The Nature of Genetic Recombination near the Third Chromosome Centromere of DROSOPHILA MELANOGASTER

Previous studies have indicated that recombination near the third chromosome centromere is associated with negative chromosome interference, a phenomenon for which Green (1975) and Sinclair (1975) suggested gene conversion as a possible mechanism. In this report, we demonstrate that negative chromosome interference is still observed when deficiencies or translocation breakpoints are scored as the middle markers in recombination experiments and the rate of recombination is increased by interchromosomal effect. We argue that these chromosomal rearrangement breakpoints are not subject to conversion. Since neither successive premeiotic and meiotic exchanges, nor negative chromatid interference, can by themselves account for the negative chromosome interference, we conclude that a greater than expected frequency of multiple exchanges actually occurs. We further suggest that negative chromosome interference may be characteristic of all chromosomal regions normally showing very little exchange in relation to physical length.     

Negative supercoils regulate meiotic crossover patterns in budding yeast

Interference exists ubiquitously in many biological processes. Crossover interference patterns meiotic crossovers, which are required for faithful chromosome segregation and evolutionary adaption. However, what the interference signal is and how it is generated and regulated is unknown. We show that yeast top2 alleles which cannot bind or cleave DNA accumulate a higher level of negative supercoils and show weaker interference. However, top2 alleles which cannot religate the cleaved DNA or release the religated DNA accumulate less negative supercoils and show stronger interference. Moreover, the level of negative supercoils is negatively correlated with crossover interference strength. Furthermore, negative supercoils preferentially enrich at crossover-associated Zip3 regions before the formation of meiotic DNA double-strand breaks, and regions with more negative supercoils tend to have more Zip3. Additionally, the strength of crossover interference and homeostasis change coordinately in mutants. These findings suggest that the accumulation and relief of negative supercoils pattern meiotic crossovers.     

The Effects of Translocations on Recombination Frequency in Caenorhabditis Elegans

In the nematode Caenorhabditis elegans, recombination suppression in translocation heterozygotes is severe and extensive. We have examined the meiotic properties of two translocations involving chromosome I, szT1(I;X) and hT1(I;V). No recombination was observed in either of these translocation heterozygotes along the left (let-362-unc-13) 17 map units of chromosome I. Using half-translocations as free duplications, we mapped the breakpoints of szT1 and hT1. The boundaries of crossover suppression coincided with the physical breakpoints. We propose that DNA sequences at the right end of chromosome I facilitate pairing and recombination. We use the data from translocations of other chromosomes to map the location of pairing sites on four other chromosomes. hT1 and szT1 differed markedly in their effect on recombination adjacent to the crossover suppressed region. hT1 had no effect on recombination in the adjacent interval. In contrast, the 0.8 map unit interval immediately adjacent to the szT1(I;X) breakpoint on chromosome I increased to 2.5 map units in translocation heterozygotes. This increase occurs in a chromosomal interval which can be expanded by treatment with radiation. These results are consistent with the suggestion that the szT1(I) breakpoint is in a region of DNA in which meiotic recombination is suppressed relative to the genomic average. We propose that DNA sequences disrupted by the szT1 translocation are responsible for determining the frequency of meiotic recombination in the vicinity of the breakpoint.     

Construction and analysis of a two-chromosome double reciprocal translocation that functions as a ‘balancer’ inNeurospora crassa

T(IIL; VL;IIR; VR) BLNC-1 is a compound chromosome rearrangement inNeurospora crassa that combines two reciprocal translocations:T(IIL; VL) AR30 which interchanges the left end of linkage group II with the left end of linkage group V, andT(IIR;VR) ALS154 which interchanges the right end of linkage group II with the right end of linkage group V.BLNC-1 acts as a crossover suppressor for most of both linkage groups II and V since single crossovers between the rearrangement breakpoints result in progeny with lethal unbalanced duplications and deficiencies. The integrity ofBLNC-1 following meiosis was tested in crosses of markedBLNC-1 by marked Normal sequence, with markers located at critical points on linkage groups II and V. Although recombination between distal markers in the four arms was reduced markedly, double crossovers in the long intervening regions occurred with a frequency of 21%. Of these double crossovers, most were coincidental crossovers, one in each of the long intervening regions, resulting in the resolution of the complex into its component rearrangements (16%), while a minority of the double crossovers (5%) were crossovers involving only one of the two component linkage groups, and resulted in the insertion of a segment between the breakpoints. - TheBLNC-1 balancer can be used for: (1) mapping new loci to linkage groups II and V, especially for identifying markers mapping near the tips of the linkage groups; (2) for isolating genetically intact chromosomes from natural populations or for quantitative genetic studies; and (3) for studying recombinational hot-spots which can be detected as escapes from crossover suppression. -Based on experience withBLNC-1, future two-chromosome balancers should be designed with two breakpoints near, but not at, the opposite ends of the chromosome to be balanced, and the other two breakpoints close to, but spanning, the centromere of a second chromosome. Such a construction when combined with appropriately placed selective markers should prevent breakdown of the complex, and should resemble an inversion in eliminating crossover products. Contribution no. 85-218-J from the Department of Plant Pathology, Kansas Agricultural Experiment Station, Kansas State University, Manhattan.     

Gene conversion and crossing over along the 405-kb left arm of Saccharomyces cerevisiae chromosome VII

Gene conversions and crossing over were analyzed along 10 intervals in a 405-kb region comprising nearly all of the left arm of chromosome VII in Saccharomyces cerevisiae. Crossover interference was detected in all intervals as measured by a reduced number of nonparental ditypes. We have evaluated interference between crossovers in adjacent intervals by methods that retain the information contained in tetrads as opposed to single segregants. Interference was seen between intervals when the distance in the region adjacent to a crossover was < approximately 35 cM (90 kb). At the met13 locus, which exhibits approximately 9% gene conversions, those gene conversions accompanied by crossing over exerted interference in exchanges in an adjacent interval, whereas met13 gene conversions without an accompanying exchange did not show interference. The pattern of exchanges along this chromosome arm can be represented by a counting model in which there are three nonexchange events between adjacent exchanges; however, maximum-likelihood analysis suggests that approximately 8-12% of the crossovers on chromosome VII arise by a separate, noninterfering mechanism.     

Evidence for negative interference: clustering of crossovers close to the am locus in Neurospora crassa among am recombinants.

In response to a conflict between two mapping studies in the predicted orientation of the allele map with respect to the centromere, Fincham proposed that recombination events at the Neurospora am locus rarely have an associated crossover. Fincham considered that the elevated levels of crossing over between flanking markers in am recombinants resulted from negative interference, an increased probability of a nearby second event, and on this basis predicted a clustering of crossing over near am in these recombinants. In this article we reevaluate the data from three mapping studies of the am locus and report molecular evidence that shows crossovers to be clustered immediately proximal to am in am recombinants.     

Heterozygous insertions alter crossover distribution but allow crossover interference in Caenorhabditis elegans

The normal distribution of crossover events on meiotic bivalents depends on homolog recognition, alignment, and interference. We developed a method for precisely locating all crossovers on Caenorhabditis elegans chromosomes and demonstrated that wild-type animals have essentially complete interference, with each bivalent receiving one and only one crossover. A physical break in one homolog has previously been shown to disrupt interference, suggesting that some aspect of bivalent structure is required for interference. We measured the distribution of crossovers in animals heterozygous for a large insertion to determine whether a break in sequence homology would have the same effect as a physical break. Insertions disrupt crossing over locally. However, every bivalent still experiences essentially one and only one crossover, suggesting that interference can act across a large gap in homology. Although insertions did not affect crossover number, they did have an effect on crossover distribution. Crossing over was consistently higher on the side of the chromosome bearing the homolog recognition region and lower on the other side of the chromosome. We suggest that nonhomologous sequences cause heterosynapsis, which disrupts crossovers along the distal chromosome, even when those regions contain sequences that could otherwise align. However, because crossovers are not completely eliminated distal to insertions, we propose that alignment can be reestablished after a megabase-scale gap in sequence homology.     

The genetics and cytology of a mutator factor in Drosophila melanogaster

A Drosophila melanogaster mutator factor is described whose effects include the induction of unique chromosomal aberrations and male crossing over. Results of experiments to map the factor suggest that genetic transmission is somehow chromosomally associated but not localizable to the X, Y, second or third chromosome. There appears to be a good correlation between the distributions of male crossover exchange points and unique aberration breakpoints for the second chromosome but not for the third chromosome. The male crossovers, which occur more frequently in the centromeric region, occur in euchromatin rather than in the centric heterochromatin. The male crossovers tend to be rather precise reciprocal exchanges, since cytologically detectable deletions and duplications are only infrequently produced. It is suggested that the present mutator may be identical to earlier reported mutators of D. melanogaster.     

Two Types of Meiotic Crossovers Coexist in Maize

We apply modeling approaches to investigate the distribution of late recombination nodules in maize (Zea mays). Such nodules indicate crossover positions along the synaptonemal complex. High-quality nodule data were analyzed using two different interference models: the “statistical” gamma model and the “mechanical” beam film model. For each chromosome, we exclude at a 98% significance level the hypothesis that a single pathway underlies the formation of all crossovers, pointing to the coexistence of two types of crossing-over in maize, as was previously demonstrated in other organisms. We estimate the proportion of crossovers coming from the noninterfering pathway to range from 6 to 23% depending on the chromosome, with a cell average of ∼15%. The mean number of noninterfering crossovers per chromosome is significantly correlated with the length of the synaptonemal complex. We also quantify the intensity of interference. Finally, we develop inference tools that allow one to tackle, without much loss of power, complex crossover interference models such as the beam film. The lack of a likelihood function in such models had prevented their use for parameter estimation. This advance will allow more realistic mechanisms of crossover formation to be modeled in the future.     

Two meiotic crossover classes cohabit in Arabidopsis: one is dependent on MER3,whereas the other one is not

BACKGROUND: Crossovers are essential for the completion of meiosis. Recently, two pathways of crossover formation have been identified on the basis of distinct genetic controls. In one pathway, crossover inhibits the occurrence of another such event in a distance-dependent manner. This phenomenon is known as interference. The second kind of crossover is insensitive to interference. The two pathways function independently in budding yeast. Only interference-insensitive crossovers occur in Schizosaccharomyces pombe. In contrast, only interference-sensitive crossovers occur in Caenorabditis elegans. The situation in mammals and plants remains unclear. Mer3 is one of the genes shown to be required for the formation of interference-sensitive crossovers in Saccharomyces cerevisiae. RESULTS: To unravel the crossover status in the plant Arabidopsis thaliana, we investigated the role of the A. thaliana MER3 gene through the characterization of a series of allelic mutants. All mer3 mutants showed low levels of fertility and a significant decrease (about 75%) but not a total disappearance of meiotic crossovers, with the number of recombination events initiated in the mutants being similar to that in the wild-type. Genetic analyses showed that the residual crossovers in mer3 mutants did not display interference in one set of adjacent intervals. CONCLUSIONS: Mutation in MER3 in Arabidopsis appeared to be specific to recombination events resulting in interference-sensitive crossovers. Thus, MER3 function is conserved from yeast to plants and may exist in other metazoans. Arabidopsis therefore has at least two pathways for crossover formation, one giving rise to interference-sensitive crossover and the other to independently distributed crossovers.     

Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements

Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.     

A Mathematical Model of Interference for Use in Constructing Linkage Maps from Tetrad Data

In determining genetic map distances it is necessary to infer crossover frequencies from the ratios of recombinant and parental progeny. To do this accurately, in intervals where multiple crossovers may occur, a mathematical model of chiasma interference must be assumed when mapping in organisms displaying such interference. In Saccharomyces cerevisiae the model most frequently used is that of R.W. Barratt. An alternative to this model is presented. This new model is implemented using a microcomputer and standard numerical methods. It is demonstrated to fit ranked tetrad data from Saccharomyces more closely than the Barratt model and thus generates more accurate estimates of map distances when used with two-point data. A computer program implementing the model has been developed for use in calculating map distances from tetrad data in Saccharomyces.     

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.     

Crossover distribution and high interference for both the X chromosome and an autosome during oogenesis and spermatogenesis in Caenorhabditis elegans

Regulation of both the number and the location of crossovers during meiosis is important for normal chromosome segregation. We used sequence-tagged site polymorphisms to examine the distribution of all crossovers on the X chromosome during oogenesis and on one autosome during both oogenesis and spermatogenesis in Caenorhabditis elegans. The X chromosome has essentially one crossover during oogenesis, with only three possible double crossover exceptions among 220 recombinant X chromosomes. All three had one of the two crossovers in the same chromosomal interval, suggesting that crossovers in that interval do not cause interference. No other interval was associated with double crossovers. Very high interference was also found on an autosome during oogenesis, implying that each chromosome has only one crossover during oogenesis. During spermatogenesis, recombination on this autosome was reduced by approximately 30% compared to oogenesis, but the relative distribution of the residual crossovers was only slightly different. In contrast to previous results with other autosomes, no double crossover chromosomes were observed. Despite an increased frequency of nonrecombinant chromosomes, segregation of a nonrecombinant autosome during spermatogenesis appears to occur normally. This indicates that an achiasmate segregation system helps to ensure faithful disjunction of autosomes during spermatogenesis.     

Plant genetics: when not to interfere

New evidence suggests that the model plant Arabidopsis has two biochemically distinct pathways that produce genetic crossovers. Studies in several organisms have revealed that one kind of crossover regulation - crossover interference - is applied differently from species to species. Arabidopsis appears to use an interference system similar to that of budding yeast.     

Mapping functions

Accurate estimation of map distance between markers is important for the construction of large-scale linkage maps because it provides reliable and useful linkage information of markers on chromosomes. How to improve accuracy of estimating map distances depends on an appropriate mapping function. We used the coefficient of coincidence to integrate the Haldane function, in which crossovers are assumed to be independent and the Morgan function, in which crossovers are assumed to be interfered, and produce a new mapping function. The mapping function based on positive interference is referred to as the positive function and that on negative interference as the negative function. In these two mapping functions, map distances between loci are determined by both recombination frequencies and the coefficient of coincidence. We applied our mapping functions to four examples and show that our map estimates have much higher goodness-of-fit to the observed mapping data than the Haldane and Kosambi functions. Therefore, they can provide much more precise estimates of map distances than the two conventional mapping functions. Furthermore, our mapping functions produced almost linear (additive) map distances.     

Crossover interference in the mouse

We present an analysis of crossover interference in the mouse genome, on the basis of high-density genotype data from two reciprocal interspecific backcrosses, comprising 188 meioses. Overwhelming evidence was found for strong positive crossover interference with average strength greater than that implied by the Carter-Falconer map function. There was some evidence for interchromosomal variation in the level of interference, with smaller chromosomes exhibiting stronger interference. We further compared the observed numbers of crossovers to previous cytological observations on the numbers of chiasmata and evaluated evidence for the obligate chiasma hypothesis.     

A high-density cytogenetic map of the Aegilops tauschii genome incorporating retrotransposons and defense-related genes: insights into cereal chromosome structure and function

Aegilops tauschii (Coss.) Schmal. (2n = 2x = 14, DD) (syn. A. squarrosa L.; Triticum tauschii) is well known as the D-genome donor of bread wheat (T. aestivum, 2n = 6x = 42, AABBDD). Because of conserved synteny, a high-density map of the A. tauschii genome will be useful for breeding and genetics within the tribe Triticeae which besides bread wheat also includes barley and rye. We have placed 249 new loci onto a high-density integrated cytological and genetic map of A. tauschii for a total of 732 loci making it one of the most extensive maps produced to date for the Triticeae species. Of the mapped loci, 160 are defense-related genes. The retrotransposon marker system recently developed for cultivated barley (Hordeum vulgare L.) was successfully applied to A. tauschii with the placement of 80 retrotransposon loci onto the map. A total of 50 microsatellite and ISSR loci were also added. Most of the retrotransposon loci, resistance (R), and defense-response (DR) genes are organized into clusters: retrotransposon clusters in the pericentromeric regions, R and DR gene clusters in distal/telomeric regions. Markers are non-randomly distributed with low density in the pericentromeric regions and marker clusters in the distal regions. A significant correlation between the physical density of markers (number of markers mapped to the chromosome segment/physical length of the same segment in microm) and recombination rate (genetic length of a chromosome segment/physical length of the same segment in microm) was demonstrated. Discrete regions of negative or positive interference (an excess or deficiency of crossovers in adjacent intervals relative to the expected rates on the assumption of no interference) was observed in most of the chromosomes. Surprisingly, pericentromeric regions showed negative interference. Islands with negative, positive and/or no interference were present in interstitial and distal regions. Most of the positive interference was restricted to the long arms. The model of chromosome structure and function in cereals with large genomes that emerges from these studies is discussed.     

The road to crossovers: plants have their say

Crossovers involve the reciprocal exchange of large fragments of genetic material between homologous chromosomes during meiosis. In this way, crossovers are the basis of genetics. Remarkably, the number and distribution of crossovers on chromosomes are closely controlled. Data from various model organisms (notably Saccharomyces cerevisiae) show that the distribution of crossovers results from a series of tightly regulated events involving the formation and repair of double-strand breaks and interference. Recent advances in genetic and cytological tools, particularly for studying Arabidopsis thaliana, have enabled crossover control in plants to be studied in more detail. In this article, we discuss the contribution of plant studies to meiosis research, particularly to our understanding of crossover control and interference, and we evaluate models of interference.     

Pch2 Links Chromosome Axis Remodeling at Future Crossover Sites and Crossover Distribution during Yeast Meiosis

Segregation of homologous chromosomes during meiosis I depends on appropriately positioned crossovers/chiasmata. Crossover assurance ensures at least one crossover per homolog pair, while interference reduces double crossovers. Here, we have investigated the interplay between chromosome axis morphogenesis and non-random crossover placement. We demonstrate that chromosome axes are structurally modified at future crossover sites as indicated by correspondence between crossover designation marker Zip3 and domains enriched for axis ensemble Hop1/Red1. This association is first detected at the zygotene stage, persists until double Holliday junction resolution, and is controlled by the conserved AAA+ ATPase Pch2. Pch2 further mediates crossover interference, although it is dispensable for crossover formation at normal levels. Thus, interference appears to be superimposed on underlying mechanisms of crossover formation. When recombination-initiating DSBs are reduced, Pch2 is also required for viable spore formation, consistent with further functions in chiasma formation. pch2Δ mutant defects in crossover interference and spore viability at reduced DSB levels are oppositely modulated by temperature, suggesting contributions of two separable pathways to crossover control. Roles of Pch2 in controlling both chromosome axis morphogenesis and crossover placement suggest linkage between these processes. Pch2 is proposed to reorganize chromosome axes into a tiling array of long-range crossover control modules, resulting in chiasma formation at minimum levels and with maximum spacing.