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Genetic diversity Assessment in ten aromatic rice varieties of bangladesh

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

Shinthia Rahman
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

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

Abstract/ Executive Summary:

The availability of molecular marker systems allowed estimating the relationships among various taxa. This study was aimed at assessing the genetic diversity among ten aromatic rice (Oryza sativa L.) pools from Bangladesh by means of randomly amplified polymorphic DNA (RAPD) markers. These varieties were evaluated for polymorphisms after amplification with 10 decamer primers. A total of 60 RAPD fragments were generated among the assessed varieties with a polymorphism percentage of 80. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that these 10 varieties could be placed into two groups with a similarity ranging from 65 to 86% depicting adjacent association between Rajbhog and Kalijira-12, whereas Maloti belongs to a separate group maintaining maximum distance from rest of the varieties. The analysis revealed that the intervarietal genetic relationship of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The present results could be used for the selection of possible parents to generate a mapping population and utilized by the breeders for assessing the genetic diversity of rice genotypes.

Keywords: Aromatic rice, Genetic variability, Molecular marker, Cluster analysis

Location: Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Rice

Objectives:

1. To evaluate the genetic variation within a collection of aromatic rice varieties, and

2. To reveal genetic relationships among them for future use in selection, hybridization, biodiversity assessment and conservation of diverse gene pools.

Methodology:

A total of 10 aromatic rice varieties viz., namely Kalguchi, Kaminisoru, Maloti, Tilkapur, Doairgura, Chinigura-2, Rajbhog, Kalijira-12, Premful and Sorukamini collected from Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh were used for this investigation. The plants were raised in the Botanical Garden of the Department of Botany, University of Dhaka. These varieties were distinctly different from each other in respect of their origin. To perform RAPD analysis, young growing tillers were collected randomly from each cultivar and fresh leaves from each tiller were used for isolation of genomic DNA. Extraction of Genomic DNA: For the extraction of genomic DNA cetyltrimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1990) was used with some modifications. Young and fresh leaves collected from 15 days old seedlings were used for this purpose. Leaves were first washed in distilled water and then with ethanol to remove spores of microorganisms and any other source of foreign DNA. They were then dried using fresh tissue paper. 100 mg leaf tissue were taken in liquid nitrogen and grinded to fine powder followed by addition of 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 then transferred to an Eppendorf tube (2.0 ml) and incubated at 60ºC in a water bath for 30 min. The samples were centrifuged at 13,000 rpm for 10 min at 4^oC then equal volume of phenol : chloroform : isoamyl alcohol (25 : 24 : 1) was added with the supernatants and centrifuged the tubes at 13,000 rpm for 10 min. The supernatant with 2/3 volume chilled isopropanol was kept for overnight at –20ºC to precipitate the DNA. The supernatants were discarded carefully after centrifugation 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 and gel electrophoresis: Ten primers of arbitrary sequence were tested. Oligonucleotide primers had 60 - 70% of (G + C) content and were 10 nucleotides long. The following protocol was used for PCR amplification: 25 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 mixture maintained at 4ºC 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, 30 cycles of 94ºC for 45 sec, 32 to 34ºC for 30 sec and 72ºC for 3 min then one cycle of 72ºC for 7 min. After completion of cycling program, the reactions were held at 4ºC. The amplification products were loaded on 2% agarose gel in tris-acetic acid EDTA (TAE) buffer and run in this buffer for at least 2 hrs and 30 min at 90 - 100V. PCR reactions were repeated at least twice for each cultivar on a first DNA extract to establish reproducibility of results. The results were confirmed using a different DNA extract of the same cultivar. Amplified products were scored as presence or absence of bands for each of the 10 cultivars with the 10 primers, faint bands were ignored. Distance estimation: The bands observed in different lanes were designated a, b, c, d and so on and scored visually. Mostly dark and prominent bands were scored, although bands of lower intensity but with high reproducibility were included for analysis. The presence of a particular DNA band was scored as “1” and its absence as “0” for each of the primers considered. These data were used to calculate a distance matrix between genotypes according to Nei (1972). The distance matrix represents the fraction of shared DNA fragments between two samples. It ranges from zero to one, corresponding to complete genetic identity. We used the clustering program ‘Popgene’ program (https://sites.ualberta.ca /~fyeh/popgene.html) to group the genotypes by the unweighted pair group method using arithmetic average (UPGMA). Genetic relationships among the tested samples were presented as a tree.

Source of Information: Plant Tissue Cult. & Biotech. 27(2): 217-225, 2017 (December)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, our current investigation revealed the genetic diversity between important aromatic rice varieties of Bangladesh- Kalguchi, Kaminisoru, Doairgura, Tilkapur, Chinigura, Maloti, Rajbhog, Kalijira-12, Premful and Sorukamini. Present study revealed that PCR based fingerprinting technique, RAPD was informative for estimating the extent of genetic diversity as well as determining the genetic relationship between different species. In future, our research work might be useful for cheap and easy RAPD analysis of new genetic species diversity.

Knowledge Management: Journal