*4.8. Statistics*

A three-factor analysis of variance (ANOVA) with factors cultivar, location, and time was used for the flesh samples. All the factors had two levels: Cultivar ('Majda' and 'Golden Delicious'), location (L1 and L2), and time (harvest and storage). For peel and leaves, the two-factor ANOVA was used (factors cultivar and location). For the determination of sugars, organic acids, and phenolic compounds, the samples were prepared in five replicates. For leaves, four replicates were used. For the enzyme activity, three replicates

were performed per treatment (each with three technical repetitions). Some variables were log-transformed before the statistical analysis to meet the assumption of constant variance between treatments. When the ANOVA showed a statistical significance, a contrast analysis was performed with user-defined contrasts. When the interactions between the factors did not show a statistically significant difference, Tukey's HSD test was used for comparison. The contrasts with statistical significances, heatmap of individual sugars, as well as the means with SE of individual phenolics are presented in supplementary materials (Tables S1–S9 and Figures S1–S3). If the *p*-value for differences between the means was less than 0.05, it was considered statistically significant.

#### **5. Conclusions**

In conclusion, cv. 'Majda' is one of the rare cultivars from traditional cross-breeding with a non-oxidation trait. We have screened some of the main actors of oxidation in apple flesh glutathione, vitamin C, phenolic content, and PPO activity. Based on the results, we conclude that the low phenolic content and high reduced glutathione content are the major reasons for the cv. 'Majda' lack of oxidation. We have upgraded the knowledge on cv. 'Majda' by also analyzing glutathione, and this feature will have to be further researched. Its high acidity is feasibly a consequence of heredity, rather than the main reason for nonoxidation, but one must not disregard that the high acidity with a low pH can contribute to a slower oxidation by reducing PPO efficiency. This will be further researched by the gene expression analysis. This work also touches upon another topic: Fruit processing. Today, the consumers seek products for a quick consumption, such as prepared snacks, but with as little as possible additives to preserve them. Cultivar 'Majda' is suitable for this role and has a great potential for use as a minimally processed product, as well as a regular apple product with no additives for the prevention of oxidation. This will also be addressed in our future research. Furthermore, cultivar 'Majda' is not an overly sensitive cultivar and is thereby undemanding for fruit growers.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/plants10071402/s1. Figure S1: Heatmap for standardized variables: Individual sugars: Sucrose, Suc; glucose, Glu; fructose, Fru; sorbitol, Sorb; organic acids: Citric acid, Cit\_ac; malic acid, Mal\_ac; and pH for cultivars 'Golden Delicious' (GD) and 'Majda' (MA) at two different locations (L1 and L2) at harvest and following storage, dendrogram based on Ward's clustering squared Euclidian distance. Figure S2: Heatmap for standardized variables: Vitamin C, C vit; methionine, Met; cysteine, Cys; reduced glutathione, GSH; oxidised glutathione, GSSG; polyphenol oxidase, PPO; peroxidase, POX for cultivars 'Golden Delicious' (GD) and 'Majda' (MA) at two different locations (L1 and L2) at harvest and following storage, dendrogram based on Ward's clustering squared Euclidian distance. Figure S3: Heatmap for standardized variables: Hydroxycinnamic acids: Cryptochlorogenic acid, X4-CQA; chlorogenic acid, X3-CQA; neochlorogenic acid, X5- CQA; and p-coumaric acid, p\_coum\_ac; dihydrochalcones: Phloridzin, Phl; phloretin 2'-O- xylosylglucoside, Phl\_xy\_phl; 3-hydroxyphloridzin, X3\_Hy\_phl; and 3-hydroxyphloretin, X3\_Hyd\_phl; flavonols: Quercetin-3-rhamnoside, Q-Rha; quercetin-3-rutinoside, Q-Rut; and quercetin-3-glycoside + quercetin-3-galactoside, Q-Glc\_Gal; flavan-3-ols: Catechin, Cat; epicatechin, Epicat; procyanidin B1, P\_B1; procyanidin B2 + B4, P\_B2\_B4 for cultivars 'Golden Delicious' (GD) and 'Majda' (MA) at two different locations (L1 and L2) at harvest and following storage, dendrogram based on Ward's clustering squared Euclidian distance. Figure S4: Daily precipitations, daily minimum and maximum air temperature for the locations L1 and L2. Table S1: The contrast analysis for total sugars with estimated differences between averages and corresponding 95% confidence intervals (lwr, lower boundary; upr, upper boundary), for time (harvest, storage), location (L1 and L2), and cultivar (GD, 'Golden Delicious'; MA, 'Majda'). Table S2: The contrast analysis for total organic acids with estimated differences between averages and corresponding 95% confidence intervals (lwr, lower boundary; upr, upper boundary), for time (harvest, storage), location (L1 and L2), and cultivar (GD, 'Golden Delicious'; MA, 'Majda'). Table S3: The contrast analysis for pH with estimated differences between averages and corresponding 95% confidence intervals (lwr, lower boundary; upr, upper boundary), for time (harvest, storage), location (L1 and L2), and cultivar (GD, 'Golden Delicious'; MA, 'Majda'). Table S4: The contrast analysis for change in color (∆E ∆t −1 ) with estimated ratios of averages and cor-

responding 95% confidence intervals (lwr, lower boundary; upr, upper boundary), for time (harvest, storage), location (L1 and L2), and cultivar (GD, 'Golden Delicious'; MA, 'Majda'). Table S5: The contrast analysis for vitamin C, methionine, cysteine, and glutathione (GSH and GSSG) with estimated ratios of averages and corresponding 95% confidence intervals (lwr, lower boundary; upr, upper boundary), for time (harvest, storage), location (L1 and L2), and cultivar (GD, 'Golden Delicious'; MA, 'Majda'). Table S6: Individual phenols (mean <sup>±</sup> SE) in flesh (mg kg−<sup>1</sup> FW), peels (mg kg−<sup>1</sup> FW), and leaves (mg kg−<sup>1</sup> DW). Hydroxycinnamic acids: Cryptochlorogenic acid, 4-CQA; chlorogenic acid, 3-CQA; neochlorogenic acid, 5-CQA; and p-coumaric acid, p\_coum\_ac; dihydrochalcones: Phloridzin, Phl; phloretin 2'-O- xylosyl-glucoside, Phl\_xy\_phl; 3-hydroxyphloridzin, 3\_Hy\_phl; and 3-hydroxyphloretin, 3\_Hyd\_phl; flavonols: Quercetin-3-rhamnoside, Q-Rha; quercetin-3-rutinoside, Q-Rut; and quercetin-3-glycoside + quercetin-3-galactoside, Q-Glc\_Gal; flavan-3-ols: Catechin, Cat; epicatechin, Epicat; procyanidin B1, P\_B1; and procyanidin B2 + B4, P\_B2\_B4. Table S7: The contrast analysis for phenolic groups dihydrochalcones and flavonols in apple flesh with estimated ratios of averages and corresponding 95% confidence intervals (lwr, lower boundary; upr, upper boundary), for time (harvest, storage), location (L1 and L2), and cultivar (GD, 'Golden Delicious'; MA, 'Majda'). Table S8: HSD test for phenolic groups, hydroxycinnamic acids, dihydrochalcones, flavonols and flavan-3-ols in apple flesh, peel and leaves with estimated ratios of averages and corresponding 95% confidence intervals (lwr, lower boundary; upr, upper boundary), for time (harvest, storage), location (L1 and L2), and cultivar (GD, 'Golden Delicious'; MA, 'Majda'). Table S9: The contrast analysis for PPO and POX with estimated ratios of averages and corresponding 95% confidence intervals (lwr, lower boundary; upr, upper boundary), for time (harvest, storage), location (L1 and L2), and cultivar (GD, 'Golden Delicious'; MA, 'Majda'). Table S10: Meteorological conditions during the vegetation period (1. 4. 2019–30. 9. 2021) on locations L1 and L2.

**Author Contributions:** Conceptualization, A.C.; methodology, A.C., A.V., and U.V.; software, D.K.; validation, U.V. and A.V., formal analysis, A.C., A.V., and U.V.; investigation, A.C. and J.H.; resources, A.C. and U.V.; data curation, A.C. and D.K.; writing—original draft preparation, A.C.; writing—review and editing, U.V.; visualization, D.K.; supervision, U.V.; project administration, A.C.; funding acquisition, A.C. and U.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Slovenian Research Agency (grants P4-0133 Sustainable agriculture) and the Republic of Slovenia (Ministry of Education, Science and Sport) and the European Regional Development Fund (project "Raziskovalci-2.1-KIS-952049", A.C.). ˇ

**Data Availability Statement:** No additional data is available.

**Acknowledgments:** The authorsare grateful to Veronika Kmecl for the help with the vitamin C analysis, Martina Perši´c for the valuable comments on the manuscript, and the FEM staff which were involved in our research.

**Conflicts of Interest:** The authors declare no conflict of interest.
