Corteva Agriscience™, 8325 NW 62nd Ave, Johnston, IA, 50131, USA.
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
Plant J. 2020 Jan;101(1):101-111. doi: 10.1111/tpj.14521. Epub 2019 Oct 25.
Type C cytoplasmic male sterility (CMS-C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS-C, is located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non-restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map-based cloning interval of 12 kb. Within this 12-kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix-loop-helix (bHLH) transcription factor with tassel-specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non-restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR-Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection.
C 型细胞质雄性不育（CMS-C）是玉米杂交种生产中最常用的 CMS 形式。育性恢复基因 4（Rf4）是 CMS-C 的主要育性恢复基因，位于 8S 染色体上。为了定位克隆 Rf4，从一个非恢复系和恢复系之间的杂交后代中筛选了一个大的 F3 群体（n=5104）用于重组体，并对穗育性进行表型分析，最终得到一个基于图谱的克隆间隔 12 kb。在这个 12-kb 间隔内，Rf4 的唯一可能候选者是 GRMZM2G021276，这是一个具有穗特异性表达的基本螺旋-环-螺旋（bHLH）转录因子。Rf4 基因产物包含一个核定位信号，可能不与线粒体直接相互作用。Rf4 序列分析显示，在恢复系和非恢复系之间有四个编码氨基酸取代，然而只有一个取代，F187Y，位于高度保守的 bHLH 结构域内。通过使用成簇规律间隔短回文重复（CRISPR）-CRISPR 相关蛋白 9（Cas9）同源定向修复（HDR）来创建 F187Y 取代不同的同基因系，测试了 Rf4 恢复是由单个氨基酸改变的假设。在对这些 CRISPR-Cas9 编辑植物（n=780）进行穗育性表型分析的群体中，含有 F187 的植物完全可育，表明育性恢复，而含有 Y187 的植物不育，表明缺乏育性恢复。结构建模表明，这个氨基酸残基 187 位于四个螺旋束核心内，这是一个稳定二聚体构象和影响相互作用伙伴选择的关键区域。
