A catalogue of recombination coldspots in interspecific tomato hybrids.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.

Critical Steps in DAPI and FISH Imaging of Chromosome Spread Preparations.

The final step in a long period of chromosome slide experiments is the publication of DAPI and multicolor fluorescence images. Quite often the result of published artwork is disappointing due to insufficient knowledge of image processing and presentation. In this chapter we describe some errors of fluorescence photomicrographs and how to avoid them. We include suggestions of processing chromosome images with simple examples of image processing in Photoshop® or the like, without the need of complex knowledge of the software programs.

Extended DNA Fibers for High-Resolution Mapping.

DNA fiber-FISH is an easy and simple light microscopic method to map unique and repeat sequences relative to each other at the molecular scale. A standard fluorescence microscope and a DNA labeling kit are sufficient to visualize DNA sequences from any tissue or organ. Despite the enormous progress of high-throughput sequencing technologies, DNA fiber-FISH remains a unique and indispensable tool to detect chromosomal rearrangements and to demonstrate differences between related species at high resolution. We discuss standard and alternative steps to easily prepare extended DNA fibers for high-resolution FISH mapping.

2D morphometric analysis of Arabidopsis thaliana nuclei reveals characteristic profiles of different cell types and accessions.

Functional changes of cells upon developmental switches and in response to environmental cues are often reflected in nuclear phenotypes, showing distinctive chromatin states corresponding to transcriptional changes. Such characteristic nuclear shapes have been microscopically monitored and can be quantified after differential staining of euchromatin and heterochromatin domains. Here, we examined several nuclear parameters (size, DNA content, DNA density, chromatin compaction, relative heterochromatin fraction (RHF), and number of chromocenters) in relation to spatial distribution of genes and transposon elements (TEs), using standard 2D fluorescence microscopy. We provide nuclear profiles for different cell types and different accessions of Arabidopsis thaliana. A variable, yet significant, fraction of TEs was found outside chromocenters in all cell types, except for guard cells. The latter cell type features nuclei with the highest level of chromatin compaction, while their chromocenters seem to contain gene-rich regions. The highest number of parameter correlations was found in the accession Cvi, whereas Ler showed only few correlations. This may point at differences in phenotype robustness between accessions. The significantly high association of NOR chromocenters in accessions Ws and Cvi corresponds to their low RHF level.

Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering.

Chromosomal inversions are recurrent rearrangements that occur between different plant isolates or cultivars. Such inversions may underlie reproductive isolation in evolution and represent a major obstacle for classical breeding as no crossovers can be observed between inverted sequences on homologous chromosomes. The heterochromatic knob (hk4S) on chromosome 4 is the most well-known inversion of Arabidopsis. If a knob carrying accession such as Col-0 is crossed with a knob-less accession such as Ler-1, crossovers cannot be recovered within the inverted region. Our work shows that by egg-cell specific expression of the Cas9 nuclease from Staphylococcus aureus, a targeted reversal of the 1.1 Mb long hk4S-inversion can be achieved. By crossing Col-0 harbouring the rearranged chromosome 4 with Ler-1, meiotic crossovers can be restored into a region with previously no detectable genetic exchange. The strategy of somatic chromosome engineering for breaking genetic linkage has huge potential for application in plant breeding.

Tidying-up the plant nuclear space: domains, functions, and dynamics.

Understanding how the packaging of chromatin in the nucleus is regulated and organized to guide complex cellular and developmental programmes, as well as responses to environmental cues is a major question in biology. Technological advances have allowed remarkable progress within this field over the last years. However, we still know very little about how the 3D genome organization within the cell nucleus contributes to the regulation of gene expression. The nuclear space is compartmentalized in several domains such as the nucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without the presence of a membrane around these domains. The role of these domains and their possible impact on nuclear activities is currently under intense investigation. In this review, we discuss new data from research in plants that clarify functional links between the organization of different nuclear domains and plant genome function with an emphasis on the potential of this organization for gene regulation.

Meiotic recombination profiling of interspecific hybrid F1 tomato pollen by linked read sequencing.

Genome wide screening of pooled pollen samples from a single interspecific F1 hybrid obtained from a cross between tomato, Solanum lycopersicum and its wild relative, Solanum pimpinellifolium using linked read sequencing of the haploid nuclei, allowed profiling of the crossover (CO) and gene conversion (GC) landscape. We observed a striking overlap between cold regions of CO in the male gametes and our previously established F6 recombinant inbred lines (RILs) population. COs were overrepresented in non-coding regions in the gene promoter and 5'UTR regions of genes. Poly-A/T and AT rich motifs were found enriched in 1 kb promoter regions flanking the CO sites. Non-crossover associated allelic and ectopic GCs were detected in most chromosomes, confirming that besides CO, GC represents also a source for genetic diversity and genome plasticity in tomato. Furthermore, we identified processed break junctions pointing at the involvement of both homology directed and non-homology directed repair pathways, suggesting a recombination machinery in tomato that is more complex than currently anticipated.

CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid.

Crossover formation during meiosis in plants is required for proper chromosome segregation and is essential for crop breeding as it allows an (optimal) combination of traits by mixing parental alleles on each chromosome. Crossover formation commences with the production of a large number of DNA double-strand breaks, of which only a few result in crossovers. A small number of genes, which drive the resolution of DNA crossover intermediate structures towards non-crossovers, have been identified in Arabidopisis thaliana. In order to explore the potential of modification of these genes in interspecific hybrids between crops and their wild relatives towards increased production of crossovers, we have used CRISPR/Cas9-mutagenesis in an interspecific tomato hybrid to knockout RecQ4. A biallelic recq4 mutant was obtained in the F1 hybrid of Solanum lycopersicum and S. pimpinellifolium. Compared with the wild-type F1 hybrid, the F1 recq4 mutant was shown to have a significant increase in crossovers: a 1.53-fold increase when directly observing ring bivalents in male meiocytes microscopically and a 1.8-fold extension of the genetic map when measured by analysing SNP markers in the progeny (F2) plants. This is one of the first demonstrations of increasing crossover frequency in interspecific hybrids by manipulating genes in crossover intermediate resolution pathways and the first to do so by directed mutagenesis. SIGNIFICANCE STATEMENT: Increasing crossover frequency during meiosis can speed up or simplify crop breeding that relies on meiotic crossovers to introduce favourable alleles controlling important traits from wild relatives into crops. Here we show for the first time that knocking out an inhibitor of crossovers in an interspecific hybrid between tomato and its relative wild species using CRISPR/Cas9-mutagenesis results in increased recombination between the two genomes.

3D genome organization: a role for phase separation and loop extrusion?

In eukaryotes, genomic information is encoded in chromosomes, which occupy distinct territories within the nucleus. Inside these territories, chromosomes are folded in a hierarchical set of topological structures, called compartments, topologically associated domains and loops. Phase separation and loop extrusion are the mechanisms indicated to mediate the 3D organization of the genome, and gene activity and epigenetic marks determine the activity level of the formed chromatin domains. The main difference between plants and animals may be the absence of canonical insulator elements in plants. Comparison across plant species indicates that the identification of chromatin domains is affected by genome size, gene density, and the linear distribution of genes and transposable elements.

Extensive nuclear reprogramming and endoreduplication in mature leaf during floral induction.

BACKGROUND: The floral transition is a complex developmental event, fine-tuned by various environmental and endogenous cues to ensure the success of offspring production. Leaves are key organs in sensing floral inductive signals, such as a change in light regime, and in the production of the mobile florigen. CONSTANS and FLOWERING LOCUS T are major players in leaves in response to photoperiod. Morphological and molecular events during the floral transition have been intensively studied in the shoot apical meristem. To better understand the concomitant processes in leaves, which are less described, we investigated the nuclear changes in fully developed leaves during the time course of the floral transition. RESULTS: We highlighted new putative regulatory candidates of flowering in leaves. We observed differential expression profiles of genes related to cellular, hormonal and metabolic actions, but also of genes encoding long non-coding RNAs and new natural antisense transcripts. In addition, we detected a significant increase in ploidy level during the floral transition, indicating endoreduplication. CONCLUSIONS: Our data indicate that differentiated mature leaves, possess physiological plasticity and undergo extensive nuclear reprogramming during the floral transition. The dynamic events point at functionally related networks of transcription factors and novel regulatory motifs, but also complex hormonal and metabolic changes.

Protocol for Chromatin Immunoprecipitation of Meiotic-Stage-Specific Tomato Anthers.

Interactions occurring between DNA and proteins across the nuclear genome regulate numerous processes, including meiosis. Meiosis ensures genetic variation and balanced segregation of homologous chromosomes. It involves complex DNA-protein interactions across the entire genome to regulate a broad range of processes, including formation and repair of double-strand DNA breaks (DSBs), chromosome compaction, homolog pairing, synapsis, and homologous recombination. The latter meiotic event, meiotic recombination, often occurs at discrete locations in a genome, within a tight time window. The identification of genomic binding sites of meiotic proteins is a major step toward understanding the molecular mechanisms underlying meiotic recombination and provides important information for plant breeding. Collecting meiotic cells from plants is challenging, tedious, and time consuming, since the meiocyte-producing organs, the anthers, are generally small and limited to certain developmental stages of plants. Here we provide a protocol to isolate meiotic-stage-specific anthers and perform ChIP on this material. We have developed a ChIP protocol specifically suited to (1) small amounts of input material and (2) proteins that bind transiently to chromatin and at very low frequency. © 2018 by John Wiley & Sons, Inc.

Fluorescence In Situ Hybridization (FISH) and Immunolabeling on 3D Preserved Nuclei.

The spatial distribution of genes in the nucleus emerges as an important factor in gene regulation and epigenetics. The position of loci relative to each other, to nuclear landmarks such as the nucleolus and chromocenters, as well as to chromatin proteins is therefore highly interesting. With fluorescent in situ hybridization (FISH) specific DNA sequences can be stained and antibodies allow the detection of specific proteins. Here, we present two protocols that preserve the 3D structure of nuclei. With whole-mount FISH, specific sequences can be stained in intact tissues and, secondly, a combined immunolabeling and FISH protocol on acrylamide-embedded nuclei makes it possible to stain DNA sequences and proteins simultaneously.

Genetic Dissection of Morphometric Traits Reveals That Phytochrome B Affects Nucleus Size and Heterochromatin Organization in Arabidopsis thaliana.

Microscopically visible chromatin is partitioned into two major components in Arabidopsis thaliana nuclei. On one hand, chromocenters are conspicuous foci of highly condensed "heterochromatic" domains that contain mostly repeated sequences. On the other hand, less condensed and gene-rich "euchromatin" emanates from these chromocenters. This differentiation, together with the dynamic nature of chromatin compaction in response to developmental and environmental stimuli, makes Arabidopsis a powerful system for studying chromatin organization and dynamics. Heterochromatin dynamics can be monitored by measuring the Heterochromatin Index, i.e., the proportion of nuclei displaying well-defined chromocenters, or the DNA fraction of chromocenters (relative heterochromatin fraction). Both measures are composite traits, thus their values represent the sum of effects of various underlying morphometric properties. We exploited genetic variation between natural occurring accessions to determine the genetic basis of individual nucleus and chromocenter morphometric parameters (area, perimeter, density, roundness, and heterogeneity) that together determine chromatin compaction. Our novel reductionist genetic approach revealed quantitative trait loci (QTL) for all measured traits. Genomic colocalization among QTL was limited, which suggests a complex genetic regulation of chromatin compaction. Yet genomic intervals of QTL for nucleus size (area and perimeter) both overlap with a known QTL for heterochromatin compaction that is explained by natural polymorphism in the red/far-red light and temperature receptor Phytochrome B. Mutant analyses and genetic complementation assays show that Phytochrome B is a negative regulator of nucleus size, revealing that perception of climatic conditions by a Phytochrome-mediated hub is a major determinant for coordinating nucleus size and heterochromatin compaction.

Molecular, genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana.

Chromosomal inversions can provide windows onto the cytogenetic, molecular, evolutionary and demographic histories of a species. Here we investigate a paracentric 1.17-Mb inversion on chromosome 4 of Arabidopsis thaliana with nucleotide precision of its borders. The inversion is created by Vandal transposon activity, splitting an F-box and relocating a pericentric heterochromatin segment in juxtaposition with euchromatin without affecting the epigenetic landscape. Examination of the RegMap panel and the 1001 Arabidopsis genomes revealed more than 170 inversion accessions in Europe and North America. The SNP patterns revealed historical recombinations from which we infer diverse haplotype patterns, ancient introgression events and phylogenetic relationships. We find a robust association between the inversion and fecundity under drought. We also find linkage disequilibrium between the inverted region and the early flowering Col-FRIGIDA allele. Finally, SNP analysis elucidates the origin of the inversion to South-Eastern Europe approximately 5000 years ago and the FRI-Col allele to North-West Europe, and reveals the spreading of a single haplotype to North America during the 17th to 19th century. The 'American haplotype' was identified from several European localities, potentially due to return migration.

Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture.

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.

Light signaling controls nuclear architecture reorganization during seedling establishment.

The spatial organization of chromatin can be subject to extensive remodeling in plant somatic cells in response to developmental and environmental signals. However, the mechanisms controlling these dynamic changes and their functional impact on nuclear activity are poorly understood. Here, we determined that light perception triggers a switch between two different nuclear architectural schemes during Arabidopsis postembryonic development. Whereas progressive nucleus expansion and heterochromatin rearrangements in cotyledon cells are achieved similarly under light and dark conditions during germination, the later steps that lead to mature nuclear phenotypes are intimately associated with the photomorphogenic transition in an organ-specific manner. The light signaling integrators DE-ETIOLATED 1 and CONSTITUTIVE PHOTOMORPHOGENIC 1 maintain heterochromatin in a decondensed state in etiolated cotyledons. In contrast, under light conditions cryptochrome-mediated photoperception releases nuclear expansion and heterochromatin compaction within conspicuous chromocenters. For all tested loci, chromatin condensation during photomorphogenesis does not detectably rely on DNA methylation-based processes. Notwithstanding, the efficiency of transcriptional gene silencing may be impacted during the transition, as based on the reactivation of transposable element-driven reporter genes. Finally, we report that global engagement of RNA polymerase II in transcription is highly increased under light conditions, suggesting that cotyledon photomorphogenesis involves a transition from globally quiescent to more active transcriptional states. Given these findings, we propose that light-triggered changes in nuclear architecture underlie interplays between heterochromatin reorganization and transcriptional reprogramming associated with the establishment of photosynthesis.

Introgression browser: high-throughput whole-genome SNP visualization.

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker-assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi-nucleotide polymorphisms (MNPs) and insertion-deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP-free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.