Allele-aware chromosome-level genome assembly and efficient transgene-free genome editing for the autotetraploid cultivated alfalfa

Haitao Chen, Yan Zeng, Yongzhi Yang, Lingli Huang, Bolin Tang, He Zhang, Fei Hao, Wei Liu, Youhan Li, Yanbin Liu, Xiaoshuang Zhang, Ru Zhang, Yesheng Zhang, Yongxin Li, Kun Wang, Hua He, Zhongkai Wang, Guangyi Fan, Hui Yang, Aike Bao, Zhanhuan Shang, Jianghua Chen (), Wen Wang () and Qiang Qiu ()
Additional contact information
Haitao Chen: Chinese Academy of Sciences
Yan Zeng: Chinese Academy of Sciences
Yongzhi Yang: Lanzhou University
Lingli Huang: Guangdong Sanjie Forage Biotechnology Co., Ltd.
Bolin Tang: Guangdong Sanjie Forage Biotechnology Co., Ltd.
He Zhang: BGI-Qingdao
Fei Hao: Northwestern Polytechnical University
Wei Liu: Chinese Academy of Sciences
Youhan Li: Xishuangbanna Tropical Botanical Garden
Yanbin Liu: Lanzhou University
Xiaoshuang Zhang: Guangdong Sanjie Forage Biotechnology Co., Ltd.
Ru Zhang: Northwestern Polytechnical University
Yesheng Zhang: Chinese Academy of Sciences
Yongxin Li: Northwestern Polytechnical University
Kun Wang: Northwestern Polytechnical University
Hua He: Xishuangbanna Tropical Botanical Garden
Zhongkai Wang: Northwestern Polytechnical University
Guangyi Fan: BGI-Qingdao
Hui Yang: Northwestern Polytechnical University
Aike Bao: Lanzhou University
Zhanhuan Shang: Lanzhou University
Jianghua Chen: Xishuangbanna Tropical Botanical Garden
Wen Wang: Chinese Academy of Sciences
Qiang Qiu: Northwestern Polytechnical University

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: Abstract Artificially improving traits of cultivated alfalfa (Medicago sativa L.), one of the most important forage crops, is challenging due to the lack of a reference genome and an efficient genome editing protocol, which mainly result from its autotetraploidy and self-incompatibility. Here, we generate an allele-aware chromosome-level genome assembly for the cultivated alfalfa consisting of 32 allelic chromosomes by integrating high-fidelity single-molecule sequencing and Hi-C data. We further establish an efficient CRISPR/Cas9-based genome editing protocol on the basis of this genome assembly and precisely introduce tetra-allelic mutations into null mutants that display obvious phenotype changes. The mutated alleles and phenotypes of null mutants can be stably inherited in generations in a transgene-free manner by cross pollination, which may help in bypassing the debate about transgenic plants. The presented genome and CRISPR/Cas9-based transgene-free genome editing protocol provide key foundations for accelerating research and molecular breeding of this important forage crop.

Date: 2020
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DOI: 10.1038/s41467-020-16338-x

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