Genome-wide discovery, characterization, and validation of novel SSR markers in clove (Syzygium aromaticum) and their application in genetic analysis

Genetic improvement of clove (Syzygium aromaticum), an economically important spice and medicinal crop, has been constrained by limited molecular resources. The recent availability of a reference genome enabled a chromosome-scale genome-wide characterization and development of simple sequence repeats (SSRs) in clove. An in silico survey of the 367.77 Mb genome assembly identified 189,127 SSRs at an overall frequency of 514.25 loci/Mb. Mononucleotides (50.30%) and dinucleotides (40.68%) were the most abundant repeat types, exhibiting a strong A/T-rich bias and a notable scarcity of CG/GC motifs. Although SSRs were uniformly distributed at the whole-chromosome level (R² = 0.99 relative to chromosome length), high-resolution analysis revealed significant intra-chromosomal heterogeneity, including distinct hotspots (> 600 loci/Mb) and coldspots (< 400 loci/Mb). A total of 11,107 hypervariable Class I loci were identified, predominantly comprising dinucleotide repeats (92.3%), highlighting them as major contributors to SSR variability. From these loci, 28,147 high-quality primer pairs targeting Class I and II SSRs were developed and curated into a publicly accessible, searchable Clove SSR Marker Database. Experimental validation of 55 primer pairs across 19 germplasm accessions demonstrated high amplification success (96.4%) and polymorphism rates (90.6%). The validated markers revealed moderate genetic diversity (mean He = 0.22; PIC = 0.18), consistent with a relatively narrow genetic base in the analyzed germplasm. Phylogenetic analysis resolved the 19 accessions into distinct clades and identified a highly divergent accession with red-coloured flower buds (Acc. 9833). The practical utility of the validated markers was further demonstrated through hybrid authentication and confirmation of co-dominant Mendelian segregation in an F₂ population. Collectively, this validated SSR marker resource and database provide a valuable genomic resource for clove genetic studies, germplasm management, and future genetic mapping efforts.