Gap-Free T2T assemblies of Micropterus salmoides identify a Y-Linked SV hotspot underlying sexual dimorphism

<h4>Background</h4>The largemouth bass (Micropterus salmoides, LMB), a globally significant aquaculture species, exhibits pronounced growth dimorphism between the sexes, yet the genetic basis of its sex determination remains unresolved. While published genomes exist, gaps, misassemblies, and unresolved repetitive regions hinder investigations into structural variations (SVs) linked to sexually dimorphic traits. High-quality telomere-to-telomere (T2T) assemblies are essential to address these limitations and advance sex determination studies.<h4>Methodology</h4>We generated the first T2T gap-free genome assemblies for male and female LMB using Oxford Nanopore (ONT), PacBio HiFi, and Hi-C sequencing. Genome quality was validated via BUSCO, Merqury, Hi-C contact maps, and synteny comparisons. Repeat content and protein-coding genes were annotated, and male-specific SVs were identified through cross-genome alignment. Transcriptomic data from gonads and muscle were integrated to analyze differential expression in previously unresolved regions (PURs).<h4>Results</h4>The female (871.07 Mb) and male (873.86 Mb) T2T assemblies achieved BUSCO completeness of 99.3% and QV scores > 50, resolving all gaps in prior references. Comparative analysis identified a 90-kb Y-specific SV-enriched region (SVER-Y) on chromosome 10, harboring male-specific insertions and marked by transposable element (TE) enrichment, with LTRs (74.9% higher) and LINEs (63.7% higher) relative to the chromosome-wide average. PURs (46.96 Mb), localizing to telomeres and centromeres, showed high TE content (LINEs: 29.6%, LTRs: 23.3%) and tissue-specific gene expression. Notably, 610 putative PUR-associated transcripts were differentially expressed between gonadal and somatic tissues, suggesting regulatory roles in sexual development.<h4>Conclusion</h4>This study provides the complete T2T genomes for LMB, correcting structural errors in prior assemblies and resolving repetitive regions critical for understanding sexual dimorphism. The SVER-Y region, enriched in male-specific SVs and TEs, offers novel insights into early Y chromosome differentiation. These resources establish a foundation for deciphering sex determination mechanisms and optimizing marker-assisted breeding strategies in LMB aquaculture.