The study was carried out at Molecular Biology Lab., Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur, Bangladesh. One hundred and fifteen collected germplasm were planted for morphological characterization in the year 2014-15. Young leaves sample has been collected from 90-95 days mature plants based on their morphological traits. As dissimilar or different plant characters were observed in the same collected accession. For this reason, a collection of leaf sample was made from the morphologically homozygous plant. Bulked DNA was isolated from 4-5 fresh leaves using following the protocol described by Saghai-Maroof et al. (1984) and also used by Rahman et al. (2007) with some modifications. Excluding usage of CTAB and liquid nitrogen, the modified protocol included digestion with homogenization buffer (Solution: Tris-50 mM, EDTA-25 mM, NaCl-300 mM, 1% SDS and deionized water) at 65ºC for 30 min, extraction with phenol: chloroform: isoamyl alcohol (25:24:1), precipitation with ice-cold and extra pure isopropyl alcohol and purification with absolute ethanol (Plus sodium acetate, 3M) and 70% ethanol chronologically. Finally, DNA sample of each chilli germplasm dissolving in 30-40 μl of TE buffer within 1.5 ml eppendorf tube was preserved separately at -20ºC. The presence of genomic DNA was confirmed on 1% agarose gel qualitatively. The gels were visualized under UV light and photographed using a photo documentation system (UV Transilluminator, Uvitec, UK). The Polymerase chain reactions was set up 10 μl volumes containing 75 ng template DNA, 0.1 U/μl Taq DNA polymerase, 0.4 mM each of the dNTPs, 1.25 μM of each of primer, 4 mM MgSO4, 20 mM KCl, 16 mM (NH4)2SO4 , 20 mM Tris-HCl, pH8.8 and a suitable amount of sterile deionized water. The reaction was performed in an oil-free eppendorf Mastercycler® nexus Gradient thermal cycler. SSRs were amplified under the following “touchdown” PCR conditions: 94oC/3 min denaturation, 11 cycles of 94oC/0.5 min, 58-60oC/1 min, decreasing by 1oC per cycle, and 72oC/1 min; 30 cycles of 94oC/0.5 min, 52-55-oC/1 min and 72oC/1 min; a final extension for 5 min. The success of the reaction product following 2% agarose gel electrophoresis was determined. PCR-products were electrophoresed on a 6% denaturing polyacrylamide gel containing 19:1 acrylamide: bis-acrylamide and 8M urea. Electrophoresis was done using the SequiGen GT Sequencing Cell (BIO-RAD Laboratories, Hercules, CA, USA) electrophoresis system. A pre-run of the gel for 30 minutes at 120W was followed by a final run at 60W and 50ºC upon loading of denatured PCR products for a specified period of time depending on the size of amplified DNA fragment (usually 1 hour for 100 bp). After completion of electrophoresis, the DNA fragments were visualized following the Promega (Madison, WI) silver-staining protocol. The bands representing particular alleles at the microsatellite loci were scored manually and designated the bands as A, B, C, etc. from the top to the bottom of the gel. The genotypes of different individuals were hypothetically scored as AA, BB, CC, etc. for homozygous or as AB, AC, BC etc. for heterozygous. The software DNA FRAG version 3.03 was used to estimate allelic length (Nash, 1991).
The amount of genomic DNA was quantified at 260nm spectrophotometrically (Spectronic GENESYS™ 10 Bio). Using the absorbance reading obtained for DNA sample of each chilli germplasm, the original DNA concentrations were determined. About fifty microsatellite primer pairs were identified and those are located at 12 chromosomes from a different publication. Preliminarily, twenty primer pairs of the different chromosome were tested for their better responsiveness in amplifying the target genomic region of template DNA and to check the expected PCR product sizes in base pairs. All twenty primer pairs with clear and expected amplified product sizes were selected and one pair were used for microsatellite analysis in the present study. The rest of the 30 primer pairs will be tested for their better responsiveness in amplifying the target genomic region of template DNA and will check the expected PCR product sizes in base pairs.