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Rapd profile analysis of single and multigrain aman rice (oryza sativa l.) varieties available in bangladesh

Shawon Mitra
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Tahmina Islam
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

R. H. Sarker
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

M. Imdadul Hoque
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Genetic variation in 14 local Aman rice varieties of Bangladesh including single grain and multigrain rice was investigated at the DNA level by RAPD molecular typing. Fifteen random primers were initially screened against DNA from five individuals that generated highly reproducible RAPD fragments, which were then subjected to further population analysis. A total 191 RAPD fragments were generated with these primers and 153 (80.1%) were polymorphic, which indicated high level of polymorphism existed within these populations. The size of amplified fragments ranged between 450 and 6000 bp. Pair-wise distance estimated the range of 0.03 to 0.59 between single and multigrain Aman rice varieties. Results illustrated the potential of RAPD markers to distinguish improved varieties and grain specificity at DNA level.

Keywords: RAPD profile analysis, Single and multigrain aman, Rice varieties

Location: Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Rice

Objectives:

To evaluate the genetic variation within a collection of local single and multigrain Aman rice varieties and to reveal genetic relationships among them for future use in selection, hybridization, biodiversity assessment and conservation of diverse gene pools.

Methodology:

A total of 14 varieties of Aman rice used in this experiment.The seeds of 11 varieties (BRRI Dhan-33, 37, 39, 40, 46, 49, 51, 53, 57, 62 and Khajur Jhupi) were released by Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh. The rest of the three varieties are local of which Biram sundari and Double rice are multigrain. DNA extraction: Young, fresh leaves were collected from 25 days old pot grown seedlings to extract genomic DNA by cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1987). The leaves were washed in distilled water and ethanol and dried on fresh tissue paper to remove spores of microorganisms and any other source of foreign DNA. Four hundred mg leaf tissue taken in liquid nitrogen and grinded to fine powder then added 1.6 ml extraction buffer (3% CTAB; 1.4 M NaCl; 100 mM Tris-HCl, pH 8.0; 20 mM EDTA, pH 8.0 and 0.2% mercaptoethanol). The homogenous paste transferred to an Eppendorf tube (2.0 ml) and incubated at 60⁰C in a water bath for 30 min. The samples were centrifuged at 13,000 rpm for 10 min at 4⁰C then equal volume of chloroform : isoamyl alcohol (24 : 1) was added with the supernatants and centrifuged the tubes at 13,000 rpm for 10 minutes. The supernatant with 2/3 vol. chilled isopropanol was kept for overnight at –20⁰C to precipitate the DNA. The supernatants were discarded carefully after centrifuged for 10 min at 13,000 rpm and DNA pellet was collected. The pellet was washed with 70% ice' cold ethanol and air dried. The dried DNA was dissolved in 50 μl of TE buffer and treated with RNase A for 30 min at 37°C and store at –20°C. The concentration of DNA was estimated with Nanodrop. DNA amplification: For RAPD analysis 15 decamer primers were applied which had 60 or 70% of G + C content. Twenty five μl reaction mixture contains 2.5 μl 10 × Taq DNA buffer, 100 μM dNTPs, 1 μM primer, 1U Taq polymerase (Thermo, scientific) and 50 ng genomic DNA. PCR amplification mixture maintained at 0ºC during its preparation. The PCR amplification mixture placed in a thermal cycler (Applied Biosystems) with the following thermal cycling conditions, these were one cycle of 94ºC for 5 min, 35 cycles of 94ºC for 45 seconds, 32 or 34ºC for 30 seconds, and 72ºC for 3 min then one cycle of 72ºC for 7 min. After completion of cycling programme, the reactions were held at 4ºC. The amplified products were separated electrophoretically on 1% agarose gel with 1 × TAE buffer and ethidium bromide. Agarose gel electrophoresis was conducted in 1 × TAE buffer at 90 volts and 250 mA for 45 min. One molecular weight marker 1 kb plus or 1 Kb DNA ladder (Gene Ruler TM) was electrophoresed alongside the amplified product. Gel was photographed by using UV Transilluminator (Cleaver Scientific Ltd.).

Source of Information: Plant Tissue Cult. & Biotech. 27(2): 195-205, 2017 (December)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Through the present work it has been possible to find out the genetic variation and relatedness among the 14 Aman rice germplasm including single and multigrain features. Genetic variability is the key component for the successful crop improvement programme (Ravi et al. 2003). The employment of RAPD markers in genetic diversity analysis helped in grouping the genotypes. Cluster analysis based on the RAPD data revealed that although the morphology of multigrain varieties was same but they exhibited little genetic distance between them. In addition, the present study showed that cultivars in first cluster possess high degree of genetic similarity possibly due to originating from closely related ancestors. The results of the present investigation have opened up a possibility for developing a molecular genetic map that will lead to the application of marker assisted selection tools in genetic improvement of Aman rice.

Knowledge Management: Journal