Targeted mutagenesis with sequence‐specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

Abstract

Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome‐editing platforms employ sequence‐specific nucleases (SSNs) to generate DNA double‐stranded breaks at their target gene. Such DNA breaks are typically repaired via the error‐prone nonhomologous end‐joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end‐use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN‐mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

Targeted genome editing in polyploids: lessons from Brassica

“…Due to a sequence resource deficit, and biased gene expression, genome sequencing is often limited to a model plant within a species. This can pose difficulties in selecting target genes, which can be addressed by using translational genomics approaches to develop genotype-phenotype connections ( May et al., 2023 ).…”

“…Medium-throughput PCR-based assays, such as cleaved amplified polymorphic sequences (CAPS), T7 endonuclease 1 (T7E1), high-resolution melting analysis (HRMA), and capillary electrophoresis, which are typically used for molecular characterization of edited genes in diploid crops, may not provide a detailed characterization of the edited alleles/genes in polyploids. High sequence depth and longer read lengths are required to distinguish between the number of edited alleles/genes, leading to increased costs and labour involved in the genotyping of polyploids ( May et al., 2023 ). Sanger and Illumina high-throughput sequencing are commonly used for the characterization of the type and extent of mutation(s) in polyploid crops.…”

Genome editing and chromosome engineering in plants

“…In plant genome engineering, however, the polyploid genomes and genetic redundancy from multiple-member gene families often present additional technical and practical challenges in gene function characterization, trait discovery, and crop improvement. May et al (2023) reviewed the progress, obstacles, and optimizations that are needed to achieve efficient targeted mutagenesis in polyploids. To address genetic redundancy issues, Zheng et al (2023) showcased the generation of single, double, and triple knockouts of 11 members in the Cytokinin oxidase/dehydrogenase (CKX) gene family in rice.…”

“…May et al. (2023) reviewed the progress, obstacles, and optimizations that are needed to achieve efficient targeted mutagenesis in polyploids. To address genetic redundancy issues, Zheng et al.…”

Strategies for delivery of CRISPR/Cas-mediated genome editing to obtain edited plants directly without transgene integration

“…Typical protocols to make gene edited (GE) varieties also use transgenes, even though the transgenes may be undesirable in the final crop product. For this reason, the techniques that are effective for crops such as maize or tomato may be impractical for crops with long generation time, that are vegetatively propagated, reproduce through apomixis, are polyploid, self-incompatible, and/or highly heterozygous ( May et al, 2023 ). Desirable allele combinations in crops such as grape, potatoes, or sugarcane would be lost due to allele segregation during meiosis ( Veillet et al, 2019 ; Krishna et al, 2023 ).…”