However, both of them still fall short for rapid generation of pure elite lines with integrated favorable traits. Here, we report the development of a Haploid-Inducer Mediated Genome Editing (IMGE) approach, which utilizes a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize inbred line and to generate genome-edited haploids in the elite maize background. Homozygous pure DH lines with the desired trait improvement could be generated within two generations, thus bypassing the lengthy procedure of repeated crossing and backcrossing used in conventional breeding for integrating a desirable trait into elite commercial backgrounds.
Figure 2: Haploid-Inducer Mediated Genome Editing of ZmLG1.
(A) Images of a B73 diploid plant, an lg1-Haploid plant (D32-17), and an unedited B73-Haploid plant (D39-3).
(B) Images showing the leaf angle of a B73 diploid plant, an lg1-Haploid plant (D32-17), and an unedited B73-Haploid plant (D39-3). The lg1-Haploid plant lacks auricle and ligules.
(C) Sequencing analysis reveals a genomic deletion of ∼8.8 kb in the ZmLG1 genomic region in the lg1-Haploid (LG1-MT) in comparison with the sequence of B73 (LG1-WT). The target sequences and protospacer-adjacent motif (PAM) are shown on the top.
(D) Sequencing analysis of the upstream flanking sequence of ZmLG1 in B73, CAU5LG1-Cas9, and lg1-Haploid (D32-17). The upstream flanking sequence of the edited ZmLG1allele in lg1-Haploid is the same as B73, but differs from that of CAU5LG1-Cas9, indicating that the edited allele of ZmLG1 in lg1-Haploids is of B73 origin.
(E–G) DNA quantification of B73 (E, diploid), an lg1-Haploid (F, D32-17), and an unedited B73-Haploid (G, D39-3) using flow cytometry.
(H) Genotyping of a B73 diploid plant, an lg1-Haploid plant (D32-17), and an unedited B73-Haploid plant (D39-3) using the marker ID4 for detecting the 4-bp insertion in MATL.