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Use of Random Pcr (rapd) Technology to Analyze Systematic Relationships in Terrestrial Bladderworts (utricularia L.)

MD. OLIUR RAHMAN
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.

Abstract/ Executive Summary:

Polymerase Chain Reaction (PCR) based technology RAPD was tested for its applicability and suitability for molecular systematics of terrestrial bladderworts. PCR was carried out and the resultant products were subjected to agarose gel electrophoresis. The banding patterns were compared among ten species of Utricularia. Out of 40 random oligonucleotide primers examined, 16 primers generated distinguishable bands. Cluster analysis, unweighted pair group method with arithmetic mean (UPGMA) and ordination approach Scatter diagram using Matrix plot were performed based on RAPD fingerprints. The UPGMA and ordination analysis revealed a clear portrayal of systematic relationships in bladderwort species which were concordant with previous studies based on morphology and molecular approaches, indicating the reliability of RAPD markers for estimation of genetic variation and species relationships in Utricularia.

Keywords: Utricularia, RAPD, PCR, Cluster analysis, Molecular taxonomy

Location: Laboratory of Plant Chromosome and Gene Stock, Graduate School of Science, Hiroshima University, Higashi Hiroshima, Japan

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

1.To study the genetic variability among different terrestrial bladderwort species and

2. To determine the relationships among them.

Methodology:

Plant materials: Ten terrestrial bladderworts viz., Utricularia alpina Jacq., U. amethystina Salzm. ex St. Hill, U. bifida L., U. caerulea L., U. calycifida Benj., U. humboldtii Schomb., U. longifoila Gardner, U. praelonga A. St. Hill & Girard, U. triflora P. Taylor and U. uliginosa Vahl were used in this study. Genomic DNA extraction: DNA was extracted from leaves of fresh materials by Cetyl Trimethyl Ammonium Bromide (CTAB) method with slight modification (Rahman and Kondo 2003a). Approximately 1.0 - 1.8 g of leaf material was ground to a fine powder in liquid nitrogen with a mortar and pestle before isolation of DNA. The isolated DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA). PCR and electrophoresis: PCR was carried out using 10-mer oligonucleotide primers. The RAPD primers used in this study are shown in Table 1. PCR amplification was carried out in 12μl reaction mixture containing 20 ng of genomic DNA, 10 pmol of primer, 1 μl X10 Taq buffer, 1 μl of dNTP mixture, 0.05 μl Taq polymerase enzyme, and deionised water to a final volume of 12 μl. Amplification was carried out in a PTC-100 thermal cycler programmed for 45 cycles with the following temperature profile: denaturation at 94º C for 1 min, annealing at 35º C for 1 min and extension at 72º C for 2 min. Cycling was concluded with a final extension at 72º C for 5 min. PCR products were electrophoresed in 1.5% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA), visualized by ethidium bromide staining and photographed under UV light. Data analysis: Electrophoretic data were scored for the presence (1) or absence (0) of PCR fragments. Rogers and Tanimoto’s coefficient was employed to detect genetic affinity among the species examined. UPGMA (Unweighted pair group method with arithmetic mean) tree was constructed by clustering the dissimilarity data based on Dist coefficient using SIMINT (Similarity for Interval data) program. A scatter diagram was constructed to view the clustering of the taxa using MXPLOT (Plots 2-way scatter diagrams of rows or columns of a matrix) where the distance matrix was transformed using the DCENTER (Performs a ‘double-centering’ of a matrix of similarities or dissimilarities among the objects) and MDSCALE (Multidimensional scaling analysis) options. All analyses were performed with the NTSYS-pc package, version 2.1.

Source of Information: Bangladesh J. Bot. 39(1): 97-102, 2010 (June)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In the study, a close relationship was detected between the species.The results obtained from the random PCR technology were in congruent with the findings generated from morphological and other molecular data. This revealed that, RAPD markers provide an accurate portrayal of relationships in Utricularia.

Knowledge Management: Journal