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Ssr Marker-based Molecular Screening of Blast Resistant Genes in Selected Rice Genotypes

Israt Yasmin
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Sourav Adhikary
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

Rice blast caused by Magnaporthe oryzae is one of the most devastating disease-causing major yield losses worldwide in every year. Use of resistant rice varieties is one of the most effective ways to control the disease and reduce yield loss. Molecular screening and allelic diversity of major rice blast resistant genes were determined in forty-eight genotypes of rice germplasms of Bangladesh with ten previously synthesized gene based SSR (Single Sequence Repeats) markers. The genetic frequencies of ten major blast resistance genes (Pi-9, Pi-1, Pi-5(t), Piz-5, Pi-b, Pi-b, Pi-ta, Pi-33, Pi27(t), Pitp(t) and Pi-kh) were ranged from 2% to 93%. The blast resistance gene Pi-kh was widely (93%) and Pi5 (2%) was sparsely distributed among the studied genotypes. Nine genotypes sonaanjana, jabedshail, karaja, pajon, IR09, BAU 39, binni, katiabagdad and kataribhug, had maximum eight blast resistance genes and only one BRRIdhan 46 had minimum two blast resistance genes. Twelve genotypes possessed seven, twelve had six, seven had five and five had four resistant genes. Out of forty-eight genotypes, forty-one genotypes occupied at least five positive fragments of expected product size. The results are useful to identify and incorporate blast resistance genes from these germplasms into different elite cultivars grown in Bangladesh through marker assisted selection.

Keywords: Rice, Blast, Resistance genes, SSR markers

Location: In Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Insects, Rice, Germplasm

Objectives:

To screen out and identify blast-resistant genes using SSR markers.

Methodology:

Genomic DNA extraction and quantification: Genomic DNA was extracted using CTAB method (Doyle and Doyle, 1987) with slight modification. Briefly, about 0.1g of young leaves was collected from each genotype grown in plant growth room, grinded using morter and pastle. Then CTAB extraction buffer was added to fine powder, and incubated at 650 C for 10 minutes. Equal amount of chloroform was added to the mixture, votexed and centrifuged. Finally, elusion and washing were done using ethanol. The extracted DNA samples were quantified spectrophotometrically by measuring A260/A280 and quality was checked by electrophoresis using 2.5 % agarose gel. Amplification of SSR Markers by PCR: Ten previously reported SSR markers (Singh et al. 2015) were used to identify the presence of blast resistance genes. The reaction mixture or PCR cocktail was prepared separately for each genotype and each SSR marker. About 10 μl of reaction mixture was prepared in individual PCR tube, each of which contained 50ng genomic DNA, 1 μl forward primer, 1μl reverse primer, 5 μl PCR master mix and double-distilled water. PCR Addbio® Taq Master Mix was used to prepare the PCR cocktail. This master mix contains DNA polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient application of a wide range of DNA template PCR. The master mix also contained two dyes (blue and yellow) that allow monitoring of progress during electrophoresis, so no additional dye was required for sample loading during electrophoresis. The PCR tubes containing the PCR reaction mixture in them were sealed and placed in a thermocycler for amplification and the PCR reaction was started immediately. The thermal cycling program involved an initial denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 2°C below Tm of respective primers for 30 sec, primer extension at 72°C for 30 sec, followed by a final extension at 72°C for 8 min. The amplified PCR products were separated using 2.5% agarose gel prepared in TAE buffer and visualized using ethidium bromide in a gel documentation system. The fragment amplified were scored (1) for presence and (0) for absence of specific resistant gene (Singh et al., 2015).

Source of Information: J Bangladesh Agril Univ 19(2): 000–000, 2021; ISSN 1810-3030 (Print) 2408-8684 (Online)

Article Link (Online Journal): https://doi.org/10.5455/JBAU.72571

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Twelve genotypes possessed seven, twelve had six, seven had five and five had four resistant genes. Out of forty-eight genotypes, forty-one genotypes occupied at least five positive fragments of expected product size. The results are useful to identify and incorporate blast resistance genes from these germplasms into different elite cultivars grown in Bangladesh through marker assisted selection.

Knowledge Management: Journal