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DNA Fingerprint and Diversity in Aromatic Rice by Gn1 Gene Linked Ssr Markers

MS Haque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

T Tabassum
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

NR Saha
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

MS Islam
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

Abstract/ Executive Summary:

Number of grains per panicle is a limiting factor for the yield of aromatic rice. To overcome this limitation, it might be helpful to screen aromatic rice using markers linked to grain number. In this study, an elementary DNA fingerprinting database of the nine aromatic rice cultivars was built using four SSR primer pairs linked to Gn1 gene responsible for grain number in rice. For genotyping of aromatic rice cultivars, DNA was extracted from leaf samples using IRRI standard protocol. Allele scoring was done by using a computer based program Alpha Ease FC 4.0 and data were analyzed by Power Marker version 3.25 software. Nei’s genetic distance value and similarity were computed. From the analysis, it was found that a total of 21 alleles were detected with an average number of 5.25 alleles per locus having PIC values ranging from 0.3402 (RM5493) to 0.7883 (RM3452) and the average value 0.6629. The highest gene diversity (0.8148) was observed in loci RM3452 and the lowest gene diversity (0.3404) was observed in loci RM5493. From the study, it can be stated that all of the aromatic rice germplasm have bands of the gene that influence the grain but they showed genetic variability. Information obtained from genotyping of varieties helped to analyze the genetic diversity within and among closely related crop varieties which has the potential for crop improvement and to meet the diverse goals like producing cultivars with increased yield of aromatic rice.

Keywords: DNA fingerprint, Aromatic rice, Gn1 gene, SSR marker

Location: Nagoya University, Japan

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

To assess the extent of variability among the aromatic rice varieties using SSR markers linked to Gn1 gene.

Methodology:

Nine aromatic rice genotypes, Chini Sagor, Kalizira, BR-34, Khirsha, LR-189, LR-53, LR-42, UkunMadhu and a Japanese line STG were used in this study. In order to extract genomic DNA for SSR analysis, young, vigorously growing fresh leaf samples were collected from 25 days old seedling of each of the 9 aromatic rice genotypes. STG, a Japanese rice line, leaves were taken from Nagoya University, Japan by the first author (courtesy, Prof. Motoyuki Ashikari). Genomic DNA extraction: DNA was extracted from the leaves of each genotype using the modified IRRI standard protocol. The simplified mini scale procedure for DNA isolation for PCR analysis was developed at IRRI. The leaf samples were cut into pieces and the sample was ground. Before and after grinding, 600 and 200µl extraction buffer was added, respectively. Then 500 µl chloroform was added and mixed well The samples were shaken at 600 rpm for 20/25 minutes and were centrifuged at 14000 rpm for 5 minutes. The supernatant (500 - 600µl) was transferred in eppendorf tube and 20 µl RNase was added. Also, 50µl PCI was added and centrifuged for 10 minutes at 14000 rpm. The supernatant was transferred into another tube. Then 900µl 100% ethanol/isopropanol was added and gently shaken. The samples were kept in freeze -20^oC overnight and the samples were taken out from freeze, vortexed and centrifuged 10 minutes at 14000 rpm. Then ethanol was removed and allowed the pellets for air drying 1 hour or more. 1X TE buffer 50µl was added for re-suspension. The samples were then kept in 4^oC overnight for chelating reaction. Then the samples were vortexed and stored at -20^oC freeze. A concentration of about 50 ng / µl was maintained in the DNA samples.DNA confirmation was done by using 0.8% agarose gel electrophoresis. Electrophoresis was carried out at 80V for 90 minutes. Four primers were selected on the basis of their association with the trait. The PCR cocktail including DNA had total volume of 10 μl/reaction based on rice protocol, was placed in the PCR tubes and run in the DNA thermal cycler.DNA amplification was performed in an oil-free thermal cycler. The reaction mix was preheated at 94^oC for 5 minutes followed by 35 cycles of 1 min denaturation at 94^oC, 1 min annealing at 55^oC and elongation or extension at 72^oC for 2 minutes. After the last cycle, a final step for 7 minutes at 72^oC to allow complete extension of all amplified fragments. After completion of cycling program, reactions were held at 4^oC. About 2 μl of each PCR product was loaded in each well. Before loading, each PCR product was mixed with 2 μl of 2X gel loading dye. DNA marker (25bp DNA ladder) was used for size determination. The electrodes were connected to the power supply and run for about 3-3.5 hours at 80 volts. After completion of electrophoresis, the gel was soaked in ethidium bromide (10mg/ ml) solution for 15-20 min. After staining, the gel was taken out carefully from the staining tray and placed on high performance ultraviolet light box (UV-trans-illuminator) of gel documentation system for checking the DNA bands. The DNA bands were observed and saved. The size of most intensely amplified fragments was determined by comparing the migrated distance of amplified fragments relative to the molecular weight of known size markers, 25 bp and 100 bp DNA ladder using Alpha-Ease FC 5.0 software (Alpha Innotech, USA). The number of alleles per locus, major allele frequency, gene diversity, PIC and Nei’s genetic identity and genetic distance values were calculated using Power Marker version 3.25. All the genotypes were scored for the presence and absence of the SSR bands for further analysis with NTSYS-pc version 2.2. MEGA software was used to construct a UPGMA (Unweighted Pair Group Method with Arithmetic Averages) dendrogram showing the distance based interrelationship among the genotypes.

Source of Information: Progressive Agriculture 29 (4): 336-344, 2018

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

PCR products of all the four primers have showed excellent bands in agarose gel. The DNA based markers offered an opportunity to detect variation among aromatic rice genotypes that can be utilized to improve aromatic rice. The findings of the study will be very useful for the future screening of aromatic rice germplasm by Gn1 marker. High yielding capacity of aromatic rice is very necessary for our country. So researchers should come forward to take steps for the improvement of aromatic rice variety by breeding for high yield using Gn1 marker.

Knowledge Management: Journal