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

TLDR

Improving traits in cultivated alfalfa is difficult because its autotetraploid genome and self‑incompatibility have prevented a reference genome and efficient editing protocol. We assembled a chromosome‑level, allele‑aware genome of 32 allelic chromosomes using high‑fidelity single‑molecule sequencing and Hi‑C, and used it to develop a CRISPR/Cas9 protocol that introduces tetra‑allelic mutations. The tetra‑allelic mutants are stably inherited transgene‑free across generations, and the genome assembly and editing protocol provide a foundation for accelerating alfalfa research and breeding.

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.