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Application of Ssr Technique for the Identification of Markers Linked to Salinity Tolerance in Rice

M. H. Kabir
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. M. Islam
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh

S. N. Begum
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

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

Abstract/ Executive Summary:

A cross was made between high yielding salt susceptible BINA variety (Binadhan-5) with salt tolerant rice landrace (Harkuch) to identify salt tolerant rice lines. Thirty six F₃ rice lines of Binadhan-5 x Harkuch were tested for salinity tolerance at the seedling stage in hydroponic system using nutrient solution. In F₃ population, six lines were found as salt tolerant and 10 lines were moderately tolerant based on phenotypic screening at the seedling stage. Twelve SSR markers were used for parental survey and among them three polymorphic SSR markers viz., OSR34, RM443 and RM169 were selected to evaluate 26 F₃ rice lines for salt tolerance. With respect to marker OSR34, 15 lines were identified as salt tolerant, 9 lines were susceptible and 2 lines were heterozygous. While RM443 identified 3 tolerant, 14 susceptible and 9 heterozygous rice lines. Eight tolerant, 11 susceptible and 7 heterozygous lines were identified with the marker RM169. Thus the tested markers could be efficiently used for tagging salt tolerant genes in marker-assisted breeding programme. Key words :

Keywords: Microsatellite markers, Rice and salt tolerance

Location: Department of Biotechnology, Bangladesh Agricultural University, Bangladesh

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Commodity: Soil salinity

Objectives:

i. To identify markers linked to salinity tolerance in rice by SSR technique and

ii. To select the salt tolerant F₃ rice lines through phenotypically and genotypically.

Methodology:

Thirty six F₃ rice lines were selected based on better agronomic performances from Binadhan-5 x Harkuch. These rice lines were screened for salinity tolerance at the seedling stage. Out of 36 F₃ rice lines, 26 F₃ rice lines were selected for genotyping based on the greenhouse evaluation of salt tolerance through marker analysis (using SSR markers). Hydroponic system with IRRI standard protocol was used at the glasshouse to evaluate for salt tolerance in F₃ rice lines using nutrient solution. Two pregerminated seeds were sown per hole on the styrofoam seedling float with tap water. The seedlings were allowed to grow for 3 days; tap water was replaced with nutrient solution. The nutrient solution was salinized at EC 12 dS/m by adding crude salt (non-refiend). Culture solution was replaced at every 8-day and the pH was maintained at 5.25 daily. The modified standard evaluation score (SES) of IRRI was used to assess the visual symptoms of salt toxicity Initial and final scoring was done at 15-day and 21-day after salinization. The modified Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep method was used to extract DNA from healthy leaf samples of 25-day old seedlings. Three polymorphic SSR markers viz., OSR34, RM443 and RM169 were selected to evaluate the 26 F₃ rice lines for salt tolerance. Each PCR reaction carried out with 15.0 μl reactions containing 1.5 μl 10X buffer, 0.75 μl dNTPs (100 mM), 1 μl primer forward (5 μM), 1 μl primer reverse (5 μM), 0.5 μl taq polymerase (3 u/μl), 8.25 μl ddH₂O and 2.0 μl of each template DNA samples. The PCR reaction were: initial denaturation at 94ºC for 5 min., denaturation at 94ºC for 1 min., primer annealing at 55ºC for 1 min., primer extension at 72ºC for 2 min., cycle to step 2 for 34 more times and incubation at 72ºC for 7 min. Then electrophoresis was done in 1.5% agarose gel and soaked in ethidium bromide (10 mg/ml) solution for 20-25 min. The gels were viewed by the Gel Documentation system. The segregating population having similar banding pattern to Harkuch were considered as tolerant, banding pattern having similar to Binadhan-5 were considered as salt susceptible and having one allele from Binadhan-5 and one allele from Harkuch was considered as heterozygous.

Source of Information: Progress. Agric. 19(2) : 57-65, 2008 ISSN 1017-8139

Article Link (Online Journal): http://www.banglajol.info/index.php/PA/article/view/16929/11885

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Evaluating and selecting salt tolerance among rice lines are not easy tasks because measurements of physiological and morphological phenotypes are highly affected by environmental factors. However, larger number of samples and adequate number of primers would be necessary to generate and construct an appropriate genetic relationship, for identification of samples with salt tolerant genes and to get more reliable markers. Molecular markers that are linked to genes controlling salinity tolerance could facilitate selection and improve rice varieties with salinity tolerance having high heritability and expressivity, which in turn increase rice production in saline environments. Thus, this study implies that SSR technique in tagging salt tolerant gene is simple, rapid and efficient and these markers (OSR34, RM443 and RM169) could be efficiently used in tagging salt tolerant genes, in marker-assisted selection and quantitative trait loci QTL) mapping. This will be useful for developing salinity tolerant variety through Marker Assisted Selection. Thus the tested markers could be efficiently used for tagging salt tolerant genes in marker-assisted breeding programme.

Knowledge Management: Journal