Plant materials Twelve elite rice genotypes of ARLs and fine rice landraces were selected for the experiment: Gondhocosturi, Gondhoraj, Hatisail, Begunbichi, Khajar, Rajasail, Basmati tap 90, Radhunipagol, Nizersail, Lalrodha dhan, Kartiksail, Tilockachari. Seeds of rice genotypes were collected from the Genetic Resource Center (GRC), BRRI for germination after dormancy breaking at 50°C for 3 days. Germination of seeds was carried out on petri dish (15 seeds of single genotype per Petri dish) with filter paper and distilled water at 28°C in dark condition. For seed germination, a minute amount of distilled water was used only to soak the filter paper under aseptic conditions and then germinated seeds were sown at specified row and column distances in pots in a glass-house. Genomic DNA isolation Prior to the isolation of genomic DNA, a healthy portion of fresh youngest leaves were harvested from a single 3-week-old plant of each line grown in pots in a glasshouse. DNA extraction was carried out following a mini-prep DNA extraction protocol which did not require liquid nitrogen and required only a very small amount of tissue samples (Zheng et al. 1995). All chemicals used for DNA extraction were purchased from Sigma-Aldrich, Germany. Leaf tissues were cut into small pieces, homogenized and digested with extraction buffer (1 M Tris, 0.5M Na2EDTA, 5M NaCl, 10% SDS and distilled H2O, pH 8.0). Leaf extracts were then placed in 65º C water bath in a tube holder for 20 min and inverted at 10-min intervals and returned to the water bath each time. Following incu-bation, 800 μl of a mixture of chloroform and isoamyl alcohol (24: 1) was added, centrifuged for 8 min at 11,000 rpm in a microcen-trifuge and 500 μl of upper aqueous layer was collected. To this supernatant, 1000 μl of cold 100% ethanol was added, mixed and centrifuged for 12 min at maximum speed (13,200 rpm). A small pellet was visible and the supernatant was decanted. The DNA pellet was washed with cold 70% ethanol and allowed to air dry. The DNA pellets were resuspended in 100 μl of 10X TE buffer and dissolved by warming in a 65°C water bath for up to 1 h (with frequent mixing or flicking of the tube). The pellet, once dissolved, was stored at -20°C. The quality of DNA was also checked by DNA quantification using a Thermo Scientific NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, USA). Microsatellite markers A total of 24 microsatellite primer pairs (Sigma-Aldrich, Ger-many) covering all 12 chromosomes were selected for the genetic diversity analysis of the 12 ARLs in Bangladesh. Primers that showed monomorphic banding patterns were excluded while only one primer with higher allele number was selected from primers showing a polymorphic banding pattern at the same chromosome based on number of allele detected. Finally, 10 microsatellite pri-mers with a distinct chromosome number were used for final poly-merase chain reaction (PCR) amplification. The original source, repeat motifs, primer sequences, expected length and chromoso-mal positions for these markers can be found in the rice genome database (http://www.gramene.org). PCR amplification Prior to DNA amplification, a PCR cocktail was prepared con-taining all required components. All reagents were purchased from Sigma-Aldrich. PCR was carried out in a DNA thermal cycler (Model: ALS1296, BioRad, USA and G- STORM, GSI, England, Serial no: GT-11620) and PCR amplification reactions were done in a total volume of 10 l containing 1.5 l of 10X buffer, 0.25 l of 10 mM dNTPs, 0.5 l of each forward and reverse primer, 2.25 l of ddH2O, 0.2 l of Ta q polymerase, 1.8 l of MgCl2 and 3 l of diluted template DNA. For PCR amplification, the thermal cyc-ler was set at 1 cycle for 5 min at 94°C (hot start and strand sepa-ration) followed by 34 cycles of denaturation (94°C), annealing (55°C) and primer elongation (72°C) for 30 sec each and a final extension in 1 cycle of 5 min at 72°C. Amplified products were stored at -20°C until further use. The reproducibility of amplification products was confirmed twice for each primer. PCR products were mixed with bromophenol blue gel loading dye at a 5: 3 ratio and resolved by polyacrylamide gel electrophoresis run on 8% polyacrylamide gels in TBE buffer. To each well, 4 μl of sample was loaded and run at 80 V for 90 min using a mini vertical poly-acrylamide gel running apparatus for high throughput manual genotyping (model: 300 series, CBS Scientific Co Inc., CA. USA). The gels were next stained in ethidium bromide solution (50 l in 500 ml distilled water) for 30-35 min, kept in the dark, and then scanned using an a UVPRO (Uvipro Platinum, EU) gel documentation unit linked to a PC (Windows). SSR data analysis The size of amplicons was estimated by comparing the migration distance of amplified fragments with that of known size fragments using 25 (bp) base pairs (Promega, Madison, USA) and 50 bp DNA ladder (Thermo Fisher Scientific, Fermentas INC., Maryland, USA). The molecular weight of distinct bands or amplified fragments was measured in bp using Alpha-Ease FC 5.0 software. Genetic diversity of cultivars by SSRs was evaluated by the number of alleles and the polymorphic information content (PIC) value, which is an estimate of the discriminatory power of a SSR marker locus. Statistics, including the number of alleles per locus, major allele frequency, gene diversity, and PIC values were calculated using PowerMarker version 3.25 (Liu and Muse 2005). SSR mar-ker alleles were analyzed using Power Marker version 3.25 and used to export the allele frequency data to a binary format in which each SSR band was scored as present (1), absent (0), or as a missing observation for analysis with NTSYS-pc version 2.2 (Rohif 2002). NTSYS-pc was used to construct a UPGMA (un-weighted pair group method with arithmetic averages) dendrogram showing the distance-based interrelationship among the genotypes. For the unrooted phylogenetic tree, genetic distance was calcu-lated using the “C.S Chord 1967” distance (Cavalli-Sforza and Edwards 1967) in PowerMarker with tree viewed using Treeview software.