A telomere‐to‐telomere gap‐free assembly integrating multi‐omics uncovers the genetic mechanism of fruit quality and important agronomic trait associations in pomegranate

Summary Pomegranate is an important perennial fruit tree distributed worldwide. Reference genomes with gaps and limit gene identification controlling important agronomic traits hinder its functional genomics and genetic improvements. Here, we reported a telomere‐to‐telomere (T2T) gap‐free genome assembly of the distinctive cultivar ‘Moshiliu’. The Moshiliu reference genome was assembled into eight chromosomes without gaps, totalling ~366.71 Mb, with 32 158 predicted protein‐coding genes. All 16 telomeres and eight centromeres were characterized; combined with FISH analysis, we revealed the atypical telomere units in pomegranate as TTTTAGGG. Furthermore, a total of 16 loci associated with 15 important agronomic traits were identified based on GWAS of 146 accessions. Gene editing and biochemical experiments demonstrated that a 37.2‐Kb unique chromosome translocation disrupting the coding domain sequence of PgANS was responsible for anthocyanin‐less, knockout of PgANS in pomegranate exhibited a defect in anthocyanin production; a unique repeat expansion in the promoter of PgANR may affected its expression, resulting in black peel; notably, the G → A transversion located at the 166‐bp coding domain of PgNST3, which caused a E56K mutation in the PgNST3 protein, closely linked with soft‐seed trait. Overexpression of PgNST3A in tomato presented smaller and softer seed coats. The E56K mutation in PgNST3 protein, eliminated the binding ability of PgNST3 to the PgMYB46 promoter, which subsequently affected the thickness of the inner seed coat of soft‐seeded pomegranates. Collectively, the validated gap‐free genome, the identified genes controlling important traits and the CRISPR‐Cas9‐mediated gene knockout system all provided invaluable resources for pomegranate precise breeding.