Two telomere-to-telomere genome assemblies and comparisons revealed the conserved key genes associated with sugar accumulation inRubusgenus

For the first time, we assembled two highly continuous, completely gap-free reference genomes of theRubussubgenus:Rubus hirsutusThunb. ‘Penglei’ (XMM) andRubus eustephanosFocke ex Diels ‘Dahongpao’ (DHP), which are widely distributed in southern China with similar phenotypic traits (Figures 1A, 1B, and Figure S1), yet ripe fruits display distinct sugar accumulation levels (Table S1), making them ideal candidates for investigating the mechanisms underlying sugar accumulation in theRubusgenus. The XMM (213.53 Mb, 28,204 genes) and DHP (218.26 Mb, 28,569 genes) genomes exhibit close evolutionary relationships, diverging approximately 3.21 Mya. Comparative genomics identified extensive synteny, interspecific structural variations (translocations, inversions, segmental duplications), and presence/absence variation (PAV). Using Hi-C interaction heatmaps and Sanger sequencing, we validated interspecific structural inversions. Additionally, we identified a sugar transporter gene (MFS1), which is present in XMM but absent in DHP. Combined analysis of the gene family expansion/contraction and transcriptome identified two conserved key genes (RhSTP13andRhSTP7) associated with sugar accumulation inRubusgenus and displayed distinct roles through transient expression assay. To facilitate functional genomics study, we also established a comprehensiveRubusdatabase, RubusDB, a freely accessible repository consolidating all genomic, transcriptomic and phenotypic data ofRubusgenus. These findings provide a foundational framework for elucidating the genetic basis of sugar accumulation, genome diversification, and trait improvement inRubusspecies.