Plant chromosome engineering – past, present and future

Puchta, Holger; Houben, Andreas

doi:10.1111/nph.19414

Cited by 5 publications

(1 citation statement)

References 141 publications

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“…However, the length of the exogenous radish chromosome was expected to be shorter than that of R2000, because R2000 was bred using irradiation to reduce the length of exogenous radish chromosome fragments [ 10 ]. As is known, subjecting organisms to X-ray irradiation can lead to chromosome fragment breakage [ 50 ]. As proven, the exogenous radish chromosome fragment can be efficiently shortened, although other methods have also been established to achieve this.…”

Section: Discussionmentioning

confidence: 99%

Cytological and Molecular Characterization of a New Ogura Cytoplasmic Male Sterility Restorer of Brassica napus L.

Huang,

Ren,

Lin

et al. 2024

Plants

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Ogura cytoplasmic male sterility (CMS) is considered the rapeseed (Brassica napus L.) with the most potential to be utilized as a heterosis system worldwide, but it lacks sufficient restorers. In this study, root tip cell (RTC) mitotic and pollen mother cell (PMC) meiosis observations were compared to ensure the number of chromosomes and the formation of a chromosomal bridge using restorer lines R2000, CLR650, and Zhehuhong (a new restorer) as the experimental material. Further, molecular markers of exogenous chromosomal fragments were detected and the sequence and expression differences of restorer genes in the three lines were determined to identify the distinctive characteristics of Zhehuhong. The results showed that the number of chromosomes in Zhehuhong was stable (2n = 38), indicating that the exogenous radish chromosome segment had been integrated into the chromosome of Zhehuhong. Molecular marker detection revealed that Zhehuhong was detected at most loci, with only the RMA05 locus being missed. The exogenous radish chromosome segment of Zhehuhong differed from R2000 and CLR650. The pollen mother cells of Zhehuhong showed chromosome lagging in the meiotic metaphase I, meiotic anaphase I, and meiotic anaphase II, which was consistent with R2000 and CLR650. The restorer gene PPRB in Zhehuhong had 85 SNPs compared with R2000 and 119 SNPs compared with CLR650, indicating the distinctive characteristic of PPRB in Zhehuhong. In terms of the spatial expression of PPRB, the highest level was detected in the anthers in the three restorer lines. In addition, in terms of temporal expression, the PPRB gene expression of Zhehuhong was highest at a bud length of 4 mm. Our results clearly indicated that Zhehuhong is a new restorer line for the Ogura CMS system, which can be used further in rapeseed heterosis utilization.

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Section: Discussionmentioning

confidence: 99%

Cytological and Molecular Characterization of a New Ogura Cytoplasmic Male Sterility Restorer of Brassica napus L.

Huang,

Ren,

Lin

et al. 2024

Plants

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CRISPR/Cas9-induced breaks are insufficient to break linkage drag surrounding the ToMV locus ofSolanum lycopersicum

Grubben,

Bijsterbosch,

Aktürk

et al. 2024

Preprint

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Despite the success of CRISPR/Cas9 in inducing DNA double-strand breaks (DSBs) for genome editing, achieving targeted recombination in somatic cells remains challenging, particularly at recombination cold spots like the Tomato Mosaic Virus (ToMV) resistance locus inSolanum lycopersicum. We investigated the potential of CRISPR/Cas9-induced targeted recombination in somatic cells to overcome linkage drag surrounding the ToMV locus. We employed two strategies: first, inducing DSBs in both alleles of F1tomato seedlings to promote non-homologous end joining (NHEJ) and homology-directed repair (HDR); second, targeting a single allele in a heterozygous background to induce HDR in seedlings. CRISPR/Cas9 activity was confirmed in F1seedlings by detecting NHEJ-mediated mutations at the target sites in ToMV. We developed a bioinformatics pipeline to identify targeted recombinants by analyzing single nucleotide polymorphisms (SNPs) between parental haplotypes, allowing precise tracking of SNP variations. A two-dimensional pooling strategy was employed to distinguish genuine recombination events from PCR artifacts. Despite these advances and the active CRISPR/Cas9 system in F1progeny, no increase in recombination frequency was observed compared to wild-type plants. We extended our research to protoplasts to assess whether CRISPR/Cas9 could induce targeted recombination under different cellular conditions at the same locus. Consistent with our findings in F1plants, we observed no increase in recombinant patterns compared to wild-type controls in protoplasts. Our findings suggest that CRISPR/Cas9-induced DSBs are insufficient to break the genetic linkage at the ToMV locus on chromosome 9 in recombination cold spots within somatic cells.

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Epigenetic state and gene expression remain stable after CRISPR/Cas-mediated chromosomal inversions

Khosravi,

Hinrichs,

Rönspies

et al. 2024

Preprint

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In Arabidopsis thaliana, the chromosome arms are DNA-hypomethylated and enriched in the euchromatin-specific histone mark H3K4me3. In contrast, pericentromeric regions are DNA-hypermethylated and enriched in H3K9me2. In order to investigate how chromosomal location affects epigenetic stability and gene activity, we induced two differently-sized inversions by CRISPR/Cas and introduced heterochromatic, pericentric sequences into an euchromatic chromosomal arm. The epigenetic status of the lines was investigated by whole genome bisulfite sequencing and chromatin immunoprecipitation. Additionally, we studied the effect of the chromosomal inversions on gene expression. Our analysis revealed that both inversions affected neither the global distribution of eu- and heterochromatin-specific histone marks nor the global DNA methylation landscape. However, minor epigenetic changes were found across the entire genome. Importantly, the inverted chromosome regions and their border regions did not change their epigenetic profile. The transcription analysis of both inversion lines revealed that 0.5 - 1 % of genes were differentially expressed across the entire genome. However, the expression activity of those genes encoded by the inverted segments, and located around the cutting sites of CRISPR/Cas was less affected. Thus, gene expression levels and the epigenetic landscape remain preserved following engineered chromosomal restructuring, at least in the following generations.

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Plant chromosome engineering – past, present and future

Cited by 5 publications

References 141 publications

Cytological and Molecular Characterization of a New Ogura Cytoplasmic Male Sterility Restorer of Brassica napus L.

Cytological and Molecular Characterization of a New Ogura Cytoplasmic Male Sterility Restorer of Brassica napus L.

CRISPR/Cas9-induced breaks are insufficient to break linkage drag surrounding the ToMV locus ofSolanum lycopersicum

Epigenetic state and gene expression remain stable after CRISPR/Cas-mediated chromosomal inversions

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