Genetic Variability of Nicotiana tabacum (Linn.) Using SSR Marker

In present study a total ten varieties of tobacco seeds Gujarat Tobacco-9 (GT-9), Mosaic Resistance Gujarat Tobacco Hybrid – 1 (MRGTH-1), Gujarat Tobacco-7 (GT-7), Gujarat Tobacco-4 (GT-4), Anand-2 (A-2), Anand BT-10 (ABT-10), Anand BD-101 (ABD-101), Anand BD-118 (ABD-118) and Anand-119 (A-119) were procured and grown on simple agar medium. The plants were harvested for further analysis after 5 to 7 days incubation in dark condition. SSR primers tested in present investigation produced fragments of different length. The minimum (150bp) sized fragment and the maximum 600 bp sized amplified fragment was obtained by primer SSR4 & SSR8 respectively. The highest (100%) polymorphism was exhibited by primer SSR1, SSR2, SSR3, SSR5, SSR6, SSR7, SSR9, and SSR10, while the lowest polymorphism (82.30%) was observed with primer SSR4. The maximum scorable bands (34) were generated by primer SSR5, SSR9, & SSR10, whereas the minimum scorable bands (5) generated by primer SSR2. In the SSR analysis, 10 primers were used for polymorphism screening. A total of 18 bands in the size range of 200 to 600 bp were produced by examining across genotypes with 10 SSR primers, with an average polymorphism of 88.8 %. The overall gene frequency analysis reveals a maximum of 1 and minimum of 0.2000. Observed number of alleles (1.3333 ± 0.4830), Effective number of alleles (1.2504 ± 0.3906), Gene diversity (0.1390 ± 0.2076) and Shannon's Information index (0.20160 ± 0.2970) was found in the Genic variation statistics for all loci. The total number of polymorphic loci was found 7 and the percentage of polymorphic loci was 33.33 and the PIC value was found in between 0.980 – 0.466. Phylogenetic trees among 10 tobacco varieties were constructed and 65% genetic similarity was found in the cluster diagram with 2 major groups. This first sub group was divided in two groups where in one group, GT-5 100% was similar with A-2. In second group, MRGTH-1 & GT-9 was 100% similar with ABD-101. Second major group was divided into two sub groups where ABD-118 was similar with A-119 & GT-4. This two tobacco varieties A-119 & GT-4 have 100% similarity. First major group was divided into four subgroups and the second major group again divided into two subgroups at 76.5% genetic similarity which contained ABD-118 and A-119 & GT-4 with 85% genetic similarity. Now, in the first major group the first subgroup was divided at 72% genetic similarity into two another subgroup first subgroups had GT-5 and A-2. The second subgroup was divided into two as MRGTH-1, ABD-101 & ABT-10 and another subgroup had GT-7. GT-9 showed maximum genetic similarity (1.0000) and A-119 showed minimum genetic similarity (0.6667). ABD-118 and GT-4 varieties showed maximum genetic distance of 0.2719 while GT-9 showed no genetic distance (0.0000). Based on study, the large range of similarity values for related cultivars using microsatellites provided greater confidence for the assessment of genetic diversity and relationships. This information (genetic variability at molecular level) will certainly be helpful to identify and develop genetically unique germplasm that compliments the existing cultivars. Further practical approaches are required to unveil the differences among these closely related species of the tobacco varieties studied in the present investigation.