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Ssr Marker Based Genetic Diversity Analysis of Modern Rice Varieties and Coastal Landraces in Bangladesh

MD. SHAHRIAR KABIR SHAKIL
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

SHAHANAZ SULTANA
Biotechnology Division, Bangladesh Rice Research Institute (BRRI), Joydebpur, Gazipur 1701, Bangladesh

MD. MEHEDI HASAN
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

MD. MUSHARAF HOSSAIN
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

MD. SHAMSHER ALI
Biotechnology Division, Bangladesh Rice Research Institute (BRRI), Joydebpur, Gazipur 1701, Bangladesh

SHAMSUL HAQUE PRODHAN
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

Abstract/ Executive Summary:

In Bangladesh, large cultivable areas lie in the coastal saline zone where rice cultivation is largely hindered by the salinity. This problem can be effectively addressed by identifying salt-tolerant genotypes using both conventional and modern biotechnological methods. In this study, the genetic relationship among 24 rice (Oryza sativa L.) genotypes including salt tolerant benchmarks Pokkali, 5 BRRI released modern varieties and some coastal landraces were assessed using 19 rice SSR markers. A total of 110 reproducible polymorphic alleles were identified from the loci with an average of 5.79 alleles per locus (range from 3-12 alleles). The polymorphism information content (PIC) values enumerated from the data obtained from allelic variation ranged from 0.3694 (RM18) to 0.8711 (RM493) with an average of 0.6533. The UPGMA clustering revealed six main genetic groups at a cut off value of 32% of similarities comprising of three separate clusters formed by BRRI dhan 47 (Cluster III), Lolo (Cluster IV) and Purbachi (Cluster V). The reference salt tolerant cultivar Pokkali and Nonabokra clustered with four other tolerant and moderately tolerant rice cultivars in cluster I. The highest genetic dissimilarity was found between Pokkali and Nona (85.29%), Pokkali and Nona kochi (85.29%),Purbachi and Nona kochi (85.29%)whereas Lona kuchi and Nona kochi (23.69%) showed the lowest genetic dissimilarity between them. DNA fingerprints of these rice cultivars by means of SSR markers provided meaningful data, which can be extended by additional SSR markers and that information will enable maximized selection of diverse parents and assist in broadening the germplasm based on future rice breeding programmes.

Keywords: Salinity, RiceLandraces, SSR (Simple Sequence Repeat), PIC (Polymorphism Information Content), Genetic diversity.

Location: BRRI, Gazipur

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Rice

Objectives:

To identify and differentiation of landraces with different genetic make-up.

Methodology:

Twenty-four rice genotypes (BRRI released modern varieties and coastal landraces of rice) including one sensitive genotype (IR 29) were selected for this study. All seeds were germinated after breaking seed dormancy (at 50 °C for 4 days) and grown in glass house. All genotypes were screened for salt tolerance at seedling stage in hydroponic system using IRRI standard protocol. Randomized complete block design was used with two replications. Nutrient solution was renewed every 7 day and was maintained the pH @ 5.0. The evaluation was done using Youshida’s nutrient solution (Yoshida et al.1976) at the glass house. The nutrient solution was salinized by adding NaCl to obtain desired EC (12 dS/m). The standard evaluation system (SES) of IRRI was followedto assess the visual symptomsof salt toxicity. This scoring discriminated the susceptible from the tolerant and the moderately tolerant genotypes.Initial and final scoring was done at 10 d and 16 d after salinization respectively. DNA extraction was carried out following the protocol as described by Zheng et al.(1995). A total of 19 polymorphic SSR primer pairs covering all 12 chromosomes were selected for the genetic diversity analysis of 24 germplasms of Bangladesh. PCR reactions were done in a total volume of 10 ml containing 1 ml of 10X buffer, 0.2 ml of 10 mM dNTPs, 0.5 ml of each forward and reverse primer, 3.4 ml of ddH₂O, 0.05 ml of Taq polymerase, 1.35 ml of MgCl₂and 3 ml of diluted template DNA. For PCR amplification, the thermal cycler was set at 1 cycle for 5 min at 94°C as an initial hot start and strand separation step. This was followed by 2^nd program having 34 cycles which comprises denaturation (94°C), annealing (55°C) and primer elongation (72°C) step for 30 sec of each. Finally, 1 cycle of 5 min at 72°C was used for final extension. The PCR products were resolved by polyacrylamide gel electrophoresis and documented using UVPRO (Uvipro Platinum, EU) Gel Documentation Unit. The molecular weight of distinct bands or amplified fragments was measured in base pair using Alpha-Ease FC 5.0 software. Genetic diversity of cultivars by SSRs was evaluated with the number of alleles and the polymorphism information content (PIC) value which is an estimate of the discriminatory power of a SSR marker locus. The summation statistics including the number of alleles per locus, major allele frequency, gene diversity, and PIC values were calculated using PowerMarker version 3.25 (Liu and Muse 2005). SSR marker alleles were analyzed using Power Marker version 3.25 and used to export the allele frequency data to binary format where each SSR band was scored as present (1), absent (0), or as a missing observation for analysis with NTSYS-pc version 2.2 (Rohif 2002). NTSYS-pc was used to construct a UPGMA (unweighted pair group method with arithmetic averages) dendrogram showing distance based interrelationship among the genotypes. For the unrooted phylogenetic tree, genetic distance was calculated using the “C.S Chord 1967” distance (Cavalli-Sfoza and Edwards 1967) in PowerMarker with tree viewed using Treeview software.

Source of Information: Indian Journal of Biotechnology.Vol 14: pp33-4

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

This information will enable maximized selection of diverse parents and selecting appropriate parental genotypes in breeding program for improving salt tolerance of elite cultivars based on genetic similarity and clustering data together with variations of tolerance to salt.

Knowledge Management: Journal