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DNA Fingerprinting of Potato Germplasms Through Rapd Markers

Ahsan-Ul Haque
Scientific Officer
Tuber Crops Research centre, BARI, Joydebpur, Gazipur

M. R. Mollah
Tuber Crops Research centre, BARI,Joydebpur, Gazipur

B. C. Kundu
Tuber Crops Research centre, BARI,Joydebpur, Gazipur

Abstract/ Executive Summary:

The programme was initiated with isolation of genomic DNA from 14 different crossing lines of potato (4.15, 4.27, 4.40, 4.5W, 4.26R, Quincy, Sagita, Spunta, 3.24, 4.11, 4.17 R, 4.32, 4.5 R and 4.26W). All genomic DNA was in condition. Six responsive RAPD primers have been tested and selected for parental survey of these lines and thermal profile were also adjusted. Characterization of all genotypes through gel electrophoresis was not possible, while RAPD markers were efficient to characterize all the genotypes.

Keywords: DNA, Potato, Germplasms, RAPD Markers

Location: Gazipur

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Potato

Objectives:

To improve the efficiency of breeding programs

Methodology:

Genomic DNA isolation DNA was extracted following standard protocol (Doyle, J. J. and J. L. Doyle. 1990.) using SDS. Required reagent Extraction buffer, Phenol: Chloroform: Isoamyl alcohol, TE buffer, Sodium acetate, ethanol. DNA Sample preparation One gram of young leaf tissue was cut into small pieces and taken into 1.5 ml Eppendorf tubes. After adding 400 µl of extraction buffer, the ground sample was incubated. Following further mixing with a vortex mixture for about 20 seconds then incubated and extract was centrifuged at 15000 rpm to allow precipitation. 600 µl of Phenol: Chloroform: Isoamyl alcohol (v: v: v = 25:24:1) was added and mixed gently and centrifuged at 15,000 rpm. The upper aqueous layer was then carefully transferred without disturbing the lower portion. DNA was precipitated first using about 800 µl of absolute alcohol and then peletted by centrifugation for 3 minutes at 15,000 rpm. After discarding the liquid completely, the DNA solution was reprecipitated by adding 400 µl of 70% ethanol with 20 µl of 3 M sodium acetate and again peletted by centrifugation for 3 minutes at 15,000 rpm. After removing the liquid completely, the pellet was then air dried and resuspended in 50 µl of TE buffer. Finally, the DNA samples were stored at -20⁰C. Confirmation of DNA preparation: 1.0 % agarose gel was used for assessing both the quantity and the quality of the genomic DNA. Polymerase Chain Reaction Polymerase Chain Reactions were done in a volume of 10 μl containing 10 x PCR Buffer, 0.25 mM each of the dNTPs, 1 μM of each of primer, 1 unit Taq DNA polymerase, 75 ng template DNA with sterile deionized water. Amplification was carried out in a thermal cycler with the following thermal profile. PCR was the confirmed by electrophoresis on 2% agarose gel.

Source of Information: TCRC Annual Report-2010-11, BARI

Article Link (Online Journal):

Funding Source:

1. Government

Budget: 3,00000/=

Total Budget (Tk.) : 3,00000/=

Achievement/Findings:

The programme was initiated with isolation of genomic DNA from 14 different crossing lines of potato (4.15, 4.27, 4.40, 4.5W, 4.26R, Quincy, Sagita, Spunta, 3.24, 4.11, 4.17 R, 4.32, 4.5 R and 4.26W). All genomic DNA was in condition. Six responsive RAPD primers have been tested and selected for parental survey of these lines and thermal profile were also adjusted. Characterization of all genotypes through gel electrophoresis was not possible, while RAPD markers were efficient to characterize all the genotypes. Electrophoregram of amplified products using the primer OPD-20 on genomic DNA of potato genotypes 4.15 (1), 4.27 (2), 4.40 (3), 4.5W (4), 4.26R (5), Quincy (6), Sagita (7), Spunta (8), 3.24 (9), 1 kb ladder molecular marker (10)

Knowledge Management: Report/Proceedings