The Relationship of Homologous Synapsis and Crossing over in a Maize Inversion

Frequency of homologous synapsis at pachytene for a relatively short heterozygous inversion was compared to the frequency of crossover occurrence within the inversion and to the frequency of the presence of a recombination nodule within the homologously synapsed inverted region. Crossover frequencies were estimated from bridge-fragment frequencies at anaphase I and anaphase II. Recombination nodules (RNs) were observed in electron micrographs. Results show very similar frequencies of homologous synapsis and the occurrence of reciprocal recombination within the inverted region, consistent with the interpretation that establishment of homologous synapsis in this case is related to at least commitment to the form of resolution of crossover intermediates which gives rise to reciprocal recombination, not conversion only, events. An RN was generally found at pachytene in homologously synapsed inverted regions.     

Synaptic initiation and extension as prerequisites for crossing over

The effect on crossover frequency in maize of three hour heat treatment was studied when treatment was applied at zygotene (substantially later than the major DNA synthetic period) and at pachytene. Crossover frequency assay was based upon bridge and fragment frequency at anaphase I in heterozygotes for a short paracentric inversion. Effect of treatment was studied in three distinguishable synaptic classes: (1) overall crossover frequency within the inversion, (2) double crossover frequency where two separate events of pairing initiation are required (coincident crossovers within and proximal to the inversion) and (3) double crossover frequency within the inversion, where spreading of synapsis over a short distance from a single event of pairing initiation can provide the requisite pairing. Evidence is reported: (1) that overall crossover frequency within the inversion was very significantly increased by treatment at zygotene but not detectably affected by treatment at pachytene; (2) that double crossover frequency within the inversion was very significantly increased by treatment at pachytene and may have been somewhat increased by treatment at zygotene. Results are consistent with the model that most crossover sites may be established at, or approximately at, events of synaptic initiation but that establishment of infrequent second crossover sites near those formed first can follow or accompany the spreading of synapsis to adjoining regions.     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Two paracentric inversions in the mouse, In(1)1 Rk and In(2)5 Rk, have been studied in surface microspreads of spermatocytes from heterozygotes. At zytogene, synaptic initiation occurs independently in three regions: within the inversion, and without, on either side. Synaptonemal complex (SC) formation is restricted to homologous regions, resulting in inversion loops in all early pachytene spermatocytes. An adjusting phase then occurs during pachytene in which the inversion loop is reduced by desynapsis of homologously synapsed SC, followed immediately by non-homologous synapsis with the alternate pairing partner, progressing from the ends toward the middle. Adjustment occurs during the first half of pachytene, but is not closely synchronized with sub-stage. It is complete by late pachytene, the loop having been eliminated in all cases and replaced by straight SCs in which the inverted region is heterosynapsed. Synapsis in the adjustment phase is evidently permitted only after the homosynaptic phase, and is indifferent to homology. It may lead to heterosynapsis, as in the inversion region, or to synapsis of homologous regions not synapsed at zytogene. The anaphase bridge frequency, a measure of crossing over within the inversion, is about 34% for both inversions studied, indicating that such crossovers do not block adjustment, that crossing over probably occurs before or during the adjustment period, and that there is some crossover suppression. The last could be the consequence of blocking by desynapsis/heterosynapsis. Synaptic adjustment appears to be a general phenomenon that occurs to varying extents in different forms. A hypothetical scheme for two phases of synapsis is proposed: at zytogene, a basic propensity for indifferent SC formation is limited by a restricting condition to synapsis between homologous regions. Subsequently, the restriction is lifted, whereupon synaptic instability is resolved by desynapsis, followed by resynapsis that is indifferent to homology, but that results in a topologically more stable structure.     

Pericentromeric chromatin reorganisation follows the initiation of recombination and coincides with early events of synapsis in cereals

The reciprocal exchange of genetic information between homologous chromosomes during meiotic recombination is essential to secure balanced chromosome segregation and to promote genetic diversity. The chromosomal position and frequency of reciprocal genetic exchange shapes the efficiency of breeding programmes and influences crop improvement under a changing climate. In large genome cereals, such as wheat and barley, crossovers are consistently restricted to subtelomeric chromosomal regions, thus preventing favourable allele combinations being formed within a considerable proportion of the genome, including interstitial and pericentromeric chromatin. Understanding the key elements driving crossover designation is therefore essential to broaden the regions available for crossovers. Here, we followed early meiotic chromatin dynamism in cereals through the visualisation of a homologous barley chromosome arm pair stably transferred into the wheat genetic background. By capturing the dynamics of a single chromosome arm at the same time as detecting the undergoing events of meiotic recombination and synapsis, we showed that subtelomeric chromatin of homologues synchronously transitions to an open chromatin structure during recombination initiation. By contrast, pericentromeric and interstitial regions preserved their closed chromatin organisation and become unpackaged only later, concomitant with initiation of recombinatorial repair and the initial assembly of the synaptonemal complex. Our results raise the possibility that the closed pericentromeric chromatin structure in cereals may influence the fate decision during recombination initiation, as well as the spatial development of synapsis, and may also explain the suppression of crossover events in the proximity of the centromeres.     

Dynamics of rye chromosome 1R regions with high or low crossover frequency in homology search and synapsis development

In many organisms, homologous pairing and synapsis depend on the meiotic recombination machinery that repairs double-strand DNA breaks (DSBs) produced at the onset of meiosis. The culmination of recombination via crossover gives rise to chiasmata, which locate distally in many plant species such as rye, Secale cereale. Although, synapsis initiates close to the chromosome ends, a direct effect of regions with high crossover frequency on partner identification and synapsis initiation has not been demonstrated. Here, we analyze the dynamics of distal and proximal regions of a rye chromosome introgressed into wheat to define their role on meiotic homology search and synapsis. We have used lines with a pair of two-armed chromosome 1R of rye, or a pair of telocentrics of its long arm (1RL), which were homozygous for the standard 1RL structure, homozygous for an inversion of 1RL that changes chiasma location from distal to proximal, or heterozygous for the inversion. Physical mapping of recombination produced in the ditelocentric heterozygote (1RL/1RL(inv)) showed that 70% of crossovers in the arm were confined to a terminal segment representing 10% of the 1RL length. The dynamics of the arms 1RL and 1RL(inv) during zygotene demonstrates that crossover-rich regions are more active in recognizing the homologous partner and developing synapsis than crossover-poor regions. When the crossover-rich regions are positioned in the vicinity of chromosome ends, their association is facilitated by telomere clustering; when they are positioned centrally in one of the two-armed chromosomes and distally in the homolog, their association is probably derived from chromosome elongation. On the other hand, chromosome movements that disassemble the bouquet may facilitate chromosome pairing correction by dissolution of improper chromosome associations. Taken together, these data support that repair of DSBs via crossover is essential in both the search of the homologous partner and consolidation of homologous synapsis.     

Meiotic checkpoints and the interchromosomal effect on crossing over in Drosophila females

During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.     

The Temporal Sequence of Synaptic Initiation, Crossing over and Synaptic Completion

Questions are raised as to the validity of arguments that crossover positions have been demonstrated to be normally established only during pachytene (after synapsis is maximal). An alternative and testable hypothesis is that crossover commitment can occur at events of synaptic initiation.-Measurements are presented of extents of pachytene synapsis and failure in and around a region of maize chromosome heterozygous for a short paracentric inversion, and these are compared to conjectured expectations from observations of crossover frequencies within the inversion. Various hypotheses consistent with the results are considered. It is pointed out that the hypothesis that increases in crossover frequency in the synapsed region of the inversion are compensatory to crossover inhibitions elsewhere requires complex assumptions: that the adjustment must take place among, not within cells and that the enhancement is preferentially expressed within the inversion instead of elsewhere in the genome. The hypothesis that the fixing and squashing procedure forces apart non-crossover regions previously synapsed but lacking a crossover also requires complex assumptions. The simplest hypothesis proposes that crossover commitment may determine synaptic expression. A role of the synaptonemal complex in the establishment of crossover sites is questioned or minimized.-Evidence is also presented with respect to conceivable function of the telomere in synaptic initiation. Restrictions on such a function, if it exists, seem to be required to account for the observations.     

Meiotic checkpoints and the interchromosomal effect on crossing over in Drosophila females

During prophase of meiosis I, genetic recombination is initiated with a Spo11-dependent DNA double-strand break (DSB). Repair of these DSBs can generate crossovers, which become chiasmata and are important for the process of chromosome segregation. To ensure at least one chiasma per homologous pair of chromosomes, the number and distribution of crossovers is regulated. One system contributing to the distribution of crossovers is the pachytene checkpoint, which requires the conserved gene pch2 that encodes an AAA+ATPase family member. Pch2-dependent pachytene checkpoint function causes delays in pachytene progression when there are defects in processes required for crossover formation, such as mutations in DSB-repair genes and when there are defects in the structure of the meiotic chromosome axis. Thus, the pachytene checkpoint appears to monitor events leading up to the generation of crossovers. Interestingly, heterozygous chromosome rearrangements cause Pch2-dependent pachytene delays and as little as two breaks in the continuity of the paired chromosome axes are sufficient to evoke checkpoint activity. These chromosome rearrangements also cause an interchromosomal effect on recombination whereby crossing over is suppressed between the affected chromosomes but is increased between the normal chromosome pairs. We have shown that this phenomenon is also due to pachytene checkpoint activity.     

Evidence for a role of the synaptonemal complex in provision for normal chromosome disjunction at meiosis II in maize

The meiotic behavior of heterozygotes from three different maize pericentric inversion stocks was quantitatively observed at a variety of stages throughout meiosis I and II. With heterozygosity for either of two of these inversions, the usual mode of pairing observed at pachytene involved synapsis of the centromere containing inverted region, and synaptic failure of the centromere region was rarely found. Abnormal chromosome behavior at subsequent meiotic stages was rare in these cases. With heterozygosity for the third inversion, however, homologous synapsis was generally found in the distal regions of the chromosome involved, the inverted region was often non-homologously synapsed, and a substantial frequency of cells apparently showed synaptic failure in the centromere containing inverted region. A substantial frequency of cells at anaphase II in this case contained two lagging monads in the plate region of the spindle. Where cells could be identified as sisters, sister cells showed identical behavior at anaphase II. Findings seem to be most simply explained by the supposition that pachytene synapsis of the centromere region is important to provision for sister centromere association until anaphase II.     

Serial-Section Analysis of Clustering within Anthers of Maize Microsporocytes with Specific Crossovers

The distribution within anthers of maize plants of microsporocytes of differing crossover class was studied by application of the serial-sectioning technique to plants heterozygous for a paracentric inversion. In such material, cells with four-strand double crossovers within the inversion are distinguishable from cells with other classes of crossovers within the inverted region and from cells with no crossovers within the inversion. Evidence was found for coarse clustering of cells with four-strand double crossovers within the inversion and for coarse clustering of cells of other crossover classes (with at least one crossover within the inversion). The local frequencies of these two major categories of crossover cells appeared to vary independently of each other, with regional increases in each occurring at the expense of noncrossover cells. There is also some suggestion that cells with four-strand doubles within the inversion may be clustered independently of other double-crossover classes within the inversion, but these other classes are not directly scorable.     

High-resolution crossover maps for each bivalent of Zea mays using recombination nodules

Recombination nodules (RNs) are closely correlated with crossing over, and, because they are observed by electron microscopy of synaptonemal complexes (SCs) in extended pachytene chromosomes, RNs provide the highest-resolution cytological marker currently available for defining the frequency and distribution of crossovers along the length of chromosomes. Using the maize inbred line KYS, we prepared an SC karyotype in which each SC was identified by relative length and arm ratio and related to the proper linkage group using inversion heterozygotes. We mapped 4267 RNs on 2080 identified SCs to produce high-resolution maps of RN frequency and distribution on each bivalent. RN frequencies are closely correlated with both chiasma frequencies and SC length. The total length of the RN recombination map is about twofold shorter than that of most maize linkage maps, but there is good correspondence between the relative lengths of the different maps when individual bivalents are considered. Each bivalent has a unique distribution of crossing over, but all bivalents share a high frequency of distal RNs and a severe reduction of RNs at and near kinetochores. The frequency of RNs at knobs is either similar to or higher than the average frequency of RNs along the SCs. These RN maps represent an independent measure of crossing over along maize bivalents.     

Synaptic Adjustment of Inversion Loops in Neurospora Crassa

Heterozygotes for three long inversions on chromosome 1 were analyzed by serial reconstruction from electron micrographs. Measurements of loop lengths at different meiotic prophase substages revealed that the homologous synapsis of the inverted region was gradually replaced by nonhomologous synapsis as loops were eliminated during pachytene. This synaptic adjustment was apparently not affected by crossovers which occurred within the 150- and 160-cM long loops.     

Synapsis and recombination in inversion heterozygotes

Inversion heterozygotes are expected to suffer from reduced fertility and a high incidence of chromosomally unbalanced gametes due to recombination within the inverted region. Non-homologous synapsis of the inverted regions can prevent recombination there and diminish the deleterious effects of inversion heterozygosity. The choice between non-homologous and homologous synapsis depends on the size of inversion, its genetic content, its location in relation to the centromere and telomere, and genetic background. In addition, there is a class of inversions in which homologous synapsis is gradually replaced by non-homologous synapsis during meiotic progression. This process is called synaptic adjustment. The degree of synaptic adjustment depends critically on the presence and location of the COs (crossovers) within the inversion loop. Only bivalents without COs within the loop and those with COs in the middle of the inversion can be completely adjusted and became linear.     

A search for the synaptic adjustment phenomenon in maize

It has recently been suggested from several laboratories that complex synaptic configurations (required for homologous synapsis in the presence of heterozygosity for chromosome rearrangements, or resulting from multivalent formation in polyploids, or even resulting from interlocking of normal bivalents) may be formed initially, but are altered by dissolution of the central element of the synaptonemal complex followed by its reinstatement in such a way that only free bivalents, typical of normal sequence homozygotes in diploids, are usually found at late pachytene. It has been suggested that the synaptic adjustment inferred may be a process of widespread occurrence. Maize microsporocytes heterozygous for a paracentric inversion were studied at early and late pachytene and also at early diplotene. Clear remnants of loop configurations typical of homologous synapsis of the inverted region were found in a number of cells at early diplotene, and synaptic failure of the inverted region was common at both early and late pachytene. In maize microsporocytes a synaptic adjustment process comparable to that which has been reported in mammals seems to be absent.     

Nonhomologous Synapsis and Reduced Crossing over in a Heterozygous Paracentric Inversion in Saccharomyces Cerevisiae

Homologous chromosome synapsis (``homosynapsis') and crossing over are well-conserved aspects of meiotic chromosome behavior. The long-standing assumption that these two processes are causally related has been challenged recently by observations in Saccharomyces cerevisiae of significant levels of crossing over (1) between small sequences at nonhomologous locations and (2) in mutants where synapsis is abnormal or absent. In order to avoid problems of local sequence effects and of mutation pleiotropy, we have perturbed synapsis by making a set of isogenic strains that are heterozygous and homozygous for a large chromosomal paracentric inversion covering a well marked genetic interval and then measured recombination. We find that reciprocal recombination in the marked interval in heterozygotes is reduced variably across the interval, on average to ~55% of that in the homozygotes, and that positive interference still modulates crossing over. Cytologically, stable synapsis across the interval is apparently heterologous rather than homologous, consistent with the interpretation that stable homosynapsis is required to initiate or consummate a large fraction of the crossing over observed in wild-type strains. When crossing over does occur in heterozygotes, dicentric and acentric chromosomes are formed and can be visualized and quantitated on blots though not demonstrated in viable spores. We find that there is no loss of dicentric chromosomes during the two meiotic divisions and that the acentric chromosome is recovered at only 1/3 to 1/2 of the expected level.     

Unexpected behavior of an inverted rye chromosome arm in wheat

Distal location of chiasmata in chromosome arms is thought to be a consequence of the distal initiation of synapsis. Observations of meiotic behavior of a rye chromosome with an inverted arm show that patterns of chiasma distribution and frequency are also inverted; therefore, the patterns of synapsis and chiasma distribution are independent, and recombination frequency along a chromosome is position-independent and segment-specific. Since cases of random distribution of chiasmata and recombination are known in rye, a genetic mechanism must be present that licenses specific chromosome regions for recombination. Large differences in the metaphase I pairing of the inversion in various combinations of two armed and telocentric chromosomes confirm the major role of the telomere bouquet in early homologue recognition. However, occasional synapsis and chiasmate pairing of the distal regions of normal arms with the proximal regions of the inversion suggest that an alternative mechanism for juxtaposing of homologues must also be present. Synapsis in inversion heterozygotes was mostly complete but in the antiparallel orientation, hence defying homology, but non-homologues never synapsed. Instances of synapsis strictly limited to the chiasma-capable segments of the arm suggest that, in rye, both recombination-dependent and recombination-independent mechanisms for homologue recognition must be present.     

Chromosome size-dependent control of meiotic reciprocal recombination in Saccharomyces cerevisiae: the role of crossover interference.

In the yeast Saccharomyces cerevisiae, small chromosomes undergo meiotic reciprocal recombination (crossing over) at rates (centimorgans per kilobases) greater than those of large chromosomes, and recombination rates respond directly to changes in the total size of a chromosomal DNA molecule. This phenomenon, termed chromosome size-dependent control of meiotic reciprocal recombination, has been suggested to be important for ensuring that homologous chromosomes cross over during meiosis. The mechanism of this regulation was investigated by analyzing recombination in identical genetic intervals present on different size chromosomes. The results indicate that chromosome size-dependent control is due to different amounts of crossover interference. Large chromosomes have high levels of interference while small chromosomes have much lower levels of interference. A model for how crossover interference directly responds to chromosome size is presented. In addition, chromosome size-dependent control was shown to lower the frequency of homologous chromosomes that failed to undergo crossovers, suggesting that this control is an integral part of the mechanism for ensuring meiotic crossing over between homologous chromosomes.     

Chiasma maintenance and terminalization across a homoeologous bivalent segment

In maize microsporocytes Heterozygous for a chromosome 2 interchange chromosome (which carries a homoeologous Tripsacum chromosome segment substituted for the distal half of its short arm), normal synapsis usually occurs at pachytene throughout the bivalent, but crossing over is almost entirely restricted to the homologous proximal region of the arm, (where it apprently occurs frequently). At diakinesis chiasmata were very often found to be located immediately proximal to an easily observable terminal knob of the Tripscacum chromosome segment. It was concluded that chiasmata, initiated in the homologous proximal region, had been maintained while terminalizing across the homoeologous region. It was also noted that heterozygosity for a telomere (and distal region) foreign to maize does not seem to inhibit pairing effective for crossing over in the homologous proximal portion of the chromosome arm.     

Plasmid Recombination in a Rad52 Mutant of Saccharomyces Cerevisiae

Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae. The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat. Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain. Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments. Reciprocal exchange occurs in fewer than 30% of events. Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells. Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length. Inverted and direct repeat plasmid recombination differ in two respects. First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination. Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids. In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events. Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles.     

Studies on crossover-specific mutants and the distribution of crossing over in Drosophila females

In Drosophila females, the majority of recombination events do not become crossovers and those that do occur are nonrandomly distributed. Furthermore, a group of Drosophila mutants specifically reduce crossing over, suggesting that crossovers depend on different gene products than noncrossovers. In mei-218 mutants, crossing over is reduced by approximately 90% while noncrossovers and the initiation of recombination remain unchanged. Importantly, the residual crossovers have a more random distribution than wild-type. It has been proposed that mei-218 has a role in establishing the crossover distribution by determining which recombination sites become crossovers. Surprisingly, a diverse group of genes, including those required for double strand break (DSB) formation or repair, have an effect on crossover distribution. Not all of these mutants, however, have a crossover-specific defect like mei-218 and it is not understood why some crossover-defective mutants alter the distribution of crossovers. Intragenic recombination experiments suggest that mei-218 is required for a molecular transition of the recombination intermediate late in the DSB repair pathway. We propose that the changes in crossover distribution in some crossover-defective mutants are a secondary consequence of the crossover reductions. This may be the activation of a regulatory system that ensures at least one crossover per chromosome, and which compensates for an absence of crossovers by attempting to generate them at random locations.