The Gap-free <i>Petunia</i> Genome Assemblies Reveal the Evolutionary Dynamics of the <i>S</i> -locus Supergene

Petunia hybrida is a key genetic model for investigating self-incompatibility (SI), a reproductive barrier governed by the multi-allelic S-locus, which encodes a pistil-specific S-RNase and multiple S-locus F-box (SLF) genes. Due to high heterozygosity and abundant repetitive sequences, previous S-locus assemblies in reference genomes have been fragmented and collapsed. Here, we present the telomere-to-telomere (T2T), haplotype-resolved genomes of two homozygous SI lines (P. hybrida S3LS3L and SVSV), enabling the complete reconstruction of both S-loci. Population genomic analyses delineated their boundaries, spanning approximately 14.01 Mb and 20.83 Mb, respectively. Remarkably, both S-loci exhibited extremely low nucleotide polymorphism and structural variation compared with the remainder of the genome. In addition to the S-RNase and the complete repertoire of SLF genes, we identified two pollen-specific genes, ubiquitin-like and MYB, which may contribute to SI regulation. Our results demonstrate that the genomic architecture of the Petunia S-locus continues to evolve dynamically while retaining the core genetic components essential for SI. Furthermore, we propose six evolutionary scenarios, providing new insights into the processes driving the generation, diversification, loss, functional maintenance, and structural reorganization of SLF genes in Petunia. Overall, the T2T genomes reported here establish P. hybrida as a premier model for comparative genomics and SI research in the Solanaceae family.