Meiotic parthenogenesis in a root-knot nematode results in rapid genomic homozygosity

Many isolates of the plant-parasitic nematode Meloidogyne hapla reproduce by facultative meiotic parthenogenesis. Sexual crosses can occur, but, in the absence of males, the diploid state appears to be restored by reuniting sister chromosomes of a single meiosis. We have crossed inbred strains of M. hapla that differ in DNA markers and produced hybrids and F(2) lines. Here we show that heterozygous M. hapla females, upon parthenogenetic reproduction, produce progeny that segregate 1:1 for the presence or absence of dominant DNA markers, as would be expected if sister chromosomes are rejoined, rather than the 3:1 ratio typical of a Mendelian cross. Codominant markers also segregate 1:1 and heterozygotes are present at low frequency (<3%). Segregation patterns and recombinant analysis indicate that a homozygous condition is prevalent for markers flanking recombination events, suggesting that recombination occurs preferentially as four-strand exchanges at similar locations between both pairs of non-sister chromatids. With this mechanism, meiotic parthenogenesis would be expected to result in rapid genomic homozygosity. This type of high negative crossover interference coupled with positive chromatid interference has not been observed in fungal or other animal systems in which it is possible to examine the sister products of a single meiosis and may indicate that meiotic recombination in this nematode has novel features.     

ZHP-3 acts at crossovers to couple meiotic recombination with synaptonemal complex disassembly and bivalent formation in C. elegans

Crossover recombination and the formation of chiasmata normally ensure the proper segregation of homologous chromosomes during the first meiotic division. zhp-3, the Caenorhabditis elegans ortholog of the budding yeast ZIP3 gene, is required for crossover recombination. We show that ZHP-3 protein localization is highly dynamic. At a key transition point in meiotic prophase, the protein shifts from along the length of the synaptonemal complex (SC) to an asymmetric localization on the SC and eventually becomes restricted to foci that mark crossover recombination events. A zhp-3::gfp transgene partially complements a null mutation and reveals a separation of function; although the fusion protein can promote nearly wild-type levels of recombination, aneuploidy among the progeny is high, indicating defects in meiotic chromosome segregation. The structure of bivalents is perturbed in this mutant, suggesting that the chromosome segregation defect results from an inability to properly remodel chromosomes in response to crossovers. smo-1 mutants exhibit phenotypes similar to zhp-3::gfp mutants at higher temperatures, and smo-1; zhp-3::gfp double mutants exhibit more severe meiotic defects than either single mutant, consistent with a role for SUMO in the process of SC disassembly and bivalent differentiation. We propose that coordination of crossover recombination with SC disassembly and bivalent formation reflects a conserved role of Zip3/ZHP-3 in coupling recombination with SC morphogenesis.     

Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway

Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.     

Homeostatic control of recombination is implemented progressively in mouse meiosis

Humans suffer from high rates of fetal aneuploidy, often arising from the absence of meiotic crossover recombination between homologous chromosomes. Meiotic recombination is initiated by double-strand breaks (DSBs) generated by the SPO11 transesterase. In yeast and worms, at least one buffering mechanism, crossover homeostasis, maintains crossover numbers despite variation in DSB numbers. We show here that mammals exhibit progressive homeostatic control of recombination. In wild-type mouse spermatocytes, focus numbers for early recombination proteins (RAD51, DMC1) were highly variable from cell to cell, whereas foci of the crossover marker MLH1 showed little variability. Furthermore, mice with greater or fewer copies of the Spo11 gene--with correspondingly greater or fewer numbers of early recombination foci--exhibited relatively invariant crossover numbers. Homeostatic control is enforced during at least two stages, after the formation of early recombination intermediates and later while these intermediates mature towards crossovers. Thus, variability within the mammalian meiotic program is robustly managed by homeostatic mechanisms to control crossover formation, probably to suppress aneuploidy. Meiotic recombination exemplifies how order can be progressively implemented in a self-organizing system despite natural cell-to-cell disparities in the underlying biochemical processes.     

Predicting the size of the progeny mapping population required to positionally clone a gene

A key frustration during positional gene cloning (map-based cloning) is that the size of the progeny mapping population is difficult to predict, because the meiotic recombination frequency varies along chromosomes. We describe a detailed methodology to improve this prediction using rice (Oryza sativa L.) as a model system. We derived and/or validated, then fine-tuned, equations that estimate the mapping population size by comparing these theoretical estimates to 41 successful positional cloning attempts. We then used each validated equation to test whether neighborhood meiotic recombination frequencies extracted from a reference RFLP map can help researchers predict the mapping population size. We developed a meiotic recombination frequency map (MRFM) for approximately 1400 marker intervals in rice and anchored each published allele onto an interval on this map. We show that neighborhood recombination frequencies (R-map, >280-kb segments) extracted from the MRFM, in conjunction with the validated formulas, better predicted the mapping population size than the genome-wide average recombination frequency (R-avg), with improved results whether the recombination frequency was calculated as genes/cM or kb/cM. Our results offer a detailed road map for better predicting mapping population size in diverse eukaryotes, but useful predictions will require robust recombination frequency maps based on sampling more progeny.     

Cooperative interactions between pairs of homologous chromatids during meiosis in Saccharomyces cerevisiae

We report a novel instance of negative interference during Saccharomyces cerevisiae meiosis, where Cre-mediated recombination between pairs of allelic loxP sites is more frequent than expected. We suggest that endogenous crossover recombination mediates cooperative pairing interactions between all four chromatids of a meiotic bivalent.     

Genetic crosses in the apicomplexan parasite Cryptosporidium parvum define recombination parameters

Recombinant progeny lines of Cryptosporidium parvum were generated by coinfecting immunosuppressed mice with two genetically distinct isolates of C. parvum. Progeny lines were obtained from a cross of parental lines MD x TU114 through targeted propagation in mice of progeny oocysts originating from populations lacking one parental allele at one or more loci. For each infection lineage this process was repeated until only a single allele remained for each marker, indicating that the progeny line was clonal. To study genetic recombination, 16 progeny clones were genotyped at 40 loci located on each of the eight chromosomes. The inheritance of parental alleles was significantly skewed towards the more virulent parent isolate MD. A contiguous 476 kb segment of chromosome V displayed MD allele in all progeny recovered, while MD and TU114 alleles were detected at other loci throughout the genome. The absence of alleles from one parental isolate in this chromosomal region may indicate phenotypic selection for the MD allele during the generation of these lines. A range for the meiotic crossover frequency was determined on the basis of 40 markers and the number of meioses estimated to have taken place during the crossing experiment. C. parvum exhibits a high rate of recombination commensurate with other Apicomplexa.     

Repeat instability at human minisatellites arising from meiotic recombination.

Little is known about the role of meiotic recombination processes such as unequal crossover in driving instability at tandem repeat DNA. Methods have therefore been developed to detect meiotic crossovers within two different GC-rich minisatellite repeat arrays in humans, both in families and in sperm DNA. Both loci normally mutate in the germline by complex conversion-like transfer of repeats between alleles. Analysis shows that inter-allelic unequal crossovers also occur at both loci, although at low frequency, to yield simple recombinant repeat arrays with exchange of flanking markers. Equal crossovers between aligned alleles, resulting in recombinant alleles but without change in repeat copy number, also occur in sperm at a similar frequency to unequal crossovers. Both crossover and conversion show polarity in the repeat array and are co-suppressed in an allele showing unusual germline stability. This provides evidence that minisatellite conversion and crossover arise by a common mechanism, perhaps by alternative processing of a meiotic recombination initiation complex, and implies that minisatellite instability is a by-product of meiotic recombination in repeat DNA. While minisatellite recombination is infrequent, crossover rates indicate that the unstable end of a human minisatellite can act as a recombination warm-spot, even between sequence-heterologous alleles.     

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.     

Dominant enhancer effect of the meiotic mei4 mutant on recombination frequencies restricted to linkage group VI in Podospora anserina.

Summary A mutant which increases second division segregation (SDS) frequency of locus 110 (linkage group VI) was isolated. It was called mei4 because of its meiotic deficiency. The present paper deals with its effect on meiotic recombination when heterozygous. mei4 then only acts on linkage group VI. The SDS frequencies were increased for all markers used, except locus 5 located very close to the centromere. This quasi general enhancement results exclusively in an enlargement of map distance on linkage group VI's proximal part. Crosses involving three mutant genes allowed to check that the distances on the distal part were constant. This is due to a real lack of crossover frequency modification in this region and not to a change of chiasma interference. Among the seven linkage groups of Podospora anserina, group VI exhibits several other particularities concerning meiotic recombination, especially a lower positive chiasma interference and a more regular crossover distribution, suggesting a particular recombination regulation.Laboratoire associé n⁰ 86 du Centre National de la Recherche Scientifique     

Genetic mapping a meiotic driver that causes sex ratio distortion in the mosquito Aedes aegypti

An endogenous meiotic driver in the dengue and yellow fever vector mosquito Aedes aegypti can cause highly male-biased sex ratio distortion in crosses from suitable genetic backgrounds. We previously selected a strain that carries a strong meiotic drive gene (D) linked with the male-determining allele (M) on chromosome 1 in A. aegypti. Here, we performed segregation analysis of the M(D) locus among backcross (BC(1)) progeny from a driver male and drive-sensitive females. Assessment of sex ratios among BC(2) progeny showed ～5.2% recombination between the M(D) locus and the sex determination locus. Multipoint linkage mapping across this region revealed consistent marker orders and recombination frequencies with the existing reference linkage map and placed the M(D) locus within a 6.5-cm interval defined by the LF159 locus and microsatellite marker 446GAA, which should facilitate future positional cloning efforts.     

Male and female recombination landscapes of diploid Arabidopsis arenosa

The number and placement of meiotic crossover events during meiosis have important implications for the fidelity of chromosome segregation as well as patterns of inheritance. Despite the functional importance of recombination, recombination landscapes vary widely among and within species, and this can have a strong impact on evolutionary processes. A good knowledge of recombination landscapes is important for model systems in evolutionary and ecological genetics, since it can improve interpretation of genomic patterns of differentiation and genome evolution, and provides an important starting point for understanding the causes and consequences of recombination rate variation. Arabidopsis arenosa is a powerful evolutionary genetic model for studying the molecular basis of adaptation and recombination rate evolution. Here, we generate genetic maps for 2 diploid A. arenosa individuals from distinct genetic lineages where we have prior knowledge that meiotic genes show evidence of selection. We complement the genetic maps with cytological approaches to map and quantify recombination rates, and test the idea that these populations might have distinct patterns of recombination. We explore how recombination differs at the level of populations, individuals, sexes and genomic regions. We show that the positioning of crossovers along a chromosome correlates with their number, presumably a consequence of crossover interference, and discuss how this effect can cause differences in recombination landscape among sexes or species. We identify several instances of female segregation distortion. We found that averaged genome-wide recombination rate is lower and sex differences subtler in A. arenosa than in Arabidopsis thaliana.     

Inheritance of Restriction Fragment Length Polymorphisms in Agaricus Brunnescens

The cultivated mushroom, Agaricus brunnescens, is secondarily homothallic; most basidia produce only two basidiospores, each of which receives two of the four post meiotic nuclei. The segregation of restriction fragment length polymorphisms (RFLPs) detected by four plasmid probes carrying single-copy nuclear DNA of Agaricus was followed in seven parental strains including commercial, wild-collected, and artificially synthesized heterokaryons. Of a total of 367 single-spore progeny examined, 351 (95.6%) were heteroallelic at all RFLP loci heteroallelic in the respective parents. Of the 16 segregant isolates, ten (2.7% of the total) were homoallelic at all segregating loci assayed, suggesting that these isolates were most probably derived from rare spores that had received only a single postmeiotic nucleus. Some of these ten isolates had recombinant genotypes. Only five isolates (1.4% of the total) showed homoallelism at one of the loci heteroallelic in the parent, while remaining heteroallelic at other loci. These five genotypes suggest that a crossover had occurred between a marker locus and its respective centromere. Taken together, the results suggest that meiosis in A. brunnescens is accompanied by low levels of recombination and that nonsister nuclei are preferentially incorporated into basidiospores after meiosis II.     

Unequal Exchange and Meiotic Instability of Disease-Resistance Genes in the Rp1 Region of Maize

The Rp1 region of maize was originally characterized as a complex locus which conditions resistance to the fungus Puccinia sorghi, the causal organism in the common rust disease. Some alleles of Rp1 are meiotically unstable, but the mechanism of instability is not known. We have studied the role of recombination in meiotic instability in maize lines homozygous for either Rp1-J or Rp1-G. Test cross progenies derived from a line that was homozygous for Rp1-J, but heterozygous at flanking markers, were screened for susceptible individuals. Five susceptible individuals were derived from 9772 progeny. All five had nonparental combinations of flanking markers; three had one combination of recombinant flanking markers while the other two had the opposite pair. In an identical study with Rp1-G, 20 susceptible seedlings were detected out of 5874 test cross progeny. Nineteen of these were associated with flanking marker exchange, 11 and 8 of each recombinant marker combination. Our results indicate that unequal exchange is the primary mechanism of meiotic instability of Rp1-J and Rp1-G.     

High‐resolution insight into recombination events at the SD1 locus in rice

Recombination during meiosis plays an important role in genome evolution by reshuffling existing genetic variations into fresh combinations with the possibility of recovery of lost ancestral genotypes. While crossover (CO) events have been well studied, gene conversion events (GCs), which represent non‐reciprocal information transfer between chromosomes, are poorly documented and difficult to detect due to their relatively small converted tract size. Here, we document these GC events and their phenotypic effects at an important locus in rice containing the SD1 gene, where multiple defective alleles contributed to the semi‐dwarf phenotype of rice in the ‘Green Revolution’ of the 1960s. Here, physical separation of two defects allows recombination to generate the wild‐type SD1 gene, for which plant height can then be used as a reporter. By screening 18 000 F₂ progeny from a cross between two semi‐dwarf cultivars that carry these different defective alleles, we detected 24 GC events, indicating a conversion rate of ~3.3 × 10^?4 per marker per generation in a single meiotic cycle in rice. Furthermore, our data show that indels and single‐nucleotide polymorphisms (SNPs) do not differ significantly in GC rates, at least at the SD1 locus. Our results provide strong evidence that GC by itself can regain an ancestral phenotype that was lost through mutation. This GC detection approach is likely to be broadly applicable to natural or artificial alleles of other phenotype‐related functional genes, which are abundant in other plant genomes.     

Genetic control of mammalian meiotic recombination. I. Variation in exchange frequencies among males from inbred mouse strains

Genetic background effects on the frequency of meiotic recombination have long been suspected in mice but never demonstrated in a systematic manner, especially in inbred strains. We used a recently described immunostaining technique to assess meiotic exchange patterns in male mice. We found that among four different inbred strains--CAST/Ei, A/J, C57BL/6, and SPRET/Ei--the mean number of meiotic exchanges per cell and, thus, the recombination rates in these genetic backgrounds were significantly different. These frequencies ranged from a low of 21.5 exchanges in CAST/Ei to a high of 24.9 in SPRET/Ei. We also found that, as expected, these crossover events were nonrandomly distributed and displayed positive interference. However, we found no evidence for significant differences in the patterns of crossover positioning between strains with different exchange frequencies. From our observations of >10,000 autosomal synaptonemal complexes, we conclude that achiasmate bivalents arise in the male mouse at a frequency of 0.1%. Thus, special mechanisms that segregate achiasmate chromosomes are unlikely to be an important component of mammalian male meiosis.     

Crossover/noncrossover differentiation, synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene transition of meiosis

Yeast mutants lacking meiotic proteins Zip1, Zip2, Zip3, Mer3, and/or Msh5 (ZMMs) were analyzed for recombination, synaptonemal complex (SC), and meiotic progression. At 33 degrees C, recombination-initiating double-strand breaks (DSBs) and noncrossover products (NCRs) form normally while formation of single-end invasion strand exchange intermediates (SEIs), double Holliday junctions, crossover products (CRs), and SC are coordinately defective. Thus, during wild-type meiosis, recombinational interactions are differentiated into CR and NCR types very early, prior to onset of stable strand exchange and independent of SC. By implication, crossover interference does not require SC formation. We suggest that SC formation may require interference. Subsequently, CR-designated DSBs undergo a tightly coupled, ZMM-promoted transition that yields SEI-containing recombination complexes embedded in patches of SC. zmm mutant phenotypes differ strikingly at 33 degrees C and 23 degrees C, implicating higher temperature as a positive effector of recombination and identifying a checkpoint that monitors local CR-specific events, not SC formation, at late leptotene.     

Etoposide exposure during male mouse pachytene has complex effects on crossing-over and causes nondisjunction

In experiments involving different germ-cell stages, we had previously found meiotic prophase of the male mouse to be vulnerable to the induction of several types of genetic damage by the topoisomerase-II inhibitor etoposide. The present study of etoposide effects involved two end points of meiotic events known to occur in primary spermatocytes--chromosomal crossing-over and segregation. By following assortment of 13 microsatellite markers in two chromosomes (Ch 7 and Ch 15) it was shown that etoposide significantly affected crossing-over, but did not do so in a uniform fashion. Treatment generally changed the pattern for each chromosome, leading to local decreases in recombination, a distal shift in locations of crossing-over, and an overall decrease in double crossovers; at least some of these results might be interpreted as evidence for increased interference. Two methods were used to explore etoposide effects on chromosome segregation: a genetic experiment capable of detecting sex-chromosome nondisjunction in living progeny; and the use of FISH (fluorescence in situ hybridization) technology to score numbers of Chromosomes X, Y, and 8 in spermatozoa. Taken together these two approaches indicated that etoposide exposure of pachytene spermatocytes induces malsegregation, and that the findings of the genetic experiment probably yielded a marked underestimate of nondisjunction. As indicated by certain segregants, at least part of the etoposide effect could be due to disrupted pairing of achiasmatic homologs, followed by precocious sister-centromere separation. It has been shown for several organisms that absent or reduced levels of recombination, as well as suboptimally positioned recombination events, may be associated with abnormal segregation. Etoposide is the only chemical tested to date for which living progeny indicates an effect on both male meiotic crossing-over and chromosome segregation. Whether, however, etoposide-induced changes in recombination patterns are direct causes of the observed malsegregation requires additional investigation.     

Domain-specific regulation of recombination in Caenorhabditis elegans in response to temperature, age and sex

It is generally considered that meiotic recombination rates increase with temperature, decrease with age, and differ between the sexes. We have reexamined the effects of these factors on meiotic recombination in the nematode Caenorhabditis elegans using physical markers that encompass >96% of chromosome III. The only difference in overall crossover frequency between oocytes and male sperm was observed at 16°. In addition, crossover interference (CI) differs between the germ lines, with oocytes displaying higher CI than male sperm. Unexpectedly, our analyses reveal significant changes in crossover distribution in the hermaphrodite oocyte in response to temperature. This feature appears to be a general feature of C. elegans chromosomes as similar changes in response to temperature are seen for the X chromosome. We also find that the distribution of crossovers changes with age in both hermaphrodites and females. Our observations indicate that it is the oocytes from the youngest mothers—and not the oldest—that showed a different pattern of crossovers. Our data enhance the emerging hypothesis that recombination in C. elegans, as in humans, is regulated in large chromosomal domains.