Understanding the genetic composition and diversity of plant collections is crucial for their effective management and utilization in breeding programs. Cultivated strawberry (Fragaria x ananassa) is a significant global crop, contributing substantially to the U.S. agricultural economy with more than $3 billion in production value. The USDA-ARS National Clonal Germplasm Repository (NCGR) maintains a collection of cultivated and wild Fragaria accessions, which provide a valuable source of disease-resistance and other quality traits for the improvement of cultivated strawberry. Despite the recognized importance of genetic diversity, an in-depth assessment of genetic relationships within the USDA-ARS collection remains incomplete. To address these gaps, we genotyped 1,876 cultivated and wild Fragaria accessions from NCGR using a strawberry 5K DArTag marker panel. The evaluation of microhaplotype-based missing data rates revealed an increase in percent of missing data with an increased distance from the cultivated strawberry. This trend is expected because the 5K panel was developed based on the octoploid cultivated strawberries. Among the species studied, the higher ploidy accessions representing F. x vescana, F. x ananassa, F. virginiana, F. cascadensis, and F. chiloensis exhibited the least missing data, with percentages of 2.6%, 5.0%, 7.5%, 10.2%, 14.7%, respectively. Conversely, diploid accessions mostly Asian F. daltoniana, F. nubicola, F. viridis (Eurasian), F. pentaphylla, and F. nilgerrensis displayed the highest percentages of missing data, with 87.1%, 82.5%, 80.2%, 80.2%, 79.9%, respectively. Utilizing the microhaplotypes derived from the 5K panel, a total of 17,925 SNPs were identified after quality filtering, which were then used to assess the ancestry and genomic diversity of the Fragaria species housed within the USDA-ARS collection. We performed PCA and UPGMA analyses, and found distinct species clusters for each putative taxonomic assignment, suggesting high concordance between sample genotype and species identity. Finally, species-specific loci were then identified and genomic diversity analyses were performed for species represented with more than five accessions. Our findings clarified the identity and diversity of Fragaria within the USDA-ARS germplasm collection, aiding in germplasm conservation efforts and informing future breeding initiatives.
