High-Throughput Fluorescent Pollen Tetrad Analysis Using DeepTetrad.

Meiotic recombination initiates from ~100-200 s of programmed DNA double stranded breaks (DSBs) in plants. Meiotic DSBs can be repaired using homologous chromosomes to generate a crossover . Meiotic crossover is critical for chromosomal segregation and increasing genetic variation. The number of crossovers is limited to one and three per chromosome pair in most plant species. Genetic, epigenetic, and environmental factors control crossover frequency and distribution. Due to the limited number of crossovers it is challenging to measure crossover frequency along chromosomes. We adapted fluorescence-tagged lines (FTLs ) that contain quartet1 mutations and linked transgenes expressing dsRed, eYFP, and eCFP in pollen tetrads into the deep learning-based image analysis tool, DeepTetrad. DeepTetrad enables the measurement of crossover frequency and interference by classifying 12 types of tetrads from three-color FTLs in a high-throughput manner, using conventional microscope instruments and a Linux machine. Here, we provide detailed procedures for preparing tetrad samples, tetrad imaging, running DeepTetrad, and analysis of DeepTetrad outputs. DeepTetrad-based measurements of crossover frequency and interference ratio will accelerate the genetic dissection of meiotic crossover control.

The effect of heat stress on sugar beet recombination.

Meiotic recombination plays a crucial role in plant breeding through the creation of new allelic combinations. Therefore, lack of recombination in some genomic regions constitutes a constraint for breeding programmes. In sugar beet, one of the major crops in Europe, recombination occurs mainly in the distal portions of the chromosomes, and so the development of simple approaches to change this pattern is of considerable interest for future breeding and genetics. In the present study, the effect of heat stress on recombination in sugar beet was studied by treating F1 plants at 28 °C/25 °C (day/night) and genotyping the progeny. F1 plants were reciprocally backcrossed allowing the study of male and female meiosis separately. Genotypic data indicated an overall increase in crossover frequency of approximately one extra crossover per meiosis, with an associated increase in pericentromeric recombination under heat treatment. Our data indicate that the changes were mainly induced by alterations in female meiosis only, showing that heterochiasmy in sugar beet is reduced under heat stress. Overall, despite the associated decrease in fertility, these data support the potential use of heat stress to foster recombination in sugar beet breeding programmes.

Direct evidence for crossover and chromatid interference in meiosis of two plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei).

Crossing over, in addition to its strictly genetic role, also performs a critical mechanical function, by bonding homologues in meiosis. Hence, it is responsible for an orderly reduction of the chromosome number. As such, it is strictly controlled in frequency and distribution. The well-known crossover control is positive crossover interference which reduces the probability of a crossover in the vicinity of an already formed crossover. A poorly studied aspect of the control is chromatid interference. Such analyses are possible in very few organisms as they require observation of all four products of a single meiosis. Here, we provide direct evidence of chromatid interference. Using in situ probing in two interspecific plant hybrids (Lolium multiflorum×Festuca pratensis and Allium cepa×A. roylei) during anaphase I, we demonstrate that the involvement of four chromatids in double crossovers is significantly more frequent than expected (64% versus 25%). We also provide a physical measure of the crossover interference distance, covering ~30-40% of the relative chromosome arm length, and show that the centromere acts as a barrier for crossover interference. The two arms of a chromosome appear to act as independent units in the process of crossing over. Chromatid interference has to be seriously addressed in genetic mapping approaches and further studies.

A phased genome based on single sperm sequencing reveals crossover pattern and complex relatedness in tea plants.

For diploid organisms that are highly heterozygous, a phased haploid genome can greatly aid in functional genomic, population genetic and breeding studies. Based on the genome sequencing of 135 single sperm cells of the elite tea cultivar 'Fudingdabai', we herein phased the genome of Camellia sinensis, one of the most popular beverage crops worldwide. High-resolution genetic and recombination maps of Fudingdabai were constructed, which revealed that crossover (CO) positions were frequently located in the 5' and 3' ends of annotated genes, while CO distributions across the genome were random. The low CO frequency in tea can be explained by strong CO interference, and CO simulation revealed the proportion of interference insensitive CO ranged from 5.2% to 11.7%. We furthermore developed a method to infer the relatedness between tea accessions and detected complex kinship and genetic signatures of 106 tea accessions. Among them, 59 accessions were closely related with Fudingdabai and 31 of them were first-degree relatives. We additionally identified genes displaying allele specific expression patterns between the two haplotypes of Fudingdabai and genes displaying significantly differential expression levels between Fudingdabai and other haplotypes. These results lay the foundation for further investigation of genetic and epigenetic factors underpinning the regulation of gene expression and provide insights into the evolution of tea plants as well as a valuable genetic resource for future breeding efforts.

High temperatures alter cross-over distribution and induce male meiotic restitution in Arabidopsis thaliana.

Plant fertility is highly sensitive to elevated temperature. Here, we report that hot spells induce the formation of dyads and triads by disrupting the biogenesis or stability of the radial microtubule arrays (RMAs) at telophase II. Heat-induced meiotic restitution in Arabidopsis is predominantly SDR-type (Second Division Restitution) indicating specific interference with RMAs formed between separated sister chromatids. In addition, elevated temperatures caused distinct deviations in cross-over formation in male meiosis. Synapsis at pachytene was impaired and the obligate cross-over per chromosome was discarded, resulting in partial univalency in meiosis I (MI). At diakinesis, interconnections between non-homologous chromosomes tied separate bivalents together, suggesting heat induces ectopic events of non-homologous recombination. Summarized, heat interferes with male meiotic cross-over designation and cell wall formation, providing a mechanistic basis for plant karyotype change and genome evolution under high temperature conditions.

DeepTetrad: high-throughput image analysis of meiotic tetrads by deep learning in Arabidopsis thaliana.

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate the genetic dissection of mechanisms that control meiotic recombination.

Linked-read sequencing of gametes allows efficient genome-wide analysis of meiotic recombination.

Meiotic crossovers (COs) ensure proper chromosome segregation and redistribute the genetic variation that is transmitted to the next generation. Large populations and the demand for genome-wide, fine-scale resolution challenge existing methods for CO identification. Taking advantage of linked-read sequencing, we develop a highly efficient method for genome-wide identification of COs at kilobase resolution in pooled recombinants. We first test this method using a pool of Arabidopsis F2 recombinants, and recapitulate results obtained from the same plants using individual whole-genome sequencing. By applying this method to a pool of pollen DNA from an F1 plant, we establish a highly accurate CO landscape without generating or sequencing a single recombinant plant. The simplicity of this approach enables the simultaneous generation and analysis of multiple CO landscapes, accelerating the pace at which mechanisms for the regulation of recombination can be elucidated through efficient comparisons of genotypic and environmental effects on recombination.

Recombination suppression in heterozygotes for a pericentric inversion induces the interchromosomal effect on crossovers in Arabidopsis.

During meiosis, recombination ensures allelic exchanges through crossovers (COs) between the homologous chromosomes. Advances in our understanding of the rules of COs have come from studies of mutations including structural chromosomal rearrangements that, when heterozygous, are known to impair COs in various organisms. In this work, we investigate the effect of a large heterozygous pericentric inversion on male and female recombination in Arabidopsis. The inversion was discovered in the Atmcc1 mutant background and was characterized through genetic and next-generation sequencing analysis. Reciprocal backcross populations, each consisting of over 400 individuals, obtained from the mutant and the wild type, both crossed with Landsberg erecta, were analyzed genome-wide by 143 single-nucleotide polymorphisms. The negative impact of inversion became evident in terms of CO loss in the rearranged chromosome in both male and female meiosis. No single-CO event was detected within the inversion, consistent with a post-meiotic selection operating against unbalanced gametes. Cytological analysis of chiasmata in F1 plants confirmed that COs were reduced in male meiosis in the chromosome with inversion. Crossover suppression on the rearranged chromosome is associated with a significant increase of COs in the other chromosomes, thereby maintaining unchanged the number of COs per cell. The CO pattern observed in our study is consistent with the interchromosomal (IC) effect as first described in Drosophila. In contrast to male meiosis, in female meiosis no IC effect is visible. This may be related to the greater strength of interference that constrains the CO number in excess of the minimum value imposed by CO assurance in Arabidopsis female meiosis.

High-resolution crossover mapping reveals similarities and differences of male and female recombination in maize.

Meiotic crossovers (COs) are not uniformly distributed across the genome. Factors affecting this phenomenon are not well understood. Although many species exhibit large differences in CO numbers between sexes, sex-specific aspects of CO landscape are particularly poorly elucidated. Here, we conduct high-resolution CO mapping in maize. Our results show that CO numbers as well as their overall distribution are similar in male and female meioses. There are, nevertheless, dissimilarities at local scale. Male and female COs differ in their locations relative to transcription start sites in gene promoters and chromatin marks, including nucleosome occupancy and tri-methylation of lysine 4 of histone H3 (H3K4me3). Our data suggest that sex-specific factors not only affect male-female CO number disparities but also cause fine differences in CO positions. Differences between male and female CO landscapes indicate that recombination has distinct implications for population structure and gene evolution in male and in female meioses.

Meiotic crossovers are associated with open chromatin and enriched with Stowaway transposons in potato.

BACKGROUND: Meiotic recombination is the foundation for genetic variation in natural and artificial populations of eukaryotes. Although genetic maps have been developed for numerous plant species since the late 1980s, few of these maps have provided the necessary resolution needed to investigate the genomic and epigenomic features underlying meiotic crossovers. RESULTS: Using a whole genome sequencing-based approach, we developed two high-density reference-based haplotype maps using diploid potato clones as parents. The vast majority (81%) of meiotic crossovers were mapped to less than 5 kb. The fine-scale accuracy of crossover detection was validated by Sanger sequencing for a subset of ten crossover events. We demonstrate that crossovers reside in genomic regions of "open chromatin", which were identified based on hypersensitivity to DNase I digestion and association with H3K4me3-modified nucleosomes. The genomic regions spanning crossovers were significantly enriched with the Stowaway family of miniature inverted-repeat transposable elements (MITEs). The occupancy of Stowaway elements in gene promoters is concomitant with an increase in recombination rate. A generalized linear model identified the presence of Stowaway elements as the third most important genomic or chromatin feature behind genes and open chromatin for predicting crossover formation over 10-kb windows. CONCLUSIONS: Collectively, our results suggest that meiotic crossovers in potato are largely determined by the local chromatin status, marked by accessible chromatin, H3K4me3-modified nucleosomes, and the presence of Stowaway transposons.

Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA.

During meiosis, homologous chromosomes undergo recombination, which can result in formation of reciprocal crossover molecules. Crossover frequency is highly variable across the genome, typically occurring in narrow hotspots, which has a significant effect on patterns of genetic diversity. Here we describe methods to measure crossover frequency in plants at the hotspot scale (bp-kb), using allele-specific PCR amplification from genomic DNA extracted from the pollen of F1 heterozygous plants. We describe (1) titration methods that allow amplification, quantification and sequencing of single crossover molecules, (2) quantitative PCR methods to more rapidly measure crossover frequency, and (3) application of high-throughput sequencing for study of crossover distributions within hotspots. We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions.

Distribution, position and genomic characteristics of crossovers in tomato recombinant inbred lines derived from an interspecific cross between Solanum lycopersicum and Solanum pimpinellifolium.

We determined the crossover (CO) distribution, frequency and genomic sequences involved in interspecies meiotic recombination by using parent-assigned variants of 52 F6 recombinant inbred lines obtained from a cross between tomato, Solanum lycopersicum, and its wild relative, Solanum pimpinellifolium. The interspecific CO frequency was 80% lower than reported for intraspecific tomato crosses. We detected regions showing a relatively high and low CO frequency, so-called hot and cold regions. Cold regions coincide to a large extent with the heterochromatin, although we found a limited number of smaller cold regions in the euchromatin. The CO frequency was higher at the distal ends of chromosomes than in pericentromeric regions and higher in short arm euchromatin. Hot regions of CO were detected in euchromatin, and COs were more often located in non-coding regions near the 5' untranslated region of genes than expected by chance. Besides overrepresented CCN repeats, we detected poly-A/T and AT-rich motifs enriched in 1-kb promoter regions flanking the CO sites. The most abundant sequence motifs at CO sites share weak similarity to transcription factor-binding sites, such as for the C2H2 zinc finger factors class and MADS box factors, while InterPro scans detected enrichment for genes possibly involved in the repair of DNA breaks.

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.

Dissecting meiotic recombination based on tetrad analysis by single-microspore sequencing in maize.

Meiotic recombination drives eukaryotic sexual reproduction and the generation of genome diversity. Tetrad analysis, which examines the four chromatids resulting from a single meiosis, is an ideal method to study the mechanisms of homologous recombination. Here we develop a method to isolate the four microspores from a single tetrad in maize for the purpose of whole-genome sequencing. A high-resolution recombination map reveals that crossovers are unevenly distributed across the genome and are more likely to occur in the genic than intergenic regions, especially common in the 5'- and 3'-end regions of annotated genes. The direct detection of genomic exchanges suggests that conversions likely occur in most crossover tracts. Negative crossover interference and weak chromatid interference are observed at the population level. Overall, our findings further our understanding of meiotic recombination with implications for both basic and applied research.

Contrasted patterns of crossover and non-crossover at Arabidopsis thaliana meiotic recombination hotspots.

The vast majority of meiotic recombination events (crossovers (COs) and non-crossovers (NCOs)) cluster in narrow hotspots surrounded by large regions devoid of recombinational activity. Here, using a new molecular approach in plants, called "pollen-typing", we detected and characterized hundreds of CO and NCO molecules in two different hotspot regions in Arabidopsis thaliana. This analysis revealed that COs are concentrated in regions of a few kilobases where their rates reach up to 50 times the genome average. The hotspots themselves tend to cluster in regions less than 8 kilobases in size with overlapping CO distribution. Non-crossover (NCO) events also occurred in the two hotspots but at very different levels (local CO/NCO ratios of 1/1 and 30/1) and their track lengths were quite small (a few hundred base pairs). We also showed that the ZMM protein MSH4 plays a role in CO formation and somewhat unexpectedly we also found that it is involved in the generation of NCOs but with a different level of effect. Finally, factors acting in cis and in trans appear to shape the rate and distribution of COs at meiotic recombination hotspots.

Haploid meiosis in Arabidopsis: double-strand breaks are formed and repaired but without synapsis and crossovers.

Two hallmark features of meiosis are i) the formation of crossovers (COs) between homologs and ii) the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype) in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.

Characterization of meiotic non-crossover molecules from Arabidopsis thaliana pollen.

Meiotic recombination is essential for proper segregation of homologous chromosomes and thus for formation of viable gametes. Recombination generates either crossovers (COs), which are reciprocal exchanges between chromosome segments, or gene conversion not associated with crossovers (NCOs). Both kinds of events occur in narrow regions (less than 10 kb) called hotspots, which are distributed along chromosomes. While NCOs may represent a large fraction of meiotic recombination events in plants, as in many other higher eukaryotes, they have been poorly characterized due to the technical difficulty of detecting them. Here, we present a powerful approach, based on allele-specific PCR amplification of single molecules from pollen genomic DNA, allowing detection, quantification and characterization of NCO events arising at low frequencies at recombination hotspots.

The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

ZIP4 in homologous chromosome synapsis and crossover formation in rice meiosis.

In budding yeast, the ZMM complex is closely associated with class I crossovers and synaptonemal complex (SC) formation. However, the relationship between the ZMM genes remains unclear in most higher eukaryotes. Here, we identify the rice ZIP4 homolog, a member of the ZMM gene group, and explore its relationship with two other characterized ZMM genes, MER3 and ZEP1. Our results show that in the rice zip4 mutant, the chiasma frequency is greatly reduced, although synapsis proceeds with only mild defects. Immunocytological analyses of wild-type rice reveal that ZIP4 presents as punctuate foci and colocalizes with MER3 in prophase I meiocytes. Additionally, ZIP4 is essential for the loading of MER3 onto chromosomes, but not vice versa. Double-mutant analyses show that zip4 mer3 displays a greater decrease in the mean number of chiasmata than either of the zip4 or mer3 single mutants, suggesting that ZIP4 and MER3 work cooperatively to promote CO formation but their individual contributions are not completely identical in rice. Although zep1 alone gives an increased chiasma number, both zip4 zep1 and mer3 zep1 show a much lower chiasma number than the zip4 or mer3 single mutants. These results imply that the normal functions of ZIP4 and MER3 are required for the regulation of COs by ZEP1.

Characterization of meiotic crossovers in pollen from Arabidopsis thaliana.

Homologous recombination processes, which occur during the prophase of the first meiotic division, while generating new allelic combinations, are mechanistically important for the regular segregation of homologous chromosomes. They generate either crossovers, which are reciprocal exchanges between chromosome segments, or gene conversions. Both kinds of events occur in narrow regions (less than 10 kb) called hotspots, which are distributed along chromosomes. Classical genetic methods for CO characterization, which rely on the building of large populations and require appropriately located markers, are not well suited to the study of meiotic recombination hotspots. Here, we present a method based on allele-specific PCR amplification of single molecules from pollen genomic DNA. It allows detection, quantification and characterization of CO events arising at low frequencies in recombination hotspots.