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Screening of Salinity Tolerance of Twenty Rice Genotypes at the Seedling Stage Through Hydroponic and Ssr Marker

Sharif Ahmad Al Muti
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Shahidul Islam
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Monirul Islam
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh- 2202, Bangladesh.

Md. Mahmud Al Noor
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh- 2202, Bangladesh.

S. M. Abdul Alim
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh- 2202, Bangladesh.

Md. Monir Hossain
Planning & Evaluation Wing, Bangladesh Agricultural Research Institute, Gazipur-1701

Pritish Chandra Paul
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh- 2202, Bangladesh.

Md. Faysal Arafatbin Siddique
Department of Agriculture Extension, Rajarhat, Kurigram-5450, Bangladesh.

Abstract/ Executive Summary:

Salinity is one of the major abiotic stresses which severely affect the production of crops across the world. In this experiment, we examined 20 rice genotypes of diverse origins and sources including few salt-tolerant varieties (Binadhan-8, Binadhan-10, Pokkali and FL478) as a check. The main objective of this study was to determine salt tolerance at the seedling stage and to evaluate genetic variation using SSR markers. IRRI standard protocol was applied to screen out salinity among those varieties, at the glasshouse of Bangladesh Institute of Nuclear Agriculture, BAU campus, Mymensingh-2202. Shoot length, root length and total dry matter were recorded at 6dS/m, 8dS/m, 10dS/m and 12 dS/m salt stress levels. According to the morphological and molecular survey of 20 rice genotypes at the seedling stage, it was evident that, Binadhan-8, Binadhan-10, Pokkali, FL478, IR64, IR4630, FR13A and Sadamota identified as salt tolerant whereas THDB, Moulata, MV-20, CPD-23, CPD-29, Pot-18, Pot-27 and Dudkalam those were found as susceptible, BRRI dhan67, Binadhan-17 and Binadhan-21 those were traced as highly susceptible. The highest Nei’s genetic distance value 1.0 was found in Moulata vs Sadamota and the lowest value 0.08 was observed in Binadhan-21 vs IR64. It will be used in the future breeding program to develop a saline tolerant variety of rice.

Keywords: Rice, Hydroponic, Genetic distance, SSR markers

Location: Division of Biotechnology, Bangladesh Institute of Nuclear Agriculture (BINA), BAU campus, Mymensingh-2202

Start Date: 00-02-2019

End Date: 00-06-2019

Program Area: Variety and Species

Commodity: Rice

Objectives:

The main objective of this study was to determine salt tolerance at seedling stage and to evaluate genetic variation using SSR markers.

Methodology:

The experiment was carried out at the glasshouse and laboratory, Division of Biotechnology, Bangladesh Institute of Nuclear Agriculture (BINA), BAU campus, Mymensingh-2202, during the period from February 2019 to June 29, 2019. Experimental materials used in the study were collected from BINA. A modified hydroponic system (Gregorio et al., 1997) was used at the glasshouse to evaluate the salt tolerance of the 20 rice genotypes using Peter's solution (Yoshida et al., 1976). To prepare the nutrient solution 1.0 gm. Peter fertilizer and 200 mg ferrous sulphate were mixed together with 1 liter of distilled water. The PH was adjusted to 5.1 by pH meter using 1N HCL and 1N NaOH when necessary. For salinization, crude salt was dissolved with nutrient solution to reach the desired salinity level. The salinization levels were EC at 6 dS/m, 8 dS/m, 10 dS/m and 12 dS/m. The salinity level was measured through EC using the EC meter. Then the old solution was replaced with the new one every 8-day and the pH was maintained at 5.1 daily. Salt stresses were applied at 7^th day old seedling. After two or three days of salinization, salt stress symptoms were obvious. The genotypes were evaluated for their tolerance to salinity under sustained water bath (hydroponic condition) using IRRI standard protocol (Gregorio et al., 1997). This scoring separated the tolerant, moderately tolerant and susceptible and highly susceptible rice genotypes. Initial and final scoring was done at 14^th day and 21^st day respectively after salinization.

The data were recorded from the screening at seedling stage in both normal and salinized conditions following SES of IRRI. Root length, shoot length and total dry matter was measured along with reduction rate at different salinity stages. Plants were separately kept into envelopes and oven-dried at 72°C for a week and weighed. Juvenile, vigorous leaves were collected from 21 days old seedlings for the isolation of genomic DNA using the mini preparation Modified Cetyl Trimethyl Ammonium Bromide (CTAB) method (IRRI, 1988). The leaf samples were cut into 2-3 cm pieces and the sample was grounded. Extraction Buffer (800 µl) and 20% SDS (50 µl) were added. Then the mixture was Vortexed for 20 seconds and incubated for 10 minutes at 65ºC in the hot water bath. 100 µl 5M NaCl was added and inverted gently to suspend the samples evenly. Then 100 µl CTAB was added and mixed well. Again, the mixture was vortexed for 20 seconds and incubated for 10 minutes at 65ºC in the hot water bath. 900µl chloroform mix (Phenol: chloroform: isoamyl alcohol = 25:24:1, v/v) was added and mixed well. The samples were centrifuged at 15000 rpm for 15 minutes. Then the supernatant was transferred into a new Eppendorf tube and 600 µl ice-cold isopropanol was added to the supernatant and shacked well. The mixture was again spinned down at 12000 rpm for 15 minutes by centrifuge. The Supernatant was discarded and the pellet was washed with 200 µl 70% ethanol. At last, the samples were spinned down again at 15000 rpm for 5 minutes, the ethanol was removed and the pellets were allowed for air-drying for 1 hour. The pellets were then suspended in 30 µl 1X TE buffer. Finally, the DNA samples were stored at -20 °C. To estimate the quantity and quality (in terms of protein and RNA contamination) of isolated genomic DNA, agarose gel electrophoresis and Nanodrop spectrophotometer were used. A set of thirty microsatellite primers developed by several investigators were used in this study. Thirty primers were screened on a sub-sample of one randomly chosen individual from twenty rice genotypes to evaluate their suitability for amplifying DNA sequences, which could be accurately scored. Primers were selected on the basis of band resolution intensity, presence of smearing, consistency within individuals and potential for population discrimination. Out of thirty primers, thirteen primers were used for further analysis. The total volume of PCR cocktails for this study was 9 µl per sample. 1 µl genomic DNA was added with 9 µl PCR cocktail and finally, the total volume was 10 µl. The PCR cocktail including DNA was placed in the PCR tubes and run in the DNA thermal cycler. The reaction mixture was preheated at 94⁰C for 5 minutes followed by denaturation at 94⁰C for 30 seconds and annealing at 55⁰C for 1 minute. The polymerization reaction was done at 72⁰C for 1 min followed by repeating those cycles 35 times. At last, the amplified products were incubated at 72⁰C for 5 minutes. The amplified products were then separated electrophoretically on polyacrylamide gel. After completion of electrophoresis, the gel was soaked in ethidium bromide (10 mg/ ml) solution for 12-15 min. After staining, the gel was taken out carefully from the staining tray and placed on a high-performance ultraviolet lightbox (UV trans-illuminator) of gel doc for checking the DNA bands. The DNA was observed as a band and the records were saved. The pattern of bands obtained after application with the primers was scored with reference to control. The size (in nucleotide base pairs) of the amplified band for each microsatellite marker was determined based on its migration relative to a molecular weight size marker with the help of Alpha Ease FC 5.0 software. The summary statistics including the number of alleles per locus, major allele frequency, gene diversity and Polymorphism Information Content (PIC) values were determined using POWER MARKER version 3.23. Allele molecular weight data also used to determine the genetic distance for phylogeny reconstruction.

Source of Information: Res. Agric., Livest. Fish.8(1): 75-88, April 2021

Article Link (Online Journal): DOI: https://doi.org/10.3329/ralf.v8i1.53270

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Binadhan-8, Binadhan-10, Pokkali, FL478, IR64, IR4630, FR13A and Sadamota can be used to develop salt stress-tolerant varieties. Observation on the reproductive stage of selected rice genotypes can be performed to assess their performance. Field trials of the selected genotypes may be examined to evaluate their production.

Knowledge Management: Journal