The study was conducted at Plant Biotechnology Division, Bangladesh
Institute of Nuclear Agriculture (BINA), Mymensingh from June 2015 to December
2015. Ten rice genotypes accessions, with diverse genetic background, were used
in this study (Table 1).
2.2. CTAB mini preparation DNA extraction
DNA isolation was done from fresh leaf tissues of 21 days old seedlings.
DNA was extracted using the mini preparation CTAB method. Grinding of leaf
sample with extraction buffer and SDS was followed by incubating the leaf sap
at 65 °C for 10 min. 100 NaCl and 100 CTAB were added sequentially and mixed well;
and incubated again at 65 °C for 10 minutes. After that the suspensions were
transferred to a new plate. 900 chloroform: isoamyl (24:1) was added and mixed
well by a shaker. The sample was then centrifuged at 11,000 rcf/g for 10
minutes. After that the supernatant were transferred into new eppendorf tubes.
Then 600 ice-cold isopropanol was added into the new
eppendorf tubes and shaken slowly and then centrifuged at 11,000 rcf/g for 15
minutes. The supernatant was decanted and air dried for at least one hour.
Pelletes were washed with 70 % ethanol (200 ), spinned for 15 minutes at 11,000
rcf/g and then air-dried for 30-60 mins. Then the ethanol was removed and air dried.
The pelletes were resuspended in 40.0 X
2.3. Primer designing, DNA amplification and SSR
A total of six SSR markers were screened (Table 2) to yield amplification
products on the total DNA obtained from the leaf tissues. The optimal reaction
for SSR analysis was set up under the following conditions: 1.5 ?l 10X PCR
buffer, 0.5 ?l Taq DNA polymerase, 1 ?l forward primer, 1 ?l reverse primer,
0.75 ?l dNTPs, 8.25 ?l sterilized ddH2O and 1 ?l template DNA for total volume
of 14 ?l. The amplification products were separated on 1.5 % agarose gels in
0.5X TBE buffer. The DNA band patterns were visualized under UV light and
photographed using a polaroid camera.
2.4. Analysis of SSR Data
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 and 1 kb DNA ladder using Alfa View
software. The summary statistics including the number of alleles per locus,
major allele frequency, gene diversity and PIC values were determined using
POWER MARKER version 3.25 (North Carolina, USA) (Liu and Muse 2005), genetic
analysis software. Molecular weights for microsatellite products, in base pairs,
were estimated with Alpha Ease FC 4.0 software. The individual fragments were
assigned as alleles of the appropriate microsatellite loci. The PIC value
described by Botstein et al. (1980) and modified by Anderson et al. (1993) for
self-pollinated species was calculated as follows
PICi = 1 – (Pij)2
Where, Pij is the freuency of jth
allele for i marker and the summation extends upto the total number of
allele for the given marker.
Ten rice genotypes were clustered based on the matrix of genetic
similarities using the unweighted pair group method with arithmetic averages
(UPGMA). The cluster analysis and dendrogram construction were performed with
NTSYS-PC (version 2.1) (Setauket, NY, USA). Genetic variation was measured in
terms of genetic diversity and was computed by averaging PIC estimates over all
loci (Weir 1996). Number of alleles, average PIC values, and average GS were
computed on the basis of different rice gene pools according to the results
from cluster analysis and origin of the accessions. Differences in average PIC
values between the three groups were evaluated by analysis of variance (SAS
Institute, Inc. 1998). PIC values were calculated for the accessions grouped in
each gene pool at each locus. Loci were used as blocks to separate the
variation among loci from the error term and increase the sensitivity of the
statistical analysis. Heterogeneity (HG) by accession and by marker was
calculated as percentage of heterogeneous loci per accession across all
accessions and loci, respectively.