# Phenotypic, Genetic, and Metabolite Variability among Genotypes of _Vicia sativa_ L.
## 2 Materials and Methods
### DNA Isolation and Markers Analysis  
The same plant material was used for the study of genetic diversity. Twenty seeds per genotype were worn to pots in a controlled growth chamber with a photoperiod of 14/10 h light/dark. The temperature was between 20 and 27 \({}^{\circ}\)C, with a mean temperature of 23 \({}^{\circ}\)C. One gram of fresh young leaves of the above plant material (ten individuals per population) was ground with liquid nitrogen and stored in \(-\)20 \({}^{\circ}\)C. The procedure described by [PERSON] (2018) was used for the isolation of total genomic DNA. The amount of DNA was quantified using a UV-Vis Spectrophotometer (Q 5000, Quawell, Sunnyvale, CA, USA) and then the samples were diluted to a 20 ng/\(\upmu\)L working concentration.  
The PCR for the SCoT analysis was performed in a total volume of 20 \(\upmu\)L containing 20 ng total genomic DNA, 1\(\times\) PCR buffer, 1 \(\upmu\)M of primer, 0.25 mM of DNTPs, and 1 U kapa Taq polymerase. A total number of 11 SCoT primers (Table 2) were selected due to their high polymorphic content and used for PCR amplification. Then, those SCoT markers were screened by using agarose gel electrophoresis in order to study intra- and inter-population diversity for _V. sativa_, and PCR amplifications were performed in a SureCyclex 8800 (Agilent Technologies, Santa Clara, CA, USA) as follows: initial denaturation of 5 min at 94 \({}^{\circ}\)C followed by 40 cycles of 30 s at 94 \({}^{\circ}\)C, annealing at 50 \({}^{\circ}\)C for 90 s, and extension at 72 \({}^{\circ}\)C for 90 s; a 5 min step at 72 \({}^{\circ}\)C was programmed as a final extension.  
Amplification products were separated by electrophoresis on 1.5% agarose gel and stained with ethidium bromide. Gel images were obtained in a UVITC Transilluminator and a 1 Kb DNA ladder (Invitrogen, Carlsbad, CA, USA) was used as a molecular weight marker.  
\begin{table}
\begin{tabular}{l l l l} \hline \hline
**A/A** & **Phenotypic Traits** & **Rating Scale** & **Type of Inheritance 1** \\ \hline
10 & Ground color of tests (GCT) & 1: whitish; 2: greyish green; 3: greyish brown; 4: brown & PQ \\ \hline
11 & Brown ornamentation (BO) & 1: absent; 2: speckles; 3: blotches; 4: speckles and blotches & PQ \\ \hline
12 & Area of brown ornamentation (ABO) & 1: very small; 3: small; 5: medium; 7: large, 9: very large & QN \\ \hline
13 & Blue-black ornamentation (BBO) & 1: absent, 2: spots, 3: blotches, 4: spots and blotches & PQ \\ \hline
14 & Area of blue-black & 1: very small; 3: small; 5: medium; 7: large, 9: very large & QN \\ \hline
15 & Color of cotyledons (CC) & 1: greyish brown; 2: orange & QL \\ \hline \hline \end{tabular} \({}^{\dagger}\)QL: qualitative characteristic; QN: quantitative characteristic; PQ: pseudo-qualitative characteristic.  
\end{table}
Table 1: _Cont._  
\begin{table}
\begin{tabular}{l l l} \hline \hline
**SCoT Primer** & **Sequence of Nucleotides (5\({}^{\prime}\)-3\({}^{\prime}\))** & **Annealing Temperature (\({}^{\circ}\)C)** \\ \hline SCoT1 & CAACAATGGCCTCCACCA & 50 \\ SCoT61 & CAACAATGGCCTACCACCG & 50 \\ SCoT30 & CCATGGTACCACCGGCG & 50 \\ SCoT13 & ACGACATGGCCGACCACCATG & 50 \\ SCoT15 & ACGACATGGCCGACCCGGA & 50 \\ SCoT66 & ACATGGTACCAGG & 50 \\ SCoT33 & CCATGGCCTACCGCGAG & 50 \\ SCoT34 & ACACATGGTACCACCGCG & 50 \\ SCoT51 & ACAATGGTACCACTGTTC & 50 \\ SCoT70 & ACCATGCTACCAGCCG & 50 \\ SCoT66 & ACCATGGTACCAGCGAG & 50 \\ \hline \hline \end{tabular}
\end{table}
Table 2: SCoT markers used in the study of six _V. sativa_ genotypes.  
Binary data points denote the presence/absence of each distinguishable band across all samples for the same primer, in both replicate sets of amplifications.