Diversity at the molecular level was studied at the Molecular Biology Lab., Plant Genetic Resources Centre of Bangladesh Agricultural Research Institute, Gazipur using SSR markers. A total of 96 germplasm of muskmelon collected from different sources were selected for the present study (Annexure 01). Young, fresh, disease and insect-free leaves were used for DNA extraction. The genomic DNA was isolated from a bulk of 3-week old seedling leaf tissues taken from 5 plants from each genotype using SDS (Sodium dodecyl sulfate) and phenol: chloroform: IAA followed by alcohol precipitation described by Saghai-Maroof et al. (1984) with some modifications. Excluding usage of 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. DNA sample of each muskmelon germplasm dissolving in 50 μl of TE buffer within 1.5 ml eppendorf tube. When the DNA pellet was totally dissolved in TE buffer, 4μl RNase (10mg/ml) was added to isolated DNA and incubated at 37°C for 1.5 hours. Finally, DNA sample was stored 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). All of the DNA samples were found to be of good quality in this study. The amount of genomic DNA was quantified using UV a spectrophotometer (Spectronic GENESYS 10 Bio) at 260 nm. Using the absorbance reading obtained for DNA sample of each muskmelon germplasm, the original DNA concentrations were determined. nNine SSR primer pairs described previously in the literature (Katzir et al. 1996, Poleg et al. 2001and Henane et al. 2015) were used for microsatellite analysis in the present study. All 9 primers pairs were shown better responsiveness with clear and expected amplified product sizes.
Amplification reactions were performed in 10-μL volumes containing 5X Green GoTaq® Reaction Buffer (Promega, USA) 15 mM MgCl2, 1.25 U Taq DNA polymerase (Thermo Scientific, USA), 0.4 mM each of the dNTPs (NEB, USA), 10 μM forward, and reverse primers and 50 ng template DNA. The mixtures were prepared at 0°C and transferred to the thermal cycler. Amplification reactions of SSR loci were carried out in a Mastercycler nexus Gradient thermal cycler (Eppendorf, Germany), using a program consisting of an initial denaturation step of 3 min at 94°C followed by 35 cycles of 45 sec at 94°C, 1 min at 48-53°C and 1 min at 72°C; the program ended with an 8 min elongation step at 72°C. PCR products were stored at 4°C prior to analysis. PCR-products were electrophoresed on a 5% denaturing polyacrylamide gel containing 19:1 acrylamide: bis-acrylamide, 10X TBE buffer, 10% APS and ultrapureTemed. Electrophoresis was done using the Triple Wide Mini-Vertical Electrophoresis System, MGV-202-33 (CBS Scientific, USA). Run the gel at 80-90V and 20ºC temperature maintained by a cooling system (Julabo, Germany) upon loading of 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 gel was stained with Ethidium bromide and the individual bands were scored for analysis. SSR markers were scored as codominant, so homozygous and heterozygous genotypes could be distinguished in individual plants. 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. A single genotypic data matrix was constructed for all loci. Statistics of genetic variation (number of observed and effective alleles, Nei’s gene diversity, Shannon’s information index, heterozygosity, and polymorphic) were calculated using allelic frequency estimates obtained from genotypic frequencies of SSR loci using the computer program POPGENE (Version 1.31) (Yeh et al., 1999). In addition, the Chi-square test (1:2:1) for Hardy–Weinberg equilibrium for each population was obtained for SSR alleles using this program. The microsatellite data matrix was used to calculate Nei’s distance (Nei 1972), and to generate the corresponding matrix of genetic distance estimates among accessions and cluster analyses were performed on the genetic distance matrix by using UPGMA method to determine the relationships among accessions (dendrograms) using POPGENE (Version 1.31) (Yeh et al., 1999). The polymorphism information content (PIC) of the SSR used or gene diversity value was calculated as PIC= 1- Σf2ij; where fij is the frequency of the ith allele for the jth SSR locus (Anderson et al. 1993). PIC values provided an estimate of the discriminatory power of any locus by considering the number of alleles per locus and the relative frequencies of those alleles in the population. The software DNA FRAG version 3.03 was used to estimate allelic length (Nash, 1991).