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DNA Fingerprinting and Genetic Diversities in Some Bangladeshi Aus Rice (oryza Sativa L.) Genotypes

M.M. Islam*
Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh Agricultural University (BAU) campus, Mymensingh-2202, Bangladesh

H.A. Begum
Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh Agricultural University (BAU) campus, Mymensingh-2202, Bangladesh

M.S. Ali
Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh Agricultural University (BAU) campus, Mymensingh-2202, Bangladesh

M. Kamruzzaman S. Hoque
Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh Agricultural University (BAU) campus, Mymensingh-2202, Bangladesh

M.I. Hoque
Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh Agricultural University (BAU) campus, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

The allelic diversity and relationship among 120 Aus rice landraces were determined through DNA fingerprinting using microsatellite (SSR) markers. A total of 85 SSR markers were used to characterize and discriminate all tested Aus rice genotypes, 45 of which were polymorphic for different chromosome numbers. The number of alleles per locus varied from 6 alleles (RM484 and RM541) to 30 alleles (RM519) with an average of 13 alleles per locus. The polymorphic information content (PIC) values varied ranged from 0.5211 (RM536) to 0.9369 (RM519) with an average 0.8217. The highest PIC value (0.9369) was obtained for RM519 followed by RM286 (0.9357). The genetic distance-based results seen in the unrooted neighbor-joining tree clustering revealed nine genetic groups. Being grouped into distant clusters and with highest genetic distance, eleven genotypes viz., Atithi dhan, Kadar chap, Pankiraj, Japanese-7, Jamri saity, Logi jota, Joba, Lada moni, Manik Mondal-2, Boilum and Brmulka-2 could be selected as potential parents for crop improvement for their distinctive characters. Panchash and Parija had closest distance in the SSR based CS-Chord distance (0.000) might have same genetic background. The highest genetic dissimilarity (1.000) was found among the nineteen Aus genotypes combinations followed by the second highest (0.9778) among 94 Aus rice combinations. Whereas lowest genetic dissimilarity was found between Kala and Kalo Hizli (0.1778) followed by Holat and Holae (0.2667). This information will be useful in the selection of diverse parents, background selection during backcross breeding programs and assist in broadening germplasm-based rice breeding programs in the near future.

Keywords: Aus rice, Genetic diversity, Microsatellite markers, DNA fingerprinting

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

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

The present investigation has been undertaken in order to find out the genetic diversities among Aus genotypes at the molecular level.

Methodology:

Plant materials and genomic DNA isolation One hundred and twenty genotypes, including twelve BRRI released Aus genotypes were used in this study. Genomic DNA was isolated from young leaves from 21 days old plants with minor modification of CTAB method. The concentration of extracted DNA was estimated by DNA confirmation test by (1.5%) agarose gel electrophoresis with lambda DNA (50ng/μl). SSR primers analysis A total of 45 primers were selected to detect polymorphic DNA alleles for discriminating the tested Aus rice genotypes. Each PCR was carried out in a 10 µl reaction volume containing 1 µl of MgCl₂ free 10X PCR buffer with (NH₄)₂SO₄, 1.2 µl of 25 mM MgCl₂, 0.2 µl of 10 mM dNTPs, 0.2 µl of 5 U/µl Taq DNA polymerase, 0.5 µl of 10 µM forward and reverse primers and 3 µl (10ng) of DNA using a 96 well thermal cycler. The mixture was overlaid with one drop (3 µl) of mineral oil to prevent evaporation. The temperature profile used for PCR amplification comprised 94° C for 5 minutes (initial denaturation) followed by 35 cycles of 94° C for 1 minute (denaturation), 55° C for 1 minute (annealing), 72° C for 2 minutes (extension) with a final extension for 7 minutes at 72° C at the end of 35 cycles. The annealing temperatures were adjusted based on the specific requirements of each primer combination. The PCR products were mixed with gel loading dye (bromophenol blue, xylene cyanol and sucrose) and electrophoresed in 8% polyacrylamide gel using vertical poly acrylamide gels for high throughput manual genotyping. Three-Four µl of amplification products were resolved by running gel in 1X TBE buffer for 1.5 hrs to 2.5 hrs depending upon the allele size at around 90 volts and 500 mA electricity. The gels were stained in 1 µg/ml ethidium bromide and were documented using UVPRO (Uvipro Platinum, EU) gel documentation unit. Data analysis Size for each amplified allele was measured in base pair using Alpha-EaseFC 5.0 software. The summary statistics including the number of alleles per locus, major allele frequency, gene diversity, Polymorphism Information Content (PIC) values were determined using Power Marker version 3.25 (Liu and Muse, 2005). The allele frequency data from Power Marker was used to export in binary format (allele presence=1 and allele absence=o) for analysis with NTSYS-pc version 2.1 (Rohlf, 2002). A similarity matrix was calculated with the Simqual subprogram using the Dice coefficient, followed by cluster analysis with the SAHN subprogram using the UPGMA clustering method as implemented in NTSYS-pc.

Source of Information: SAARC J. Agri., 15(1): 123-137 (2017)

Article Link (Online Journal): DOI: http://dx.doi.org/10.3329/sja.v15i1.33162

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The allelic diversity revealed by 45 SSR primers was sufficient enough to distinguish among the tested Aus rice genotypes. The allelic variation was lower within the genotypes group than the other genotypes, indicating the possibility to exploit distant relatives to broaden the genetic base of rice.

Knowledge Management: Journal