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

• DOI: 10.1038/s41467-020-16338-x
• Publication Date: 2020-05-19T10:04:11Z
• Authors: 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; Qiang Qiu
• Keywords: Crops, Agricultural; Gene Editing; 0301 basic medicine; 2. Zero hunger; 0303 health sciences; Science; Q; Article; Chromosomes, Plant; Tetraploidy; Plant Breeding; 03 medical and health sciences; Phenotype; Mutation; Transgenes; CRISPR-Cas Systems; Alleles; Genome, Plant; Medicago sativa

AbstractArtificially improving traits of cultivated alfalfa (Medicago sativaL.), 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.