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 28 to 10 29 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 offtarget 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. PLANT GENETIC VARIABILITY Genetic differences between individuals are the basis of adaptation and evolution. Plant breeding, as a form of directed evolution, has a long history of using genetic diversity for crop improvement. During the process of crop domestication, humans selected individual plants with favorable traits that resulted from novel mutations or standing variation in the ancestral species. The process of selecting plant varieties with favorable characteristics for cultivation and consumption continues to the present day. Modern plant breeding is a more directed process than the crop improvement that occurred through the history and prehistory of most

Plant minichromosomes have the potential for stacking multiple traits on a separate entity from the remainder of the genome. Transgenes carried on an independent chromosome would facilitate conferring many new properties to plants and using minichromosomes as genetic tools. The favored method for producing plant minichromosomes is telomere-mediated chromosomal truncation because the epigenetic nature of centromere function prevents using centromere sequences to confer the ability to organize a kinetochore when reintroduced into plant cells. Because haploid induction procedures are not always complete in eliminating one parental genome, chromosomes from the inducer lines are often present in plants that are otherwise haploid. This fact suggests that minichromosomes could be combined with doubled haploid breeding to transfer stacked traits more easily to multiple lines and to use minichromosomes for massive scale genome editing.

Site‐specific recombinase enzymes function in heterologous cellular environments to initiate strand‐switching reactions between unique DNA sequences termed recombinase binding sites. Depending on binding site position and orientation, reactions result in integrations, excisions, or inversions of targeted DNA sequences in a precise and predictable manner. Here, we established five different stable recombinase expression lines in maize through Agrobacterium‐mediated transformation of T‐DNA molecules that contain coding sequences for Cre, R, FLPe, phiC31 Integrase, and phiC31 excisionase. Through the bombardment of recombinase activated DsRed transient expression constructs, we have determined that all five recombinases are functional in maize plants. These recombinase expression lines could be utilized for a variety of genetic engineering applications, including selectable marker removal, targeted transgene integration into predetermined locations, and gene stacking.

Minichromosomes have been generated in maize using telomere‐mediated truncation. Telomere DNA, because of its repetitive nature, can be difficult to manipulate. The protocols in this unit describe two methods for generating the telomere DNA required for the initiation of telomere‐mediated truncation. The resulting DNA can then be used with truncation cassettes for introduction into maize via transformation. © 2016 by John Wiley & Sons, Inc.

Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium‐mediated transformation. However, the large size of BiBACs and low‐copy number in Escherichia coli (DH10B) and Agrobacterium tumefaciens make cloning experiments more difficult than other available binary vector systems. Therefore, a protocol that outlines preparation, modification, and transformation of high‐molecular weight (HMW) constructs is advantageous for researchers looking to use BiBACs in plant genomics research. This unit does not cover the cloning of HMW DNA into BiBAC vectors. Researchers looking to clone HMW DNA into BiBACs can refer to Zhang et al. (2012; doi: https://doi.org/10.1038/nprot.2011.456). © 2017 by John Wiley & Sons, Inc.