Development of Single Nucleotide Polymorphism and Phylogenetic Analysis of Species in Zhejiang Province, China, Using ddRAD-Seq Technology.

The genus presents significant challenges for systematic classification due to extensive hybridization and adaptive radiation. Here, we employed double-digest restriction site-associated DNA sequencing (ddRAD-seq) to resolve phylogenetic relationships among nine ecologically significant species (34 accessions) endemic to Zhejiang Province, China, a biodiversity hotspot for this genus. Using as the reference genome, we generated 39.40 Gb of high-quality sequencing data with a Q30 score of 96.65% and a GC content of 39.63%, achieving an average alignment rate of 92.79%. Through stringent filtering (QD ≥ 2, MQ ≥ 40), we identified 14,048,702 genome-wide single nucleotide polymorphism (SNP), predominantly characterized by the mutation types T:A>C:G and C:G>T:A. The widespread and var. exhibited significant genetic diversity, whereas the low-altitude widespread and the endemic exhibited lower genetic diversity. Moderate genetic differentiation ( = 0.097) was observed between and var. , while substantial genetic differentiation was detected among the other species. Principal component analysis (PCA), combined with phylogenomic reconstruction, demonstrated that the genus can be stratified into six well-supported genetic clades. Furthermore, this study provides the first genomic validation of the sibling relationship between and its variety, var. , and clarifies the systematic position of , suggesting that it should be classified as a new subgenus. This study establishes ddRAD-seq as a cost-effective tool, providing both a theoretical framework for SNP-based phylogenetics and critical insights for conserving China's azalea biodiversity.