Recombination landscape divergence between populations is marked by larger low-recombining regions in domesticated rye.

The genomic landscape of recombination plays an essential role in evolution. Patterns of recombination are highly variable along chromosomes, between sexes, individuals, populations, and species. In many eukaryotes, recombination rates are elevated in sub-telomeric regions and drastically reduced near centromeres, resulting in large low-recombining (LR) regions. The processes of recombination are influenced by genetic factors, such as different alleles of genes involved in meiosis and chromatin structure, as well as external environmental stimuli like temperature and overall stress. In this work, we focused on the genomic landscapes of recombination in a collection of 916 rye (Secale cereale) individuals. By analysing population structure among individuals of different domestication status and geographic origin, we detected high levels of admixture, reflecting the reproductive biology of a self-incompatible, wind-pollinating grass species. We then analysed patterns of recombination in overlapping subpopulations, which revealed substantial variation in the physical size of LR regions, with a tendency for larger LR regions in domesticated subpopulations. Genome-wide association scans (GWAS) for LR region size revealed a major quantitative-trait-locus (QTL) at which, among 18 annotated genes, an ortholog of histone H4 acetyltransferase ESA1 was located. Rye individuals belonging to domesticated subpopulations showed increased synaptonemal complex length, but no difference in crossover frequency, indicating that only the recombination landscape is different. Furthermore, the genomic region harbouring rye ScESA1 showed moderate patterns of selection in domesticated subpopulations, suggesting that larger LR regions were indirectly selected for during domestication to achieve more homogeneous populations for agricultural use.

Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells.

The 3D organization of chromatin plays an important role in genome stability and many other pivotal biological programs. Therefore, the establishment of imaging methods, which enable us to study the dynamics of chromatin in living cells, is necessary. Although primary live cell imaging methods were a breakthrough, there is a need to develop more specific labeling techniques. With the discovery of programmable DNA binding proteins, such zinc finger proteins (ZFP), transcription activator-like effectors (TALE), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a major leap forward was made. Here, we review the applications and potential of fluorescent repressor-operator systems, programmable DNA binding proteins with an emphasis on CRISPR-based chromatin imaging in living and fixed cells, and their potential application in plant science.

Variation in Recombination Rate Is Shaped by Domestication and Environmental Conditions in Barley.

Meiotic recombination generates genetic diversity upon which selection can act. Recombination rates are highly variable between species, populations, individuals, sexes, chromosomes, and chromosomal regions. The underlying mechanisms are controlled at the genetic and epigenetic level and show plasticity toward the environment. Environmental plasticity may be divided into short- and long-term responses. We estimated recombination rates in natural populations of wild barley and domesticated landraces using a population genetics approach. We analyzed recombination landscapes in wild barley and domesticated landraces at high resolution. In wild barley, high recombination rates are found in more interstitial chromosome regions in contrast to distal chromosome regions in domesticated barley. Among subpopulations of wild barley, natural variation in effective recombination rate is correlated with temperature, isothermality, and solar radiation in a nonlinear manner. A positive linear correlation was found between effective recombination rate and annual precipitation. We discuss our findings with respect to how the environment might shape effective recombination rates in natural populations. Higher recombination rates in wild barley populations subjected to specific environmental conditions could be a means to maintain fitness in a strictly inbreeding species.

Natural variation in meiotic recombination rate shapes introgression patterns in intraspecific hybrids between wild and domesticated barley.

Meiotic recombination rates vary considerably between species, populations and individuals. The genetic exchange between homologous chromosomes plays a major role in evolution by breaking linkage between advantageous and deleterious alleles in the case of introgressions. Identifying recombination rate modifiers is thus of both fundamental and practical interest to understand and utilize variation in meiotic recombination rates. We investigated recombination rate variation in a large intraspecific hybrid population (named HEB-25) derived from a cross between domesticated barley and 25 wild barley accessions. We observed quantitative variation in total crossover number with a maximum of a 1.4-fold difference between subpopulations and increased recombination rates across pericentromeric regions. The meiosis-specific α-kleisin cohesin subunit REC8 was identified as a candidate gene influencing crossover number and patterning. Furthermore, we quantified wild barley introgression patterns and revealed how local and genome-wide recombination rate variation shapes patterns of introgression. The identification of allelic variation in REC8 in combination with the observed changes in crossover patterning suggest a difference in how chromatin loops are tethered to the chromosome axis, resulting in reduced crossover suppression across pericentromeric regions. Local and genome-wide recombination rate variation is shaping patterns of introgressions and thereby directly influences the consequences of linkage drag.

RNA-guided endonuclease - in situ labelling (RGEN-ISL): a fast CRISPR/Cas9-based method to label genomic sequences in various species.

Visualising the spatio-temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two-part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN-ISL (RNA-guided endonuclease - in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans-activating crRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry. Real-time visualisation of the CRISPR/Cas9-mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN-ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology.

Live-cell CRISPR imaging in plants reveals dynamic telomere movements.

Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of the regulation of genes and non-coding sequences during development and environmental changes. Emerging techniques of chromatin imaging promise to bridge the long-standing gap between sequencing studies, which reveal genomic information, and imaging studies that provide spatial and temporal information of defined genomic regions. Here, we demonstrate such an imaging technique based on two orthologues of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal dynamic telomere movements of up to 2 µm over 30 min during interphase. Furthermore, we show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. By simultaneously using two dCas9 orthologues, we pave the way for the imaging of multiple genomic loci in live plants cells. CRISPR imaging bears the potential to significantly improve our understanding of the dynamics of chromosomes in live plant cells.

The low-recombining pericentromeric region of barley restricts gene diversity and evolution but not gene expression.

The low-recombining pericentromeric region of the barley genome contains roughly a quarter of the genes of the species, embedded in low-recombining DNA that is rich in repeats and repressive chromatin signatures. We have investigated the effects of pericentromeric region residency upon the expression, diversity and evolution of these genes. We observe no significant difference in average transcript level or developmental RNA specificity between the barley pericentromeric region and the rest of the genome. In contrast, all of the evolutionary parameters studied here show evidence of compromised gene evolution in this region. First, genes within the pericentromeric region of wild barley show reduced diversity and significantly weakened purifying selection compared with the rest of the genome. Second, gene duplicates (ohnolog pairs) derived from the cereal whole-genome duplication event ca. 60MYa have been completely eliminated from the barley pericentromeric region. Third, local gene duplication in the pericentromeric region is reduced by 29% relative to the rest of the genome. Thus, the pericentromeric region of barley is a permissive environment for gene expression but has restricted gene evolution in a sizeable fraction of barley's genes.

Barley stripe mosaic virus-mediated somatic and heritable gene editing in barley (Hordeum vulgare L.).

Genome editing strategies in barley (Hordeum vulgare L.) typically rely on Agrobacterium-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants that constitutively express Cas9. Here, we explored virus-induced genome editing (VIGE) based on barley stripe mosaic virus (BSMV) in Cas9-transgenic barley. Somatic and heritable editing in the ALBOSTRIANS gene (CMF7) resulting in albino/variegated chloroplast-defective barley mutants is shown. In addition, somatic editing in meiosis-related candidate genes in barley encoding ASY1 (an axis-localized HORMA domain protein), MUS81 (a DNA structure-selective endonuclease), and ZYP1 (a transverse filament protein of the synaptonemal complex) was achieved. Hence, the presented VIGE approach using BSMV enables rapid somatic and also heritable targeted gene editing in barley.

Quantification of Recombination Rate and Segregation Distortion by Genotyping and Sequencing of Single Pollen Nuclei.

Meiosis is a specialized cell division during which homologous chromosomes can exchange genetic material through recombination. This mechanism generates novel allelic combinations, which can be exploited by plant breeders to achieve crop improvement. Pollen grains are the haploid products of meiosis required in fertilization. Here, we describe two approaches to measure meiotic recombination in single haploid pollen nuclei. Pollen nuclei are first separated by fluorescence-activated cell-sorting. Afterwards, the DNA of single pollen nuclei can be amplified by multiple-displacement-amplification using Phi29 DNA polymerase and meiotic recombination events can be measured using KASP markers. Alternatively, the PicoPLEX DNA-seq kit can be used to amplify the DNA of single pollen nuclei followed by library preparation for whole-genome sequencing and subsequent bioinformatic analysis.

Live-Cell CRISPR Imaging in Plant Cells with a Telomere-Specific Guide RNA.

Chromatin organization is highly dynamic in living cells. Therefore, it might have a regulatory role over biological mechanisms like transcription, replication, and DNA repair. To elucidate how these mechanisms are regulated, it is required to establish imaging methods to visualize the chromatin dynamic in living cells. Here, we provide a protocol for a live plant cell imaging technique based on application of two orthologs of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) from Streptococcus pyogenes and Staphylococcus aureus. This technique uses the inactive variants of Cas9 combined with different fluorescent proteins (GFP and mRuby) and telomere-specific guide RNA to target telomeric repeats in Nicotiana benthamiana. Our immuno-FISH data revealed that signals arising from the CRISPR/dCas9 method are specifically belonging to telomeric regions.

Analysis of Crossover Events and Allele Segregation Distortion in Interspecific Citrus Hybrids by Single Pollen Genotyping.

In citrus, a classical method of studying crossovers and segregation distortion (SD) is the genetic analysis of progenies. A new strategy combining fluorescence-activated cell sorting and whole genome amplification of haploid pollen nuclei with a large set of molecular markers, offers the opportunity to efficiently determine the frequency of crossovers and the identification of SD without the need to generate segregating populations. Here we have analyzed meiotic crossover events in a pollen nuclei population from "Eureka" lemon and the allelic SD was evaluated in a pollen nuclei population from a clementine × sweet orange hybrid ("CSO"). Data obtained from the "CSO" pollen nuclei population were compared to those obtained from genotyping of a segregating population ("RTSO") arising from a hand-made sexual hybridization between diploid non apomictic selected tangor (mandarin × sweet orange; "RTO" tangor) as female parent pollinated with "CSO" tangor as male parent. The analysis of crossovers rates on chromosome 1 revealed the presence of up to five crossovers events on one arm and four on the corresponding other arm, with an average of 1.97 crossovers per chromosome while no crossover events were observed in five "Eureka" lemon pollen nuclei. The rate of SD observed in "CSO" pollen nuclei (13.8%) was slightly lower than that recovered in the "RTSO" population (20.7%). In the pollen nuclei population, SD was found on linkage group (LG) 2, while the "RTSO" population showed SD on LGs 2 and 7. Potential male gametic selection mechanisms were distinguished in pollen grains, while in the population, mechanisms of gametophytic selection and/or zygotic selection were observed. This methodology is a very useful tool to facilitate research focused on the reproductive biology of citrus and study the mechanisms that affect crossovers and SD.

More Filtering on SNP Calling Does Not Remove Evidence of Inter-Nucleus Recombination in Dikaryotic Arbuscular Mycorrhizal Fungi.

Evidence for the existence of dikaryote-like strains, low nuclear sequence diversity and inter-nuclear recombination in arbuscular mycorrhizal fungi has been recently reported based on single nucleus sequencing data. Here, we aimed to support evidence of inter-nuclear recombination using an approach that filters SNP calls more conservatively, keeping only positions that are exclusively single copy and homozygous, and with at least five reads supporting a given SNP. This methodology recovers hundreds of putative inter-nucleus recombination events across publicly available sequence data from individual nuclei. Challenges related to the acquisition and analysis of sequence data from individual nuclei are highlighted and discussed, and ways to address these issues in future studies are presented.

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.

Unequal contribution of two paralogous CENH3 variants in cowpea centromere function.

In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the  diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.

Together But Different: The Subgenomes of the Bimodal Eleutherine Karyotypes Are Differentially Organized.

Bimodal karyotypes are characterized by the presence of two sets of chromosomes of contrasting size. Eleutherine bulbosa (2n = 12) presents a bimodal karyotype with a large chromosome pair, which has a pericentric inversion in permanent heterozygosity with suppressed recombination, and five pairs of three to four times smaller chromosomes. Aiming to understand whether high copy number sequence composition differs between both chromosome sets, we investigated the repetitive DNA fraction of E. bulbosa and compared it to the chromosomal organization of the related Eleutherine latifolia species, not containing the pericentric inversion. We also compared the repetitive sequence proportions between the heteromorphic large chromosomes of E. bulbosa and between E. bulbosa and E. latifolia to understand the influence of the chromosome inversion on the dynamics of repetitive sequences. The most abundant repetitive families of the genome showed a similar chromosomal distribution in both homologs of the large pair and in both species, apparently not influenced by the species-specific inversions. The repeat families Ebusat1 and Ebusat4 are localized interstitially only on the large chromosome pair, while Ebusat2 is located in the centromeric region of all chromosomes. The four most abundant retrotransposon lineages are accumulated in the large chromosome pair. Replication timing and distribution of epigenetic and transcriptional marks differ between large and small chromosomes. The differential distribution of retroelements appears to be related to the bimodal condition and is not influenced by the nonrecombining chromosome inversions in these species. Thus, the large and small chromosome subgenomes of the bimodal Eleutherine karyotype are differentially organized and probably evolved by repetitive sequences accumulation on the large chromosome set.

Assessing Ploidy Level Analysis and Single Pollen Genotyping of Diploid and Euploid Citrus Genotypes by Fluorescence-Activated Cell Sorting and Whole-Genome Amplification.

Flow cytometry is widely used to determine genome size and ploidy level in plants. This technique, when coupled with fluorescence-activated cell sorting (FACS), whole genome amplification and genotyping (WGA), opens up new opportunities for genetic studies of individualized nuclei. This strategy was used to analyze the genetic composition of single pollen nuclei of different citrus species. The flow cytometry and microscope observations allowed us to differentiate the populations of pollen nuclei present in the diploid and euploid genotypes analyzed, showing that citrus has binuclear pollen. We have identified in the "CSO" tangor an additional nuclei population composed by the vegetative plus generative nuclei. Genotyping of this nuclei population revealed that vegetative and generative nuclei show the same genetic configuration. In addition, we have demonstrated the presence of unreduced gametes in the diploid genotype "Mexican lime." Genomic amplification is a robust method for haploid nuclei genotyping with several molecular markers, whereas in diploid nuclei using heterozygous markers showed a bias towards one of the two alleles, limiting the use of this tool in this type of nuclei. We further discuss the importance and applications of single pollen genotyping in citrus genetic studies.

Single nucleus sequencing reveals evidence of inter-nucleus recombination in arbuscular mycorrhizal fungi.

Eukaryotes thought to have evolved clonally for millions of years are referred to as ancient asexuals. The oldest group among these are the arbuscular mycorrhizal fungi (AMF), which are plant symbionts harboring hundreds of nuclei within one continuous cytoplasm. Some AMF strains (dikaryons) harbor two co-existing nucleotypes but there is no direct evidence that such nuclei recombine in this life-stage, as is expected for sexual fungi. Here, we show that AMF nuclei with distinct genotypes can undergo recombination. Inter-nuclear genetic exchange varies in frequency among strains, and despite recombination all nuclear genomes have an average similarity of at least 99.8%. The present study demonstrates that AMF can generate genetic diversity via meiotic-like processes in the absence of observable mating. The AMF dikaryotic life-stage is a primary source of nuclear variability in these organisms, highlighting its potential for strain enhancement of these symbionts.

Sequencing of Single Pollen Nuclei Reveals Meiotic Recombination Events at Megabase Resolution and Circumvents Segregation Distortion Caused by Postmeiotic Processes.

Meiotic recombination is a fundamental mechanism to generate novel allelic combinations which can be harnessed by breeders to achieve crop improvement. The recombination landscape of many crop species, including the major crop barley, is characterized by a dearth of recombination in 65% of the genome. In addition, segregation distortion caused by selection on genetically linked loci is a frequent and undesirable phenomenon in double haploid populations which hampers genetic mapping and breeding. Here, we present an approach to directly investigate recombination at the DNA sequence level by combining flow-sorting of haploid pollen nuclei of barley with single-cell genome sequencing. We confirm the skewed distribution of recombination events toward distal chromosomal regions at megabase resolution and show that segregation distortion is almost absent if directly measured in pollen. Furthermore, we show a bimodal distribution of inter-crossover distances, which supports the existence of two classes of crossovers which are sensitive or less sensitive to physical interference. We conclude that single pollen nuclei sequencing is an approach capable of revealing recombination patterns in the absence of segregation distortion.

Measuring Meiotic Crossovers via Multi-Locus Genotyping of Single Pollen Grains in Barley.

The detection of meiotic crossovers in crop plants currently relies on scoring DNA markers in a segregating population or cytological visualization. We investigated the feasibility of using flow-sorted haploid nuclei, Phi29 DNA polymerase-based whole-genome-amplification (WGA) and multi-locus KASP-genotyping to measure meiotic crossovers in individual barley pollen grains. To demonstrate the proof of concept, we used 24 gene-based physically mapped single nucleotide polymorphisms to genotype the WGA products of 50 single pollen nuclei. The number of crossovers per chromosome, recombination frequencies along chromosome 3H and segregation distortion were analysed and compared to a doubled haploid (DH) population of the same genotype. The number of crossovers and chromosome wide recombination frequencies show that this approach is able to produce results that resemble those obtained from other methods in a biologically meaningful way. Only the segregation distortion was found to be lower in the pollen population than in DH plants.