Exploring Transcriptomic Microsatellite Markers in Hydrangea petiolaris (Hydrangeaceae): A Resource for Population...

Hydrangea petiolaris Siebold & Zucc., commonly known as climbing hydrangea, is a vine native to the woodlands of Korea, Japan, and Sakhalin Island. It is an economically important ornamental species characterized by both fertile and sterile flowers. Despite growing interest in Hydrangea breeding and germplasm conservation, genomic resources and informative molecular markers remain limited for H. petiolaris, constraining population genetic and functional studies. To address this, we employed Illumina NovaSeq 6000 sequencing to generate a total of 39,945,480 RNA-seq reads, which were assembled into 137,715 contigs. From 109,092 filtered transcripts, we identified 54,587 microsatellite repeat motifs across 33,556 contigs. Among these, 16,739 microsatellite-containing transcripts were functionally annotated using Gene Ontology analysis, highlighting gene families such as those encoding pentatricopeptide repeat proteins, aldehyde dehydrogenases, and basic helix-loop-helix= transcription factors, which have been implicated in fertility restoration and floral development in plants. To develop molecular markers, we designed 45,073 primer pairs targeting microsatellite-containing transcripts. Of the 50 tested primer pairs, 41 successfully amplified across 52 individuals, and genotyping yielded 30 polymorphic microsatellite loci with an average of 4.13 alleles per locus (ranging from 2 to 8). Two loci (HAN33 and HAN35) showed evidence of null alleles and significant deviations from Hardy-Weinberg equilibrium and were treated with caution in downstream analyses. Using validated loci, population genetic analyses of two island populations (Jeju-do and Ulleungdo) revealed strong genetic differentiation and clear clustering, illustrating the utility of these transcriptomic SSR markers for population-level analyses and conservation-oriented studies in H. petiolaris. Overall, this study provides a large genic SSR resource and an experimentally validated marker set that can be extended to broader sampling and applied to related Hydrangea taxa.