Plant Genome Editing and the Relevance of Off-Target Changes.

Site-directed nucleases (SDNs) used for targeted genome editing are powerful new tools to introduce precise genetic changes into plants. Like traditional approaches, such as conventional crossing and induced mutagenesis, genome editing aims to improve crop yield and nutrition. Next-generation sequencing studies demonstrate that across their genomes, populations of crop species typically carry millions of single nucleotide polymorphisms and many copy number and structural variants. Spontaneous mutations occur at rates of ∼10-8 to 10-9 per site per generation, while variation induced by chemical treatment or ionizing radiation results in higher mutation rates. In the context of SDNs, an off-target change or edit is an unintended, nonspecific mutation occurring at a site with sequence similarity to the targeted edit region. SDN-mediated off-target changes can contribute to a small number of additional genetic variants compared to those that occur naturally in breeding populations or are introduced by induced-mutagenesis methods. Recent studies show that using computational algorithms to design genome editing reagents can mitigate off-target edits in plants. Finally, crops are subject to strong selection to eliminate off-type plants through well-established multigenerational breeding, selection, and commercial variety development practices. Within this context, off-target edits in crops present no new safety concerns compared to other breeding practices. The current generation of genome editing technologies is already proving useful to develop new plant varieties with consumer and farmer benefits. Genome editing will likely undergo improved editing specificity along with new developments in SDN delivery and increasing genomic characterization, further improving reagent design and application.

Use of selection with recurrent backcrossing and QTL mapping to identify loci contributing to southern leaf blight resistance in a highly resistant maize line.

B73 is a historically important maize line with excellent yield potential but high susceptibility to the foliar disease southern leaf blight (SLB). NC292 and NC330 are B73 near-isogenic lines (NILs) that are highly resistant to SLB. They were derived by repeated backcrossing of an elite source of SLB resistance (NC250P) to B73, with selection for SLB resistance among and within backcross families. The goal of this paper was to characterize the loci responsible for the increased SLB resistance of NC292 and NC330 and to determine how many of the SLB disease resistance quantitative trait loci (dQTL) were selected for in the development of NC292 and NC330. Genomic regions that differentiated NC292 and NC330 from B73 and which may contribute to NC292 and NC330s enhanced SLB resistance were identified. Ten NC250P-derived introgressions were identified in both the NC292 and NC330 genomes of which eight were shared between genomes. dQTL were mapped in two F(2:3) populations derived from lines very closely related to the original parents of NC292 and NC330--(B73rhm1 x NC250A and NC250A x B73). Nine SLB dQTL were mapped in the combined populations using combined SLB disease data over all locations (SLB AllLocs). Of these, four dQTL precisely colocalized with NC250P introgressions in bins 2.05-2.06, 3.03, 6.01, and 9.02 and three were identified near NC250P introgressions in bins 1.09, 5.05-5.06, and 10.03. Therefore the breeding program used to develop NC292 and NC330 was highly effective in selecting for multiple SLB resistance alleles.