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Molecular Characterization and Genetic Diversity in Fine Grain and Aromatic Landraces of Rice (oryza Sativa L.) Using Microsatellite Markers

M. Z. Hossain
Seed Certification Agency, Ministry of Agriculture, Joydebpur, Gazipur 1701

M. G. Rasul
Department of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh

M. S. Ali
Biotech Division, Bangladesh Rice Research Institute, Joydebpur, Gazipur 1701, Bangladesh

K. M. Iftekharuddaula
Plant Breeding Division, Bangladesh Rice Research Institute, Joydebpur, Gazipur 1701, Bangladesh

M. A. K. Mian
Department of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh

Abstract/ Executive Summary:

A total of thirty microsatellite molecular markers were used across 21 rice genotypes for their characterization and discrimination. The number of alleles per locus ranged from three (RM165, RM219, RM248, RM463, RM470 and RM517) to nine (RM223), with an average of 4.53 alleles across the 30 loci obtained in the study. The polymorphism information content (PIC) values ranged from 0.30 (RM219) to 0.84 (RM223) in all 30 loci. RM223 was found the best marker for the identification of 21 genotypes as revealed by PIC values. The frequency of the most common allele at each locus ranged from 24% (RM223 and RM334) to 81% (RM219). A two dimensional principal coordinate analysis (PCoA) with 21 genotypes showed that the genotypes Supper Basmoti, Basmati370, BasmatiD, Keora, Chinisakkora, Thakurbhog, Benaful, Kolgochi, Buchi, Awnedtapl and Kalijira-11 were found far away from centroid of the cluster than rest of the genotypes which placed around the centroid. The pair-wise genetic dissimilarity coefficients indicated that the highest genetic distance was obtained between Thakurbhog and Supper Basmoti (0.81) as well as between Benaful and Keora (0.81). Basmati (Basmoti D, Super Basmati, Basmati 370) and Kalijira (Kalijira 11, 12, 13, 14) genotypes had close similarity among them but showed wide dissimilarity with other local genotypes. Being grouped into distant clusters, SupperBasmoti, Thakurbhog, Keora, and Benaful could be utilized as potential parents for the improvement of fine grain aromatic rice varieties. Genotypes Kolgochi and Buchi (having zero dissimilairty) might be possessed same genetic background. The microsatellite marker based molecular fingerprinting could serve as a sound basis in the identification of genetically distant accessions as well as in the duplicate sorting of the morphologically close accessions.

Keywords: Rice (Oryza sativa L), Genetic diversity, Microsatellite marker, SSR

Location: Biotechnology Division of BRR

Start Date: 00-00-2006

End Date: 00-00-2007

Program Area: Variety and Species

Commodity: Rice

Objectives:

This study is involved with the aromatic and fine grain traditional landraces of rice locally cultivated by farmers those were either not used, or poorly used as parents in the Bangladeshi rice-breeding program. Objective of these germplasm can function as the source of alternative genetic pools to the improved varieties.

Methodology:

Plant materials: The 18 genotypes of aromatic and fine grain landraces of rice and three basmati types were selected from the rice germplasm collection at the Bangladesh Rice Research Institute (BRRI) gene bank. The whole experiment was conducted at Biotechnology Division of BRRI during 2006-7. Five grams of germinated seed from each genotype was sown in the pot. Genotyping protoco: DNA was extracted from young leaves of three-week-old plants following a simple and modified protocol to isolate total genomic DNA for PCR analysis which did not require liquid nitrogen and required only a very small amount of tissue samples as described by Zheng et al. (1995) . 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 in an MJ Research single 96-well thermal cycler. The mixer was overlaid with one drop of mineral oil to prevent evaporation. After initial denaturation for 5 min at 94°C, each cycle comprised of 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). Two and half 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 0.5mg/ml ethidium bromide and were documented using UVPRO (Uvipro Platinum, EU) gel documentation unit. Microsatellite or Simple Sequence Sepeat (SSR) markers were used for selection (Temnykh et al. 2001; McCouch et al., 2002; IRGSP 2005). Thirty SSR markers (distributed in the 12 chromosomes) with clear amplifications were selected for genetic diversity analysis of 21 genotypes.

Data analysis: Molecular weight for each amplified allele was measured in base pair using Alpha-EaseFC 5.0 software. The summary statistics including the number of alleles per locus, major allele frequency, gene diversity, polymorphism information content (PIC) values were determined using PowerMarker version 3.25 (Liu and Muse 2005). For the unrooted phylogenetic tree, genetic distance was calculated using the “C.S. Chord 1967” distance (Cavalli-Sfoza and Edwards 1967) followed by phylogeny reconstruction using neighbor-joining as implemented in PowerMarker with tree viewed using Treeview (Page, 1996). 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 2.1 (Rohlf, 2002). A similarity matrix was calculated with the Simqual subprogram using the Dice coefficient, followed by cluster analysis with the SAHN subprogram using the UPGMA clustering method as implemented in NTSYS-pc. The similarity matrix was also used for principal coordinate analysis (PCoA) with the DCenter, Eigen, Output, and MXPlot subprograms in NTSYS-pc.

Source of Information: Bangladesh J. Genet Pl. Breed., 20(2): 01-10, 2007

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In crop improvement program these genetically diverse genotypes could be chosen as parents in the crossing program to create genetic variability. On the other hand BasmotiD, Basmoti 370 and SuperBasmoti were the most similar genotype (21%-27% dissimilar) followed by Kalijira 11, 12, 13, 14 (24% dissimilar). Though three Basmoti genotypes were more similar with each other but most dissimilar with other landraces. Likewise, Kalijira 11, 12, 13, 14 were more similar with each other but dissimilar with other landraces rice genotypes. It was also obtained that two aromatic landraces (Kolgochi and Buchi) were found exactly same in this analysis (0% dissimilar). Hence, microsatellite marker based molecular fingerprinting could serve as a potential basis in the identification of genetically distant accessions as well as in identification of the morphologically close accessions.

Knowledge Management: Journal