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Front. Plant Sci. | doi: 10.3389/fpls.2018.00842

Simultaneous editing of two copies of Gh14-3-3d confers enhanced transgene-clean plant defense against Verticillium dahliae in allotetraploid upland cotton

zhennan zhang1,  Xiaoyang Ge2, xiaoli luo3, peng wang2, qiang fan1, guang hu1, juanli xiao3, fuguang li2* and  Jia-He Wu1*
  • 1The State Key Laboratory of Plant Genomics, Institute of Microbiology (CAS), China
  • 2The State Key Laboratory of Cotton Biology, Cotton Research Institute (CAAS), China
  • 3Institute of Cotton Research, Shanxi Academy of Agricultural Sciences, China

Gossypium hirsutum is an allotetraploid species, meaning that mutants that are difficult to generate by classical approaches due to gene redundancy. The CRISPR/Cas9 genome editing system is a robust and highly efficient tool for generating target gene mutants, by which the genes of interest may be functionally dissected and applied through genotype-to-phenotype approaches. In this study, the CRISPR/Cas9 genome editing system was developed in G. hirsutum through editing the Gh14-3-3d gene. In T0 transgenic plants, lots of insertions and deletions (indels) in Gh14-3-3d at the expected target site were detected in the allotetraploid cotton At or Dt subgenomes. The results of the PCR, T7EI digestion and sequencing analyses showed that the indels of Gh14-3-3d gene can be stably transmitted to the next generation. Additionally, the indels in the At and Dt subgenomes were segregated in the T1 transgenic plants following Mendelian law, independing on the T-DNA segregation. Two homozygous Gh14-3-3d-edited plants free of T-DNA were chosen by PCR and sequencing assays in the T1 plants, which were called transgene-clean editing plants and were designated ce1 and ce2. ce1 and ce2 in the T2 lines had high resistance to Verticillium dahliae infestation compared to the wild-type plants. Thus, the two transgene-clean edited lines can be used as a germplasm to breed disease-resistant cotton cultivars, possibly avoiding complex and expensive safety assessments of the transgenic plants.

Keywords: Gossypium hirsutum L., Allotetraploid cotton, Simultaneous editing, transgene-clean, CRISPR/Cas9, Verticillium dahliae

Received: 31 Jan 2018; Accepted: 30 May 2018.

Edited by:

Junhua Peng, Center for Life Sci&Tech of China National Seed Group Co. Ltd.

Reviewed by:

Kaijun Zhao, Institute of Crop Science, Chinese Academy of Agricultural Sciences
Junkang Rong, Zhejiang Agriculture and Forestry University, China  

Copyright: © 2018 zhang, Ge, luo, wang, fan, hu, xiao, li and Wu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Prof. fuguang li, Cotton Research Institute (CAAS), The State Key Laboratory of Cotton Biology, Anyang, China, aylifug@163.com
Prof. Jia-He Wu, Institute of Microbiology (CAS), The State Key Laboratory of Plant Genomics, Beijing, China, wujiahe@im.ac.cn