Cold damage poses a significant challenge to the cultivation of soft-seeded pomegranate varieties, hindering the growth of the pomegranate industry. The genetic basis of cold tolerance in pomegranates has remained elusive, largely due to the lack of high-quality genome assemblies for cold-tolerant varieties and comprehensive population-scale genomic studies. In this study, we addressed these challenges by assembling a high-quality chromosome-level reference genome for 'Sanbai', a pomegranate variety renowned for its freezing resistance, achieving an impressive contig N50 of 15.93 Mb. This robust assembly, enhanced by long-read sequencing of 38 pomegranate accessions, facilitated the identification of 14 239 polymorphic structural variants, revealing their critical roles in genomic diversity and population differentiation related to cold tolerance. Of particular significance was the discovery of a ~ 5.4-Mb inversion on chromosome 1, which emerged as an important factor affecting cold tolerance in pomegranate. Moreover, through the integration of bulked segregant analysis, differential selection analysis, and genetic transformation techniques, we identified and validated the interaction between the PgNAC12 transcription factor and PgCBF1, disclosing their pivotal roles in response to cold stress. These findings mark a significant advancement in pomegranate genomics, offering novel insights into the genetic mechanisms of cold tolerance and providing valuable resources for the genetic improvement of soft-seeded pomegranate varieties.

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