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Evaluation of F3 Rice Lines of Jangliboro × BRRI Dhan40 for Salt Tolerance Using Ssr Markers

M. Alauddin Ahmed
MS Student
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Dr. Mirza Mofazzal Islam
Principal Scientific Officer
Plant Breeding Division and Head, Biotechnology Division, Bangladesh Institute of Nuclear Agriculture,Bangladesh Agricultural University campusMymensingh 2202, Bangladesh

Dr. Shamsun Nahar begum
Senior Scientific Officer
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture,Bangladesh Agricultural University campusMymensingh 2202, Bangladesh

J Halder
MS Student
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M H Ahmed
MS Student
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

Selected 22 F₃ lines of Jangliboro × BRRI Dhan40 were tested for salt tolerance and genotyped with SSR markers. Salinity screening was done at the seedling stage using hydroponic system at BINA glasshouse following IRRI standard protocol. Among the tested entries, 4 lines (line 07, 17, 26 and 40) were found as salt tolerant, 5 were moderately tolerant, 10 were susceptible and rest of the lines were highly susceptible. Parental polymorphism survey was done with 7 SSR primers and out of them 3 primers viz. RM18, RM127 and RM169 were selected to characterize F3 lines for salt tolerance. In respect of RM18, 11 lines were found as salt tolerant and 11 lines were susceptible. Primer RM127 resembles 8 tolerant, 7 susceptible and 7 heterozygous lines. Moreover, 6 tolerant, 11 susceptible and 5 heterozygous lines were identified when 22 F3 lines were genotyped with RM169 primer. Line 17 was shown tolerant and line 08 and line 12 were shown susceptible in comparison to salt tolerant parent BRRI Dhan40 and salt susceptible parent Jangliboro considering 3 selected markers. On the other hand, under salt stress at seedling stage, line 17 and line 8 were tolerant and susceptible respectively, similar to marker analysis, but line 12 showed different result, i.e., moderately tolerant at seedling stage and susceptible in marker survey. The identified tolerant line can be used in further study to evaluate salinity tolerance. Thus, these markers could be efficient for tagging salt tolerant genes, in marker assisted selection (MAS) and quantitative trait loci (QTL) mapping.

Keywords: Rice, Salt tolerance, SSR Markers

Location: Glass house and Biotechnology lab, Plant Breeding Division, BINA, Mymensingh

Start Date: 01-06-2007

End Date: 30-06-2008

Program Area: Variety and Species

Commodity: Rice

Objectives:

To evaluate the performance of F₃ rice lines for salt tolerance and identify the salt tolerant lines using SSR markers

Methodology:

Evaluation of rice genotypes at the seedling stage: The salinity screening was done in hydroponic system using IRRI standard protocol (Gregorio, 1997) at seedling stage. 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 (Yoshida et al. 1976). At the same time, EC level 12dS/m and pH 5.2 were maintained up to the final scoring. The modified standard evaluation score of IRRI was used to assess the visual symptoms of salt toxicity. This scoring discriminates the highly tolerant, tolerant, moderately tolerant, susceptible and highly susceptible genotypes. Initial scoring was started at 15 day after salinization and final scoring was done at 21 day after salinization.

Genotypic study of salinity tolerance: Extraction of DNA was done using the mini preparation cetyl trimethylammonium bromide (CTAB) method from young leaf tissues of 14-day old seedlings. Seven random primers were used for surveying and among them, three primers (RM18, RM127 and RM169) were showed polymorphisms during parental survey. These three primers were then utilized for amplification of the DNA sequences of segregating F₃lines.

Amplification of SSR markers: PCR amplification of simple sequence repeats (SSR) was performed with 1.5 µl 10X PCR buffer, 0.75 µl dNTPs, 1.0 µl forward, 1.0 µl reverse primers, 0.5 µl Taq polymerase, 2.0 µl of each template DNA and 8.25 µl ddH₂O using DNA thermal cycler. The PCR reactions were: initial denaturation at 94°C for 5 minutes, then final denaturation at 94°C for 1 minute and annealing at 55°C for one minute. Polymerization was carried out at 72°C for 2 minutes to complete a cycle and cycle was repeated for 34 times. The final extension was at 72°C for 7 minutes. After PCR, products were mixed with 3 µl of 2X gel loading dye. Polymorphisms in the PCR products were detected by ethidium bromide staining after electrophoresis on 1.5% agarose gel using UV transilluminator.

Source of Information: Int. J. BioRes. 1 (1): 01-05, 2010.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Screening of F₃rice lines for salt tolerance at the seedling stage: Salt stress symptoms started to develop after two or three days of salinzation. Seedlings grown in salt stress showed several symptoms of salt injury like-yellowing of leaves, drying of leaves, and reduction in root growth, reduction of shoot growth and stem thickness and in many cases dying of seedlings. Overall, the seedling growth was suppressed under salinity stress. The observed plant population in the non-salinized condition showed normal seedling growth. The salinity injury symptoms like rolling, tip whitening and reduction in leaf area were the first symptoms. In salinized setup, F₃ rice lines showed wide variation. Four lines were identified as salt tolerant, 5 moderately tolerant and 10 susceptible and rest of the lines was highly salt susceptible.

Screening of salt tolerance of 22 F₃rice lines using SSR markers: Primer RM18 tagged 11 lines (line 1, 7, 10, 15, 17, 18, 24, 33, 36, 40 and 43) tolerant and rest of lines were susceptible. Eight tolerant (line 4, 5, 9, 17, 20, 22, 40 and 43), 7 susceptible and 7 heterozygous lines were identified when F₃lines were genotyped with primer RM127. Primer RM 169 exhibited 6 tolerant (line 1, 4, 9, 17, 18 and 33), 11 susceptible and 5 heterozygous lines in comparison with parents Jangliboro and BRRI Dhan40.

Considering phenotypic and genotypic data, Line17 was tolerant against primers RM18, RM127 and RM169 and also shown salt tolerant under salt stress at the seedling stage. Likewise, line 8 and line 12 were found as susceptible against these markers while phenotypically these lines were susceptible and moderately tolerant under salt stress, respectively.

Knowledge Management: Journal