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Detection of Rb Gene in Selected Transgenic Late Blight Resistant Potato Clones Through Pcr

M.Mahmuda Khatun
Biotechnology Division, BARI, Joydebpur, Gazipur

M. R. Kabir
Biotechnology Division, BARI, Joydebpur, Gazipur

D. Khanam
Biotechnology Division, BARI, Joydebpur, Gazipur

Abstract/ Executive Summary:

Six selected transgenic late blight resistant potato clones were used to find out the RB gene integration. DNA was extracted from 6 clones along with non-transgenic control variety Diamant. Through PCR method RB gene confirmation test was done. Among the 6 potato clones, all showed positive band which indicates that RB gene was present of those clones

Keywords: PCR, Potato, Transgenic

Location:

Start Date: 00-00-2014

End Date: 00-00-2015

Program Area: Variety and Species

Commodity: Potato

Objectives:

To generate molecular data of those clones, needs to be done PCR confirmation for RB gene integration of desired clones of potato.

Methodology:

Six LBR potato clones D951 (2), D951 (3), D951 (12), D951 (13), D951 (137) and D951(304) along with control variety Diamant were used in this study. Isolation of genomic DNA was done from fresh leaf samples. N terminal primers were used. DNA amplification by PCR was done using Thermal Cycler. The amplified DNA products were separated by electrophoresis in 1% agarose gel.

Isolation of genomic DNA

Fresh leaf samples were used for DNA isolation. Isolation of genomic DNA was done using Geneaid protocol.

Amplification of RB gene by PCR

This technique utilized Polymerase Chain Reaction (PCR) amplification of template DNA using N terminal forward and reverses primer.

Primer:

N-terminal

Forward: 5’-CTCATTTTACCCCTACAA-3’

Reverse: 5’-GCGTTTTGGACCCTTTTA-3’

After PCR reaction, 1% agarose gel with 1X TAE buffer was prepared and loading dye was added to each PCR amplification product and the samples were loaded into the gel and ran at 80 volt (for 20 cm long gel casting tray) until the loading dye reaches the gel front and the image was taken through polaroid camera fixed on a UV transilluminator.

Source of Information: Annual Research Report 2014-2015, Biotechnology Division, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The genomic DNA was obtained following the Genaid protocol using fresh leaf samples. Then the PCR products were obtained using N terminal primers. After running the gel, the bands were obtained. Result showed that all the hybrid clones gave positive band at 600-700 bp while control variety did not produce any band. Using N terminal primer, among the six potato clones, all showed positive band with RB gene. This indicated that the selected potato clones possess the desired gene in their genome.

Knowledge Management: Report/Proceedings