*4.3. Selective Pressure Analysis on UGlcAE Proteins in the Tomato*

The ratio of non-synonymous to synonymous substitutions (dN/dS; termed ω) at each codon site of each protein was identified, according to an empirical Bayesian method using the Server for the identification of site-specific positive selection and purifying selection (SELECTON version 2.4, Tel Aviv University, Tel Aviv, Israel [44,45]. Selection pressure analysis can be used to identify purifying or positive selection of specific areas in a sequence, and the sites that ω values significantly >1 or <1 suggest positive (Darwinian) or purifying, respectively [46]. The selection pressure acting on the coding sequences of the *SlUGlcAE* genes was recognized with the M8 model (extra category ωs ≥ 1, beta distribution, and positive selection allowed). In order to ensure the accuracy of the results, a likelihood ratio test was used to test the significance of the ω values [47], which compares two nested models: a null model that assumes no positive selection (M8a) and an alternative model that assumes positive selection (M8). Non-nested models, including M8a (extra category ωs set to 1) and MEC (positive selection allowing model), were also used in the pressure analysis.
