Past innovations and future possibilities in plant chromosome engineering

Abstract

Plant chromosome engineering has emerged as a pivotal tool in modern plant breeding, facilitating the transfer of desirable traits through the incorporation of alien chromosome fragments into plants. Here, we provide a comprehensive overview of the past achievements, current methodologies and future prospects of plant chromosome engineering. We begin by examining the successful integration of specific examples such as the incorporation of rye chromosome segments (e.g. the 1BL/1RS translocation), Dasypyrum villosum segments (e.g. the 6VS segment for powdery mildew resistance), Thinopyrum intermedium segments (e.g. rust resistance genes) and Thinopyrum elongatum segments (e.g. Fusarium head blight resistance genes). In addition to trait transfer, advancements in plant centromere engineering have opened new possibilities for chromosomal manipulation. This includes the development of plant minichromosomes via centromere-mediated techniques, the generation of haploids through CENH3 gene editing, and the induction of aneuploidy using KaryoCreate. The advent of CRISPR/Cas technology has further revolutionized chromosome engineering, enabling large-scale chromosomal rearrangements, such as inversions and translocations, as well as enabling targeted insertion of large DNA fragments and increasing genetic recombination frequency. These advancements have significantly expanded the toolkit for genetic improvement in plants, opening new horizons for the future of plant breeding.

Keywords: CRISPR/Cas technology; Plant chromosome engineering; centromere; minichromosome.

Figure 1

Integration of alien chromosome segments into wheat. The phenotypes of S. cereale (a), D. villosum (b), Th. intermedium (c) and Th. elongatum (d) have been instrumental in wheat genetic improvement. This includes the development of key translocations such as 1BL/1RS from S. cereale (a), 6VS/6AL from D. villosum (b), and the creation of wheat varieties like Zhongke15 (c) and Zhongke166 (d) derived, respectively, from Th. intermedium and Th. elongatum. The genomic DNA of S. cereale (green), D. villosum (red), Th. intermedium (green) and Th. elongatum (green) was used as a probe.

Figure 2

Methods for developing wheat‐related species translocation lines. (a) Ionizing radiation: This method induces chromosome breaks and facilitates the exchange of segments between wheat and related species. (b) A schematic representation of chromosome pairing regulated by Ph1. (c) Gametocidal chromosomes from related species induce chromosome breakage in wheat, leading to the preferential retention and incorporation of alien chromosome segments during gametogenesis.

Figure 3

Manipulating centromeres for the generation of haploid inducers, minichromosomes and aneuploidy. (a) Haploid generation using CENH3 variants: Haploids are achieved by employing CENH3 variants that either modify or compromise centromere integrity. When these variant lines are crossed with wild‐type counterparts, the resulting offspring exhibit haploid formation due to the elimination of chromosomes from one parent. (b) Top‐Down strategy for synthetic chromosome generation using the LexO‐LexA system. A LexA‐CENH3 fusion protein is tethered to a chromosomal LexO repeat array, initiating the formation of a de novo centromere. By combining de novo centromere formation with targeted telomere seeding near the tandem repeats, a minichromosome can be released. (c) Schematic representation of the KaryoCreate system for generating chromosome‐specific aneuploidies. The system utilizes co‐expression of sgRNA targeting chromosome‐specific CENPA‐binding α‐satellite repeats and a dead Cas9 (dCas9) fused to a mutant KNL1. Upon introduction into cells, the system facilitates controlled mis‐segregation of the targeted chromosomes, resulting in precise gains or losses of the specific chromosome in the cellular progeny.

Figure 4

CRISPR‐mediated plant chromosome engineering. (a) Targeted chromosomal inversions can effectively disrupt genetic linkage between two closely associated genes. This process involves inducing a DSB at a specific location between the two linked genes. Following the induction of the inversion, the physical separation of these genes occurs, breaking their genetic linkage. (b) The PrimeRoot system is utilized for the precise insertion of large DNA segments, including genes of interest, into a target genome. (c) CRISPR‐Cas9‐induced DSBs effectively separate linked genes, enabling independent trait segregation and enhancing plant agronomic characteristics.