Development and Characterization of 14 Novel Genome-Derived SSR Markers for Genetic Diversity and Population Structure Analyses of Two Agroathelia Species

Agroathelia (syn. Sclerotium) is a global soil-borne pathogen with a broad host range, causing significant agricultural losses in diverse crops. However, genomic and population genetic resources of this genus remain limited. To develop genome-based molecular tools, we newly sequenced two Korean isolates (A. rolfsii KACC 93004P and A. delphinii KACC 93031P) and compared them with the reference genome of A. rolfsii GP3. Comparative genome analysis identified 723 polymorphic simple sequence repeat (SSR) loci, from which 14 were selected and validated across 34 Korean isolates representing multiple host plants. Genetic diversity was assessed using the number of alleles (NA), observed heterozygosity (Ho), unbiased expected heterozygosity (He), and polymorphic information content (PIC). Most SSRs were moderately to highly informative (PIC = 0.341 to 0.541 in A. rolfsii; 0.367 to 0.612 when including A. delphinii). Unweighted pair group method with arithmetic mean (UPGMA) clustering based on SSR allele profiles clearly separated the two species and revealed a distinct intraspecific structure within A. rolfsii. Principal coordinates analysis (PCoA) also revealed clear species-level separation, while A. rolfsii isolates were partitioned into two intraspecific clusters with one divergent isolate, indicating structured genetic variation without a host-associated population structure. The developed SSR markers provide useful tools for studying genetic diversity, population structure, and epidemiology of Agroathelia species and isolates.