Strawberries are a highly sought-after fruit for their flavor, color, and nutritional benefits. In this study, strawberry fruit from 268 commercial cultivars and advanced selections from the North Carolina breeding program were evaluated to determine and characterize genotype diversity, relationships, and overall trends in fruit composition. Fifteen marketable, fully red, firm fruit were harvested per genotype between April and June of 2022 from a greenhouse core collection and held at -20°C until analysis. Juice from thawed fruit was used to collect total soluble solids (SSC), total titratable acidity (TTA), and pH data. Strawberry juice was also extracted with acidified methanol (0.1ml/1.6 ml total) and supernatants used to determine monomeric anthocyanin content (TA) using the pH differential method. Agglomerative hierarchical clustering (AHC) and multivariate analysis of variance (MANOVA) sorted genotypes into four clusters, highlighting significant differences in fruit composition profiles. The majority of commercial cultivars, and 38 advanced selections, were grouped together in Cluster 1. This cluster was distinguished by lowest SSC (7.0%), TTA (0.72%), and TA (31.22 mg/100g) values. In contrast, Cluster 2 containing 41.4% of germplasm had fruit highest in TA (54.57 mg/100g). Fruit from genotypes in Cluster 3 had high pH and %SSC, and the smallest group (Cluster 4), with 13.1% of the germplasm, had low pH and was significantly greater in %TTA. Regional strawberry breeding programs were found to group together. The Florida germplasm were grouped in Cluster 1, North Carolina commercial genotypes in Cluster 2, and two advanced New Jersey genotypic lines were found in Cluster 3. California day-neutrals were found in Cluster 1, while short-day genotypes were split between the first two clusters. Additionally, the first two principal components in PCA analysis captured 64.88% of the total variance, with both pH and TTA largely contributing to PC1 (91.1%), SSC in PC2 (71.1%), and TA associated with PC3 (77.4%). Variation in pH and %TTA had a more pronounced effect on genotype profiles and were the primary driving force for germplasm separation and differentiation. This work represents the first instance where an entire germplasm collection was assessed together in a single evaluation. The determination of genotype diversity, characterization of relationships among the genotypes and the fruit composition parameters, and visualization of trends within the collection will help guide future breeding decisions for developing new strawberry cultivars with improved quality.
