*2.4. Data Analysis*

The sequences were analyzed and compared with sequences of CS (*Wx-A*1: AB019622, *Wx-B1*: AB019623, and *Wx-D1*: AB019624), two-rowed barley cv. Vogelsanger Gold (*Wx-H*1: X07931) and six-rowed barley cv. Morex (*Wx-H1*: AF474373) available in the databases using Geneious Pro version 5.0.4 software (Biomatters Ltd., Auckland, New Zealand). The synonymous substitution rate (*Ks*) and non-synonymous substitution rate (*Ka*), as well as the *Ka*/*Ks* ratios, were computed using DNAsp ver. 5.0 [35]. Divergence times were calculated by the mean divergence time, 2.7 million years ago (MYA) between the A and D genomes estimated by Dvorak and Akhunov [36]. Predicted proteins, as well as secondary structure predictions, were also obtained with this software, using the EMBOSS tool Garnier [37]. Amino acid substitutions between the predicted proteins obtained from the new alleles and reference proteins were analyzed using the PROVEAN (Protein Variation Effect Analyzer) software tool to predict whether these amino acid substitutions or InDels have an impact on their biological function [38,39].

A phylogenetic tree was constructed with the software MEGA6 [40] using the complete coding regions of the two sequences obtained, together with the following sequences of the *Wx* genes: common wheat CS (*Wx*-*A*1: AB019622, *Wx*-*B*1: AB019623, and *Wx*-*D*1: AB019624), two-rowed barley "Vogelsanger Gold" (*Wx*-*H*1: X07931) and six-rowed barley 'Morex' (*Wx-H*1: AF474373). A neighbor-joining cluster of all of the analyzed sequences was generated using the Poisson correction method for amino acid sequences [41] with one bootstrap consensus from 1,000 replicates [42].
