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Molecular Characterization of BSRI Stenia Using Papd Markers

N. Islam
Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh.

M. A. Hossain
Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh.

M. S. Arefin
Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh.

M. S. Islam
Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh.

M. K. Begum
Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna, Bangladesh.

Abstract/ Executive Summary:

For molecular characterization. an easy and efficient method was developed for isolation of quality DNA from shoot tip, stem segment and mature leaf of Stevia rebaudiana. The quality and quantity of DNA were determined by visual estimation of agarose gel electrophoreses and by UV spectrophotometer. The yield of DNA was 1200 to 5600 ng/µl. Sufficient amount of quality DNA isolation was possible without use of liquid nitrogen, tissue homogenizer and using a simple micro-centrifuge. The amount of recovered DNA was enough for Polymerase Chain Reaction (PCR) amplification and marker studies such as random amplified polymorphic DNA (RAPD). Molecular characterization of Stevia was performed using RAPD primers as markers. Among the 10 primers the primer OPB-09 was reproducible for PCR amplification for BSRI Stevia DNA. The results could be used for protection of the variety using OPB-09 as molecular marker. The technique could be efficiently used for identification of the variety and molecular characterization for development. The results give the positive indications of the further possibilities of linkage map analysis and Marker Assisted Selection for Stevia.

Keywords: Stevia. molecular characterization. RAPD marker.

Location: Biotechnology Laboratory, Bangladesh Sugarcane Research Institute (BSRI), Ishurdi, Pabna

Start Date: 01-04-2007

End Date: 30-06-2007

Program Area: Pest Management

Commodity: Sugarcane

Objectives:

To tea and coffee, cooked or baked goods, processed foods and beverages. It is also used as a table top sweetener, in soft drinks, pickles, fruit juices, tobacco products, confectionery uses, jams and jellies, candies, yogurts, pastries, chewing gum, sherbets, etc.

Methodology:

The samples were cut into small pieces (about 1 cm long) with sterile scissors and weighted required amount (O.2g) using a fine balance which was then washed with ddH20. Care was taken during sampling to avoid bare hand touch to the sample. For this hand gloves were worn.PCR amplification was done in an oil-free thermal cycler (Master Cycler Gradient, Eppendorf) following the PCR profile of 94°C for 4 minutes followed by 35 cycles of 1 minute denaturation at 94⁰C, 1 minute annealing at 36°C and elongation or extension at 72⁰C for 2 minutes. After the last cycle, a final step of 7 minutes at 72°c was added to allow complete extension of all amplified fragments. After completion of cycling programme, reactions were held at 4°C. The PCR reactions were performed on each DNA sample in a 10 III reaction mixture containing 1µl of 10X Ampli Taq polymerase buffer, 1 µl of 10 µM Primer, 1 µl of 2.5mM DNTPs, 0.2 µl of 3U/ µl Ampli Taq DNA polymerase (Bangalore Genei Pvt. Ltd., India), 4 µl of 25ng/ µl genomic DNA and a suitable amount (2.8 µl) of sterile deionized water. After amplification 2 µl loading dye was added to the amplification product and separated on 1.4% agarose gel (SRL, India) containing ethidium bromide (0.8 µg/ml from 10mg/ml in 1 xTBE buffer). Electrophoresis was performed at 100V for 2 hours. The DNA ladder 100 bp (Bangalore Genei Pvt. Ltd., India) was run alongside the RAPD reactions. DNA bands were observed on UV-transilluminator in Gel Documentation System (uvitec, DBT-2000LS), printed and saved on floopy disket (3.5") for lateral use.

Source of Information: Bangladesh Journal of Sugarcane, Vol: 31, pp:52-57, 2009, ISSN 1991-8925

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Results of this investigation revealed that sufficient amount of quality DNA isolation was possible without use of liquid nitrogen, tissue homeginizer, using a simple micro-centrifuge and using the minimum amount of chemicals. Isolated DNA well preformed for PCR amplification and DNA Fingerprinting using RAPD markers.

Knowledge Management: Journal