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

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.