Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081, China.
Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081, China; Institute of Biotechnology Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Mol Plant. 2020 Sep 7;13(9):1262-1269. doi: 10.1016/j.molp.2020.06.003. Epub 2020 Jul 8.
The next-generation hybrid seed technology enables the successful production of sortable hybrid seeds from genic male sterile (GMS) lines and maintainers; however, it requires multiple laborious and complicated steps. Here, we designed a simple next-generation hybrid seed production strategy that takes advantage of the CRISPR/Cas9 technology to create a Manipulated GMS Maintainer (MGM) system via a single transformation. Under this schema, the maize male fertility gene ZmMS26 was nullified by removal of its fifth exon using the CRISPR/Cas9 system on a vector, and a second vector carrying a functional ZmMS26 cDNA was co-transformed to restore fertility. The second vector also contains a male gametophyte inactivation gene (ZmAA1) encoding maize α-amylase driven by the pollen-specific promoter PG47 and an endosperm fluorescent marker (DsRED) driven by the barley endosperm aleurone-specific promoter Ltp2. The derived single-copy hemizygous MGM lines bore a mutated MS26 gene, leading to complete male sterility but normal vegetative growth and grain yield. The MGM system could prevent genetic transmission of the MGM elements via male gametophytes, providing an efficient method for sorting maintainer seeds labeled by DsRED. This strategy can be extended to any GMS gene and to hybrid crops other than maize.
下一代杂种种子技术使从基因雄性不育（GMS）系和保持系成功生产可分拣的杂种种子成为可能；然而，它需要多个费力且复杂的步骤。在这里，我们设计了一种简单的下一代杂种种子生产策略，利用 CRISPR/Cas9 技术通过一次转化创建一个操纵的 GMS 保持系（MGM）系统。在这种方案下，通过在载体上使用 CRISPR/Cas9 系统去除玉米雄性育性基因 ZmMS26 的第五个外显子，使 ZmMS26 基因失活，并使用第二个载体携带功能正常的 ZmMS26 cDNA 来恢复育性。第二个载体还包含一个编码玉米α-淀粉酶的雄性配子体失活基因（ZmAA1），该基因由花粉特异性启动子 PG47 驱动，以及一个由大麦糊粉层特异性启动子 Ltp2 驱动的内胚乳荧光标记物（DsRED）。衍生的单拷贝杂合 MGM 系携带突变的 MS26 基因，导致完全雄性不育，但正常的营养生长和籽粒产量。MGM 系统可以通过雄性配子体阻止 MGM 元件的遗传传递，为标记有 DsRED 的保持系种子的分拣提供了一种有效方法。该策略可扩展到任何 GMS 基因和除玉米以外的杂交作物。
