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Molecular Marker Based (ssr) Genetic Diversity Analysis in Deep Water Rice Germplasms of Bangladesh

Sadia Matin
Department of Genetic Engineering and Biotechmology, School of Life Sciences, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

M. Ashrafuzzaman
Department of Genetic Engineering and Biotechmology, School of Life Sciences, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

Md. Monirul Islam
Biotechnology Division, Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh

Saif U. Sikdar
Department of Genetic Engineering and Biotechmology, School of Life Sciences, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

Nayem Zobayer
Department of Genetic Engineering and Biotechmology, School of Life Sciences, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

Abstract/ Executive Summary:

The study was undertaken to assess the genetic diversity among deep water rice genotypes using Simple Sequence Repeat (SSR) markers through marker aided selection (MAS). Twelve deep water rice (Oryza sativa L.) germplasms of Bangladesh was selected for genetic diversity analysis using eighteen SSR markers. Upon PCR amplification the alleles were separated on Polyacrylamide Gel Electrophoresis (PAGE) system. Initial polymorphism detection was conducted using eighteen primer pairs distributed on twelve rice chromosomes. The chosen microsatellite marker panel consisted of RM₁, RM₄₅₂, RM₁₃₀, RM₂₅₂, RM₁₃, RM₂₀₄, RM₁₁, RM₂₅, RM₂₀₅, RM₂₄₄, RM₂₀₆, and RM₄₆₃ with one representative from each chromosome. A total of 79 alleles were detected with an average of 4.38 alleles per locus. The polymorphism information content (PIC) reflections of alleles diversity frequency among the varieties, which is ranged from 0.477 to 0.782, with an average of 0.634. RM₁₃ was found as the best marker for identification of genotypes as revealed by PIC values. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram revealed 2 major groups with 4 clusters and the wide range of dissimilarity values (0.14-0.89) which showed a high degree of diversity among the cultivars. The results of the genetic diversity will be useful for the selection of the parents for developing submergence tolerant and flash flood tolerant rice variety through molecular breeding program.

Keywords: SSR markers, Genetic diversity, Deep water rice

Location: Biotechnology Laboratory of BRRI (Bangladesh Rice Research Institute)

Start Date: 00-07-2011

End Date: 00-12-2011

Program Area: Variety and Species

Commodity: Rice

Objectives:

To identify the suitable SSR primers for genetic analysis of deep-water rice and to measure the genetic diversity and relatedness among twelve deep-water rice genotypes using SSR markers.

Methodology:

The whole experiment was conducted at Biotechnology Laboratory of BRRI (Bangladesh Rice Research Institute) during the period of July/2011- December/2011. Plant materials - Seeds of twelve deep water rice varieties were collected from BRRI. Seeds were germinated at aseptic condition by incubating them at 300 C and grown in glass house. Isolation of genomic DNA - Genomic DNA was isolated from young leaves of 21 days old plants following the mini preparation modified CTAB method. DNA samples were evaluated both quantitatively and qualitatively using spectrophotometer and λ (lamda) DNA (concentration marker) respectively. SSR analysis - PCR amplification of SSR markers was carried out using eighteen primer pairs listed in table 2. Each reaction tube contained 25 ng of template DNA, 1 x PCR buffer, 2 mM MgCl2, 0.4 mM of dNTPs, 0.4 μM each of forward and reverse primers and 1.6 units of Taq DNA polymerase. Amplification was performed using the following conditions: denaturation at 94ºC for 5 min; 35 cycles of 1 min denaturation at 94ºC, 1 min annealing at 55ºC, 1 min extension at 72ºC and a final extension at 72ºC for 5 min. The SSR amplification products were separated in a vertical denaturing 8% polyacrylamide. DNA fragments were revealed using the ethidium bromide staining procedure. The gels were stained for 30-35 minutes and were documented using UVPRO (Uvipro Platinum EU) gel documentation unit. Data analysis - Polymorphic information content (PIC) values were calculated for each SSR locus. Major allele frequency, gene diversity, polymorphism information content (PIC) values were determined using Power Marker Version 3.25. The amplified bands were scored for each SSR primer pairs based on the presence or absence of bands, generating a binary data matrix of 1 and 0 for each marker system. Both matrices were then analyzed using the NTSYS pc statistical package version 2.2. The data matrices were used to calculate genetic similarity based on Jaccard’s similarity coefficients, and two dendograms displaying relationships among 12 rice cultivars were constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). The Pearson’s correlation between similarity coefficients based on SSR markers was determined from data among all twelve rice cultivars.

Source of Information: International Journal of Biosciences (IJB); ISSN: 2220-6655 (Print) 2222-5234 (Online); Vol. 2, No. 10(2), p. 64-72, 2012

Article Link (Online Journal): http://www.innspub.net

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The results revealed that highest genetic distance for one pair of landraces can be utilized as potential parents for improvement of varieties. The SSR markers based molecular fingerprinting could serve as a sound basis in the identification of genetically distant accessions as well as sorting of duplicate germplasm of morphologically close accessions.

Knowledge Management: Journal