**2.6. Alignment and phylogeny**

Mega7.0.14 software was used for alignment (Clustal W algorithm with Blossom 62 matrix) and reconstruction of the phylogenetic pattern (Neighbor Joining model with JTT matrixbased model for distance computing and 1000 replicates as bootstrap test).


**Table 1.** Primers used in semi-quantitative PCR.

#### **2.7. Semi-quantitative RT-PCR**

The RNA from different seed stages (1, 4 and 8 months of development) digested with DNAse I amplification grade (Invitrogen) was used as a template in sqPCR reactions, and the synthesis of cDNAs was carried out with the First Strand cDNA Synthesis Kit (Thermo Scientific). In Veriti 96-well Thermal cycler (Applied Biosystems), in reactions of 10 μl (100 ng/μl cDNA,10X Reaction Buffer, 2 mM dNTPs, 50 mM MgCl<sup>2</sup> , 1 U Taq DNA polymerase), sqPCR primers (**Table 1**) were designed by Primer3 webtool (http://bioinfo. ut.ee/primer3-0.4.0/); the primers for bHLH transcription factor, three metallothioneins, snakin and ubiquitin as reference gene were selected. The amplification procedures were 95°C - 10min., 30 cycles (95°C - 45 s, annealing 30 s, amplification 72°C - 45 s), 72°C - 7 min. Gene expression ratio between selected gene and endogenous control was calculated using band intensity measured with GelAnalyzer 2010. The semi-quantitative PCR was performed with three repeats.
