Integration of phenotype characterization and DNA barcoding for comprehensive genetic diversity assessment in Hydrangea germplasm

Hydrangea has become a focus of horticultural research owing to its exceptional ornamental value. However, analysis of genetic diversity in Hydrangea using DNA barcode markers is limited. This study systematically investigated the genetic diversity and phylogenetic relationships among Hydrangea varieties using a combination of distinctness, uniformity, and stability (DUS) phenotypic analysis and DNA barcode markers. Diversity analysis, correlation analysis, and principal component analysis (PCA) were conducted on 24 phenotypic traits for 50 accessions. The coefficient of variation for the DUS traits ranged from 12.37% to 132.34%. The simpson diversity index indicated that floral organ traits showed the most significant variation and highlighted the substantial genetic diversity within the Hydrangea population. PCA identified 11 core traits explaining 81.97% of the total variation, demonstrating their utility as primary evaluation indicators. Compared to rbc L, psb A– trn H, and their combinations, mat K exhibited superior discriminatory potential, making it a more effective specific DNA barcode marker; consistent with this, amplification of 267 DNA sequences from 89 samples revealed that mat K and psb A- trn H displayed the highest mutation rates and optimal identification efficiency. PCA, phylogenetic reconstruction, and the Mantel test identified mat K and psb A– trn H as optimal markers for cultivar identification, which showed significant correlations with phenotypic traits. Haplotype analysis of the three DNA fragments revealed 13 haplotypes. The haplotype comprised three dominant and 10 unique haplotypes. The results revealed substantial genetic diversity among the tested cultivars and the utility of multiple genetic markers for variety differentiation. The molecular marker and genetic diversity information provide crucial insights useful for future Hydrangea breeding programs.