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

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.

Fig. 1. Overview of the cultivated alfalfa genome.

The tracks indicate (moving inwards): a density of LTR transposons, b density of LINE transposons, c density of SINE transposons, d density of DNA transposons, e gene density, f gene expression levels, g Ka/Ks of syntenic gene pairs identified between chrX.1 and one of chrX.2, chrX.3, chrX.4, h Ka/Ks of syntenic gene pairs identified between chrX.2 and one of chrX.1, chrX.3, chrX.4, i Ka/Ks of syntenic gene pairs identified between chrX.3 and one of chrX.1, chrX.2, chrX.4, and j Ka/Ks of syntenic gene pairs identified between chrX.4 and one of chrX.1, chrX.2, chrX.3. Links in the core connect synteny blocks, blue ribbons indicate synteny blocks between chrX.1 and chrX.2, chrX.3, chrX.4, green ribbons indicate synteny blocks between chrX.2 and chrX.3, chrX.4, red ribbons indicate synteny blocks between chrX.3 and chrX.4. Source data are provided as a Source data file.

Fig. 2. Assembly, similarity, and divergence of allelic chromosomes.

a Overview of Hi-C heatmap for assembled chromosomes. Each allelic group contains four chromosomes, there are few linkages between allelic groups, indicating high quality chromosome-level scaffolding. b Chr1.1 and chr1.2 were chosen as examples to illustrate the assembly quality. The Hi-C heatmap shows the contiguity within chromosomes and the linkage between allelic chromosomes, the depth shows the even coverage of assembled sequence. The syntenic blocks show good synteny between chr1.1 and chr1.2, and two inversions were detected by both Hi-C heatmapping (red rectangles) and synteny (red ribbons). c Frequencies of synonymous distances between syntenic gene pairs, cross-comparing M. sativa (Msa) and M. truncatula (Mtr).

Fig. 3. CRISPR/Cas9-mediated genome editing in autotetraploid cultivated alfalfa.

a Schematic illustration of the Cas9 and sgRNA expression cassettes in the pMS-CRISPR/Cas9 vector (LB left border, RB right border, genes are represented by rectangles and promoters by arrows). The sgRNA consists of a guide sequence (blue rounded rectangle) and a scaffold, and the region between two AarI restriction endonuclease sites (light red) is used for the ligation of guide sequences. b Pipeline for designing guide sequences based on the cultivated alfalfa genome. c Guide sequence for MsPDS. Dark blue boxes and gray lines represent exons and introns, respectively. PAM is shown in red. d, e Genome editing of MsPDS. d Photos of three representative MsPDS mutants (mspds-1, mspds-4, and mspds-5), with mspds-1 exhibiting a wild type phenotype, and mspds-4 and mspds-5 exhibiting dwarf, albino phenotypes. Scale bar, 1 cm. e Sequencing of all screened mutants confirmed the presence of mutations at the target sites (light blue). The PAM regions are shown in black and lowercase. Nucleotide deletions, insertions or substitutions are shown in red, with details given in the right panel.

Fig. 4. Genome editing of MsPALM1, and generating transgene-free and stably inherited palm1-type progenies.

a A guide sequence for MsPALM1. Dark blue box represents exon. PAM is shown in red. The BstUІ site is underlined and shown in light blue. b Leaf morphologies of three representative T0 plants. Scale bar, 1 cm. c Results of PCR-RE analyses for identifying mutants among T0 plants. In the gel, wt and wt-dg lanes contain DNA samples from wild-type plants without and with digestion by the BstUІ restriction endonuclease, respectively. Red arrowheads indicate bands used to identify mutations. Notably, paT0-19 (highlighted with a red rectangle) yields dim digested bands (indicated by white arrows), although it develops palmate-like pentafoliate leaves (b). d Genotyping of the corresponding mutants in b confirmed the presence of mutations at the target sites (light blue). The PAM regions are shown in black and lowercase. Nucleotide deletions, insertions, or substitutions are shown in red, with details in the right panel. e Three representative T1 plants with two showing anticipated palm1-type leaf morphologies like their parents. Scale bar, 1 cm. f, g Results of PCR-RE analyses (f) and sequencing analyses (g) confirmed the parental MsPALM1 mutations in corresponding T1 progenies in e. h Outcome of tests for transgene-free mutants in 20 corresponding T1 progenies in f. Lanes without bands (indicated by red arrowheads) identify transgene-free mutants. Lanes labeled wt, paT0-19 and paT0-46 show PCR fragments amplified from a WT plant, and two T0 mutants (paT0-19 and paT0-46), respectively. Source data underlying Fig. 4c, f, h are provided as a Source Data file.