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Coordinated Sub Project on Characterization of Important Plant Genetic Resources: BRRI Component

Mohammad Khalequzzaman
Chief Scientific Officer
GRS Division, BRRI, Gazipur 1701, Bangladesh

Dr. Md. Shamsher Ali
Former Director (Research)
Former Director (Research), BRRI, Gazipur-1701 (Now at BARC), Bangladesh,

Md. Abubakar Siddique
Scientific Officer
Scientific Officer, GRS Division, BRRI, Gazipur- 1701, Bangladesh,

Abstract/ Executive Summary:

Experiments were conducted to characterize rice germplasm in molecular and morph-agronomic levels to protect biopiracy and establish varietal rights. In field, 270 germplasm were characterized in different seasons using rice germplasm descriptor. Besides, 20 geographical indication (GI) rice germplasm were also characterized. It revealed from morphoagronomic characters that most of the germplasm are different from each other showing their identity. Molecular characterization was done with 100 SSR markers. For GI rice, eight primers were used to find out polymorphism and four primers were found polymorphic. Aus season, 48 landraces were characterized with 30 primers which 14 found polymorphic. Similarly, Aman season, 96 germplasm were characterized using 22 primers and eight found polymorphic. The test germplasm are grouped into six different clusters after evaluation for diversity analysis. Among these 96 T. Aman rice were grouped into six clusters, 48 Aus genotypes into six clusters and 20 GI rice into seven clusters. It observed from molecular characterization that RM163 is the best primer to identify GI and T. Aman genotypes. It is apparent that diversity prevails in the germplasm both in morpho-agromonic and molecular levels, which is helpful to protect our rice germplasm from biopiracy and establish the sovereignty.

Keywords: Rice, Molecular characterization, Geographical Indication

Location: BRRI, Gazipur

Start Date: 13-01-2012

End Date: 30-06-2014

Program Area: Variety and Species

Commodity: Rice

Objectives:

To analyze the genetic diversity and population structure of Bangladeshi germplasm
To characterize important local germplasm both phenotypically and at molecular level.
To evaluate the potential usefulness of Bangladeshi germplasm for association mapping.
To protect our important local germplasm from any biopiracy.

Methodology:

A total of 270 germplasm accessions were grown in Aus, T. Aman and Boro seasons for characterization using the rice germplasm descriptors and evaluation form. Single seedling per hill with a spacing of 25 x 20 cm between rows and plants, respectively were grown using single row of 5.4 m long per entry. Fertilizers were applied @ 60:60:40 kg NPK/ha in T. Aman & Aus, and @ 100:60:40 kg NPK/ha in Boro seasons, respectively. Appropriate control measures were taken for pests, diseases and weeds when necessary. Data were recoded on 45 morpho-agronomic characters. Molecular Characterization: plant materials- Ninety-six T. Aman, 48 Aus, 96 Boro landraces and 20 Geographical Indication (GI) rice landraces were used as test materials. Five gram seeds from each of the entry was first germinated and then sown in the earthen pots. Genotyping- DNA was extracted from young leaves of 3 weeks old plants . PCR was performed in 12.5 µl reaction containing 5-25 ng of DNA template, 1.25 µl of MgCl₂ free 10X PCR buffer (100 mM Tris-HCl pH 9.0 at 25°C, 500 mM KCl, 0.1% Triton^® X-100 and H₂O), 1.5 µl of 25 mM MgCl₂, 0.25 µl of 10mM dNTP, 0.25 µl of 5 U/µl Taq polymerase enzyme, 0.625 µl each of 10 µM forward and reverse primers using a MJ Research single 96 well thermal cycler. The mixture was overlaid with one drop of mineral oil to prevent evaporation. After initial denaturation for 5 minutes at 94°C, each cycle comprised 1 min denaturation at 94°C, 1 min annealing at 55°C, and 2 min extension at 72°C with a final extension for 7 min at 72°C at the end of 35 cycles. The PCR products were mixed with bromophenol blue gel loading dye and were analyzed by electrophoresis on 8% polyacrylamide gel using mini vertical polyacrylamide gels for high throughput manual genotyping (CBS Scientific Co. Inc., CA, USA). 2.5 µl of amplification products were resolved by running gel in 1xTBE buffer for 2-2.5 hrs depending upon the allele size at around 75 volts and 180 mA current. The gels were stained in 0.5 mg/ml ethidium bromide and were documented using UVPRO (Uvipro Platinum, EU) gel documentation unit. Microsatellite or Simple Sequence Repeat (SSR) markers were used for DNA analysis (Temnykh et al. 2001; McCouch et al. 2002; IRGSP 2005). Eight SSR markers for T. Aman, 14 for Aus and 12 for GI with known amplifications were used for genetic diversity analysis. Data analysis: Molecular weight for each amplified allele was measured in base pair (bp) using Alpha-Ease 5.0 software. The summary statistics including the number of alleles per locus, major allele frequency, gene diversity, polymorphism information content values were determined using Power Marker version 3.25. For the unrooted phylogenic tree, genetic distance was calculated using the “C.S. Chord 1967” distance implemented in PowerMarker with tree viewed using Treeview. The allele frequency data from Powermarker was used to export the data in binary format (allele presence=“1” and allele absence = “0”) for analysis with NTSYS-pc version 2.2.

Source of Information: Project completion report

Article Link (Online Journal):

Funding Source:

1. World Bank	Budget: TK ninety six lakh and eighty three thousand tk only
2. Government	Budget: TK ninety six lakh and eighty three thousand tk only

Total Budget (Tk.) : TK ninety six lakh and eighty three thousand tk only

Achievement/Findings:

Morphological characterization of 20 GI rice, 100 Aus, 98 T. Aman and 98 Boro landraces has been completed.
Molecular characterization of 20 GI rice, 48 Aus, 96 T. Aman and 96 Boro landraces of rice has been completed.
GI rice characterized at molecular level using eight (8) primers, 4 identified as polymorphic. Similarly, 48 Aus rice using thirty (30) primers, revealed 14 as polymorphic, whereas 22 primers used for 96 T-Aman materials eight showed polymorphism.

Knowledge Management: Report/Proceedings