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Research Detail

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Abdul M. Sajib
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

Md. Musharaf Hossain
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

A.T.M.J. Mosnaz
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

Hosneara Hossain
Biotechnology Division, Bangladesh Rice Research Institute, Joydebpur, Gazipur 1701, Bangladesh.

Md. Monirul Islam
Biotechnology Division, Bangladesh Rice Research Institute, Joydebpur, Gazipur 1701, Bangladesh.

Md. Shamsher Ali
Biotechnology Division, Bangladesh Rice Research Institute, Joydebpur, Gazipur 1701, Bangladesh.

Shamsul H. Prodhan
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh.

Molecular characterization of the genotypes gives precise information about the extent of genetic diversity which helps in the development of an appropriate breeding program. In the present study, a total of 24 SSR markers were used across 12 elite aromatic rice genotypes for their characterization and discrimination. Among these 24 markers 9 microsatellite markers were showed polymorphism. The number of alleles per locus ranged from 2 alleles (RM510, RM244, and RM277) to 6 alleles (RM 163), with an average of 3.33 alleles across 9 loci obtained in the study. The polymorphic information content values ranged from 0.14 (RM510) to 0.71 (RM163) in all 9 loci with an average of 0.48. RM163 was found the best marker for the identification of 12 genotypes as revealed by PIC values. The frequency of most common allele at each locus ranged from 41% (RM163, RM590, and RM413) to 91% (RM510). The pairwise genetic dissimilarity co-efficient indicated that the highest genetic distance was obtained between Basmati PNR 346 and Deepa; Basmati PNR 346 and Patnai-23; Dolargura and Sugandha; Bhogganijia and Sugandha; and finally between Dolargura and Chinikani (88.89%). Opchaya, Basmati PNR 346 and Sugandha had close similarity among them but showed wide dissimilarity with other genotypes. Being grouped into distant clusters Dolargura and Opchaya could be utilized as potential parents for the improvement of fine grain aromatic rice varieties. Genotypes Deepa and Patnai-23 (having zero dissimilarity) might have possessed somewhat similar genetic background and more markers are needed to discriminate them. The microsatellite marker based molecular fingerprinting could serve as a sound basis in the identification of genetically distant accessions as well as in the duplicate sorting of the morphologically close accessions.

  Molecular characterization, Genetic diversity, Aromatic rice landraces, Oryza sativa, SSR
  
  
  
  Crop-Soil-Water Management
  Rice

In the present study, twelve aromatic landraces of rice were analyzed for genetic variation using SSR markers. Specially, the objective of the study was DNA fingerprinting and genetic diversity analysis of aromatic landraces to measure the extent of genotypic differences, genetic relationship and to assist in broadening the germplasm base of future aromatic rice breeding programs.

Germplasm collection and genomic DNA extraction A total of 12 rice genotypes were evaluated in this study including Jamaisohagi, Sugandha, Darsail, Chinikani, Bhogganijia, Dolargura, Depa, Patnai-23, Opchaya, Holdijorun, Jingasail and Basmati PNR 346. All seeds those collected from the Genetic Resource Center (GRC), Bangladesh Rice Research Institute (BRRI) were germinated at aseptic condition by keeping them at 30ºC for 1 day and raised in pots in a net house. At 3 weeks of age, about 2 cm of leafs from each plant was harvested and bulked for each genotype. Total genomic DNA was extracted from the bulked leaf samples by following a miniprep DNA extraction protocol, which did not require liquid nitrogen and required only a small amount of tissue samples (Zheng et al., 1995). The quality of DNA was also checked by DNA quantification using a Thermo Scientific NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, USA). All chemicals used for DNA extraction were purchased from Sigma-Aldrich, Germany. SSR markers and PCR amplification Twenty four SSR primer pairs (Promega Corporation, USA) were selected on the basis of the published rice microsatellite framework map for the genetic diversity analysis of the 12 aromatic rice cultivars in Bangladesh. Primers that showed polymorphic banding patterns were selected whereas primers that showed monomorphic banding patterns were excluded. Finally, 9 microsatellite primers with a distinct chromosome number were used for final polymerase chain reaction (PCR) amplification. Information regarding the original source, repeat motifs, primer sequences, expected length, chromosomal localizations and repeat types of the SSRs can be found in the Web database (http://www.gramene.org). Prior to DNA amplification, a PCR cocktail was prepared containing all required components. All reagents were purchased from Sigma Aldrich. PCR amplification reactions were done in 10 l reaction mixtures, containing 3 ?l of diluted template DNA, 0.5 ?l of each forward and reverse primer, 0.25  of 10 mM dNTPs, 1.5  of 10x buffer, 0.2 l of Taq polymerase, 1.8 l of MgCl2 and 2.25 of ddH2O. An DNA thermal cycler (Model: ALS 1296, BioRad, USA and G-STORM, GSI, England, Serial no: GT-11620) was used along with the following PCR profile: an initial denaturation step for 5 min at 94°C (hot start and strand separation), followed by 34 cycles of denaturation (94°C), annealing (55°C) and primer elongation (72°C) for 30 seconds each and then a final extension at 72°C for 5 min. Amplified products were stored at -20°C until further use. Electrophoretic separation and visualization of amplified products Prior to electrophoresis, each PCR product was mixed with gel loading dye (bromophenol blue, xylene cyanol and sucrose) and electrophoresis was carried out in a mini vertical electrophoresis tank (CBS Scientific Co Inc., CA. USA), run on 8% polyacrylamide gels in TBE buffer. Four microliters of the sample were loaded in each well and run at 80 V for 90 minutes. The gel, after electrophoresis, was stained with ethidium bromide for 30-35 min, kept in dark, and then scanned using an UVPRO (Uvipro Platinum, EU) gel documentation unit linked to a PC. The reproducibility of amplification products was confirmed twice for each primer. SSR data analysis The size of most intensely amplified fragments was determined by comparing the migration distance of amplified fragments relative to the molecular weight of known size markers, 50 base pairs (bp) DNA ladder using Alpha-Ease FC 5.0 software (Alpha Innotech, USA). The number of alleles per locus, major allele frequency, gene diversity and PIC values were calculated using Power Marker version 3.25 (Liu & Muse, 2005). All the genotypes were scored for the presence and absence of the SSR bands throughout all 12 genotypes and the data were exported to binary data for the presence (1) or absence (0) or as a missing observation for further analysis with NTSYS-pc version 2.2 (Rohif, 2002). NTSYS-pc was used to construct a UPGMA (unweighted pair group method with arithmetic averages) dendrogram showing the distance-based interrelationship among the genotypes. For the unrooted phylogenetic tree, genetic distance was calculated using the “C.S Chord 1967” distance (Cavalli-Sforza & Edwards, 1967) in Power Marker with tree viewed using Tree view software.

  J. BioSci. Biotech. 2012, 1(2): 107-116. ISSN: 1314-6246
  
Funding Source:
1.   Budget:  
  

In summary, the present study revealed a wide variation among the germplasms. The result indicated that the SSR markers are neutral and co-dominant and could be a powerful tool to assess the genetic variability of the cultivars. The information about the genetic diversity will be very useful for proper identification and selection of appropriate parents for breeding programs, including gene mapping, and ultimately for emphasizing the importance of marker-assisted selection (MAS) in aromatic rice improvement worldwide.

  Journal
  


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