CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements.

DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole-genome sequencing and molecular and cytological analyses supports a model in which a breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.

Evolution and origin of bread wheat.

Bread wheat (Triticum aestivum, genome BBAADD) is a young hexaploid species formed only 8,500-9,000 years ago through hybridization between a domesticated free-threshing tetraploid progenitor, genome BBAA, and Aegilops tauschii, the diploid donor of the D subgenome. Very soon after its formation, it spread globally from its cradle in the fertile crescent into new habitats and climates, to become a staple food of humanity. This extraordinary global expansion was probably enabled by allopolyploidy that accelerated genetic novelty through the acquisition of new traits, new intergenomic interactions, and buffering of mutations, and by the attractiveness of bread wheat's large, tasty, and nutritious grain with high baking quality. New genome sequences suggest that the elusive donor of the B subgenome is a distinct (unknown or extinct) species rather than a mosaic genome. We discuss the origin of the diploid and tetraploid progenitors of bread wheat and the conflicting genetic and archaeological evidence on where it was formed and which species was its free-threshing tetraploid progenitor. Wheat experienced many environmental changes throughout its evolution, therefore, while it might adapt to current climatic changes, efforts are needed to better use and conserve the vast gene pool of wheat biodiversity on which our food security depends.

Genome sequences of five Sitopsis species of Aegilops and the origin of polyploid wheat B subgenome.

Common wheat (Triticum aestivum, BBAADD) is a major staple food crop worldwide. The diploid progenitors of the A and D subgenomes have been unequivocally identified; that of B, however, remains ambiguous and controversial but is suspected to be related to species of Aegilops, section Sitopsis. Here, we report the assembly of chromosome-level genome sequences of all five Sitopsis species, namely Aegilops bicornis, Ae. longissima, Ae. searsii, Ae. sharonensis, and Ae. speltoides, as well as the partial assembly of the Amblyopyrum muticum (synonym Aegilops mutica) genome for phylogenetic analysis. Our results reveal that the donor of the common wheat B subgenome is a distinct, and most probably extinct, diploid species that diverged from an ancestral progenitor of the B lineage to which the still extant Ae. speltoides and Am. muticum belong. In addition, we identified interspecific genetic introgressions throughout the evolution of the Triticum/Aegilops species complex. The five Sitopsis species have various assembled genome sizes (4.11-5.89 Gb) with high proportions of repetitive sequences (85.99%-89.81%); nonetheless, they retain high collinearity with other genomes or subgenomes of species in the Triticum/Aegilops complex. Differences in genome size were primarily due to independent post-speciation amplification of transposons. We also identified a set of Sitopsis genes pertinent to important agronomic traits that can be harnessed for wheat breeding. These newly assembled genome resources provide a new roadmap for evolutionary and genetic studies of the Triticum/Aegilops complex, as well as for wheat improvement.

Targeted Inter-Homologs Recombination in Arabidopsis Euchromatin and Heterochromatin.

Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both "hot and cold" regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding.

Plant genome engineering from lab to field-a Keystone Symposia report.

Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On March 8 and 9, 2021, experts in plant genome engineering and breeding from academia and industry met virtually for the Keystone eSymposium "Plant Genome Engineering: From Lab to Field" to discuss advances in genome editing tools, plant transformation, plant breeding, and crop trait development, all vital for transferring the benefits of novel technologies to the field.

Independent evolution of transcript abundance and gene regulatory dynamics.

Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.

Homoeologous exchanges occur through intragenic recombination generating novel transcripts and proteins in wheat and other polyploids.

Recombination between homeologous chromosomes, also known as homeologous exchange (HE), plays a significant role in shaping genome structure and gene expression in interspecific hybrids and allopolyploids of several plant species. However, the molecular mechanisms that govern HEs are not well understood. Here, we studied HE events in the progeny of a nascent allotetraploid (genome AADD) derived from two diploid progenitors of hexaploid bread wheat using cytological and whole-genome sequence analyses. In total, 37 HEs were identified and HE junctions were mapped precisely. HEs exhibit typical patterns of homologous recombination hotspots, being biased toward low-copy, subtelomeric regions of chromosome arms and showing association with known recombination hotspot motifs. But, strikingly, while homologous recombination preferentially takes place upstream and downstream of coding regions, HEs are highly enriched within gene bodies, giving rise to novel recombinant transcripts, which in turn are predicted to generate new protein fusion variants. To test whether this is a widespread phenomenon, a dataset of high-resolution HE junctions was analyzed for allopolyploid Brassica, rice, Arabidopsis suecica, banana, and peanut. Intragenic recombination and formation of chimeric genes was detected in HEs of all species and was prominent in most of them. HE thus provides a mechanism for evolutionary novelty in transcript and protein sequences in nascent allopolyploids.

CRISPR/Cas9 Induced Somatic Recombination at the CRTISO Locus in Tomato.

Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.

Redistribution of Meiotic Crossovers Along Wheat Chromosomes by Virus-Induced Gene Silencing.

Meiotic recombination is the main driver of genetic diversity in wheat breeding. The rate and location of crossover (CO) events are regulated by genetic and epigenetic factors. In wheat, most COs occur in subtelomeric regions but are rare in centromeric and pericentric areas. The aim of this work was to increase COs in both "hot" and "cold" chromosomal locations. We used Virus-Induced gene Silencing (VIGS) to downregulate the expression of recombination-suppressing genes XRCC2 and FANCM and of epigenetic maintenance genes MET1 and DDM1 during meiosis. VIGS suppresses genes in a dominant, transient and non-transgenic manner, which is convenient in wheat, a hard-to-transform polyploid. F1 hybrids of a cross between two tetraploid lines whose genome was fully sequenced (wild emmer and durum wheat), were infected with a VIGS vector ∼ 2 weeks before meiosis. Recombination was measured in F2 seedlings derived from F1-infected plants and non-infected controls. We found significant up and down-regulation of CO rates along subtelomeric regions as a result of silencing either MET1, DDM1 or XRCC2 during meiosis. In addition, we found up to 93% increase in COs in XRCC2-VIGS treatment in the pericentric regions of some chromosomes. Silencing FANCM showed no effect on CO. Overall, we show that CO distribution was affected by VIGS treatments rather than the total number of COs which did not change. We conclude that transient silencing of specific genes during meiosis can be used as a simple, fast and non-transgenic strategy to improve breeding abilities in specific chromosomal regions.

The Israeli-Palestinian wheat landraces collection: restoration and characterization of lost genetic diversity.

BACKGROUND: For over a century, genetic diversity of wheat worldwide was eroded by continual selection for high yields and industrial demands. Wheat landraces cultivated in Israel and Palestine demonstrate high genetic diversity and a potentially wide repertoire of adaptive alleles. While most Israeli-Palestinian wheat landraces were lost in the transition to 'Green Revolution' semi-dwarf varieties, some germplasm collections made at the beginning of the 20th century survived in gene banks and private collections worldwide. However, fragmentation and poor conservation place this unique genetic resource at a high risk of genetic erosion. Herein, we describe a long-term initiative to restore, conserve, and characterize a collection of Israeli and Palestinian wheat landraces (IPLR). RESULTS: We report on (i) the IPLR construction (n = 932), (ii) the historical and agronomic context to this collection, (iii) the characterization and assessment of the IPLR's genetic diversity, and (iv) a data comparison from two distinct subcollections within IPLR: a collection made by N. Vavilov in 1926 (IPLR-VIR) and a later one (1979-1981) made by Y. Mattatia (IPLR-M). Though conducted in the same eco-geographic space, these two collections were subjected to considerably different conservation pathways. IPLR-M, which underwent only one propagation cycle, demonstrated marked genetic and phenotypic variability (within and between accessions) in comparison with IPLR-VIR, which had been regularly regenerated over ∼90 years. CONCLUSION: We postulate that long-term ex situ conservation involving human and genotype × environment selection may significantly reduce accession heterogeneity and allelic diversity. Results are further discussed in a broader context of pre-breeding and conservation. © 2019 Society of Chemical Industry.

Single Molecule Imaging of T-DNA Intermediates Following Agrobacterium tumefaciens Infection in Nicotiana benthamiana.

Plant transformation mediated by Agrobacterium tumefaciens is a well-studied phenomenon in which a bacterial DNA fragment (T-DNA), is transferred to the host plant cell, as a single strand, via type IV secretion system and has the potential to reach the nucleus and to be integrated into its genome. While Agrobacterium-mediated transformation has been widely used for laboratory-research and in breeding, the time-course of its journey from the bacterium to the nucleus, the conversion from single- to double-strand intermediates and several aspects of the integration in the genome remain obscure. In this study, we sought to follow T-DNA infection directly using single-molecule live imaging. To this end, we applied the LacO-LacI imaging system in Nicotiana benthamiana, which enabled us to identify double-stranded T-DNA (dsT-DNA) molecules as fluorescent foci. Using confocal microscopy, we detected progressive accumulation of dsT-DNA foci in the nucleus, starting 23 h after transfection and reaching an average of 5.4 and 8 foci per nucleus at 48 and 72 h post-infection, respectively. A time-course diffusion analysis of the T-DNA foci has demonstrated their spatial confinement.

The effects of AtRad52 over-expression on homologous recombination in Arabidopsis.

AtRad52 homologs are involved in DNA recombination and repair, but their precise functions in different homologous recombination (HR) pathways or in gene-targeting have not been analyzed. In order to facilitate our analyses, we generated an AtRad52-1A variant that had a stronger nuclear localization than the native gene thanks to the removal of the transit peptide for mitochondrial localization and to the addition of a nuclear localization signal. Over-expression of this variant increased HR in the nucleus, compared with the native AtRad52-1A: it increased intra-chromosomal recombination and synthesis-dependent strand-annealing HR repair rates; but conversely, it repressed the single-strand annealing pathway. The effect of AtRad52-1A over-expression on gene-targeting was tested with and without the expression of small RNAs generated from an RNAi construct containing homology to the target and donor sequences. True gene-targeting events at the Arabidopsis Cruciferin locus were obtained only when combining AtRad52-1A over-expression and target/donor-specific RNAi. This suggests that sequence-specific small RNAs might be involved in AtRad52-1A-mediated HR.

Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system.

Current breeding relies mostly on random mutagenesis and recombination to generate novel genetic variation. However, targeted genome editing is becoming an increasingly important tool for precise plant breeding. Using the CRISPR-Cas system combined with the bean yellow dwarf virus rolling circle replicon, we optimized a method for targeted mutagenesis and gene replacement in tomato. The carotenoid isomerase (CRTISO) and phytoene synthase 1 (PSY1) genes from the carotenoid biosynthesis pathway were chosen as targets due to their easily detectable change of phenotype. We took advantage of the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene, via homologous recombination (HR). Mutagenesis experiments, performed in the Micro-Tom variety, achieved precise modification of the CRTISO and PSY1 loci at an efficiency of up to 90%. In the gene targeting (GT) experiments, our target was a fast-neutron-induced crtiso allele that contained a 281-bp deletion. This deletion was repaired with the wild-type sequence through HR between the CRISPR-Cas-induced DSB in the crtiso target and the amplified donor in 25% of the plants transformed. This shows that efficient GT can be achieved in the absence of selection markers or reporters using a single and modular construct that is adaptable to other tomato targets and other crops.

Genomic features shaping the landscape of meiotic double-strand-break hotspots in maize.

Meiotic recombination is the most important source of genetic variation in higher eukaryotes. It is initiated by formation of double-strand breaks (DSBs) in chromosomal DNA in early meiotic prophase. The DSBs are subsequently repaired, resulting in crossovers (COs) and noncrossovers (NCOs). Recombination events are not distributed evenly along chromosomes but cluster at recombination hotspots. How specific sites become hotspots is poorly understood. Studies in yeast and mammals linked initiation of meiotic recombination to active chromatin features present upstream from genes, such as absence of nucleosomes and presence of trimethylation of lysine 4 in histone H3 (H3K4me3). Core recombination components are conserved among eukaryotes, but it is unclear whether this conservation results in universal characteristics of recombination landscapes shared by a wide range of species. To address this question, we mapped meiotic DSBs in maize, a higher eukaryote with a large genome that is rich in repetitive DNA. We found DSBs in maize to be frequent in all chromosome regions, including sites lacking COs, such as centromeres and pericentromeric regions. Furthermore, most DSBs are formed in repetitive DNA, predominantly Gypsy retrotransposons, and only one-quarter of DSB hotspots are near genes. Genic and nongenic hotspots differ in several characteristics, and only genic DSBs contribute to crossover formation. Maize hotspots overlap regions of low nucleosome occupancy but show only limited association with H3K4me3 sites. Overall, maize DSB hotspots exhibit distribution patterns and characteristics not reported previously in other species. Understanding recombination patterns in maize will shed light on mechanisms affecting dynamics of the plant genome.

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication.

Wheat (Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat's domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer (T. turgidum ssp. dicoccoides). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 (TtBtr1) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties.

T-DNA-genome junctions form early after infection and are influenced by the chromatin state of the host genome.

Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of previously isolated sites of T-DNA integration in Kanamycin-selected transgenic plants showed an association with extremely low methylation and nucleosome occupancy. Conversely, non-selected junctions from this study showed no correlation with methylation and had chromatin marks, such as high nucleosome occupancy and high H3K27me3, that correspond to three-dimensional-interacting heterochromatin islands embedded within euchromatin. Such structures may play a role in capturing and silencing invading T-DNA.

Bread Affects Clinical Parameters and Induces Gut Microbiome-Associated Personal Glycemic Responses.

Bread is consumed daily by billions of people, yet evidence regarding its clinical effects is contradicting. Here, we performed a randomized crossover trial of two 1-week-long dietary interventions comprising consumption of either traditionally made sourdough-leavened whole-grain bread or industrially made white bread. We found no significant differential effects of bread type on multiple clinical parameters. The gut microbiota composition remained person specific throughout this trial and was generally resilient to the intervention. We demonstrate statistically significant interpersonal variability in the glycemic response to different bread types, suggesting that the lack of phenotypic difference between the bread types stems from a person-specific effect. We further show that the type of bread that induces the lower glycemic response in each person can be predicted based solely on microbiome data prior to the intervention. Together, we present marked personalization in both bread metabolism and the gut microbiome, suggesting that understanding dietary effects requires integration of person-specific factors.

Targeted recombination between homologous chromosomes for precise breeding in tomato.

Homologous recombination (HR) between parental chromosomes occurs stochastically. Here, we report on targeted recombination between homologous chromosomes upon somatic induction of DNA double-strand breaks (DSBs) via CRISPR-Cas9. We demonstrate this via a visual and molecular assay whereby DSB induction between two alleles carrying different mutations in the PHYTOENE SYNTHASE (PSY1) gene results in yellow fruits with wild type red sectors forming via HR-mediated DSB repair. We also show that in heterozygote plants containing one psy1 allele immune and one sensitive to CRISPR, repair of the broken allele using the unbroken allele sequence template is a common outcome. In another assay, we show evidence of a somatically induced DSB in a cross between a psy1 edible tomato mutant and wild type Solanum pimpinellifolium, targeting only the S. pimpinellifolium allele. This enables characterization of germinally transmitted targeted somatic HR events, demonstrating that somatically induced DSBs can be exploited for precise breeding of crops.