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Molecular Characterization and Genetic Diversity in Rice (oryza Sativa L.) Landraces Using Microsatellite Markers During 2013-14

Md. Abubakar Siddique,
Scientific Officer,
Genetic Resources and Seed Division, BRRI, Gazipur-1701, Bangladesh.

Dr. Mohammad Khalequzzaman,
Chief Scientific Officer and Head,
Genetic Resources and Seed Division, BRRI, Gazipur-1701, Bangladesh.

Kaniz Fatema
Scientific Officer,
NATP-SPGR Project, Genetic Resources and Seed Division, BRRI, Gazipur-1701, Bangladesh.

M M Islam
Senior Scientific Officer,
IAPP, BRRI Regional Station Barisal, Barisal, Bangladesh.

Abstract/ Executive Summary:

Ninety six landraces, selected and collected from Rice Genebank, Bangladesh Rice Research Institute, Gazipur were used as test materials for molecular characterization and genetic diversity study using 12 microsatellite markers during 2013-14 at BRRI, Gazipur. A total of 252 alleles were detected at 12 microsatellite markers across 96 rice genotypes. The number of alleles per locus ranged from 11 alleles (RM163) to 41 alleles (RM283), with an average of 21 alleles across the 12 loci obtained in the study. The polymorphism information content (PIC) values ranged from 0.75 (RM163) to 0.95 (RM283), were identified in all 12 loci. PIC values revealed that RM283 was considered as the best marker for 96 genotypes. The genetic distance-based results seen in the unrooted neighbor-joining tree revealed that the 96 genotypes were clustered clearly into seven different groups. The highest numbers of genotypes (25) were found in cluster I followed by clusters VII (19), V (17), II and III (10), IV (9) and the lowest in cluster VI (6).

Keywords: Molecular characterization, SSR, Rice, Landraces.

Location: Molecular laboratory, GRSD, BRRI, Gazipur.

Start Date: 01-07-2013

End Date: 30-06-2014

Program Area: Conservation and Biodiversity

Commodity: Rice

Objectives:

To characterize the rice germplasm through molecular tools for varietal identification and establishment of property right

Methodology:

Ninety six landraces, selected and collected from Rice Genebank, Bangladesh Rice Research Institute, Gazipur were used as test materials for molecular characterization and genetic diversity study using 12 microsatellite markers. But, first one hundred markers were surveyed to find out the polymorphic marker. Based on polymorphism, twelve SSR markers (RM 6, RM 11, RM 30, RM 44, RM 125, RM 147, RM 163, RM 273, RM 277, RM 278, RM 283 and RM 287) were selected for genetic diversity analysis at GRS Division of BRRI during 2013-14. DNA was extracted from young leaves of 3-week-old plants following a simple and modified protocol as described by Zheng et al. (1995) for PCR analysis. 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). The 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. All data were analyzed in computer using Alpha-Ease 5.0 software, Power Marker version 3.25 (Liu and Muse, 2005) and NTSYS-pc version 2.2 (Rohlf, 2002).

Source of Information: Proceedings of the BRRI Annual Internal Review for 2013-14, Genetic Resources and Seed Division, BRRI, Gazipur-1701.

Article Link (Online Journal):

Funding Source:

1. Government	Budget:
2. Bangladesh Agricultural Research Council	Budget:

Total Budget (Tk.) :

Achievement/Findings:

A total of 252 alleles were detected at 12 microsatellite markers across 96 rice genotypes. The number of alleles per locus ranged from 11 alleles (RM163) to 41 alleles (RM283), with an average of 21 alleles across the 12 loci obtained in the study. The polymorphism information content (PIC) values ranged from 0.75 (RM163) to 0.95 (RM283), were identified in all 12 loci. The allele frequency ranged from 8.33% (RM283) to 34.38% (RM163) with an average of 17.62 alleles. The PIC values for other markers were 0.93 (RM6, RM11), 0.92 (RM278), 0.91 (RM44), 0.90 (RM273), 0.89 (RM125, RM287), 0.87 (RM147, RM277) and 0.83 (RM30), respectively. PIC values revealed that RM283 was considered as the best marker for 96 genotypes. The genetic distance-based results seen in the unrooted neighbor-joining tree revealed that the 96 genotypes were clustered clearly into seven different groups. The highest numbers of genotypes (25) were found in cluster I followed by clusters VII (19), V (17), II and III (10), IV (9) and the lowest in cluster VI (6).

Knowledge Management: Report/Proceedings