Chen Lina, Wang Hao, Xu Tingtao, Liu Ruitao, Zhu Juanli, Li Haoxian, Zhang Huawei, Tang Liying, Jing Dan, Yang Xuanwen, Guo Qigao, Wang Peng, Wang Luwei, Liu Junhao, Duan Shuyun, Liu Zhaoning, Huang Mengchi, Li Xiaolong, Lu Zhenhua
National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, China.
Zhongyuan Research Center, Chinese Academy of Agricultural Sciences, Xinxiang, China.
Plant Biotechnol J. 2025 Jul;23(7):2852-2870. doi: 10.1111/pbi.70107. Epub 2025 May 3.
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 PgNST3 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.
石榴是一种重要的多年生果树，分布于世界各地。带有缺口的参考基因组以及限制对重要农艺性状进行基因鉴定的因素，阻碍了其功能基因组学研究和遗传改良。在此，我们报道了独特品种‘墨石榴’的端粒到端粒（T2T）无缺口基因组组装。墨石榴参考基因组被组装成8条无缺口的染色体，总计约366.71 Mb，有32,158个预测的蛋白质编码基因。所有16个端粒和8个着丝粒都得到了表征；结合荧光原位杂交分析，我们揭示了石榴中典型的端粒单元为TTTTAGGG。此外，基于对146份种质的全基因组关联研究（GWAS），共鉴定出16个与15个重要农艺性状相关的位点。基因编辑和生化实验表明，一个37.2-Kb的独特染色体易位破坏了PgANS的编码域序列，导致无花青素，石榴中PgANS的敲除表现出花青素产生缺陷；PgANR启动子中的一个独特重复扩增可能影响其表达，导致果皮变黑；值得注意的是，位于PgNST3的166-bp编码域的G→A颠换，导致PgNST3蛋白发生E56K突变，与软籽性状密切相关。在番茄中过表达PgNST3会使种皮更小更软。PgNST3蛋白中的E56K突变消除了PgNST3与PgMYB46启动子的结合能力，进而影响了软籽石榴内种皮的厚度。总的来说，经过验证的无缺口基因组、鉴定出的控制重要性状的基因以及CRISPR-Cas9介导的基因敲除系统，都为石榴的精准育种提供了宝贵资源。
