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DNA Fingerprinting of BRRI Released Varieties

Md. Shamsher Ali
Chief Scientific Officer
Biotechnology Division, BRRI Head Office, Gazipur 1701.

Enamul Hoque
Principal Scientific Officer
Biotechnology Division, BRRI Head Office, Gazipur 1701.

Nasrin Sultana
Scientific Officer
Biotechnology Division, BRRI Head Office, Gazipur 1701.

Hisham Al Rabbi
Scientific Officer
Biotechnology Division, BRRI Head Office, Gazipur 1701.

Abstract/ Executive Summary:

To establish intellectual property right of BRRI released varieties, DNA fingerprinting is necessary. Diversity analysis of these varieties by molecular markers is also required to carry out breeding programmes. DNA was extracted from leaves of 21 days old plants by mini DNA preparation method. The concentration of extracted DNA was estimated by DNA confirmation test by (1.5%) agarose gel electrophoresis. PCR was carried out and the PCR products were electrophoresed in 8% polyacrylamide gel using vertical polyacrylamide gels for high throughput manual genotyping. Fifty BRRI released varieties have been screened against 55 SSR markers for DNA fingerprinting. Among them 24 primers showed polymorphism. One hundred and five alleles were detected at the loci of 24 microsatellite markers across 50 rice genotypes. Genetic dissimilarity co-efficient was found by which differences between every two variety is got on DNA basis. The highest genetic dissimilarity (0.8612) was found between BRRI dhan37 and BRRI dhan50. However, most BRRI varieties have narrow genetic base as they showed less genetic dissimilarity co-efficient. The genetic similarity analysis using UPGMA clustering system generated 7 major genetic clusters at a cut-off similarity co-efficient level of 0.46. Unfortunately the primers used could not differentiate BR22 from BR23.

Keywords: Rice, DNA, Fingerprinting.

Location: Biotechnology Division, BRRI Head Office, Gazipur 1701.

Start Date: 01-07-2007

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

To establish intellectual property right of BRRI released varieties DNA fingerprinting and diversity analysis were done by SSR markers.

Methodology:

Fifty BRRI released varieties have been screened against 55 SSR markers for this study. DNA was extracted from young leaves of 21 days old plants by mini DNA preparation method (modified CTAB). The concentration of extracted DNA was estimated by DNA confirmation test by (1.5%) agarose gel electrophoresis. Each PCR was carried out in a 10 µL reaction volume containing 1 µL of MgCl₂free 10X PCR buffer, 1.2 µL of 25 mM MgCl₂_,0.2 µL of 10 mM dNTPs, 0.2 µL of 5 U/µL Taq DNA polymerase, 0.5 µL of 10 µM forward and reverse primers and 2 µL (10ng) of DNA using a 96 well thermal cycler. The temperature profile use for PCR amplification comprised 94° C for 5 min (initial denaturation) followed by 35 cycles of 94° C for 1 min (denaturation), 55° C for 1 min (annealing), 72° C for 2 min (extension) with a final extension for 7 min at 72° C at the end of 35 cycles. The annealing temperatures were adjusted based on the specific requirements of each primer combination. The PCR products were mixed with gel loading dye and electrophoresed in 8% polyacrylamide gel using vertical polyacrylamide gels for high throughput manual genotyping. Four µL of amplification products were resolved by running gel in 1X TBE buffer for 1.5 - 2.5 hrs depending upon the allele size at around 75 volts and 180 mA electricity. The gels were stained in 1 µg/ml ethidium bromide solution (500ml) and were documented using UVPRO (Uvipro Platinum, EU) gel documentation unit.

Source of Information: Proceedings of the BRRI Annual Internal Review for 2007-2008

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Fifty BRRI released varieties were screened against 55 SSR markers for DNA fingerprinting. Among 55 SSR markers 24 markers showed polymorphism and 105 alleles were detected across 50 rice genotypes. Genetic dissimilarity co-efficient was found by which differences between every two variety was got on DNA basis. The highest genetic dissimilarity (0.8612) was found between BRRI dhan37 and BRRI dhan50. However, most BRRI varieties have narrow genetic base as they showed less genetic dissimilarity co-efficient. The genetic similarity analysis using UPGMA clustering system generated 7 major genetic clusters at a cut-off similarity co-efficient level of 0.46. Unfortunately the primers used could not differentiate BR22 from BR23.

Knowledge Management: Report/Proceedings