Lorinda K. Anderson

Recombination: an underappreciated factor in the evolution of plant genomes

Our knowledge of recombination rates and patterns in plants is far from being comprehensive. However, compelling evidence indicates a central role for recombination, through its influences on mutation and selection, in the evolution of plant genomes. Furthermore, recombination seems to be generally higher and more variable in plants than in animals, which could be one of the primary reasons for differences in genome lability between these two kingdoms. Much additional study of recombination in plants is needed to investigate these ideas further.

Male Mouse Recombination Maps for Each Autosome Identified by Chromosome Painting

Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure.

Two-dimensional spreads of synaptonemal complexes from solanaceous plants

By using serial sectioning and a new hypotonic bursting technique on primary microsporocytes of tomato (Lycopersicon esculentum), relatively large numbers of recombination nodules (RNs) are observed on the synaptonemal complexes forming during zygonema. In pachynema most, but not all, of these RNs are lost. If RNs represent sites of potential crossing over during zygonema and sites of actual crossing over during late pachynema, the observed temporal and spatial distribution of RNs may provide answers for some classic cytogenetic questions such as: how is at least one crossover per bivalent assured? How are crossovers localized? What is the basis for positive chiasma interference?

A model for chromosome structure during the mitotic and meiotic cell cycles

The chromosome scaffold model in which loops of chromatin are attached to a central, coiled chromosome core (scaffold) is the current paradigm for chromosome structure. Here we present a modified version of the chromosome scaffold model to describe chromosome structure and behavior through the mitotic and meiotic cell cycles. We suggest that a salient feature of chromosome structure is established during DNA replication when sister loops of DNA extend in opposite directions from replication sites on nuclear matrix strands. This orientation is maintained into prophase when the nuclear matrix strand is converted into two closely associated sister chromatid cores with sister DNA loops extending in opposite directions. We propose that chromatid cores are contractile and show, using a physical model, that contraction of cores during late prophase can result in coiled chromatids. Coiling accounts for the majority of chromosome shortening that is needed to separate sister chromatids within the confines of a cell. In early prophase I of meiosis, the orientation of sister DNA loops in opposite directions from axial elements assures that DNA loops interact preferentially with homologous DNA loops rather than with sister DNA loops. In this context, we propose a bar code model for homologous presynaptic chromosome alignment that involves weak paranemic interactions of homologous DNA loops. Opposite orientation of sister loops also suppresses crossing over between sister chromatids in favor of crossing over between homologous non-sister chromatids. After crossing over is completed in pachytene and the synaptonemal complex breaks down in early diplotene (= diffuse stage), new contractile cores are laid down along each chromatid. These chromatid cores are comparable to the chromatid cores in mitotic prophase chromosomes. As an aside, we propose that leptotene through early diplotene represent the ‘missing’ G2 period of the premeiotic interphase. The new chromosome cores, along with sister chromatid cohesion, stabilize chiasmata. Contraction of cores in late diplotene causes chromosomes to coil in a configuration that encourages subsequent syntelic orientation of sister kinetochores and amphitelic orientation of homologous kinetochore pairs on the spindle at metaphase I.

Recombination nodules in plants

The molecular events of recombination are thought to be catalyzed by proteins present in recombination nodules (RNs). Therefore, studying RN structure and function should give insights into the processes by which meiotic recombination is regulated in eukaryotes. Two types of RNs have been identified so far, early (ENs) and late (LNs). ENs appear at leptotene and persist into early pachytene while LNs appear in pachytene and remain into early diplotene. ENs and LNs can be distinguished not only on their time of appearance, but also by such characteristics as shape and size, relative numbers, and association with unsynapsed and/or synapsed chromosomal segments. The function(s) of ENs is not clear, but they may have a role in searching for DNA homology, synapsis, gene conversion and/or crossing over. LNs are well correlated with crossing over. Here, the patterns of ENs and LNs during prophase I in plants are reviewed.

The relationship between genome size and synaptonemal complex length in higher plants

There appears to be only a weak correlation between genome size and the corresponding total length of a complete set of synaptonemal complexes (SCs) based on published evidence for several fungal, plant, and animal species. This result is unexpected, considering the strong positive correlations between genome size (DNA amount) and total chromosome length and volume and between relative lengths of chromosomes and SCs. Because the observed weak correlation was based on limited data, we systematically investigated the relationship between genome size and SC length, using ten higher plant species. Two-dimensional spreads of SCs from primary microsporocytes at pachytene were prepared using a hypotonic bursting technique. The SC spreads were examined either by light or electron microscopy, and the lengths of at least ten complete sets of SCs were measured for each of the ten species. Additionally, the genome size of each species was determined from pollen tetrad protoplasts using flow cytometry. A strong correlation (r = 0.97) between total SC length and genome size was observed for higher plants, indicating a constant amount of DNA is associated with a given length of SC, at least when averaged over the whole genome.

Predicting and testing physical locations of genetically mapped loci on tomato pachytene chromosome 1

Predicting the chromosomal location of mapped markers has been difficult because linkage maps do not reveal differences in crossover frequencies along the physical structure of chromosomes. Here we combine a physical crossover map based on the distribution of recombination nodules (RNs) on Solanum lycopersicum (tomato) synaptonemal complex 1 with a molecular genetic linkage map from the interspecific hybrid S. lycopersicum × S. pennellii to predict the physical locations of 17 mapped loci on tomato pachytene chromosome 1. Except for one marker located in heterochromatin, the predicted locations agree well with the observed locations determined by fluorescence in situ hybridization. One advantage of this approach is that once the RN distribution has been determined, the chromosomal location of any mapped locus (current or future) can be predicted with a high level of confidence.

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome

The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome–fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.

Nodules associated with axial cores and synaptonemal complexes during zygotene in Psilotum nudum

Two-dimensional spreads of synaptonemal complexes (SCs) from the lower vascular plant Psilotum nudum were examined after staining with uranyl acetate-lead citrate (UP). Staining with UP allows visualization of lateral elements/axial cores (ACs), central elements, kinetochores, and nodules. Numerous darkly stained nodules were associated with forming SCs. In addition to nodules found on the central element of SC segments, other nodules were found at points of convergence between two adjacent ACs. Of these latter nodules, some were obviously associated with a fiber that connected adjacent ACs. No central element material was visible between the ACs, and the nodule complex appeared to be the only structure holding the ACs together. Although the function(s) of nodules during zygotene is unknown, the presence of a nodule-fiber complex that connects adjacent ACs before central element formation suggests that at least some of the nodules may be involved in synaptic initiation.

Meiotic recombination and spatial proximity in the etiology of the recurrent t(11;22).

Although balanced translocations are among the most common human chromosomal aberrations, the constitutional t(11;22)(q23;q11) is the only known recurrent non-Robertsonian translocation. Evidence indicates that de novo formation of the t(11;22) occurs during meiosis. To test the hypothesis that spatial proximity of chromosomes 11 and 22 in meiotic prophase oocytes and spermatocytes plays a role in the rearrangement, the positions of the 11q23 and 22q11 translocation breakpoints were examined. Fluorescence in situ hybridization with use of DNA probes for these sites demonstrates that 11q23 is closer to 22q11 in meiosis than to a control at 6q26. Although chromosome 21p11, another control, often lies as close to 11q23 as does 22q11 during meiosis, chromosome 21 rarely rearranges with 11q23, and the DNA sequence of chromosome 21 appears to be less susceptible than 22q11 to double-strand breaks (DSBs). It has been suggested that the rearrangement recurs as a result of the palindromic AT-rich repeats at both 11q23 and 22q11, which extrude hairpin structures that are susceptible to DSBs. To determine whether the DSBs at these sites coincide with normal hotspots of meiotic recombination, immunocytochemical mapping of MLH1, a protein involved in crossing over, was employed. The results indicate that the translocation breakpoints do not coincide with recombination hotspots and therefore are unlikely to be the result of meiotic programmed DSBs, although MRE11 is likely to be involved. Previous analysis indicated that the DSBs appear to be repaired by a mechanism similar to nonhomologous end joining (NHEJ), although NHEJ is normally suppressed during meiosis. Taken together, these studies support the hypothesis that physical proximity between 11q23 and 22q11--but not typical meiotic recombinational activity in meiotic prophase--plays an important role in the generation of the constitutional t(11;22) rearrangement.