# Genetic Variation of a Lentil (_Lens culinaris_) Landrace during Three Generations of Breeding
## 3 Results  
Data were analyzed with the Roche Light Cycler 96 software with the appropriate adjustments. The pre- and post- melt range of melting curves were selected for optimum discrimination considering the pedigree of the lines, with settings for normalization to proportional and sensitivity for Delta Tm and curve shape discrimination adjusted to 50%. Grouping of the samples was automatic depending on normalized melting curves (Figure 1a) and normalized melting peaks (Figure 1b), while the presence of a group was also examined comparing the banding patterns on the agarose gel. In several SSRs it was possible to confirm the presence of different alleles from the gel band patterns (Supplementary Figure S1). Each group was depicted with a different color. A numeric matrix was created assuming that each group represented a different allele of the respective SSR locus. A total number of 48 alleles were detected for the 15 SSR primer sets in the 14 ICSLs. Nine alleles were lost in the second cycle of selection (18.75%), and from the remaining 39 alleles, five were lost (12.82%) in the third cycle. Fourteen alleles were lost in total representing 29.1% of the allelic richness of the 1 st GSLs. Two to five alleles per locus were detected (Figure 2a). Analysis of allelic patterns across breeding generations indicated a decrease of mean numbers of alleles (Na), alleles with a frequency higher than 5% (Na Freq. \(\geq\) 5%), and the number of private alleles (No. Pr. Al.) that was zero at the 3 GSLs, while effective alleles (Ne), information index (I), and haploid diversity increased from 1 GSL to 2 GSL and then declined from 2 GSL to 3 GSL (Figure 2b). Figure 1: Graphs of (**a**) Normalized melting peaks and (**b**) normalized melting curves generated by analysis of SSR156 with Roche Light Cyber 96 software from data of the 40 lentil lines. Lines of family 8 are in red, lines of family 5 in orange, and all the rest in blue. Figure 2: Cont. Genetic diversity parameters for the 15 SSRs were calculated to assess their overall informativeness (Table 3) finding that the number of different alleles (Na) varied from two to five, the number of effective alleles (Ne) from 1.225 to 3.236, information index (I) between 0.381 and 1.272, PIC ranged from 0.664 to 0.925, diversity (h) from 0.184 to 0.691 and unbiased diversity (uh) from 0.188 to 0.710. In general, all markers were polymorphic and had high discrimination capacity. The most informative was SSR59 with 5 alleles and the highest values for all indices while less informative was SSR154 with the lowest values. Genetic diversity parameters at population level indicated that the number of different alleles (Na) varied from 2.13 to 2.87, the number of effective alleles (Ne) varied from 1.65 to \begin{table}
\begin{tabular}{c c c c c c c} \hline
**SSR** & **Na** & **Ne** & **I** & **PIC** & **h** & **uh** \\ \hline
**SSR19** & 5 & 2.548 & 1.118 & 0.906 & 0.608 & 0.623 \\
**SSR33** & 4 & 1.656 & 0.722 & 0.848 & 0.396 & 0.406 \\
**SSR59** & 5 & 3.236 & 1.272 & 0.925 & 0.691 & 0.710 \\
**SSR80** & 2 & 1.690 & 0.598 & 0.677 & 0.408 & 0.420 \\
**SSR107** & 4 & 2.930 & 1.179 & 0.889 & 0.659 & 0.676 \\
**SSR113** & 4 & 2.164 & 0.924 & 0.877 & 0.538 & 0.552 \\
**SSR154** & 3 & 1.225 & 0.381 & 0.723 & 0.184 & 0.188 \\
**SSR156** & 3 & 1.831 & 0.719 & 0.790 & 0.454 & 0.465 \\
**SSR167** & 3 & 1.831 & 0.719 & 0.790 & 0.454 & 0.465 \\
**SSR184** & 2 & 1.724 & 0.611 & 0.664 & 0.420 & 0.431 \\
**SSR199** & 2 & 1.782 & 0.631 & 0.683 & 0.439 & 0.450 \\
**SSR204** & 2 & 1.724 & 0.611 & 0.664 & 0.420 & 0.431 \\
**SSR302** & 3 & 2.897 & 1.081 & 0.859 & 0.655 & 0.672 \\
**SSR317** & 3 & 1.225 & 0.381 & 0.727 & 0.184 & 0.188 \\
**SSR323** & 3 & 1.831 & 0.719 & 0.790 & 0.454 & 0.465 \\ \hline
**Average** & 3.2 & 2.2 & 0.778 & 0.787 & 0.454 & 0.465 \\ \hline
**SE** & 0.262 & 0.158 & 0.071 & 0.024 & 0.039 & 0.040 \\ \hline \end{tabular}
\end{table}