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Screening of F3 Rice Families Under Salinity Condition and Polymorphism Study Using Rapd Primer

M. Arifunnahar
Professor
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

J. Halder
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M.M. Islam
Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh 2202, Bangladesh

S.N. Begum
Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh 2202, Bangladesh

A.C. Manidas
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

Twenty three F₃ rice lines of Pokkali × Mut-1-1 were used to evaluate salinity tolerance at the seedling stage and based on their performance 17 lines including the parents were selected for polymorphism study using RAPD markers. Among the 23 lines, 4 lines were found as salt tolerant, 6 moderately salt tolerant, 8 susceptible and rest of the lines were highly susceptible. Three out of five decamer random primers were selected to amplify genomic DNA and produced a total of 17 scorable bands. Among them, 15 (88.24%) were polymorphic and 2 (11.76%) were monomorphic bands. The highest intra-genotype similarity indices (Si) value was found in Pokkali, Mut-1-1 and line-15 (100%) and lowest was in line-12 (77.77%). The highest inter-genotype similarity indices (Sij) were found in Pokkali vs Line-10 (93.45%) and the lowest band sharing value was observed in Pokkali vs Mut-1-1 (52.38%) and Mut-1-1 vs Line-3 (52.37%). The co-efficient of gene differentiation (Gst) was 0.8402 reflecting the existence of high level of genetic diversity among the rice lines. The Unweighted Pair Group Method of Arithmetic Means (UPGMA) dendrogram constructed from Nei’s (1972) genetic distance produced 2 main clusters of the 17 rice lines. Only Mut-1-1 grouped in cluster 1 and Pokkali and remaining lines were grouped in cluster 2. The highest genetic distance (0.7338) was found between Pokkali and Mut-1-1 where they were remaining in different cluster. The genetic distance between line-10 and Pokkali was the lowest (0.0668) and they grouped together in the same cluster. All moderately tolerant and tolerant lines were grouped with Pokkali in cluster one and the genetic distance between the lines in group one was less. Indicating they were more or less genetically similar. Thus, RAPD offers a potentially simple, rapid and reliable method for genetic diversity analysis.

Keywords: RAPD, genetic diversity, salt tolerant, Rice.

Location: BAU Campus

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Non-commodity: Soil salinity

Objectives:

To study at screening F3 rice lines of Pokkali × Mut-1-1 at the seedling stage under salt condition and molecular characterization of F3 lines using RAPD markers.

Methodology:

Twenty three F3 rice lines of a cross between Pokkali (Salt tolerant Indian rice variety) × Mut-1-1 (high yielding advanced mutant line) were used to evaluate salt tolerance at seedling stage. Hydroponic system was used to evaluate for salt tolerance using nutrient solution (Yoshida et al., 1976) at the glasshouse. Two setups viz. Salinized and non-salinized with 3 replications were maintained. Pregerminated seeds were sown in hydroponic system with tap water. After 3 days; tap water was replaced with nutrient solution. The nutrient solution was salinized at EC 12 dS/m by adding crude salt (non-refined) and since then, PH was monitored every day and maintained at 5.25 up to the final scoring (24DAS). The modified Evaluation System (SES) of IRRI was followed to assess the visual symptoms of salt toxicity. According to the SES of IRRI a score from 1-9 was used for grading the genotypes on the basis of visual salt injury where, 1= highly tolerant and 9= highly susceptible. Initial scoring was started at 15 day after salinization and final scoring was done at 21 day after salinization. Fresh samples from twenty-one day old seedlings were used for DNA extraction. Modified CTAB mini-prep method was followed to extract DNA from leaf samples. Quantification of the DNA was done on a 0.8% agarose gel. Five primers of random sequence were screened on each sample for their ability to amplify scorable and reproducible DNA fragments. Three primers which produced clear and visible bands were selected for this study. Two replications were used for each sample. Each RAPD reaction were done in a volume of 10 µl containing 10X PCR buffer 1 µl, 250 µM dNTP (mix) 1.0 µl, 10 µM primer 2.5 µl, 25 ng/ µl DNA template 2 µl, Taq DNA polymerase 0.2 μl, sterile deionized water 3.3 μl. DNA amplification was performed in an oil-free thermal cycle with the following program: preheated at 94oC for 3 minutes followed by 40 cycles of 1 minute denaturation at 94oC, 1 min annealing 34oC and elongation or extension at 72oC for 2 minutes. Final step for 7 minutes at 72oC to allow complete extension of all amplified fragments. Then agarose gel electrophoresis was conducted in 0.5X TBE buffer at 120 V for 1 hr. After electrophoresis, the gel was taken out carefully from the gel chamber and transferred in a prepared ethidium bromide solution for staining and placed on the UV transilluminator in the dark chamber of the Gel Documentation System. The amplified bands were visually scored as present (1) and absent (0) separately for each individual and each primer. The scores obtained were pooled to create a single data matrix. This was used to estimate polymorphic loci, Nei’s (1973), genetic diversity, genetic distance (D) and a UPGMA (unweighted Pair Group Method with Areathmetic Means) dendogram using a computer program, POPGENE (Version 1.31).

Source of Information: Bangladesh J. Seed Sci. & Tech. 15 (1 &2): 203-210 (2011), ISSN 1029 - 8800

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The present study was undertaken to assess the polymorphism among the F3 rice lines of a cross between Pokkali and Mut-1-1. In the salinized setup at seedling stage, 23 rice lines showed wide variation in phenotypes under salt stress. Among the 23 lines, 4 lines were found as salt tolerant viz. Pokkali, line-7, line-10 and line-13, while 6 lines were moderately salt tolerant, 8 lines were susceptible and rest of the lines were highly susceptible. This scoring discriminated the susceptible genotypes from the tolerant and moderately tolerant genotypes. The wide variation in phenotypes from tolerant (score 3) to highly susceptible (score 9) using modified SES of IRRI standard protocol. In the present study the mean percentage of polymorphic loci was 88.57%. A diverse level of polymorphism in rice genotypes, 83.5% and 95.33%. The present experiment produced 5.66 scorable bands per primer and 5 polymorphic RAPD markers per primer. The highest polymorphic loci (29.41%) was found in Line-5 which gave 5 polymorphic bands, whereas the lowest values (00.00) of these traits was recorded in the varieties of Pokkali, Mut-1-1 and Line-15 which produced no polymorphic band. Average gene diversity across all varieties for all loci was found to be 0.2960. High level of gene diversity value and Shanon’s Information index was found in locus OPA01-1, OPC01-5, and OPA02-4 (0.4969, 0.4899 and 0.4891 respectively). Lowest value of gene diversity (0.000) and Shanon’s Information index (0.000) was found in locus OPA02-5 and OPC01-7. The tolerant and moderately tolerant lines (based on seedling stage performance) were grouped in same cluster and showed lower genetic distance among them and with Pokkali. On the other hand, Mut-1-1 remains in a different cluster having highest genetic distance (0.7338) with Pokkali and which was also susceptible under salt stress at seedling stage. Finally, the identified tolerant and moderately tolerant lines could be used for yield trials in saline prone areas.

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