PRAGGA SAHA SHARMI
Genetic diversity, ISSR marker, Population, Shorea robusta
MD ABUL KASHEM
Genetic diversity, ISSR marker, Population, Shorea robusta
RIFAT SAMAD
Genetic diversity, ISSR marker, Population, Shorea robusta
MOHAMMAD ZABED HOSSAIN*
Genetic diversity, ISSR marker, Population, Shorea robusta
Genetic diversity, ISSR marker, Population, Shorea robusta
Madhupur tract under the districts of Tangail and Gazipur and those in the districts of Cumilla and Dinajpur
Conservation and Biodiversity
For the collection of leaf samples, natural populations of Sal were selected from the forests in the Madhupur tract under the districts of Tangail and Gazipur and those in the districts of Cumilla and Dinajpur. A total of 13 locations, 7 from Madhupur tract (Gazipur and Tangail) and each three from Cumilla and Dinajpur forests were selected in the present study to collect samples. The three forests varied in average precipitation, humidity and temperature; the precipitation rate of Madhupur tract, Cumilla and Dinajpur were 1872, 2295 and 1728 mm, respectively, likewise the humidity was 70, 75 and 69%. The forestlands also differed in soil type: Madhupur tract is clayey, Cumilla is sandy loam and Dinajpur is rich in lime (Banglapedia 2015). Fully expanded youngest leaf samples were collected from the plants for the extraction of DNA. During leaf sample collection, minimum 50 m distance was maintained among sampling locations within each of the three populations. After completion of sampling, leaves were labeled separately and kept at -20ºC until DNA extraction was carried out.
For the extraction of DNA, 500 mg of fresh leaf material was used. At first, leaves were washed and sterilized with distilled water and 70% alcohol, respectively and then rinsed with distilled water to remove alcohol. Leaves were ground using liquid nitrogen. DNA was extracted following a modified CTAB method (Milligan 1992). Eight ISSR primers were selected based on their ability to produce distinct and maximum polymorphic amplified products (Surabhi et al. 2017). DNA sequence, annealing temperature and number of alleles of each primer.
The PCR reaction mixture consisted of 50 ng of template DNA, 12.5 μl of PCR master mix (1 mM of each dNTP, 1.5 mM MgCl2, 1-unit Taq polymerase), 1μl primer and 8.5 μl of PCR-grade sterile de-ionized distilled water. The PCR amplification was done using a thermal cycler (Applied Biosystems 2720 Thermal cycler, USA) as following 40 cycle of initial denaturing step of 2 minutes at 94ºC, denaturation step at 94ºC for 30 sec, annealing step of 1 min and extension and final extension at 72ºC for 2 and 7 min, accordingly. After the completion of PCR, gel electrophoresis was performed by separating the DNA in 1.5 % (w/v) agarose gel stained with ethidium bromide. Solidified gel was placed into gel-running kit 1x TAE buffer. Molecular marker of 1.0 kb Plus DNA ladder was electrophoresed alongside the reaction samples to compare and score band size. DNA bands were observed on UV-transilluminator and photographed by a gel documentation system (Cleaver Scientific’s MultiSUBTM, UK).
Each experiment was run twice in case of every primer to avoid any dubious result. Clear and reproducible bands were scored as binary data on the basis of their presence (1) and absence (0). Observed number of alleles (No), effective number of alleles (Ne), percentage of polymorphism (%), allelic richness, number of polymorphic loci, Nei’s gene diversity (Nei 1973) and Shannon's Information index (Lewontin 1972) were calculated using Popgene version 1.32 software. Analysis of molecular variance (AMOVA) was used to estimate the partitioning of genetic variance among and within populations using GenAlEx6.5 (Peakall and Smouse 2006). Principal Coordinate (PCO) analysis was also performed to find out the association among individuals from different geographic populations. A measure of Isolation by Distance (IBD) was obtained by plotting genetic distance values against log transformed geographic distance values and by performing a mantel test using the software GenAlEx 6.5 (Peakall and Smouse 2006). Nei's unbiased measures of genetic identity and genetic distance (Nei 1973) were performed by PopGen32 software.
Bangladesh J. Bot. 50(2): 405-412, 2021 (June)
Journal