



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