Pangenome Analysis Reveals Structural Variations Associated With Citric Acid Accumulation in Prunus mume

Pangenome can reveal a large number of variations, providing a more comprehensive view of the genetic diversity of species that a single reference genome cannot surpass. Here, we assembled the haplotype telomere-to-telomere genome and 10 chromosome-level genomes, integrated with two previously reported genomes, and constructed a graph pangenome for P. mume with 'PmNH_Hap1' T2T genome as the reference. Core gene families accounted for 64.47%, while non-essential gene families comprised 35.53%. The total length of the graph pangenome reaches 412.41 Mb, which is a significant increase of 179.60 Mb compared with the 'PmNH_Hap1' reference genome, and these unique insertion sequences contain 5918 genes. Additionally, by combining with five Prunus armeniaca genomes for pangenome variation detection, we identified a total of 51 461 non-redundant SVs, including 15 217 insertions, 21 683 deletions, 13 509 translocations and 1025 inversions. Among these, 60.50% of presence/absence variations were formed by transposons. Pangenome analysis revealed that a 376 bp SV insertion was universally present in the promoter of this gene in P. mume, PmAGL30 can bind to this SV to enhance PmPH4 gene expression and promoted citric acid accumulation in P. mume fruits, leading to extremely significant differences in citric acid content compared with other stone fruits. Additionally, we developed SV molecular markers for the early screening of germplasm with high citric acid content in P. mume fruits. In summary, we constructed a high-quality graph pangenome that reveals abundant genetic variations, providing valuable insights for variety improvement and molecular breeding in P. mume.