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 significant spice and medicinal crop, has been constrained by limited molecular resources. The recent availability of a reference genome enabled the first comprehensive, genome-wide characterization and development of simple sequence repeats (SSRs). 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 (> 600loci/Mb) and coldspots (< 400 loci/Mb). A total of 11,107 hypervariable Class I loci were identified, which were uniquely dominated by dinucleotide repeats (92.3%), pinpointing them as the primary source of hypervariability. From these findings, a comprehensive resource of 28,147 high-quality primer pairs targeting Class I and II loci was 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), indicative of a potential introductory founder effect in the analyzed germplasm. These markers successfully resolved the 19 accessions into distinct phylogenetic clades, identifying a highly divergent accession (Acc. 9833). The practical utility of the markers was further confirmed by verifying the F ₁ hybrid status of high-yielding plants, identifying hybrids at the seedling stage within open-pollinated progeny, and confirming co-dominant 1:2:1 Mendelian segregation in an F ₂ population. This comprehensive, validated SSR marker toolkit and database provide a foundational resource to accelerate clove breeding programs, germplasm management, and future genetic mapping studies.