Commonwealth Scientific and Industrial Research Organisation (CSIRO) Agriculture and Food, Glen Osmond, SA 5064, Australia.
Genes (Basel). 2020 Sep 10;11(9):1064. doi: 10.3390/genes11091064.
Most subgenus species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic by targeting mutations in the endogenous () gene using -mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify gene edits. Up to 86% of seeds produced albino seedlings with complete knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing segregation from the target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic , enabled development of the discussed strategies for future identification of causal apomixis genes.
大多数亚属物种是自交不亲和的。有些是兼性无融合生殖，其中大多数种子通过母本基因型进行无性繁殖。大多数胚囊通过有丝分裂发育，没有减数分裂，种子形成不需要受精。无融合生殖受显性基因座控制，其中重组受到抑制。γ-射线辐照导致基因座缺失会导致向有性生殖的回复。在鉴定的基因座上对基因进行靶向诱变将有助于确定因果基因。在这项研究中，通过使用 -介导的叶片圆盘转化靶向内源性 () 基因中的突变，检查了 CRISPR/Cas9 在无融合生殖中的编辑效果。在三个实验中，在 11%的 T0 植物中观察到预期的白化矮化致死表型，这是 基因敲除的特征，31.4%是扇形白化嵌合体，其余为绿色。嵌合体植物开花。从两个嵌合体的无融合生殖中萌发的 T1 种子进行表型分析和测序，以鉴定 基因编辑。多达 86%的种子产生了具有完整 基因敲除的白化幼苗。这归因于在无融合生殖种子形成过程中，嵌合体中的 Cas9 介导的编辑持续进行，阻止了从 目标的 分离。这一成功证明了 CRISPR/Cas9 在无融合生殖中的高效基因编辑，为未来鉴定因果无融合生殖基因的讨论策略的发展提供了依据。
