*3.2. Phylogenetic Analysis*

The complete amino acid sequences of the two novel variants from *H. chilense* obtained in this study, together with other waxy protein sequences present in the NCBI database, were used to construct a phenogram based on the Poisson correction method for amino acid sequences (Figure 4). Three main clusters were observed, representing the correlations between the *H. chilense* sequences and the common barley sequences used.

**Figure 4.** Neighbor-joining tree based on the Poisson correction method for amino acid sequences analyzed. Number above nodes indicates bootstrap estimates from 1000 replications.

These data were corroborated when the genomic nucleotide sequences of these variants were analyzed. Furthermore, the *Ks* and *Ka* substitution rates among *Wx* genes were calculated using the coding sequences of the complete genes. The comparison value between the genes from *H. chilense* and *H. vulgare* was high (*Ks* = 0.127), which suggested that the divergence time between species was ~2.3 MYA based on the mean divergence rate (0.0533 *Ks* per MY) obtained for this gene in a previous study [45].
