Liang Siyi, Jia Sisi, Lu Wenyu, Wang Jiafeng, Huang Ming, Chen Chun, Huang Cuihong, Zhou Danhua, Guo Tao, Liu Hong
National Engineering Research Center of Plant Aerospace-Mutation Breeding, South China Agricultural University, Guangzhou, 510642, China.
College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou, Guangdong, China.
Plant Cell Rep. 2025 Aug 4;44(8):190. doi: 10.1007/s00299-025-03581-z.
CRISPR/Cas9-mediated modification of OsCENH3 induces aneuploidy but fails to trigger haploid formation in rice, underscoring limited efficiency and the need for strategy refinement. The centromeric histone H3 variant (CENH3) is essential for kinetochore assembly and accurate chromosome segregation during cell division. Alterations in CENH3 have been shown to trigger haploid induction in various plant species; however, its utility in rice remains largely unexplored. In this study, six OsCENH3 mutant lines were generated through CRISPR/Cas9-mediated genome editing, complemented by a GFP-tagged construct. To assess their haploid induction potential, both self pollination and outcrossing with the cultivar Nipponbare (NIP) were performed. Flow cytometry analysis revealed that none of the mutants produced true haploids. However, two aneuploid individuals (Het-C1-1-1 and Het-C1-5-1) were identified among the hybrid progeny derived from tail domain mutants C1-1 and C1-5, suggesting partial chromosome missegregation. These aneuploid plants exhibited reduced pollen viability, abnormal morphology, and compromised agronomic performance. In addition, significantly elevated rates of embryo/endosperm abortion were observed across different crosses, which far exceeded the frequency of aneuploid production, implying that early-stage chromosomal instability may result in embryo lethality. Collectively, while specific OsCENH3 mutations can induce limited chromosomal instability, their haploid induction efficiency remains low in rice. Further refinement of editing strategies and exploration of favorable genetic backgrounds will be essential for developing effective centromere-based haploid induction systems. This study provides a theoretical framework and technical reference for engineering haploid inducers in rice through centromere manipulation.
CRISPR/Cas9介导的OsCENH3修饰诱导了水稻非整倍体的产生，但未能触发单倍体的形成，这突出了其有限的效率以及策略优化的必要性。着丝粒组蛋白H3变体（CENH3）对于细胞分裂过程中的动粒组装和准确的染色体分离至关重要。已表明CENH3的改变可在多种植物物种中触发单倍体诱导；然而，其在水稻中的效用在很大程度上仍未得到探索。在本研究中，通过CRISPR/Cas9介导的基因组编辑产生了六个OsCENH3突变系，并用绿色荧光蛋白标记的构建体进行了补充。为了评估它们的单倍体诱导潜力，进行了自花授粉以及与品种日本晴（NIP）的异花授粉。流式细胞术分析表明，没有一个突变体产生真正的单倍体。然而，在来自尾部结构域突变体C1-1和C1-5的杂交后代中鉴定出了两个非整倍体个体（Het-C1-1-1和Het-C1-5-1），这表明存在部分染色体错分离。这些非整倍体植物表现出花粉活力降低、形态异常以及农艺性能受损。此外，在不同杂交组合中观察到胚/胚乳流产率显著升高，这远远超过了非整倍体产生的频率，这意味着早期染色体不稳定性可能导致胚胎致死。总体而言，虽然特定的OsCENH3突变可诱导有限的染色体不稳定性，但它们在水稻中的单倍体诱导效率仍然很低。进一步优化编辑策略和探索有利的遗传背景对于开发有效的基于着丝粒的单倍体诱导系统至关重要。本研究为通过着丝粒操作培育水稻单倍体诱导系提供了理论框架和技术参考。
