Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding

Sukumar Biswas; Jiaqi Tian; Rong Li; Xiaofei Chen; Zhijing Luo; Mingjiao Chen; Xiangxiang Zhao; Dabing Zhang; Staffan Persson; Zheng Yuan; Jianxin Shi

doi:10.1016/j.jgg.2020.04.004

Volume 47 Issue 5

May 2020

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Article Navigation > Journal of Genetics and Genomics > 2020 > 47(5): 273-280

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Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding

doi: 10.1016/j.jgg.2020.04.004

a
Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University-University of Adelaide Joint Centre for Agriculture and Health, State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China
b
Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, Huaian, 223300, China
c
School of Biosciences, University of Melbourne, Parkville VIC, 3010, Melbourne, Australia

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Corresponding author: E-mail address: zyuan@sjtu.edu.cn (Zheng Yuan); E-mail address: jianxin.shi@sjtu.edu.cn (Jianxin Shi)
Publish Date: 2020-05-25

Abstract

Abstract

Although Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has been widely used for basic research in model plants, its application for applied breeding in crops has faced strong regulatory obstacles, due mainly to a poor understanding of the authentic output of this system, particularly in higher generations. In this study, different from any previous studies, we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1 (semi-dwarf 1) mutants from T₂ to T₄ generations, of which the selection of T₁ and T₂ was done only by visual phenotyping for semidwarf plants. Our data revealed not only on- and off-target mutations with small or lager indels but also exogenous elements in T₂ plants. All indel mutants passed stably to T₃ or T₄ without additional modifications independent on the presence of Cas9, while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements. In addition, effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds. Taken together, our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis, but it may be not as precise as expected in rice, and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.
- CRISPR/Cas9,
- Plant height,
- Yield

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References(44)

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