Fengyuan Xu, Tongbing Su, Xiaochen Zhang, Lei Qiu, Xiaodong Yang, Nobuya Koizuka, Shin-ichi Arimura, Zhongyuan Hu, Mingfang Zhang, Jinghua Yang

Plant Biotechnology Journal; First published: 11 January 2024

https://doi.org/10.1111/pbi.14268

Summary

Cytoplasmic male sterility (CMS), encoded by the mitochondrial open reading frames (ORFs), has long been used to economically produce crop hybrids. However, the utilization of CMS also hinders the exploitation of sterility and fertility variation in the absence of a restorer line, which in turn narrows the genetic background and reduces biodiversity. Here, we used a mitochondrial targeted transcription activator-like effector nuclease (mitoTALENs) to knock out ORF138 from the Ogura CMS broccoli hybrid. The knockout was confirmed by the amplification and re-sequencing read mapping to the mitochondrial genome. As a result, knockout of ORF138 restored the fertility of the CMS hybrid, and simultaneously manifested a cold-sensitive male sterility. ORF138 depletion is stably inherited to the next generation, allowing for direct use in the breeding process. In addition, we proposed a highly reliable and cost-effective toolkit to accelerate the life cycle of fertile lines from CMS-derived broccoli hybrids. By applying the k-mean clustering and interaction network analysis, we identified the central gene networks involved in the fertility restoration and cold-sensitive male sterility. Our study enables mitochondrial genome editing via mitoTALENs in Brassicaceae vegetable crops and provides evidence that the sex production machinery and its temperature-responsive ability are regulated by the mitochondria.

See https://onlinelibrary.wiley.com/doi/10.1111/pbi.14268

Figure 1

Knockout of CMS-associated ORF138 from Ogura cytoplasmic male sterile hybrid of broccoli via mitoTALENs. (a) Assembly of the mitochondrial genome from CMS hybrid of broccoli cultivar YX. (b) Top, the target sites on ORF138. Red triangles indicate mitoTALENs (TAL1 and TAL2) and their target sites, respectively. Bottom, Identification of ORF138 knockout lines by PCR amplification. NPTII is used as a marker gene for the mitoTALENs expression cassette in the nucleus. COX1 is used as a positive control gene on the mitochondrial genome. (c) The short read coverage in the flanking region of ORF138 of six independent ORF138 knockout lines and YX.