In the South Khorasan province of Iran, the seedless barberry variety ‘Zereshk Bidaneh’ is cultivated extensively and is noted for its exceptional resilience to adverse climatic conditions. As part of a breeding program to develop new barberry cultivars, 90 F1 hybrid progenies were produced from crosses between ‘Zereshk Bidaneh’ and selected wild genotypes. This study investigates the biochemical and phenological diversity within this F1 population. Phenological assessments revealed a broad range in flowering time among genotypes, with statistically significant differences (p < 0.05). The earliest flowering was observed on April 15 in genotype ‘0506’, while the latest occurred on May 5 in ‘0202’, resulting in a 40-day interval. Biochemical evaluations indicated substantial and significant variability (p < 0.05) in total anthocyanin content (TAC), with values spanning from 133.37 mg/L (‘0203’) to 2063.55 mg/L (‘0601’). The average TAC was 614.44 mg/L, and the high coefficient of variation (67%) emphasized the extent of diversity. Notably, four genotypes (‘0601’, ‘0606’, ‘0607’, and ‘0617’) exhibited TAC levels above 1250 mg/L. Vitamin C concentrations also showed significant differences (p < 0.05), averaging 108.50 mg/100 mL, with several genotypes exceeding 130 mg/100 mL. Total antioxidant activity (TAA) ranged markedly from 7.89 to 90%, while total phenolic content (TPC) had a mean of 1770 mg GAE/100 mL (p < 0.05), underscoring the presence of bioactive compoundrich genotypes. Total soluble solids (TSS) varied between 14 and 38 °Brix, with a mean of 29.27 °Brix (p < 0.05). Measured pH values ranged from 2.66 to 3.76. The fruit flavor index (FI) also varied widely, ranging from 0.98 to 7.68 (p < 0.05). Principal component analysis (PCA) revealed that the first two principal components accounted for 50.04% of the total variance, with TAC, TAA, titratable acidity (TA), and FI identified as the most influential variables. Heat map analysis further classified the genotypes into seven distinct biochemical clusters, reinforcing the diversity patterns observed. The results reveal extensive phenotypic and biochemical diversity among the F1 barberry progenies, with several traits showing statistically significant variation (p < 0.05). These findings provide a valuable foundation for selecting superior genotypes and advancing breeding programs aimed at developing improved barberry cultivars with enhanced nutritional and horticultural qualities. Among the evaluated progenies, genotypes such as ‘0601’ and ‘0203’ stood out due to their exceptionally high total anthocyanin content (2063.55 mg/L) and antioxidant activity (90%), respectively, highlighting their potential value in future breeding strategies.
