Rhododendron plants have ornamental, commercial, and medicinal value to people. Flavonoids are one of the components used in traditional remedies, and Rhododendron plants are found to be rich in flavonoids. Flavonoids can reduce the risk of human disease and participate in the regulation of antioxidant defense systems in response to heat stress. Rhododendron prefers cold climates, so the relatively high temperatures of cities affect the extraction of medicinal ingredients and limit the cultivation environment. Recent studies found that the exogenous application of calcium acts to alleviate heat stress in Rhododendron plants. This study explores the mechanism by which exogenous calcium alleviates heat stress and the role of flavonoids in regulating the antioxidative system in Rhododendron × pulchrum Sweet using combined transcriptomic and metabolomic methods. The activities of peroxidase, catalase and superoxide enzymes were found to increase in response to heat stress and external CaCl₂ in the leaves of R. × pulchrum. In total, 433 metabolic components and 370 DEGs were identified as being differentially expressed in response to heat stress and external calcium chloride (CaCl₂) in the leaves of R. × pulchrum. These results illustrate that heat stress induces oxidative stress and that external CaCl₂ can enhance the heat tolerance of Rhododendron. Flavonoid compounds are responsible for the antioxidant scavenging of reactive oxygen species in R. × pulchrum leaves exposed to heat stress and external calcium.

The genus Rhododendron Linnaeus, comprising over 1,000 species distributed across temperate and alpine regions of Asia, Europe, and North America, holds significant value for its ornamental characteristics, ecological adaptability, and medicinal potential. Despite its importance, the genus presents persistent challenges in taxonomic classification and phylogenetic resolution, compounded by the current insufficiency of genomic SSR markers (g-SSRs) for comprehensive genetic studies. In this study, we conducted the first comprehensive genome-wide identification of SSR markers using whole-genome sequencing data of Rhododendron × pulchrum Sweet. Our analysis identified 324,674 SSR loci across the 509.49 Mb genome. From 190 synthesized primer pairs, 124 (65.3 %) demonstrated successful amplification, with 30 (24.2 %) showing high polymorphism. These polymorphic g-SSR markers generated 597 polymorphic bands, exhibiting polymorphic information content (PIC) values ranging from 0.240 to 0.949 (mean = 0.748), reflecting substantial genetic diversity among the studied accessions. Genetic structure, cluster analysis, and principal coordinates analysis of 96 Rhododendron accessions revealed three distinct clades that correlated with geographic origins, hybrid relationships, and utilization values. Association mapping identified 17 SSR markers significantly linked (P < 0.05) to 10 key morphological traits, explaining 5.68-13.35 % of phenotypic variance. The number of significantly associated markers for each trait was as follows: leaf length (1), remontant (3), corolla lobe shape (2), initial flowering period (3), flower diameter (3), leaf shape (1), petal type (3), petiole length (2), stamen number (4), and flower shape (1). This study provides valuable genomic resources for marker-assisted breeding and establishes a foundation for systematic characterization, conservation, and utilization of Rhododendron genetic resources.