The *Allium* genus, encompassing economically important crops like bunching onions, bulb onions, garlic, and shallots, is characterized by large genomes and a pungent flavor derived from alk(en)yl cysteine sulfoxides (ACSOs). Despite their global cultivation and recognized health benefits, the genomic basis of their unique flavor and the evolutionary processes shaping *Allium* genomes remain poorly understood. Previous research has identified alliin and isoalliin as major ACSOs, with alliin spontaneously converting to allicin upon tissue damage and isoalliin requiring alliinase (ALL) and lachrymatory factor synthase (LFS) for hydrolysis. However, the genomic mechanisms underlying ACSO biosynthesis and the diversity in ACSO profiles among *Allium* species have not been comprehensively elucidated. Furthermore, the genetic relationships within the *Allium* genus are complex due to morphological similarities and interspecific hybridization, leading to ambiguities in taxonomy and hindering the progress of genome-assisted breeding. This study aims to address these knowledge gaps by assembling a high-quality chromosome-level genome of bunching onion (*A. fistulosum*), a key *Allium* crop, and utilizing this resource to investigate *Allium* genome evolution, the genomic basis of flavor formation, and the domestication history of bunching onions. The availability of a high-quality reference genome is crucial for advancing *Allium* research and breeding programs, especially given the challenges posed by their large and repetitive genomes.

Existing research on *Allium* crops has primarily focused on characterizing their bioactive compounds and exploring their potential health benefits. Studies have identified various ACSOs, including alliin, isoalliin, and methiin, and their enzymatic conversion into pungent and lachrymatory factors. However, detailed knowledge about the genetic regulation of ACSO biosynthesis and the evolutionary dynamics of flavor-related genes has been limited by the lack of high-quality genome assemblies. Previous attempts to characterize the *Allium* genome faced challenges due to large genome size and high repetitive content. Traditional taxonomy relies heavily on morphology, leading to ambiguities in species identification and evolutionary relationships, particularly among closely related *Allium* species. Interspecific hybridization is common within the *Allium* genus, creating both challenges and opportunities for breeding programs. The development of a chromosome-level genome assembly for a representative *Allium* species will significantly enhance our understanding of *Allium* genetics and evolution.

This study employed a multi-platform sequencing approach to generate a high-quality chromosome-level genome assembly of bunching onion (*A. fistulosum*). Genomic DNA was extracted from young leaves of the accession 'SXSJC'. PacBio long-read sequencing was employed to generate a draft assembly, which was subsequently polished using both PacBio and Illumina short-reads. Bionano optical mapping data provided additional scaffolding information, improving the accuracy of the assembly. Finally, Hi-C sequencing data were used to anchor contigs to chromosomes, resulting in a chromosome-level assembly. The genome assembly was assessed using various metrics, including BUSCO and CEGMA scores for completeness, k-mer analysis for genome size and heterozygosity, and mapping rates of Illumina reads. Gene prediction was performed using a combination of ab initio methods, protein homology searches, and RNA-Seq data. Repeat annotation was performed using RepeatMasker, RepeatModeler, and TRF. Comparative genomics analysis was conducted by comparing the *A. fistulosum* genome with the genomes of 13 other plant species, including other *Allium* species, to infer evolutionary relationships and identify gene family expansions and contractions. The phylogenetic relationships among *Allium* crops were further investigated by re-sequencing 135 diverse *Allium* accessions, including bunching onions, shallots, Chinese red onions, and *A. altaicum*. SNP and InDel calling were performed on these re-sequencing data. Phylogenetic trees were constructed using maximum likelihood methods, and population structure analysis was conducted using a Bayesian clustering approach. Finally, the isoalliin content of 91 *A. fistulosum* accessions was quantified using ultra-high-performance liquid chromatography (UHPLC), and genome-wide association studies were used to identify genes associated with isoalliin variation.

The study produced a high-quality chromosome-level genome assembly of bunching onion (*A. fistulosum*) with a size of 11.27 Gb and a contig N50 of 7.34 Mb. Comparative genomics revealed that bursts of Gypsy-type LTR retrotransposons, particularly the Tekey and Tat clades, and dispersed duplication events were the main drivers of genome expansion in *Allium* crops. A total of 62,255 genes were predicted in the *A. fistulosum* genome. Phylogenetic analysis based on single-copy genes estimated that *A. fistulosum* and *A. cepa* diverged approximately 7.4 million years ago (MYA), and their common ancestor diverged from *A. sativum* around 16.7 MYA. Analysis of gene family expansions revealed that sulfur metabolism-related genes, including those involved in ACSO biosynthesis and hydrolysis, were significantly expanded in the three major *Allium* species, potentially contributing to their characteristic pungent flavor. The ALL and LFS gene families showed extensive expansion and differentiation, with the LFS gene family being unique to *Allium* crops among the species examined. The study further revealed that China is the origin and domestication center for bunching onion. Population structure analysis of 96 *A. fistulosum* accessions identified five subgroups with distinct geographical distributions, suggesting migration routes from western China to Central Asia and from southeastern China to Japan, America, and Europe. Genome-wide association studies (GWAS) identified several genes associated with variations in isoalliin content, further supporting the contribution of sulfur metabolism genes to flavor diversity. A positive correlation between isoalliin content and the expression levels of several ACSO biosynthesis and hydrolysis genes was observed.

The high-quality genome assembly of *A. fistulosum* provides a valuable resource for understanding *Allium* genome evolution and the genetic basis of flavor formation. The findings highlight the significant role of transposable elements, particularly LTR retrotransposons, in shaping *Allium* genome size and structure. The extensive expansion and diversification of genes involved in sulfur metabolism, particularly ALL and LFS, likely underlies the species' unique flavor profiles. The identification of China as the origin and domestication center for bunching onion provides valuable insights into the crop's history and potential for future breeding efforts. The GWAS analysis of isoalliin content revealed the involvement of specific genes in this trait, providing targets for future crop improvement. The identification of genes under selection pressure across different geographical subgroups suggests adaptation to local environmental conditions. These results also highlight the limitations of relying solely on morphological traits for *Allium* taxonomy and demonstrate the utility of genomic approaches for clarifying evolutionary relationships.

This study generated a high-quality chromosome-level genome assembly for bunching onion, providing a significant resource for *Allium* research. The findings reveal the impact of transposable elements and gene duplication in shaping *Allium* genomes and the key role of sulfur metabolism genes in flavor formation. The study confirms China as the origin and domestication center for bunching onion, revealing migration patterns from this center. Future research could focus on functional characterization of identified genes to further elucidate the mechanisms of ACSO biosynthesis and hydrolysis and to explore the potential for developing improved *Allium* cultivars with enhanced flavor and other desirable traits.

While the study provides a comprehensive analysis of *Allium* genome evolution and flavor formation, some limitations exist. The analysis was limited to a specific bunching onion accession ('SXSJC') for genome assembly, and further studies incorporating additional accessions would enhance the understanding of genetic diversity. The re-sequencing data analysis focuses mainly on SNPs and InDels, potentially overlooking other forms of structural variation that may play important roles in genome evolution and phenotypic variation. The functional characterization of candidate genes identified in the GWAS analysis is required to confirm their role in isoalliin accumulation.