The study was conducted at Plant Biotechnology Division, BangladeshInstitute of Nuclear Agriculture (BINA), Mymensingh from June 2015 to December2015. Ten rice genotypes accessions, with diverse genetic background, were usedin 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 leafsample with extraction buffer and SDS was followed by incubating the leaf sapat 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 weretransferred to a new plate. 900 chloroform: isoamyl (24:1) was added and mixedwell by a shaker. The sample was then centrifuged at 11,000 rcf/g for 10minutes. After that the supernatant were transferred into new eppendorf tubes.Then 600 ice-cold isopropanol was added into the neweppendorf tubes and shaken slowly and then centrifuged at 11,000 rcf/g for 15minutes.
The supernatant was decanted and air dried for at least one hour.Pelletes were washed with 70 % ethanol (200 ), spinned for 15 minutes at 11,000rcf/g and then air-dried for 30-60 mins. Then the ethanol was removed and air dried.The pelletes were resuspended in 40.
0 XTE buffer. 2.3. Primer designing, DNA amplification and SSR A total of six SSR markers were screened (Table 2) to yield amplificationproducts on the total DNA obtained from the leaf tissues. The optimal reactionfor SSR analysis was set up under the following conditions: 1.5 ?l 10X PCRbuffer, 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 volumeof 14 ?l. The amplification products were separated on 1.5 % agarose gels in0.
5X TBE buffer. The DNA band patterns were visualized under UV light andphotographed using a polaroid camera.2.4. Analysis of SSR DataThe size of most intensely amplified fragments was determined bycomparing the migration distance of amplified fragments relative to themolecular weight of known size markers and 1 kb DNA ladder using Alfa Viewsoftware. The summary statistics including the number of alleles per locus,major allele frequency, gene diversity and PIC values were determined usingPOWER MARKER version 3.
25 (North Carolina, USA) (Liu and Muse 2005), geneticanalysis software. Molecular weights for microsatellite products, in base pairs,were estimated with Alpha Ease FC 4.0 software. The individual fragments wereassigned as alleles of the appropriate microsatellite loci. The PIC valuedescribed by Botstein et al.
(1980) and modified by Anderson et al. (1993) forself-pollinated species was calculated as follows PICi = 1 – (Pij)2Where, Pij is the freuency of jthallele for i marker and the summation extends upto the total number ofallele for the given marker. Ten rice genotypes were clustered based on the matrix of geneticsimilarities using the unweighted pair group method with arithmetic averages(UPGMA). The cluster analysis and dendrogram construction were performed withNTSYS-PC (version 2.1) (Setauket, NY, USA). Genetic variation was measured interms of genetic diversity and was computed by averaging PIC estimates over allloci (Weir 1996). Number of alleles, average PIC values, and average GS werecomputed on the basis of different rice gene pools according to the resultsfrom cluster analysis and origin of the accessions.
Differences in average PICvalues between the three groups were evaluated by analysis of variance (SASInstitute, Inc. 1998). PIC values were calculated for the accessions grouped ineach gene pool at each locus. Loci were used as blocks to separate thevariation among loci from the error term and increase the sensitivity of thestatistical analysis.
Heterogeneity (HG) by accession and by marker wascalculated as percentage of heterogeneous loci per accession across allaccessions and loci, respectively.