Pan-SSR architecture in <i>Acrossocheilus fasciatus</i> reveals copy-number–dominated variation and gene-associated repeat organization

Microsatellites, or simple sequence repeats (SSRs), are abundant and highly polymorphic genomic features, yet comparative analyses of genome-wide SSR landscapes remain difficult because repeat discovery is detector-dependent and multi-assembly integration lacks a coherent coordinate system. Here, we characterized pan-SSR architecture in the non-model cyprinid Acrossocheilus fasciatus by aligning four public assemblies to the AF_xajfe reference and merging lifted loci into core, dispensable, and private components, enabling direct comparison of homologous loci. We systematically compared four detectors—TRF, PyTRF, Ultra, and our de Bruijn graph-based GraphSSR—on synthetic ground-truth datasets using stringent interval-plus-period matching. TRF showed the highest accuracy and parameter stability for pan-SSR inference, whereas GraphSSR provided a complementary route for detecting imperfect repeats. Across pan-categories, SSR variation was dominated by copy-number polymorphism, consistent with slippage-driven length dynamics, whereas motif mutations and mixed polymorphisms were comparatively rare. Pan-SSR categories showed strong local genomic stratification: core SSRs preferentially occurred near genes and promoters, whereas private SSRs were enriched in transposable element-dense regions and tightly coupled with DNA transposons. At megabase resolution, SSR density was robustly and inversely associated with GC content, indicating scale-dependent constraints in which local functional context and macro-scale compositional background jointly shape the repeat landscape. Independent resequencing showed that filtered neutral/core and gene-associated/core panels remained tractable in low-depth short-read data and retained population-informative SSR dosage variation. Together, these analyses connect pan-SSR architecture with gene-associated candidate prioritization and provide a basis for studying repeat evolution and genome organization across heterogeneous public assemblies in non-model fishes.