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Morpho-molecular Characterization of Bangladeshi Local Boro Rice (oryza Sativa L.) Genotypes

T. Halder
Bangladesh Rice research Institute Gazipur 1701, Bangladesh.

M. E. Hoque
Bangladesh Rice research Institute Gazipur 1701, Bangladesh.

M. M. Islam
Bangladesh Rice research Institute Gazipur 1701, Bangladesh.

Liakat Ali
Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh.

A. K. Chowdhury
Patuakhali Science and Technology University, Patuakhali, Bangladesh

Abstract/ Executive Summary:

An investigation was carried out to analysis the genetic diversity of 12 Bangladeshi local Boro rice (Oryza sativa L.) germplasm using morphological traits and molecular markers. Eight morphological traits (viz., days to 50 percent flowering, growth duration, plant height, filled grain/panicle, 1000 grain weight and grain yield) and eight Simple Sequence Repeat (SSR) markers were used for this analysis. The plant morphological traits exhibited more variation among the genotypes tested. Several traits were found to be significantly positive in correlation coefficient analysis and thus those traits can be considered stable as demonstrated by their coefficient of variability. A set of eight SSR primer pairs was used for molecular characterization resulting 49 alleles, where average of allele number was 6.13. The polymorphic information content (PIC) values ranged from 0.67 (RM1) to 0.86 (RM314) with an average of 0.76. The highest PIC value (0.86) was obtained for RM314 which also gave maximum alleles. The PIC value revealed that RM314 was the best marker for 12 genotypes tested. The cluster analysis based on UPGMA system grouped 12 genotypes into four clusters.

Keywords: Rice genotypes; Morphological traits; Molecular marker; Genetic diversity

Location: Bangladesh Rice Research Institute (BRRI), Gazipur

Start Date: 00-00-2012

End Date: 00-00-2013

Program Area: Variety and Species

Commodity: Rice

Objectives:

The objective of the study is to characterize and to evaluate the genetic diversity of 12 Bangladeshi local Boro rice germplasm using morphological traits and molecular markers

Methodology:

Twelve local Boro rice genotypes were used as experimental materials in this study. The germplasm were collected from Bangladesh Rice Research Institute (BRRI) gene bank. The accessions were grown in the field during the Boro season of 2012-13 in randomized blocks design with three replications. The accessions were grown in the field during the Boro season of 2012-13 in randomized blocks design with three replications. Thirty five days old seedling were transplanted in 3m × 3m plot with 20cm × 20cm spacing. The experiment was subjected to standard agronomic management includes aspects regarding plant geometry, fertilizer application and insect management. (BRRI Adhunik Dhaner Chash, 2012). Data were recorded on five competitive plants per genotype from the middle row for yield and yield components such as days to 50% flowering (DFL), growth duration (GD), plant height (PLH), number of fertile tiller per hill (NFT), panicle length (PNL), filled grain per panicle (FGN), 1000-grain weight (TGW), and yield (YLD). Days to 50% flowering and total growth duration were measured from the date of seed germination to 50 % plant were heading at stage and 80% grain at hard dough stage of each plot respectively. Plant height was the distance from the ground level to the tip of the tallest panicle. NFT was counted from the number of fertile tillers per plant when single plants have been used. Grain yield (t ha^-1) was estimated from harvests from sampling units of 5 m² within the middle rows in each plot. Statistical analysis was done for quantitative traits by using CROPSTAT 7.2 and SPSS 11.5. Leaves were collected at seedling stage (15 days) to extract the genomic DNA. Extractions of genomic DNA was carried out following miniprep DNA extraction protocol, which did not require liquid nitrogen and required only a small amount of tissue samples. The quality of DNA was also checked by DNA quantification using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). Eight SSR primer pairs were used for polymerase chain reaction (PCR). 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). PCR reaction was carried out in a volume of 10 μl reaction mixture, containing 3 μl of diluted template DNA, 0.5 μl of each forward and reverse primer, 0.25 μl of 10 mM dNTPs, 1.5 μl of 10x buffer, 0.2 μl of Taq polymerase, 1.8 μl of MgCl₂ and 2.25 μl of ddH₂O and the PCR reaction was carried out in a DNA thermal cycler (G-STORM, GSI, England). The following condition was performed for PCR amplification of SSR marker: 94⁰C for 5 min (initial denaturation) followed by 35 cycles of 94⁰C for 1 min (denaturation), 55⁰C for 1 min (annealing), 72⁰C for 2 min (extension) with a final extension for 7 min at 72⁰C. After amplification PCR product was mixed with gel loading dye (bromophenol blue, xylene cyanol and sucrose) and electrophoresis was carried out in a mini vertical polyacrylamide gel (8% denatured polyacrylamide gel containing 19:1 acrylamide : bisacrylamide) in TBE buffer. Three micro liters of the sample were loaded in each well and run at 80 volt for 90 minutes. To estimate the PCR product size, 50 bp ladders was used. After completing gel electrophoresis, the gel was stained with ethidium bromide for 30-35 min, kept in dark, and then visualized using gel documentation unit linked to a PC. Diversity Analysis Clearly observed unambiguous bands were scored visually for their presence or absence with each primer 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). The scores were obtained in the form of matrix with ‘1’ and ‘0’, which indicate the presence and absence of bands in each variety respectively. This observation was 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.

Source of Information: Bangladesh J. Pl. Breed. Genet., 29(2): 01-09, 2016

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Achievement/Findings:

In summary, morphological and molecular characterization both could serve as a potential basis in the identification of genetically distant accessions as well as in identification of the morphologically close accessions. This characterization also provided valuable information about genetic diversity of 12 local Boro rice germplasm. Therefore, it is highly necessary not only to conserve germplasm, but also to reveal the gene-pool of rice germplasm and unlock valuable genes for breeding purposes. However, the use of more number of markers would be efficient to characterize the germplasm than used for the study, which highlighted the presence of diversity at genomic level among the genotypes studied.

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