# Genetic Variation of a Lentil (_Lens culinaris_) Landrace during Three Generations of Breeding
## 2 Materials and Methods
### PCR Amplification and HRM Analysis  
Fifteen Simple Sequence Repeats (SSR) primer sets designed by [14] were used for genetic differentiation analysis of 40 lentil samples. The PCR amplification and HRM analysis on a Light Cyler 96 System (Roche, Mannheim, Germany) for real-time PCR and HRM analysis were performed in a total volume of 11 \(\upmu\)L, containing 20 ng genomic DNA, 1\(\times\)KAPA HRM FAST master mix, 2.5 mM MgCl\({}_{2}\), 0.2 \(\upmu\)M forward and reverse primer. The PCR protocol included the following steps: preincubation of 95 \({}^{\circ}\)C for 180 s, followed by 45 cycles of 95 \({}^{\circ}\)C for 5 s (denaturation), 51-58 \({}^{\circ}\)C (depending on the primer pair) for 20 s (annealing) and 72 \({}^{\circ}\)C for 45 s (extension). The optimal annealing temperature for each primer pair (Table 2) was defined testing them in gradient Real-time PCR with a specific range of temperatures (data not shown). The PCR was followed by HRM analysis using SYBR Green I dye with the following steps: 95 \({}^{\circ}\)C for 60 s, 40 \({}^{\circ}\)C for 60 s, and then 65 \({}^{\circ}\)C to 97 \({}^{\circ}\)C allowing 25 data acquisitions per 1 \({}^{\circ}\)C. PCR products were visualized in 2% agarose gel electrophoresis in 1\(\times\) TBE buffer using 1 kb ladder as a size marker.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{2}{c}{**1 GSLs**} & \multicolumn{2}{c}{**2 GSLs**} & \multicolumn{2}{c}{**3 GSLs**} \\ \hline
**Sample Code** & **Line Code** & **Sample Code** & **Line Code** & **Sample Code** & **Line Code** \\ \hline
1 & 1 & 15 & 20-13 & 30 & 8-2-2 \\
2 & 2 & 16 & 2-4 & 31 & 8-11-7 \\
3 & 3 & 17 & 2-10 & 32 & 15-5-10 \\
4 & 4 & 18 & 18-15 & 33 & 2-4-6 \\
5 & 5 & 19 & 15-5 & 34 & 8-2-4 \\
6 & 6 & 20 & 1-14 & 35 & 8-2-3 \\
7 & 7 & 21 & 8-1 & 36 & 8-1-9 \\
8 & 8 & 22 & 8-3 & 37 & 15-5-11 \\
9 & 9 & 23 & 2-9 & 38 & 8-3-8 \\
10 & 10 & 24 & 15-6 & 39 & 8-2-1 \\
11 & 11 & 25 & 8-2 & 40 & 15-7-5 \\
12 & 12 & 26 & 15-7 & & \\
13 & 13 & 27 & 8-11 & & \\
14 & 15 & 28 & 2-8 & & \\  & & 29 & 15-12 & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: The genealogy of 40 lentil lines examined in the present study, obtained through single-plant selection for three successive generations within a landrace. Numerical code indicates the line’s origin, i.e., the 3 GSL coded 8-2-2 was originated from a single plant of the 2 GSL coded 8-2, stemmed from a single plant of the 1 GSL coded 8, ancestor of which was a single plant of the initial landrace.  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Primer** & **Tm (\({}^{\circ}\)C)** \\ \hline
**SSR19** & 58 \\
**SSR33** & 58 \\
**SSR59-2** & 58 \\
**SSR80** & 58 \\
**SSR107** & 54 \\
**SSR113** & 54 \\
**SSR154** & 57 \\
**SSR156** & 57 \\
**SSR167** & 56 \\
**SSR184** & 56 \\
**SSR199** & 58 \\
**SSR204** & 56 \\
**SSR302** & 56 \\
**SSR317-2** & 56 \\
**SSR323** & 58 \\ \hline \hline \end{tabular}
\end{table}
Table 2: The annealing temperatures (\({}^{\circ}\)C) of the SSR primer sets used in the research; further information on primer’s sequence, band size and inheritance are given in [14].