During meiosis homologous chromosomes undergo crossover recombination. Sequence differences between homologs can locally inhibit crossovers. Despite this, nucleotide diversity and population-scaled recombination are positively correlated in eukaryote genomes. To investigate interactions between heterozygosity and recombination we crossed Arabidopsis lines carrying fluorescent crossover reporters to 32 diverse accessions and observed hybrids with significantly higher and lower crossovers than homozygotes. Using recombinant populations derived from these crosses we observed that heterozygous regions increase crossovers when juxtaposed with homozygous regions, which reciprocally decrease. Total crossovers measured by chiasmata were unchanged when heterozygosity was varied, consistent with homeostatic control. We tested the effects of heterozygosity in mutants where the balance of interfering and non-interfering crossover repair is altered. Crossover remodeling at homozygosity-heterozygosity junctions requires interference, and non-interfering repair is inefficient in heterozygous regions. As a consequence, heterozygous regions show stronger crossover interference. Our findings reveal how varying homolog polymorphism patterns can shape meiotic recombination.https://doi.org/10.7554/eLife.03708.001
The genomes of plants and animals consist of several long DNA molecules that are called chromosomes. Most organisms carry two copies of each chromosome: one inherited from each parent. This means that an individual has two copies of each gene. Some of these gene copies may be identical (known as ‘homozygous’), but other gene copies will have sequence differences (or be ‘heterozygous’).
The sex cells (eggs and sperm) that pass half of each parent's genes on to its offspring are made in a process called meiosis. Before the pairs of each chromosome are separated to make two new sex cells, sections of genetic material can be swapped between a chromosome-pair to produce chromosomes with unique combinations of genetic material.
The ‘crossover’ events that cause the genetic material to be swapped are less likely to happen in sections of chromosomes that contain heterozygous genes. However, in a whole population of organisms, the exchange of genetic material between pairs of chromosomes tends to be higher when there are more genetic differences present.
Here, Ziolkowski et al. sought to understand these two seemingly contradictory phenomena by studying crossover events during meiosis in a plant known as Arabidopsis. The plants were genetically modified to carry fluorescent proteins that mark when and where crossovers occur. Ziolkowski et al. cross-bred these plants with 32 other varieties of Arabidopsis. The experiments show that some of these ‘hybrid’ plants had higher numbers of crossover events than plants produced from two genetically identical parents, but other hybrid plants had lower numbers of crossovers.
Ziolkowski et al. found that crossovers are more common between heterozygous regions that are close to homozygous regions on the same chromosome. The boundaries between these identical and non-identical regions are important for determining where crossovers take place. The experiments also show that the heterozygous regions have higher levels of interference—where one crossover event prevents other crossover events from happening nearby on the chromosome. In future, using chromosomes with varying patterns of heterozygosity may shed light on how this interference works.https://doi.org/10.7554/eLife.03708.002
Sexual reproduction via meiosis is highly conserved within eukaryotes and allows recombination of genetic variation within populations (Barton and Charlesworth, 1998). During meiosis homologous chromosomes pair and undergo crossover recombination, which together with independent chromosome segregation and gamete fusion increases genetic diversity between progeny (Barton and Charlesworth, 1998; Villeneuve and Hillers, 2001). Meiotic crossovers form via the repair of DNA double-strand breaks (DSBs) generated by the SPO11 endonuclease (Bergerat et al., 1997; Keeney et al., 1997). Nucleolytic resection of DSBs generates 3′ single-stranded DNA (ssDNA), which is bound by the RAD51 and DMC1 recombinases (Bishop et al., 1992; Shinohara et al., 1992). The resulting nucleoprotein filament then invades a homologous chromatid to form a heteroduplex intermediate (Hunter and Kleckner, 2001). The invading ssDNA 3′-ends undergo DNA synthesis using the homologous duplex as a template and after second-end capture forms double Holliday junctions (dHJs) (Szostak et al., 1983; Schwacha and Kleckner, 1995). The dHJs can then be resolved as crossovers, which are cytologically evident as chiasmata (Page and Hawley, 2003; Janssens et al., 2012). Chiasmata hold chromosomes together and ensure that homologous pairs segregate to opposite cell poles, so that gametes inherit a balanced chromosome number (Page and Hawley, 2003).
Crossover numbers are under tight control, with many eukaryote species experiencing 1–2 per chromosome, despite large variation in genome size (Villeneuve and Hillers, 2001; Smukowski and Noor, 2011; Henderson, 2012; Mercier et al., 2014). In Arabidopsis ∼200 DSBs form per meiosis and proceed to form strand invasion intermediates, of which ∼10 are repaired as crossovers, with the excess being repaired as non-crossovers, or via intersister recombination (Giraut et al., 2011; Ferdous et al., 2012; Lu et al., 2012; Sun et al., 2012; Yang et al., 2012; Drouaud et al., 2013; Wijnker et al., 2013; Qi et al., 2014). 80–85% of wild type crossovers are dependent on the ZMM pathway (MSH4, MSH5, MER3, HEI10, ZIP4, SHOC1, PTD) and show interference, that is, they are spaced further apart than expected at random (Copenhaver et al., 2002; Higgins et al., 2004, 2008a; Chen et al., 2005; Mercier et al., 2005; Chelysheva et al., 2007, 2010, 2012; Macaisne et al., 2008). The remaining minority of crossovers are non-interfering and require MUS81 (Berchowitz et al., 2007; Higgins et al., 2008b). However, as chiasmata are still observed in msh4 mus81 double mutants, additional crossover pathways must exist (Higgins et al., 2008b). The majority of interhomolog strand invasion intermediates are dissolved by the FANCM helicase, which acts with the MHF1 and MHF2 co-factors (Crismani et al., 2012; Knoll et al., 2012; Girard et al., 2014). Mutations in FANCM, MHF1 and MHF2 cause dramatic increases in non-interfering crossovers (Crismani et al., 2012; Knoll et al., 2012; Girard et al., 2014). It is presently unclear whether non-interfering crossovers occurring in fancm are generated by the same pathway as in wild type, as a direct test of MUS81 dependence is precluded by fancm mus81 lethality (Crismani et al., 2012; Knoll et al., 2012). Both crossovers and non-crossovers can be accompanied by gene conversion events, which in the case of non-crossovers form via the synthesis-dependent strand annealing pathway (Allers and Lichten, 2001; McMahill et al., 2007; Lu et al., 2012; Sun et al., 2012; Yang et al., 2012; Drouaud et al., 2013; Wijnker et al., 2013; Qi et al., 2014).
Meiotic recombination is sensitive to DNA polymorphism between homologous chromosomes, that is, heterozygosity. For example, insertion-deletion (indel) and single nucleotide polymorphisms (SNPs) suppress crossovers at the scale of hotspots (kb) in fungi, plants and mammals (Dooner, 1986; Borts and Haber, 1987; Jeffreys and Neumann, 2005; Baudat and de Massy, 2007; Cole et al., 2010). This is thought to occur due to heteroduplex base-pair mismatches inhibiting recombination, following interhomolog strand invasion. Large scale chromosome rearrangements, such as inversions or translocations, also suppress crossovers (Schwander et al., 2014; Thompson and Jiggins, 2014). Despite the inhibitory effects of polymorphism on crossovers, nucleotide diversity and population-scaled recombination estimates are positively correlated in many plant and animal genomes (Begun and Aquadro, 1992; Hellmann et al., 2003; Spencer et al., 2006; Gore et al., 2009; Paape et al., 2012; Cutter and Payseur, 2013). For example, linkage disequilibrium-based crossover estimates and sequence diversity (π) are positively correlated in Arabidopsis at varying physical scales (Figure 1A and Table 1) (Cao et al., 2011; Choi et al., 2013). Multiple processes contribute to these relationships. For example, positive or negative directional selection can reduce diversity at linked sites, with a greater effect in regions of low recombination, known as hitchhiking and background selection (Hill and Robertson, 1966; Hudson and Kaplan, 1995; Nordborg et al., 1996; Smith and Haigh, 2007; Cutter and Payseur, 2013; Campos et al., 2014). These phenomena will cause regions of low recombination under selection to have low diversity, consistent with data in Drosophila (Aguade et al., 1989; Begun and Aquadro, 1992; Wiehe and Stephan, 1993; Campos et al., 2014). Recombination may also be mutagenic and increase diversity, for example via mismatch repair enzymes showing a mutational bias for A:T > G:C transversions (Duret and Galtier, 2009; Webster and Hurst, 2012; Glémin et al., 2014).
Here we use natural variation in Arabidopsis to directly investigate the influence of heterozygosity on meiotic recombination. Extensive evidence exists for cis and trans modification of crossover frequency by plant genetic variation (Barth et al., 2001; Yao and Schnable, 2005; Yandeau-Nelson et al., 2006; Esch et al., 2007; McMullen et al., 2009; López et al., 2012; Salomé et al., 2012; Bauer et al., 2013). We define trans modifiers as loci encoding diffusible molecules that control recombination on other chromosomes, and elsewhere on the same chromosome, as exemplified by mammalian PRDM9 (Baudat et al., 2010; Berg et al., 2010; Myers et al., 2010; Parvanov et al., 2010; Fledel-Alon et al., 2011; Sandor et al., 2012; Kong et al., 2013). We define cis modification as variation that influences recombination only on the same chromosome, for example, the inhibitory effects of high SNP density, inversions and translocations (Dooner, 1986; Borts and Haber, 1987; Jeffreys and Neumann, 2005; Baudat and de Massy, 2007; Cole et al., 2010; Schwander et al., 2014; Thompson and Jiggins, 2014). Regional patterns of chromatin and epigenetic information can also cause significant cis effects, for example loss of either H2A.Z deposition or DNA methylation alters crossover frequency in Arabidopsis (Colomé-Tatché et al., 2012; Melamed-Bessudo and Levy, 2012; Mirouze et al., 2012; Yelina et al., 2012; Choi et al., 2013).
In this study we crossed Arabidopsis lines carrying fluorescent crossover reporters generated in a common background (Col-0) to 32 diverse accessions. We observed extensive variation in F1 hybrid recombination rates, with both significantly higher and lower crossovers than homozygous backgrounds. We further analysed Col × Ct F2 recombinant populations using three independent crossover reporter intervals (420, CEN3 and I2f). We did not detect trans modifiers in these crosses, but observed a novel cis modification effect caused by heterozygosity. Specifically, juxtaposition of heterozygous and homozygous regions is associated with increased crossover frequency in the heterozygous region and a reciprocal decrease in the homozygous region. To investigate this phenomenon mechanistically we repeated analysis in mutants where the balance of interfering and non-interfering crossover repair is altered (fancm, zip4 and fancm zip4). This analysis demonstrates that remodelling of crossovers across heterozygosity/homozygosity junctions is dependent on interference. We also show that the non-interfering repair is less efficient in heterozygous regions. As a consequence, interference measurements are stronger in heterozygous regions. Our findings show how varying polymorphism patterns can differentially influence meiotic recombination along chromosomes.
To test the effect of heterozygosity on meiotic recombination we crossed transgenic Arabidopsis with fluorescent crossover reporters generated in the Col-0 background to 32 diverse accessions that represent global genetic diversity within this species (Figure 1, Tables 2, 3) (Melamed-Bessudo et al., 2005; Berchowitz and Copenhaver, 2008; Yelina et al., 2013). The 5 intervals tested (I1b, I1fg, I2f, 420 and CEN3) range from 0.67–5.40 megabases (Mb), represent 11.5% of the genome (14.34 Mb) in total and are located in sub-telomeric, interstitial and centromeric regions (Figure 1A and Table 2). The intervals vary in experimental recombination rate, with the centromeric interval CEN3 being the lowest (2.11 cM/Mb) and the sub-telomeric interval I2f being the highest (13.02 cM/Mb) (Table 2). As Arabidopsis male meiosis shows elevated sub-telomeric recombination, this likely contributes to the high I2f crossover frequency, which is measured in pollen (Giraut et al., 2011). Low recombination in CEN3 is also expected, as the centromeric regions are heterochromatic and known to show suppressed crossover frequency (Figure 1A) (Copenhaver et al., 1999; Giraut et al., 2011; Salomé et al., 2012; Yelina et al., 2012). To asses relative heterozygosity levels we analysed pairwise sequence differences relative to Col-0 using the 19 genomes dataset, which was generated from a subset of the accessions used in our crosses (Gan et al., 2011). CEN3 shows the highest heterozygosity levels, followed by the interstitial and sub-telomeric intervals (Table 2). Therefore, the regions analysed represent diverse chromosomal environments with varying levels of recombination and inter-accession sequence polymorphism.
The crossover reporter systems utilize fluorescent proteins encoded by linked, heterozygous transgenes that are expressed from the pollen-specific LAT52, or seed-specific NapA promoters (Melamed-Bessudo et al., 2005; Francis et al., 2006; Yelina et al., 2013). Fluorescent measurements of gametes or progeny are used to asses segregation of the transgenes through meiosis and thereby measure crossover rates (Melamed-Bessudo et al., 2005; Berchowitz and Copenhaver, 2008; Yelina et al., 2013). Previously, we developed flow cytometry protocols to increase scoring-throughput using fluorescent pollen, allowing up to 80,000 gametes to be scored per individual plant (Yelina et al., 2012, 2013). To increase throughput when measuring fluorescent seed we adapted CellProfiler image analysis software, allowing us to rapidly score ∼2000 seed per individual (Figure 2A–F) (Carpenter et al., 2006). This method gives recombination measurements not significantly different from manually collected data (Figure 2F, Figure 2—source data 1) (generalized linear model (GLM), hereafter GLM, p = 0.373). To test for significant differences between recombinant and non-recombinant counts using replicate groups we used a GLM assuming a binomial count distribution. Replicate heterozygous F1 individuals were analysed for each cross and 13,264,943 gametes were scored in total, to provide an extensive survey of the influence of polymorphism heterozygosity on crossover frequency (Figure 3 and Table 3).
We observed substantial variation in crossovers between F1 crosses, although the interstitial intervals varied less than those in sub-telomeric and centromeric locations (Figure 3A–E, Figure 3—source data 1–5). F1 heterozygotes showed both significantly higher and lower total recombination compared to Col homozygotes (Figure 3 and Table 3) (GLM with 113° of freedom p < 2.0 × 10−16). F1 genetic distances and polymorphism levels within the intervals were poorly correlated, consistent with previous observations (Table 4) (Barth et al., 2001; Gan et al., 2011; Salomé et al., 2012). This weak correlation may be partially explained by unknown structural rearrangements. For example, the Shahdara (Sha) accession has a sub-telomeric inversion (3–5.1 Mb) on chromosome 3 relative to Col (Loudet et al., 2002; Simon et al., 2008; Salomé et al., 2012), and Col/Sha F1s show consistently low crossovers in 420, which overlaps the inversion (Figure 3D and Table 3). Hence the contribution of unknown structural polymorphisms to variation in recombination rates could be significant. Further evidence of the complex effect of polymorphism is evident from the CEN3 interval, which spans the repetitive and structurally diverse centromeric region of chromosome 3 (Figure 1A) (Copenhaver et al., 1999; Clark et al., 2007; Ito et al., 2007; Cao et al., 2011; Gan et al., 2011; Horton et al., 2012; Long et al., 2013), and showed high variability in F1 crossover frequency (Figure 3E and Table 3). Unexpectedly, some of the most diverged crosses, for example two accessions from Atlantic islands Cvi-0 and Can-0, showed highest CEN3 crossovers (Figure 3E and Table 3) (Ito et al., 2007). 10 of 26 F1s showed significantly higher summed crossover frequency compared with Col homozygotes, consistent with previous reports that recombination can increase in heterozygous backgrounds in Arabidopsis (Barth et al., 2001) (Figure 3F and Table 3). Both cis and trans modification of crossovers by genetic variation has been observed in plants (Barth et al., 2001; Yao and Schnable, 2005; Yandeau-Nelson et al., 2006; Esch et al., 2007; McMullen et al., 2009; López et al., 2012; Salomé et al., 2012; Bauer et al., 2013). Therefore, the variation in F1 crossover frequency observed here is likely caused by complex interactions between cis and trans modifying effects.
To investigate the extent of cis and trans modification of crossover frequency by heterozygosity we generated a 420 Col × Ct recombinant F2 population (n = 139) (Figure 4A). We selected F2 individuals that were heterozygous for linked T-DNAs expressing red and green fluorescent proteins and Col/Ct heterozygous within 420, but genetically mosaic elsewhere in the genome (Figure 4A,E). The 420/++ Col/Ct F2 population showed significantly greater variation in recombination rates than Col/Col homozygotes (F-test p = 0.0129) (Figure 4D, Figure 4—source data 1). We genotyped 51 Col/Ct markers throughout the genome and tested for their association with 420 crossover frequency using QTL analysis. We detected no association using markers on chromosomes 1, 2, 4 or 5 (Figure 4B). However, on chromosome 3 itself homozygosity (Col/Col or Ct/Ct) outside of 420 was associated with high recombination (FDR corrected chi-square test p = 3.29 × 10−31) (Figure 4B,E–F and Table 5). For each marker we used the heterozygous and homozygous counts in the hottest quartile vs the coldest quartile to construct 2 × 2 contingency tables and performed chi-square tests, followed by FDR correction for multiple testing (Table 5).
To test an additional chromosome for the effect of heterozygosity/homozygosity juxtaposition we measured recombination in an I2f Col × Ct F2 population (n = 78) (Figure 4G–I). The I2f interval is 0.67 Mb and located sub-telomerically on the long arm of chromosome 2 (Figure 1A and Table 2). The I2f/++ Col/Ct F2 population also showed significantly greater variation in recombination rates than Col/Col homozygotes (F-test, p = 0.04) (Figure 4G, Figure 4—source data 2). We performed QTL analysis for Col/Ct markers on chromosomes 2 and 3 and again observed a significant effect on the same chromosome and no trans effect from chromosome 3. An identical trend to that seen for 420 was observed, with the highest recombination F2 quartile showing significantly greater marker homozygosity (both Col/Col and Ct/Ct) outside I2f on chromosome 2 (FDR corrected chi-square test p = 1.44 × 10−10) (Figure 4C,G–I and Table 6). The most distal marker showing a significant difference between hot and cold quartiles was of comparable megabase distance for 420 (10.60 Mb) and I2f (10.12 Mb).
To test whether the effect of heterozygosity/homozygosity juxtaposition is dependent on chromosomal location we measured crossovers in a CEN3 Col × Ct F2 population (n = 121) (Figures 4A and 5C, Figure 4—figure supplement 1, Figure 4—source data 3). As for 420 and I2f, CEN3 F2 recombination rates were significantly more variable than Col/Col homozygotes (F-test p = 0.01268) (Figure 4A, Figure 4—figure supplement 1). We genotyped 9 Col/Ct markers on chromosome 3 and observed that 5 markers in proximity to CEN3 were significantly more homozygous in the hottest compared to the coldest F2 quartile (FDR corrected chi-square test p = 1.14 × 10−07) (Figure 4D–F, Figure 4—figure supplement 1 and Table 7). The physical extent of the effect was less (2.62 Mb) on the long arm of chromosome 3 for CEN3 than observed for 420 and I2f, potentially due to heterozygosity effects acting independently from both arms across the centromere. Together this shows that juxtaposition of heterozygous and homozygous regions in various chromosomal locations can modify local crossover frequency.
We reasoned that if heterozygous regions increase recombination when juxtaposed with homozygous regions, then the linked homozygous regions may show compensatory decreases, due to crossover interference (Copenhaver et al., 2002; Zhang et al., 2014a). To test this idea we constructed a three-colour pollen FTL interval termed I3bc that overlaps the 420 seed interval on chromosome 3 (Figure 5 and Table 2). Three-colour FTL configurations allow simultaneous measurement of crossover frequency in adjacent intervals and measurement of crossover interference (Berchowitz and Copenhaver, 2008; Yelina et al., 2013) (Figure 5—figure supplement 1). To calculate interference, the observed double crossover (DCO) classes (N-Y- + NB-R) are compared to the number expected in the absence of interference: (I3b cM/100) × (I3c cM/100) × Ntotal (Figure 5A). The Coefficient of Coincidence (CoC) is calculated by dividing Observed DCOs by Expected DCOs, and interference strength calculated as 1-CoC (Figure 5A).
I3bc wild type genetic distance was greater than that measured from 420 self-fertilization data, as expected due to increases observed in sub-telomeric regions in male meiosis (Table 2—Figure 5—source data 1) (Giraut et al., 2011). I3b crossover frequency was also higher than I3c, consistent with a telomeric gradient in male crossover frequency (Figure 5B and Table 2) (Giraut et al., 2011). We compared crossovers in plants that were entirely Col homozygous (HOM-HOM) vs plants that were Col/Ct heterozygous within I3b, but Col/Col homozygous in I3c and for the rest of chromosome 3 (HET-HOM) (Figure 5A). Dense genotyping markers were used to confirm the location of homozygous and heterozygous regions (Figure 5A). We observed that I3b crossovers significantly increased in HET-HOM compared to HOM-HOM plants, and there was a reciprocal decrease in I3c crossovers (Figure 5B, Figure 5—source data 2) (both GLM p < 2.0 × 10−16). Together this is consistent with reciprocal crossover changes in juxtaposed heterozygous and homozygous regions being driven by crossover interference.
The effect of heterozygosity/homozygosity juxtaposition on crossovers extends over megabase distances, which is similar to the scale of crossover interference in Arabidopsis (Copenhaver et al., 2002; Giraut et al., 2011; Salomé et al., 2012). We therefore next used mutations in meiotic recombination pathways to analyse the genetic requirements of these effects. Specifically, we generated plants carrying the linked chromosome 3 fluorescent crossover reporters 420 and CEN3 (420-CEN3), with varying Col/Ct genotype and that were wild type, fancm or fancm zip4 (Figure 6—Figure 6—figure supplement 1). Crossover frequency in 420 and CEN3 can be scored in the same individuals, as these intervals use fluorescent proteins expressed in seed and pollen respectively. In fancm DSBs that would normally be repaired as non-crossovers enter a non-interfering pathway leading to a substantial increase in crossovers, although the interfering pathway remains active (Crismani et al., 2012). In fancm zip4 only non-interfering crossovers occur, due to mutation of the ZMM gene ZIP4 (Chelysheva et al., 2007; Crismani et al., 2012). In wild type, both interfering and non-interfering pathways are active, but interfering crossovers predominate and constitute ∼85% of total crossovers (Copenhaver et al., 2002; Higgins et al., 2004; Mercier et al., 2005). Therefore, by comparing genetic distances in wild type, fancm and fancm zip4, where the relative proportions of interfering and non-interfering repair vary dramatically, we can investigate the sensitivity of different recombination pathways to heterozygosity.
When chromosome 3 is Col/Col homozygous (HOM-HOM) genetic distance in the 420 interval significantly increased in fancm and fancm zip4 mutants compared with wild type (both GLM p < 2.0 × 10−16) (Figure 6A, Figure 6—source data 1), consistent with repair of the majority of DSBs via a non-interfering crossover pathway (Crismani et al., 2012). However, the CEN3 interval experienced a smaller yet significant increase in genetic distance in fancm and decreased in fancm zip4 (both GLM p < 2.0 × 10−16), indicating that non-interfering crossover repair is less efficient in this region (Figure 6A, Figure 6—source data 2). We next generated plants that were Col/Ct heterozygous (HET-HET) on chromosome 3 and observed that the previous increase in 420 crossovers was strongly suppressed in fancm and fancm zip4 (GLM p = 1.24 × 10−06 and p < 2.0 × 10−16), whereas wild type Col/Ct were slightly but significantly higher than wild type Col/Col (GLM p = 0.0126) (Figure 6A–B). CEN3 crossovers were also significantly suppressed by Col/Ct heterozygosity in fancm and nearly eliminated in fancm zip4 compared to Col/Col (both GLM p < 2.0 × 10−16) (Figure 6A–B). Together this indicates that the non-interfering crossover repair pathway that predominates in fancm and fancm zip4 is less efficient in heterozygous regions and particularly within the centromeric region, which shows high polymorphism levels (Table 2).
We next tested the effect of juxtaposing heterozygous and homozygous regions in fancm and fancm zip4 mutants. We first generated lines that were Col/Ct heterozygous within 420 and Col/Col homozygous outside (HET-HOM) (Figure 6—figure supplement 1). As expected, wild type HET-HOM lines show a significant increase in 420 and a reciprocal decrease in CEN3 crossovers compared to wild type HOM-HOM (both GLM p < 2.0 × 10−16) (Figure 6A,C), indicating compensatory changes between the two intervals in the HET-HOM lines. As the HET-HOM lines are heterozygous within 420, this again inhibited crossovers in fancm compared to fancm HOM-HOM (GLM p = 2.38 × 10−15) (Figure 6A,C). HET-HOM lines in fancm zip4 showed lower 420 crossovers than wild type HOM-HOM (GLM p < 2.0 × 10−16), which demonstrates that the interfering pathway is required for the heterozygosity-homozygosity juxtaposition effect (Figure 6A,C). We also generated HOM-HET lines that were homozygous within 420 and heterozygous outside, which significantly reduced 420 crossovers compared to wild type HOM-HOM as expected (GLM p = 7.60 × 10−11) (Figure 6A,D). HOM-HET lines in fancm and fancm zip4 showed high 420 crossovers comparable to HOM-HOM, as the non-interfering crossover repair active in these backgrounds is efficient in homozygous regions (Figure 6A,D). CEN3 genetic distance was again strongly suppressed in fancm and fancm zip4 HOM-HET lines compared with HOM-HOM (both GLM p < 2.0 × 10−16), consistent with heterozygosity inhibiting non-interfering crossover repair (Figure 6A,D). Together these data demonstrate that juxtaposition of heterozygous and homozygous regions causes reciprocal changes in crossover frequency via interference.
As we observed regional changes in crossover frequency with varying patterns of heterozygosity, we next sought to test whether total recombination events were different. When homologous chromosomes align on the metaphase-I plate, crossovers can be cytologically visualized as chiasmata (Sanchez-Moran et al., 2002). To estimate the number of crossovers per meiotic nucleus we performed chromosome spreads of pollen mother cells (PMCs), followed by fluorescence in situ hybridization using a 45S rDNA probe (Figure 7, Figure 7—source data 1). We counted total chiasmata in metaphase-I nuclei in Col/Col homozygotes, Ct/Ct homozygotes and Col/Ct F1 heterozygotes. In addition, we counted chiasmata in recombinant 420-CEN3 lines showing high (HET-HOM, 27.96 cM) and low (HOM-HET, 13.83 cM) 420 crossover frequency (Figure 7C,D). Adjacent chiasmata count categories were combined to give a minimum expected value of five for the purposes of a chi-square test with 8° of freedom. This test gave no significant differences in chiasmata between the genotypes (p = 0.3365) (Figure 7). Together this is consistent with homeostatic maintenance of crossover numbers, despite local crossover changes caused by juxtaposition of heterozygous and homozygous regions.
Our analysis of 420-CEN3 recombination rates implicated interference as driving crossover changes across homozygosity/heterozygosity junctions. We therefore sought to directly measure interference in lines with varying heterozygosity. We generated I3bc lines that varied in Col/Ct genotype and that were wild type, fancm, zip4 or fancm zip4 (Figure 8—figure supplement 1). We first compared I3bc plants that were Col/Col homozygous (HOM-HOM) with Col/Ct heterozygotes (HET-HET). In wild type, genetic distances did not significantly change between HOM-HOM and HET-HET (GLM p = 0.352 and p = 0.666), but crossover interference significantly increased (GLM p < 2.0 × 10−16) (Figure 8A,B, Figure 8—source data 1). Consistent with previous observations, fancm and fancm zip4 showed a significant reduction and an absence of interference respectively, in a HOM-HOM background (GLM p < 2.0 × 10−16 and p = 4.94 × 10−16) (Figure 8A, Figure 8—source data 2) (Crismani et al., 2012; Yelina et al., 2013). In HET-HET plants the crossover frequency increases seen in fancm and fancm zip4 were again greatly suppressed, or eliminated, relative to HOM-HOM, as observed previously for 420-CEN3 (GLM both p < 2.0 × 10−16) (Figure 8B). Unexpectedly, interference measurements significantly increased in both fancm and fancm zip4 mutants in a HET-HET background compared to HOM-HOM (GLM p < 2.0 × 10−16 and p = 4.94 × 10−16) (Figure 8B). We propose that in the absence of the ZMM pathway alternative repair pathways exist which are differentially sensitive to polymorphism and interference. Multiple, redundant repair pathways are consistent with the residual crossovers observed in msh4 mus81 double mutants (Higgins et al., 2008b). Finally, we measured I3bc cM in zip4 mutants alone (HOM-HOM) and observed significantly decreased crossovers compared with wild type HOM-HOM (GLM p < 2.0 × 10−16) (Figure 8E, Figure 8—source data 1). Importantly, zip4 genetic distances were further significantly reduced when comparing HOM-HOM to HET-HET backgrounds (GLM p = 1.79 × 10−10 and p = 1.53 × 10−9) (Figure 8E). This provides additional evidence that the non-interfering repair pathway remaining in zip4 is inefficient in heterozygous regions. Interference measurements using I3bc are reliant on the relatively rare double crossover classes (N-Y- + NB-R) (Figure 5A). Due to low zip4 fertility it was difficult to obtain sufficient DCO counts to make reliable interference measurements, although the observed counts are consistent with an absence of interference in this mutant (Figure 8—source data 4).
To test the effects of heterozygosity/homozygosity juxtaposition we next generated lines that were Col/Ct heterozygous within I3bc and Col/Col homozygous outside (HET-HOM). As expected, wild type I3b and I3c genetic distances both significantly increase in HET-HOM lines relative to HOM-HOM (GLM both p < 2.0 × 10−16), consistent with our previous 420 experiments, and this was associated with a significant increase in crossover interference (GLM p < 2.0 × 10−16) (Figure 8A,C). As shown earlier, we observed that Col/Ct (HET-HOM) heterozygosity suppressed the crossover increases seen in fancm and fancm zip4 (GLM p < 2.0 × 10−16), with the same significant increases in crossover interference strength (GLM p < 2.0 × 10−16 and p = 4.94 × 10−16) (Figure 8A,C). The reciprocal situation was observed in HOM-HET plants where I3bc was Col/Col homozygous and the rest of the chromosome Col/Ct heterozygous. I3b and I3c genetic distances were significantly decreased in wild type HOM-HET compared with wild type HOM-HOM plants (GLM both p < 2.0 × 10−16) (Figure 8A,D). HOM-HET fancm and fancm zip4 plants showed high crossovers, as the non-interfering pathway is efficient in the homozygous I3bc interval (Figure 8A,D). We also generated HET-HOM zip4 lines, which unlike wild type showed significantly lower I3b and I3c cM than HOM-HOM zip4 (GLM both P= p < 2.0 × 10−16) (Figure 8E). This again demonstrates that crossover remodelling at heterozygosity/homozygosity junctions requires interference and that non-interfering repair is inefficient in heterozygous regions.
As an independent test of the effect of heterozygosity on crossover interference we analysed four three-colour FTL intervals distributed throughout the genome (Figure 1A and Table 2). We measured crossover frequency and interference in Col/Col homozygotes vs Col/Ler F1 heterozygotes using meiotic pollen tetrads (Tables 8, 9). This approach is possible as the FTL crossover reporter system was generated in the qrt1-2 mutant background, where sister pollen grains remain physically attached as meiotic tetrads (Berchowitz and Copenhaver, 2008). We scored a total of 49,801 tetrads for Col/Col (an average of 6225 per interval) and 42,422 tetrads for Col/Ler (an average of 5302 per interval) (Tables 8, 9). Compared to Col/Col, genetic distance significantly decreased in Col/Ler for six of the eight intervals measured and the remaining two intervals were not significantly changed (Table 8). To calculate interference strength we compared cM values in each interval from tetrads that had a crossover in the adjacent interval, to the same intervals in tetrads lacking a crossover in the adjacent interval, and detected significant positive interference in all cases (Table 9) (Berchowitz and Copenhaver, 2008). The resulting interference ratios were then compared between Col/Col and Col/Ler using Fisher's combined probability test, which revealed a significant increase in interference strength in Col/Ler (χ2.001 = 39.26) (Table 9). Therefore, the effect of heterozygosity increasing the interference strength is evident in both Col × Ct and Col × Ler crosses.
We demonstrate reciprocal crossover increases and decreases when heterozygous and homozygous regions are juxtaposed and further demonstrate that this process requires crossover interference. The mechanism of interference is presently unclear, but a Beam-Film model has been developed where crossovers are patterned via forces similar to mechanical stress and which predicts experimental data (Kleckner et al., 2004; Zhang et al., 2014a, 2014b). In this model each chromosome begins with an array of precursor interhomolog strand invasion events, one of which becomes crossover designated via a stress-related force (Designation Driving Force DDF). This causes a local reduction and redistribution of stress in both directions that dissipates with increasing distance (Kleckner et al., 2004; Zhang et al., 2014a, 2014b). At the point where stress increases sufficiently precursor events can again become crossover designated. Any remaining precursors then mature into other fates including non-crossovers and non-interfering crossovers (Kleckner et al., 2004; Zhang et al., 2014a, 2014b).
We considered the effect of juxtaposition of heterozygous/homozygous regions in the context of the Beam-Film model (Kleckner et al., 2004; Zhang et al., 2014a, 2014b). Detection of heterozygosity most likely occurs downstream of interhomolog strand invasion and the formation of base pair mismatches. Therefore, we assume that the initial distribution of meiotic DSBs is unchanged in homozygous or heterozygous states. Mismatches are observed to have a local inhibitory effect on meiotic crossovers (Dooner, 1986; Borts and Haber, 1987; Jeffreys and Neumann, 2005; Baudat and de Massy, 2007; Cole et al., 2010). Therefore, one possibility is that mismatched precursors in heterozygous regions are slowed in maturation and trigger feedback mechanisms that cause further DSBs, for example via ATM/ATR kinase signalling (Carballo et al., 2008; Lange et al., 2011; Zhang et al., 2011; Kurzbauer et al., 2012; Garcia et al., 2015). As a consequence, heterozygous regions would receive more ‘late’ DSBs, leading to more precursors and a higher chance of receiving a crossover designation event. An increased chance of crossover designation would lead to spreading of interference into adjacent homozygous regions causing reciprocal crossover decreases. An alternative model is that mismatched precursors are more sensitive to crossover designation and thus heterozygous regions have a higher chance of an interfering crossover, leading to similar effects. These potential models could be distinguished by measurement of non-crossover (NCO) levels, which should increase in heterozygous regions if more DSBs occur. Our data also demonstrate that non-interfering repair is less efficient in heterozygous regions, which will further contribute to the changes we see across homozygosity/heterozygosity junctions.
Sequence polymorphism has been observed to suppress crossover recombination at the hotspot (kilobase) scale in diverse eukaryotes (Dooner, 1986; Borts and Haber, 1987; Jeffreys and Neumann, 2005; Baudat and de Massy, 2007; Cole et al., 2010). For example, at the mouse A3 hotspot an indel polymorphism within an inverted repeat overlaps a crossover refractory zone (Cole et al., 2010). However, this zone forms significant numbers of non-crossovers, indicating that the repeat/indel does not inhibit DSB formation, but inhibits downstream progression to crossover recombination (Cole et al., 2010). In yeast addition of SNPs to the MAT-URA3 hotspot decreased crossovers and increased the frequency of gene conversions, further indicating that polymorphism can inhibit crossovers at fine-scale (Borts and Haber, 1987). Finally, intragenic mapping of the maize Bronze hotspot demonstrated that transposon insertions suppress crossovers more strongly than single nucleotide changes (Dooner, 1986; Fu et al., 2001; Dooner and He, 2008), again consistent with progression to crossover repair being inhibited by local sequence polymorphisms. Several heteroduplex joint molecules with distinct properties form during meiosis, including displacement-loops and dHJs (Keeney and Neale, 2006). It is possible that these joint molecules and their interactions with recombinases are sensitive to base-pair mismatches. The mismatch repair protein MutS directly recognizes mismatched base-pairs and serves as a paradigm for this type of function (Lamers et al., 2000; Obmolova et al., 2000).
The reciprocal crossover changes we observe when heterozygous regions are juxtaposed with homozygous regions are reminiscent of other homeostatic effects characterized during meiosis (Hillers and Villeneuve, 2003; Martini et al., 2006; Robine et al., 2007; Libuda et al., 2013; Thacker et al., 2014). For example, multiple levels of interference have been detected in mice (de Boer et al., 2006; Cole et al., 2012), Zip3 foci with distinct timing and properties are observed in budding yeast (Serrentino et al., 2013), and ‘upstream’ DSB patterns are altered in ‘downstream’ ZMM mutants (Thacker et al., 2014). As plants, fungi and mammals share the presence of interfering and non-interfering crossover repair pathways similar effects over heterozygosity/homozygosity junctions may be generally important (Stahl et al., 2004). However, when assessing the significance of such effects it is also important to consider how outcrossing vs selfing will influence patterns of homozygosity and heterozygosity within different species. Together our data show how varying patterns of sequence polymorphism along chromosomes can have a significant effect on distributions of meiotic recombination.
Flow cytometry of pollen can be used to rapidly measure meiotic segregation of heterozygous transgenes encoding distinct colours of fluorescent protein (Yelina et al., 2012, 2013). cM were calculated from flow cytometry data using the formula:
Where R5 is a number of green-alone fluorescent pollen grains and R3 is a number of green and red fluorescent pollen grains (Yelina et al., 2012, 2013). We previously observed that the number of red-alone pollen exceeded that of green-alone pollen when lines heterozygous for both eYFP and dsRed (eYFPDsRed/++) were analysed (Yelina et al., 2012, 2013). Using pulse-width/SSC (side scatter) analysis and back-gating we demonstrated that the excess counts come primarily from non-hydrated pollen (Yelina et al., 2012, 2013). Therefore to avoid this artifact we multiply the green-alone counts by two to obtain the number of recombinant pollen.
To increase measurement throughput using fluorescent seed we adapted CellProfiler image analysis software (Carpenter et al., 2006) (Figure 2). This program identifies seed boundaries in micrographs and assigns a RFP and GFP fluorescence intensity to each seed object (Figure 2A–B). Three pictures of the seed are acquired at minimum magnification (×0.72) using a charge coupled device (CCD) camera; (i) brightfield, (ii) UV through a dsRed filter and (iii) UV through a GFP filter (Figure 2A). As seed are diploid, there are nine possible fluorescent genotypes when a RFP-GFP/++ heterozygote is self-fertilized, in contrast to four possible states for haploid pollen (Yelina et al., 2013) (Figure 2E). Histograms of seed fluorescence can be used to classify fluorescent and non-fluorescent seed for each colour (Figure 2C–D). Although it is possible to distinguish seed with one vs two T-DNA copies, there is greater overlap between the groups (Figure 2C–E). Therefore, we use fluorescent vs non-fluorescent seed counts for crossover measurement. Using this method it is possible to score 2000–6000 meioses per self-fertilized individual. When plants have been self-fertilized, genetic distance is calculated using the formula:
Where NG is a number of green-alone fluorescent seeds, NR is a number of red-alone fluorescent seed and NT is the total number of seeds counted. During generation of 420/++ F2 populations we selected for individuals that are heterozygous for transgenes expressing red and green fluorescent proteins (RFP-GFP/++). The majority of these individuals receive a chromosome with linked RFP and GFP transgenes over a non-transgenic chromosome (RFP-GFP/++) (Figure 2—figure supplement 1). In a minority of cases F2 plants receive recombined RFP-+ and +-GFP chromosomes (Figure 2—figure supplement 1). In the progeny of these individuals the fluorescent seed classes representing parental and crossover genotypes are reversed (Figure 2—figure supplement 1). As R+/+G plants also have variable heterozygosity/homozygosity patterns within 420 depending on crossover positions we excluded these plants from further analysis.
To test whether recombinant and non-recombinant counts were significantly different between replicate groups we used a GLM. We assumed the count data is binomially distributed:
where Yi represents the recombinant counts, are the total counts, and we wish to model the proportions Yi/ni. Then:
Thus, our variance function is:
and our link function must map from (0,1) → (−∞, ∞). We used a logistic link function which is:
where . Both replicates and genotypes are treated as independent variables (X) in our model. We considered p values less than 0.05 as significant.
Measurements of interference within the I3bc interval were carried out as described previously with minor modifications (Yelina et al., 2013). Inflorescences were collected in polypropylene tubes and pollen was extracted by vigorous shaking in 30 ml of freshly prepared pollen sorting buffer (PSB: 10 mM CaCl2, 1 mM KCl, 2 mM MES, 5% wt/vol sucrose, 0.01% Triton X-100, pH 6.5). The pollen suspension was filtered through a 70 µM cell strainer to a fresh 50 ml polypropylene tube and centrifuged at 450×g for 3 min. The supernatant was removed and the pollen pellet washed once with 20 ml of PSB without Triton. The pollen suspension was centrifuged at 450×g for 3 min and the supernatant discarded and the pollen pellet resuspended in 500 µl of PSB without Triton. A CyAn ADP Analyser (Beckman Coulter, California, USA) equipped with 405 nm and 488 nm lasers and 530/40 nm, 575/25 nm and 450/50 nm band-pass filters was used to analyse the samples. Polygons were used for gating pollen populations and for each sample eight pollen class counts were obtained (Figure 5—figure supplement 1). I3b and I3c genetic distances were calculated using the following formula:
where N-Y-, NB-R, N-YR, NB--, NBY-, N--R, NBYR, and N--- are pollen grain counts in each of the eight populations (Figure 5—figure supplement 1). For example, NBYR is the number of pollen that were blue, yellow and red fluorescent.
Crossover interference was calculated using the following formulas:
At least three biological replicates, constituting 3–5 individual plants were analysed for each sample (Yelina et al., 2013). Statistical tests for genetic distances were performed as described above using a GLM. To test for significant differences in interference we compared observed and expected double crossovers using the same approach.
Col-0 420 and Ct-1 lines were crossed to fancm-1 zip4-2 double mutant lines in the Col-0 background (Crismani et al., 2012) (Figure 6—figure supplement 1). The resulting F1 plants were crossed together and progeny identified that were fancm zip4 heterozygous, and 420/++ Col/Ct heterozygous on chromosome 3. Chromosome 3 genotypes were tested in all cases using 13 Col/Ct indel markers (Supplementary file 1). These plants were self-fertilized and 420 homozygous individuals identified (all seed were red and green fluorescent) that were also Ct homozygous outside of 420 and that were fancm zip4 heterozygous (Figure 6—figure supplement 1 and Figure 8—figure supplement 1). These plants were then crossed to CEN3 or I3bc in wild type, fancm and fancm zip4 mutants to obtain scorable progeny with a HOM-HET genotype (Figure 6—figure supplement 1). The selfed progeny of 420/++ Col/Ct fancm zip4 heterozygous plants were also selected for plants with no fluorescent T-DNAs and either chromosome 3 in a Ct homozygous state, or with Ct homozygosity within 420 and Col homozygosity outside (Figure 6—figure supplement 1). These plants were crossed with doubly marked 420-CEN3 or I3bc lines in either wild type, fancm or fancm zip4 mutant backgrounds to obtain HET-HET and HET-HOM scorable plants respectively (Figure 6—figure supplement 1 and Figure 8—figure supplement 1). Equivalent genetic crosses were performed during analysis of I3bc (Figure 8—figure supplement 1). At least three independent lines were generated and analysed for each combination, apart from HOM-HET 420-CEN3 where two were analysed.
To genotype zip4-2 (Salk_068052) the following primers were used:
The resulting PCR products were ∼680 bp for wild type (zip4-2-F + zip4-2-R) and ∼340 bp for zip4-2 mutant (zip4-2-F + Lbb1.3) (Crismani et al., 2012).
To genotype the fancm mutation we amplified using the following primers:
The resulting PCR product was digested with MboII, which yields a ∼215 bp product for wild type and ∼180 bp for fancm (Crismani et al., 2012).
Chiasmata counting was performed as previously described (Sanchez-Moran et al., 2002).
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Detlef WeigelReviewing Editor; Max Planck Institute for Developmental Biology, Germany
eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]
Your full submission, “Heterozygosity promotes interfering and inhibits non-interfering crossovers during Arabidopsis meiotic recombination”, has been evaluated by Detlef Weigel (Senior editor), a Reviewing editor, and two peer reviewers, and the decision was reached after discussions between the reviewers. We regret to inform you that your work will not be considered further for publication.
There were two types of concerns. First, the reviewers were not convinced that heterozygosity generally increases crossovers (independently of genetic background and physical location in the genome), and that crossovers were recruited from homozygous regions of the genome to adjacent heterozygous regions. Similarly, the reviewers found that the mutant analysis was not fully developed. Second, there was agreement that, while your findings are provocative, they are not appropriately framed vis à vis alternative explanations for the observed relationships between recombination rates and diversity along genomes.
The authors argue that recombination may be recruited from homozygous regions to nearby heterozygous regions. Overall I found the results interesting but I have some significant concerns about the presentation and interpretation of the results.
One of my major concerns about the article is that the authors frame it as a possible explanation for the positive correlation between crossover frequency and diversity that is seen across a number of the species examined to date. However, there is relatively good support, in population genetics literature, for the idea that this pattern reflects the fact that linked selection (e.g. hitchhiking and background selection) have effects over relatively large physical regions in regions of low crossing over (see Cutter and Payseur 2013). The authors do not seem to mention this dominant, and relatively well supported, group of hypotheses and instead seem to frame the positive correlation as an empirical observation in search of a mechanistic hypothesis (that they provide). Obviously these hypotheses are not mutually exclusive, and so this does not rule out the idea that the authors' mechanistic explanation could also play a role. The authors need to be much clearer about the background to these observations, if they are to contribute to the larger debate about determinants of levels of polymorphism.
Along these lines it is not totally clear to me whether the observations could possibly explain a bulk of the correlation between recombination and polymorphism in many species. Much of the broad variation in recombination rates, in many systems, is explained by proximity to centromeres and telomeres (presumably a mechanistic constraint). Thus a lot of the variation in broad-scale recombination rates is not tied to polymorphism, but rather to large-scale chromosomal architecture. Once again the authors' proposed mechanism could contribute to the strength of the correlation, but this argument does suggest that this contribution may be somewhat minor.
The authors state, in the Abstract, that “using recombinant populations we show that heterozygous regions attract crossovers from homozygous regions on the same chromosome at the megabase scale”, and in one of the subsection of the Results that “Heterozygosity recruits crossovers over homozygous regions”.
I apologize if I've missed something, but I do not think the authors’ results actually demonstrate this. To my reading of these statements it sounds like the authors are saying that the extra recombination events in heterozygous regions come at the expense of fewer recombination events in the homozygous regions. But I do not see any results supporting for the second part of this statement, i.e. measuring recombination rates in homozygous regions. The authors' statements are one possible explanation of the results, but another is that the homozygous regions promote recombination with no “cost” to themselves. The authors may have evidence in favor of their hypothesis, perhaps this is the argument being made in the subsection headed “Differential sensitivity of interfering and non-interfering crossover repair to polymorphism”, but I found it hard to follow.
One concern I had was that recombination may be increased over broad chromosomal regions due to homozygosity in a specific region, because the individual would have reduced heterozygosity for structural rearrangements. This increase in recombination would not be associated with either parental allele, but with homozygosity itself. Presumably the authors have thought this through, and perhaps these lines are known to have no structural variation of suitable size in these regions, but it is worth clarifying this point.
At the end of the subsection headed “Modification of crossover frequency by Arabidopsis natural variation”, the authors state: “therefore, in many cases heterozygosity promotes Arabidopsis recombination relative to homozygosity, which is inconsistent with a purely suppressive effect of polymorphism.” I don't think this is correct as stated. The authors have shown that when Col is crossed to other lines the F1s sometimes have higher recombination rates than Col homozygotes. However, this could be because the other lines crossed to harbor recombination modifiers that increase recombination rates. For the authors claim that heterozygosity promotes recombination to be true they would need to show that the F1s often have higher recombination rates than either of the parental lines.
This paper presents evidence for three conclusions.
1) In one well-studied test interval, in a particular heterozygous state, the frequency of crossovers is higher when the region adjacent to the test interval is homozygous rather than heterozygous. This is a “cis” effect. This is shown in Figure 4 and in the non-mutant background of Figure 7 (compare panels A and C).
There are some limitations to this observation. First, it is shown for only one interval. Second, interval is sub-telomeric and thus likely not to be representative of most of the genome. Third, it is shown for only one pair of lines (Col vs. Ct).
It is also important to note that in this test interval, there is no difference in crossover level between the homozygous and full F1 hybrid strains (Figure 7 compare panels A and B in the non-mutant case). Thus, there is no general effect of heterozygosity to increase the number of crossovers. This is also seen in the overall evaluation of Col/Col vs Col/Ct F1's. Thus, the identified phenomenon is some type of unusual cis interaction which may or may not be widely general.
The basis for this cis interaction proposed by the authors does not seem to make sense to me. This is in part because it seems to invoke result (2) below in an inappropriate way and in part because the statement is made that crossovers are “recruited” from homozygous regions. But this conclusion cannot be drawn. There was no analysis of the effects of a heterozygous region on a flanking homozygous region.
It seems that this conclusion of some type of “competition” between homozygous and heterozygous regions is drawn largely from Figure 6. But the results in this Figure do not support this conclusion. What this figure shows is that when the test region is homozygous and the flanking region is heterozygous, the level of crossovers is reduced in the test region as compared to the case where the test region is heterozygous and the flanking region is homozygous. This does not imply that there is a reciprocal effect of heterozygosity to reduce crossovers in the homozygous region. There is no control to show what the frequency of crossovers is in a fully homozygous case where there is no heterozygous region adjacent to the test region. There is also no control to show what happens if the Col/Col homozygous test region is flanked by the same homozygous region as when the test region is heterozygous (i.e. Ct/Ct).
Finally, the authors suggest that the cis phenomenon is general because some F1 hybrid strains show higher levels of recombination than one particular reference homozygous strain. However, this finding is just as easily explained by general genetic background effects on the recombination process rather than anything to do with heterozygosity at the DNA level, as the authors admit. Thus, this is not really supporting evidence.
2) The authors show that heterozygosity is accompanied by a change in the sensitivity of crossovers to fancm and zip4 mutations. The analysis is not rigorously correct because there was no test of a zip4 mutation by itself; the only test was in a fancm zip4 background.
The most important point is that this is a general characteristic of heterozygosity: it is observe in a pure F1 hybrid. Thus there is no reason to link this phenomenon to the cis effect, as the authors seem to do.
A second important point is that the total frequency of crossovers in the F1 hybrid is the same as in the homozygous reference strain. Thus, there is no “recruitment of crossovers” as the authors also seem to suggest.
Finally, comparison implies a likely situation is that there is a change in the proportion of recombination events that are “interfering crossovers” and versus crossovers that arise as a minority population from the “non-crossover” pathway. To put it another way, in the wild type case, there is an increase in crossovers and a decrease in non-crossovers (more of which turn into crossovers in fancm than in wild type). The basis for this effect is unclear. However, it is strongly reminiscent of crossover homeostasis where a reduction in DSBs leads to a differential loss of non-crossovers as compared to crossovers. Since it is unlikely that heterozygosity will be recognized at the DSB level (although this is not totally excluded given diverse indications of trans effects on DSBs in yeast), it would instead imply that there is a reduced chance that a DSB actually giving a recombination intermediate that could lead to a crossover or non-crossover. This is entirely possible since heterozygosity could be sensed at the time of establishment of such an interaction or concomitant with crossover/non-crossover decision making.
3) The authors show that an F1 hybrid appears to have increased crossover interference as compared to the homozygous reference strain. There are two problems with this result and linking it to other results. First, this is a different hybrid from the one tested for all other phenomena. Second, there is no evidence that this effect is the result of heterozygosity at the DNA level rather than some trans-acting strain background effect. Third, in this hybrid the total crossover level is lower than in the reference homozygote; thus, this is not related to the finding that F1 hybrids can exhibit higher levels of recombination.
That being said: there is a possible way that this result could be relevant to the sub-telomeric effect described above. In Arabidopsis, as in several other organisms, interference distance goes with physical chromosome length. Thus, in the hybrid, increased interference per Mb (which is what is assayed) could result from a decrease in physical chromosome length. Furthermore, the cis effect described in the results in prior sections could be explained if the longer length characteristic of the homozygous region can spread into the adjacent sub-telomeric region, thus increasing physical length, decreasing interference per Mb and thus increasing crossovers per Mb.
Given the above considerations, the statements in the Summary need some amendment, as follows:
A) “We… found hybrids with higher recombination than homozygotes, demonstrating that polymorphism can promote crossovers”. If this is a reference to the finding of F1 hybrids with higher recombination, it is not accurate. There is no basis for the conclusion that the higher recombination is due to DNA polymorphism rather than trans-acting genetic effects. The cis effect is not evidence. Moreover, in the Col/Ct F1 hybrid used for most of the analysis, recombination frequencies are generally not higher than in Col/Col and in the Col/Ler hybrid used for interference analysis, recombination frequencies are lower than in the Col/Col homozygote.
B) “Using recombinant populations we show that heterozygous regions attract crossovers from homozygous regions on the same chromosome at a megabase scale”. This is definitively not shown by these data (see discussion in point 1 above).
C) “We demonstrate that this polymorphism cis-effect is dually mediated by promotion of interfering crossovers and inhibition of non-interfering crossovers in heterozygous regions”. This is not correct. As discussed in detail for point (2) above, the changes in interfering and non-interfering crossovers are not specific to the cis effect: they are a general feature of the heterozygous cases analyzed (even assuming no general genetic background issues). Furthermore, the observation does not imply two mechanistically distinct effects as the above statement suggests. Rather, there is a change in the distribution of undifferentiated recombination intermediates into crossover versus non-crossover outcomes.
D) “This reveals an unanticipated mechanism whereby DNA polymorphism can recruit crossovers, contribute to positive correlations between recombination and diversity and influence the action of selection.” For this reviewer, there is no positive correlation between recombination and diversity shown in this paper, as described in detail above. The F1 hybrid data are not evidence. Hybrids can have higher or lower levels than a particular reference homozygote. A F1 hybrid strain shows a higher proportion of interfering crossovers among total crossovers but no difference in total crossover levels. And the one case in which crossover levels are increased in a heterozygous region is not shown to involve “recruitment” of crossovers to the heterozygous region from a homozygous region. There is a cis effect in which presence of an adjacent homozygous region increases crossovers in the heterozygous test region, but this is an increase above the level seen in the heterozygous region in the pure F1 hybrid, so it does not represent a simple effect of DNA polymorphism. Finally, there is no mechanism revealed. There is the finding that F1 hybrids have altered crossover/non-crossover ratios, but this does not increase the overall level of crossovers, so the “mechanism” alluded to does not increase crossing-over.
E) The Discussion culminates with the following point:
“We propose that detection of sequence mismatches occurs during strand invasion or heteroduplex/dHJ formation and differentially inflluences the activity of interfering and non-interfering recombination proteins coincident with crossover/non-crossover repair choice. Therefore, as interfering and non-interfering repair pathways compete for DSBs, their activities are simultaneously modulated by heterozygosity, causing the cis effect.”
There are several problems, touched on above, which converge here. (i) There is not really a competition between interfering and non-interfering pathways. There is a crossover designation process and the leftovers become mostly non-crossovers but occasionally become crossovers, and the level of those latter crossovers are increased by fancm (as the authors of the fancm study state). (ii) Most importantly, the relevant effect does not cause the cis effect—it is seen in a pure F1 hybrid. The cis effect must come from something else. (iii) There is no reason to think that the crossover/non-crossover choice is made during dJH formation; all that can happen at this stage is that the process can be degraded to give fewer crossovers and more non-crossovers, which is the opposite of the effect observed here.
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Thank you for resubmitting your work entitled “Heterozygosity promotes interfering and inhibits non-interfering crossovers during Arabidopsis meiotic recombination” for further consideration at eLife. Your revised article has been favorably evaluated by Detlef Weigel (Senior editor) and three reviewers. The manuscript is improved, but there are still concerns whether your interpretation of the data goes too far. Therefore, we are asking you to rewrite the manuscript as much as possible to be a fair description of the unsuspected phenomena, without making too many claims regarding crossovers being attracted from one kind of region to another. In other words: please accommodate the reviewers' comments as much as possible. We realize that there are differences in interpretation of the data between you and specifically reviewer 2, but we felt that the phenomenon is important enough that it deserves prominent publication. One of our board members also stated that the “phenomenon is interesting, but the point is not to find a model (it does not add much to invoke the beam film model, since there is no way to know if it is late DSBs and/or mismatches, and the authors are certainly far from understanding the molecular mechanism) but to validate the general principle.”
Detlef Weigel has made specific comments in the manuscript, as attached.
Over all I found the manuscript to be much improved in terms of it presentation of its results, and the addition of the new analyses made the findings a more general statement of the effect of heterozygosity on recombination patterns. I note, however, that the other reviewer's original concerns were much more substantive than mine. As such, I view their opinion as carrying more weight than mine in this appeal.
The last paragraph of the Discussion is problematic:
“We propose that the biological function of the heterozygosity cis-effect is to recruit crossovers to variable regions of the chromosomes, acting as a feed-forward mechanism to increase diversity.”
“… meiotic recombination has been selected to promote…”
Arguments about the evolution of recombination modulators are very slippery (as recombination unlinks the fate of the modifier and the recombinant haplotype it creates). As such these proposals are unsupported speculation, perhaps changing “propose” to “speculate” would help. Arabidopsis thaliana has not been a selfing lineage for very long (like most selfers), and the authors' argument does not seem super convincing evolutionary mechanism for outcrossers (as homozygosity runs will be broken up across generations, by segregation). So their explanation seems somewhat shaky.
In outbred organisms very long blocks of homozygosity are rare. The authors should caution that they know little about how long a block of homozygosity is needed to promote this effect, so the importance of this effect in other systems (e.g. mammals) is unclear.
In general the authors have done a better job of acknowledging other likely contributors to the recombination/heterozygosity relationships. Except for:
“However, the cis-effect is unlikely to explain all of this relationship and genetic hitchhiking, background selection and recombination associated mutagenesis may play important roles”.
The contribution of these other effects has been subject to quantitative investigation for over a decade. While the authors’ findings are very interesting, it is a disservice to use “all” and “may” in this way. I'd say that the contribution of linked selection is much more established than their mechanism, and should be acknowledged as such.
Title: Adjacent homozygous and heterozygous regions reciprocally enhance and suppress crossing-over in an interference-mediated process.
Summary: Analysis of meiosis in mosaically-hybrid Arabidopsis lines reveals that a heterozygous region suppresses crossing-over in an adjacent homozogous region while, reciprocally, the homozygous region increases crossing-over in the adjacent heterozygous region. This interplay requires crossover interference: it is absent in a fancm zip4 background where crossovers occur but interference is absent. Two new features specific to recombination in heterozygous regions are also revealed: an effective increase in crossover interference and a decreased effect of a fancm mutation, which normally increases crossovers that do not exhibit interference. Potential mechanisms and evolutionary implications are discussed.
Part I. Summary. The authors have identified an interesting phenomenon that takes place at the junctions between homozygous and heterozygous regions, particularly when one of the involved regions is sub-telomeric: the frequency of recombination (crossing-over) in the homozygous region goes down while the frequency of recombination in the heterozygous region goes up (relative to the fully homozygous and fully heterozygous cases, respectively). This phenomenon implies reciprocal interplay between the two regions. This phenomenon applies specifically to junction regions. Fully heterozygous regions exhibit almost the same recombination frequency as fully homozygous regions, at least in the situation examined here. This could be of genetic/evolutionary significance, although that would depend, particularly since the role of crossing-over for evolution is hotly debated. It is not 100% clear whether the phenomenon applies generally throughout the genome and/or why it is particularly prominent in sub-telomeric regions.
The authors go on to suggest a specific mechanism for this phenomenon. One point is clear: this phenomenon requires crossover interference. In a mutant situation where there are crossovers, but no interference, the phenomenon is absent. This is interesting and sensible because crossover interference is, by its nature, a process in which adjacent regions communicate with one another.
Beyond this point, however, the authors’ assertions regarding mechanism are not supported by the data.
1) The authors say that non-interfering COs are suppressed in heterozygous regions. This features prominently in the Title and Summary. This is not shown in this paper. What is shown is that the COs which occur in a fancm background (which do not exhibit interference) are reduced in heterozygous regions. There is no evidence that these COs are occurring by the same molecular mechanism as the “canonical non-interfering COs” that arise in wild type meiosis.
2) The authors show that interference, as classically defined, appears to be stronger in heterozygous regions than in homozygous regions. This is also an interesting observation, which is documented not only for the specific situation analyzed in detail, but more broadly. This is the first time that interference has been examined in heterozygous situations, as far as I know.
However, further consideration of the implications of this finding have some problems: (a) the phenomenon of increased interference may not mean what the authors think it means; and (b) the authors wish to say that this increased interference is responsible for the interplay between heterozygous and homozygous regions. This is a possible model. But I think it is wrong and there is actually evidence in the paper against it.
The strongest direct argument against the authors’ model is that the reciprocal interplay between heterozygous and homozygous regions is observed in a situation where crossovers in the two regions exhibit the level of interference characteristic of the homozygous region (Figure 6F and 8; details below).
More generally: it is clear that, at junctions, crossovers go down in homozygous regions and go up in heterozygous regions, relative to the pure homozygous or pure heterozygous cases, respectively, and that interference is required (above). The question is: what is the basis for this asymmetry? In my opinion, the underlying effect could be that there are more DSBs (or total inter-homolog interactions) in heterozygous regions. More DSBs means more COs, which means more interference emanating from that region across the border to the homozygous region which means fewer COs in the homozygous region than in the pure homozygous case. Oppositely, fewer DSBs in homozygous regions means fewer COs which means less interference emanating from than region across the border to the heterozygous region which means fewer COs in the heterozygous region than would be observed in the pure heterozygous case.
This model is further supported by two interrelated considerations. (1) Fully heterozygous regions show the same level of COs as fully homozygous regions (Figures 7, 8 wild type cases) even though there is more interference. Clearly there must be some other effect that counterbalances the apparent increase in interference. (2) The apparent increase in interference could, in principle, reflect either of two effects (Zhang et al., 2014 PLoS Genetics and PNAS): (a) an increase in the distance over which interference spreads; or (b) a decrease in the “strength” of CO-designation. The first would imply a more robust process in the heterozygous case, which seems peculiar a priori; the second would imply a less robust process in the heterozygous case, which makes more sense. (3) If phenomenologically increased CO interference reflects model (b), then the level of COs can be restored by increasing the number of DSBs.
By my alternative model, the phenomenological increase in interference in heterozygous regions is actually irrelevant (as shown by the data mentioned above). The real effect would be more DSBs.
Another problem with the authors' model is that it requires that the “increased interference in a heterozygous region” spreads across the boundary into the homozygous region and that the “decreased interference in a homozygous region” spreads across the boundary into the heterozygous region. This is a priori unlikely if the basis for the altered interference is heterozygosity per se.
It would actually not be so difficult to provide at least some evidence for more DSBs/interactions in heterozygous regions (e.g. by Dmc1 focus analysis). However, this opens up another can of worms and is beyond the scope of the current presentation.
The basic point is that it is not proven that there is a role for increased interference in heterozygous regions in the observed junction phenomenon and there seems to be evidence against it.
Importantly also: the documentation, description and presentation in this paper requires significant improvement as described below. There are problems of logic, vocabulary, data, controls, explanation and interpretation. This paper is not really readable by a general audience in its present form.
There is redundancy in the comments below, for which I apologize, but hopefully this is useful.
Part II. Specific Issues with Title and Summary.
The title says: “Heterozygosity promotes interfering and inhibits non-interfering crossovers during Arabidopsis meiotic recombination”. I do not think that either of these conclusions is warranted.
(i) The latter conclusion, that heterozygosity inhibits non-interfering crossovers, is wrong because it is based on the assumption that the extra COs that arise in a fancm mutant (and do not exhibit interference) are biochemically the same as the extra COs that arise in wild type (and are defined as “non-interfering COs”). Heterozygosity does decrease the COs seen in a fancm mutant (Figures 7, 8), but this cannot be extrapolated to wild type.
(ii) Heterozygosity does, phenomenologically, increase CO interference. This is an interesting observation. But the interpretations placed on it are not proven and, in my opinion, are not correct. This finding could perhaps be interpreted as “promoting the interfering CO pathway” (as in the Title). However: (a) In comparisons between fully homozygous and fully heterozygous regions, the frequency of COs is essentially the same (Figures 7AB, 8AB). This cannot be explained by an increase in CO interference alone. It implies some other effect. (b) The observation of increased CO interference by CoC analysis can be explained either by an increase in the “spreading distance” of the interference signal or by a decrease in the strength of CO-designation; both effects decrease the CoC at short inter-interval distances (See Zhang et al PLoS Genetics 2014 and PNAS 2014). If the relevant effect is to decrease the strength of CO-designation the observed effect is actually not promoting the interfering CO pathway but making it worse. (c) The fact that CO number does not change in a fully heterozygous region even though interference effectively increases (a, above) suggests/implies the existence of another effect, which could/should be upstream of any recombination fate decision. Most simply: if there is a decrease in the efficiency of CO-designation, the resultant decrease in COs can be overcome by an increase in the frequency of DSBs (or DSB-mediated pre-CO interactions). Perhaps mismatched chromatin structure has such an effect, maybe especially in sub-telomeric regions which might be influenced by pre-DSB homologous pairing interactions.
The end of the Summary says: “Using recombinant populations we show that heterozygous regions attract crossovers from homozygous regions on the same chromosome at the megabase scale. Interference inhibits formation of adjacent crossovers over similar physical scales, and we demonstrate that this polymorphism cis-effect is dually mediated by promotion of interfering crossovers and inhibition of non-interfering crossovers in heterozygous regions. This reveals an unanticipated mechanism whereby DNA polymorphism can recruit crossovers, contribute to positive correlations between recombination and diversity and influence the action of selection.”
I do not agree with any of these statements. Let us first define “polymorphism cis effect”. What was analyzed were constructs in which recombination was assayed in a sub-telomeric test region, which could be homozygous or heterozygous, and had an adjacent internal region that could be homozygous or heterozygous. Assuming experimental problems (below) can be ignored, what is observed (assayed region underlined) is, essentially, the following: (a) the frequency of COs is lower in HOM-HET than in HOM-HOM; and (b) the frequency of COs in HET-HOM is higher than in HET-HET. That is, an adjacent heterozygous region decreases COs in a homozygous region relative to what would have been seen in a fully homozygous situation while an adjacent homozygous region increases COs in a heterozygous region relative to what would have been seen in a fully heterozygous situation. But… HOM-HOM and HET-HET both show the same frequency of COs as one another (see Figures 6CD, 7 and 8 for the clearest examples). [There is one exception (Figure 8; see below) which I will simply ignore]. This is not: “heterozygous regions attract crossovers from homozygous regions on the same chromosome at the megabase scale”. There is no attraction; this is not a zero-sum game; the effect is reciprocal, not unidirectional; and stating the result in this way is not accurate and is prejudicial to thinking. “Recruitment” to heterozygous regions is simply wrong. So what is the explanation for the observed effects? The paper also shows (Figures 7, 8) that this interplay is not observed in a fancm zip4 double mutant where there are still COs, but of a variety that does not set up interference. Fine. Interference is required. And if everything were the same in HOM and HET regions, there would be no effect of having one next to the other, so just saying that there is interference is not enough. There has to be some asymmetry. The authors seem to think that the difference is that interference is “stronger” in HET regions and “weaker” in HOM regions. If this were true, and if that effect crossed the border between the two regions, COs in HET regions would tend to decrease COs in HOM regions relative to the effect from normal HOM interference; oppositely, HET regions will experience less interference from an adjacent HOM region than from an adjacent HET region. However: I do not think that this is correct. To reiterate some of the points made above:
(A) This model assumes that interference specific to HOM or HET can cross a HET/HOM border. This is a priori unlikely. Furthermore, there is data in the paper, which says that you can see the diagnostic reciprocal effects on CO levels in three-factor crosses where the HOM-type CO interference level is observed (Figures 6F and 8; see below).
(B) An alternative explanation is the same one required to explain interference patterns above (and thus is more likely): more DSBs in HET regions vs HOM regions. More DSBs in HET regions will imply more COs. More COs means more interference signals, which means fewer COs in the adjacent region. Oppositely, in HOM regions, fewer COs means fewer interference signals which implies more COs in the adjacent region.
To return to the Summary: “we demonstrate that this polymorphism cis-effect is dually mediated by promotion of interfering crossovers and inhibition of non-interfering crossovers in heterozygous regions. This reveals an unanticipated mechanism whereby DNA polymorphism can recruit crossovers, contribute to positive correlations between recombination and diversity and influence the action of selection.” Inhibition of non-interfering COs is not shown (above).
Enhanced interference in heterozygous regions (which is apparently what the authors mean by “promotion of interfering COs”) is not relevant. The likely relevant effect (more DSBs and thus more COs in HET regions) is not mentioned or discussed as a possibility.Thus: there is, therefore, no “unanticipated mechanism”, and no effect in which “DNA polymorphosms recruit COs and thus increasing genetic diversity in hybrid situations”.
Instead: what has been shown? There is a phenomenon that is observed when HOM and HET regions are side-by-side. A “junction” phenomenon, if you will. There is a reciprocal interplay in which COs are decreased in HOM regions and increased in HET regions and this interplay requires interference. This is likely correct, although there are a lot of technical issues pertaining to the data (below). NB that this is specific to adjacent HET/HOM regions, because HOM/HOM and HET/HET recombination are very similar. Thus, this is not a general effect of global heterozygosity, but an effect specific to juxtaposed HET/HOM regions (junctions). Thus any evolutionary implications must derive from this specific situation, not from heterozygosity in general.
The basis for this effect, about which the authors are quite specific in the title and the Summary, is probably not what the authors think it is, as described above. To repeat (again): There is no data on interplay between HET regions and “non-interfering COs” in wild type, only in a mutant situation that might be different. The observed increase in interference is real and per se interesting, but this does not imply a more robust interfering CO pathway; it could as easily and more probably imply a weaker pathway. And regardless of that point, the increase in interference in HET regions is not responsible for the “junction interplay”, as described above. This leaves the actual basis for the asymmetry between the two types of regions to be determined, but increased DSBs is an attractive possibility, which is not considered (described above).
It is also notable that most of the data come from analysis of a sub-telomeric interval(s) which could be special for any number of reasons. This maybe ok, but should be discussed more.
The above considerations address the paper on the assumption that all data are valid and conclusions fully supported by the observations. But there are quite a few issues that need to be addressed before one is really sure that these criteria are met. These are discussed below. Maybe I'm just slow. But for me, this paper was extremely difficult to read and understand. This is, in part, because the underlying effects and issue are complicated. But in addition, there are a variety of problems with presentation and analysis and interpretation and logic/assumptions. Important experimental details are absent or buried or written “in plant language”. There is a mixture of ideas and experiments. There is a historical/narrative presentation, rather than a consideration of the data per se irrespective of how the authors came to some ideas. This latter feature makes it extremely difficult to think about what could be going on.
Part III: More comments of various types.
1) It is a well-established fact that heterozygous lines may have higher or lower recombination rates than (more) homozygous lines. This is reiterated by the authors, in considerable detail. As the authors state, these could reflect differences in the natures of the diffusible molecules produced, which the authors call “trans” effects.
There is a major vocabulary problem. In opposition to trans, there is cis. There a major confusion with regard to the word “cis”, which is used in two different ways at different places in the paper. The most general way is in opposition to “trans” as defined above. That is: the effects of heterozygosity per se, irrespective of differences in diffusible factors. However, the word cis can/is also used to refer to the effect of the nature of one region on events in an adjacent region on the same bivalent, that is an effect that is in cis along a given chromosome. The two different uses make reading this paper really difficult. For example: “dual cis effects” probably uses the word “cis” in the first sense; and “cis effects of heterozygosity” probably uses the word in the second sense.
2) There is additionally what appears to be a fundamental experimental problem. The entire paper (after Figure 4) presents the “genotypes” of tester bivalents, and analyzes events on those bivalents, without ever discussing anywhere that I could find in the text what is known about the other chromosomes in those same cells and whether variations in trans-acting factors might be relevant or controlling for such variations in any way. The problem begins with Figure 4D, E and its relatives. It is stated that the pattern described in that figure, which involves the presence of homo/heterozygosity adjacent to a heterozygous test interval, is independent of the nature of the homozygous region. But this is not correct. Genotypes at the top of the pattern tend to have Col/Col (red) while genotypes at the bottom tend to have Ct/Ct (green). This is seen also in the other analyzed sub-telomeric interval (Figure 4GH). This should be specifically investigated. More generally, there has to be some attempt to deal with this as a general issue for all experiments. Perhaps this was actually done by analyzing multiple lines with the same critical genotype but, necessarily, different complements of information on other chromosomes. Are these the multiple data points in various figures? If so, it is just not obvious in the text of the paper or the figures or figure legends and the reader should not have to go searching for it. If not, this issue remains as an underlying problem throughout the paper. There are arguments against trans effects as a big issue in several places, but they are never made in the paper.
Summary: this issue has to be specifically addressed for Figure 4 in order to draw the conclusion in that figure and it needs to be discussed as an issue in all other cases.
3) The paper makes many comparisons among different regions without much emphasis on the fact that things are different or why. Of course part of the difference between the CEN interval and the 420 interval is the location; but also there is the fact that CEN is measured in male meiosis where in Arabidopsis, there are more COs, and they are more sub-telomeric. 420, which is sub-telomeric, is measured in female meiosis, where CO rates are generally lower and COs tend to be LESS sub-telomeric. It is frustrating that the authors did not discuss how these differences, particularly the M/F differences, might affect their results and/or interpretation. To the only partly initiated, it seems like apples and oranges.
4) At the most general level, this paper is among the first, in a plant system, to consider cis effects in either sense defined above. In the first category of cis, there is a long history of studies on the effects of basepair mismatches on recombination, both biochemically (they are sensed by RecA and RecA homologs, in combination with mismatch repair proteins, such that the ongoing strand exchange is rejected) and genetically, where recognition of mismatches by the mismatch repair system is known, in meiosis, to specifically eliminate crossovers (e.g. Hunter and Borts). I actually don't know whether anyone has examined DSB levels in heterozygous regions. I am not aware of any previous study that examines either interference or the effects of fancm-type or similar mutations in heterozygous regions. I am also not aware of any other studies that address cis effects in the second sense. So, as phenomena, the reported observations will be of interest to the meiosis field.
5) To set a baseline, it is easiest to begin with consideration of general properties of heterozygous regions. The authors make two new findings.
A) Fancm mutations have different effects on CO frequency in heterozygous versus homozygous regions. However: the interpretation of this finding in the paper is incorrect. The authors assume that the COs that emerge specifically in a fancm mutant are going by the same “non-interfering crossover pathway” that is argued to exist at a low level in wild type cells. If this were true, then effects seen in fancm mutant backgrounds would also be occurring in wild type. As stated above, this is possible, but it is not known and is not shown by this paper.
More specifically: is generally agreed (e.g. by Mercier and supported indirectly by yeast studies) that there is a set of DSB-initiated intermediates, among which a subset are specified to be “interfering COs”, ie Zip4-dependent COs that show classical interference. The remaining interactions are then matured to other fates, mostly non-crossovers and inter-sister events but also, at a low level to COs. These are the “non-interfering COs”. In a fancm mutant, the fates of the interactions that are not specified to be “interfering COs” are different, with a much larger proportion becoming COs than in the wild type case. The reason for this is unclear, but presumably is some modulation of biochemistry. What is shown in this paper is that, in a fancm mutant, the level of “extra” COs that arise in heterozygous regions is less than in homozygous regions. This does not speak to what might or might not be going on in wild type.
Another problem with this analysis is that wild type, fancm and fancm zip4 strains are analyzed but zip4 single mutant strains are not. This is not acceptable for a properly controlled analysis, although I understand that it did not seem “relevant” in the context of what the authors were trying to address. Moreover, the missing mutant might actually be directly informative about what is going on in a wild type background.
There is the always-underlying issue of possible trans variations; however, it can be argued that a consistent picture emerges in several types of constructs that should (apparently?) have different types of trans effects.
B) The authors find that heterozygous regions show higher levels of (phenomenological) CO interference than homozygous regions. This is shown by a three-factor cross for one sub-telomeric region for Col/Ct heterozygosity and by a different method for a broader range of heterozygous lines. The latter analysis suggests that it is general.
This is an interesting and novel finding. It implies that, as an observational fact, within a heterozygous region, if there is a CO in one region, the probability that a CO will occur nearby is reduced even more than in the normal homozygous case.
[As summarized above: there are two ways to think about what this means in reality. The “normal” way might be to think that the interference signal spreads out for a longer distance in the heterozygote. However, there is another way to think about it. The same result can be obtained if there is a reduced propensity for a recombinational interaction to be specified to be a crossover. This is shown by the “beam-film” model (or any scenario involving designation of a CO and spreading interference) by shifts in CoC curves to the left or to the right according to variations in CO-desigation probability, with a constant “interference distance”.] This is an intriguing finding per se. Its significance for other phenomena is more complicated, as discussed above.
6) The authors spend a lot of time discussing situations in which they believe that events in a heterozygous region are influenced by events in a homozygous region, and/or vice versa. First, comments on Figures 4-6:
Figure 4A. The left part shows that a pure homozygous line, heterozygous for the red and green tester markers, is produced by crossing a marked Col/Col line with an unmarked accession line (top) and then that when this line is selfed to give an F2, various types of progeny can result.
Comment. The authors show only cases in which the R and G markers are in cis, which implies that during selfing, there has been no crossing-over between them. This may be the majority of outcomes. However, the criterion for selection of F2 progeny to analyze was heterozygosity for R and G. By fluorescence, this implies that there is one copy of each. For all of the examples shown, R and G are “in cis” to one another. But 15% of meioses will give a crossover between the two markers; and if the appropriate products unite (R+G- with R-G+), the resulting F2 seed will ALSO have only one copy of each marker. This may be rare (15% x 15%). Nonetheless, such cases would be scored as having the highest recombination rates if they were not somehow identified and removed from further analyses. Do the authors know from genotyping whether their F2s include such cases and if so, which ones are they among the 139?
Other “missing information”: (i) Were the F2's obtained from a single F1? Or from multiple F1's with results pooled? (ii) Is it completely obvious that the fluorescence detection could distinguish RRG and RGG from RG genotypes? This is clearly a critical point and it does not seem to be addressed explicitly, though I might have missed it.
Minor presentation issue: the meaning of the box on the right is obscure without working at figuring it out. Apparently it means that when the F2s are taken through meiosis, they may generate recombinants, but this is hardly obvious in the cartoon or the legend. (Again assuming that they started out with the cis configuration.)
Figure 4B. In this figure, the authors select a set of F2s that are heterozygous at the R and G markers. They then ask, at each of 51 other positions, whether the frequencies of the two parental alleles is different from what you expect from an absence of correlation with the selected “phenotype”. What is detected is a deviation from expectation for chromosome 3 (the chromosome on which phenotype is selected).
Comment: We can start with the fact that this analysis is essentially not described at all in the paper. The non-plant reader would have no idea what is going on from the half a sentence in the text.
If I have understood correctly, this analysis appears to have a fundamental flaw. If you select for F2's that are heterozygous for R and G, most of these will have the cis configuration, meaning that they have not undergone a CO between R and G on that chromosome in the previous meiosis. Since there must be at least one CO, there will be a CO somewhere else on chromosome 3. And there will be a non-random tendency for marker disposition to occur just because there is already a selection that restricts localization on this chromosome specifically. More specifically: since the average per chromosome is just a little above one, and since the CO is not sub-telomeric at the marked end, it will tend to occur at non-random position (or positions if there are 2 COs) away from the marked end, e.g. towards the middle of the chromosome. This effect explains the peak in the graph, which is centered in the middle of chromosome 3: it reflects the non-random consequences of having the CO in a non-random position along the marked chromosome. If this interpretation is correct: this data do not provide evidence for a cis effect. There is only the fact that you have pre-selected a population that, by its nature, will have a non-random arrangement of markers along chromosome 3.
Two further points: (i) 139 is a very small number of F2s from which to draw a conclusion. (ii) There should be a control showing that if you look at the entire population, you do not see any peak. Of course, given the basis for the peak (above), the control will show that it is specific to the selected sub-population, which is not really any help, but still, if such analysis is to be shown, the control must be there.
Figure 4C. Figure 4C shows the recombination frequencies in the 420 interval observed for the 139 F2's. The conclusion is that the range is greater than for the Col/Col wild type strain or for a Col/Ct F1 full genome heterozygote. This is likely true, but it is hardly surprising under any model. It likely reflects trans effects as well as cis effects. It is unclear why this is valuable information, unless it sets the stage for later panels.
Minor point: it is not appropriate to compare the rank plot for the 139 F2s with averages and ranges for Col/Col and Col/Ct. The ranges for the two compared lines are a statistical measure; they do not give the same impression as the rank plots. Thus one is comparing “apples and oranges”. The rank plots for the two comparison lines should also be shown, and then the averages and ranges for all three sample sets should be compared.
Figures 4D, E, G, H; Figure 5. Figure 4D displays the natures of the 139 F2s, shown from top to bottom in rank order of recombination frequency in the 420 interval, with respect to whether the markers indicated on the X-axis are heterozygous (black), homozygous Col, or homozygous Ct. This plot shows a gradient of “black”, emanating from the test interval towards the end of the chromosome, to greater and greater distances from bottom to top. The authors interpret this data as follows: “However, we detected a significant association on chromosome 3 itself, where the hottest F2 quartile had significantly higher homozygosity outside 420, compared to the coldest quartile. This cis-effect was observed when the rest of the chromosome was either Col or Ct homozygous, indicating that it is non-allele specific and caused by polymorphism per se, rather than by specific sequence variants.”
The restatement of the result above does not accurately describe the entire picture. The fact that the black portion emanates from the selected region is not mentioned (and is presumably significant). An accurate restatement of the result, which admits to all interpretations, is that F2s with higher 420 recombination tend to be homozygous for flanking material while F2s with lower 420 recombination tend to be heterozygous. Moreover, the “extra heterozygosity” towards the bottom of the rank plot tends to occur specifically adjacent to the 420 region. The term “cis effect” should not be used, as this is an interpretation. It has become lab jargon for the phenomenon the authors think is going on, but it is confusing given the various versions of this phenomenon (including its reciprocity), as well as the fact that there should be a formal description of the data, not an interpretive one. Better to call it a “junction phenomenon” or Hom-Het interplay.
More importantly, contrary to what the authors say, there is a clear tendency for more “red” at the top and more “green” at the bottom, not only in Figure 4D but in the corresponding figure for the other sub-telomeric region in Figure 4G. This would likely appear in an appropriate plot, although a larger data set may be required to see it with very strong statistical significance. It suggests that having more Col information promotes a higher frequency of COs in general versus more Ct information. This is directly supported by the fact that, overall, Col/Col recombination rates are higher than Col/Ct recombination rates genome-wide (Figure 4C). This has to be dealt with.
Also importantly: the authors cannot, in principle, say that they see a cis effect unless they do a control to show that the sample subset of 139 F2s have similar frequencies of recombination throughout the genome and that there are or are not any associations of recombination with homozygosity, and/or Col vs Ct content on a genome-wide basis. I appreciate that this is not trivial. But the authors' hoped-for conclusion is not trivial either. It cannot be made from this figure without more supporting (or contradicting) data.
Secondarily (perhaps): there are general uncertainties. (i) There is the possible problem that the hottest F2s might have their markers in trans, with unknown consequences. (ii) The top-ranked F2 in the 4D data set is very pecuIiar. It appears to be Col for the entire length of one chromosome and to have had a single CO exactly at the border of the marked region in the other. How is this possible? (iii) As in Figure 4B, there could be unknown effects from selecting the subset of F2s that did not have a CO in the sub-telomeric region. Are there markers all the way to the end of the chromosomes or are sub-telomeric crossovers going to be missed (which is a problem for the analysis since many COs will be there specifically in the selected population, thus biasing the sample. There is actually a hint of such a problem in the fact that the observed “effect” is absent for the centromere (a central marker) and weak for an interstitial marker. One interpretation is that observation of an effect is a consequence of selecting a sub-telomeric region for analysis (rather than being an actual phenomenon is specific to such regions). The authors have only presented data saying whether each F2 is homozygous or heterozygous for the markers tested. But in principle, they could figure out the exact arrays of markers on the two chromosomes of the F2 and thus were crossovers occurred in the preceding F1 cross(es). While perhaps difficult, this might reveal additional informative information to further guide interpretation.
Figure 6A-C. Panels A-C show that two lines that are heterozygous for 420 and Ct/Ct for all or most of rest of the chromosome exhibit higher 420-region recombination than a line that is homozygous Col/Col in 420 and heterozygous for the rest of the chromosome. The authors think that the relevant difference is that heterozygosity adjacent to homozygous 420 depresses recombination in 420. They use this data to argue that the presence of homozygosity adjacent to heterozygous 420 “increases” recombination, and thus “eliminates the cis effect”.
First: this is not “eliminating the cis effect”. It is a different type of cis effect. It is not clear that the authors appreciate that they are seeing reciprocal effects. They are totally focused on the effects of heterozygous regions on homozygous regions.
In any case, without controls, there are several hypotheses that are equally consistent with the data. What is the relevant difference(s)? Heterozygosity in 420? Col/Col in 420? Heterozygosity or Ct/Ct in the rest of the chromosome? Some combination of the two? There is no way to know without more lines with different genotypes. What is needed (minimally) is Heterozygous 420 plus homozygous Col/Col and fully homozygous Col/Col. (Homozygous Ct/Ct is desirable but difficult to construct). No conclusion can be drawn without controls. This is just not acceptable. To a first approximation, this data should be omitted.
And to repeat: it is assumed that the only relevant differences among the different lines is on the chromosome of interest. But all of the chromosomes are different in the different strains. Is there evidence that the variations are not due to trans effects.
Another thing that is very unclear is why these particular lines were picked for testing. Both of the HL lines chosen for 6 A/B seem to be the rare type in the top quartile in Figure 4; most of the lines in this quartile are homozogous Col/Col outside of 420. Why were these chosen rather than the more common type; or, better, why not test both types? And specifically, exactly which of the 139 F2s are HL1 and HL2? It is not obvious.
Figure 6 D, E, F. This figure shows a 3-factor cross in which there are two intervals that comprise most of the single 420 interval in (AB). (DE) shows that HOM adjacent to HET results in an increase COs in the HET region and that HET adjacent to HOM results in a decrease in COs in the HOM region. This points to the reciprocal effect shown further in Figures 7/8. (F) shows that, despite these changes, there is no change in interference between the two intervals. To anticipate later results, this implies that the nature of interference in the HOM region predominates (below).
Figures 7 and 8. Figures 7 and 8 have three components (1) They provide information about fancm effects and interplay of heterology with fancm recombination. These were discussed above. [The observed effects pertain specifically to heterozygous regions regardless of what is or is not adjacent to them. They do not say anything about what is going on in wild type non-interfering COs.] (2) They provide more data for wild type (FANCM+) strains in several situations. (3) They show that interference is required for the so-called “cis effects”. The exposition in the paper was hard to follow. Here is my own restatement of the situation.
Issue (2). Figure 7. For the 420 interval, there are four CO frequencies: HOM/HOM 18cM; HOM/HET 15cM; HET/HET 21cM; HET/HOM 28cM. It is unclear what the multiple data points represent. If these are multiple different lines (and thus different “trans” effects, then the data, are more meaningful). Just looking at these numbers, some conclusions emerge:
Adjacent HET reduces COs in HOM (in HOM/HET 15cm relative to HOM/HOM 18cm). This is the effect emphasized above.
Oppositely, however, adjacent HOM increases COs in HET (in HET/HOM 28cM vs HET HET 21cM). So the effects are reciprocal, which is not emphasized enough.
For the CEN interval, HOM/HOM 12cM; HOM/HET 12cM; HET/HET 10cM; HET/HOM 9cM. Clearly the centromere behaves differently, for whichever reasons, as suggested by Figure 5 data.
Figure 8 addresses the same to issues above, but for a three-factor cross specifically in the sub-telomeric interval and with the addition of interference analysis.
Left interval: HOM/HOM 16cM; HOM/HET 11cM; HET/HET 16cM; HET/HOM 18cM.
Right interval: HOM/HOM 5cM; HOM/HET 4cM; HET/HET 5cM; HET/HOM 6cM.
In this case:
HET reduces COs in HOM: HOM/HOM 16cM; HOM/HET 11cM; HOM increases COs in HET: HET/HOM 18cM; HET/HET 16cM.
The right interval behaves oppositely, but with small effects;
HET increases COs in HOM: HOM/HOM 5cM; HET/HOM 6cM;
HOM reduces COs in HET: HOM/HET 4cM; HET/HET 5cM.
The right interval is small. Apparently the “left side dominates”. I have no idea what this means.
Figure 8. Interference is also analyzed in the wild type background. NB: there is nowhere in this paper (except the Methods) a description of what the interference metric is, even on the figure. Axis must be labeled (1-CoC) and this must be explained in the text.
Results: HOM/HOM 0.6; HOM/HET 0.6cM; HET/HET 0.8; HET/HOM 0.8.
(a) HET/HET has “more interference” than HOM/HOM, the effect discussed above Importantly, this is despite the fact that the frequencies of COs in both intervals are the same in these two situations! This would not be expected if the only difference were in interference. Thus, in the pure heterozygous situation, there must be two effects, one that increases COs plus interference that decreases COs, e.g. more DSBs and counterbalancing interference increase. (b) The two reciprocal “mixed” cases are not the same. HOM/HET looks like HOM/HOM while HET/HOM looks like HET/HET. Formally, the nature of the left interval dominates, as also seen with respect to effects on CO levels (above).
Overall, it seems that the ability of a HET region to depress COs in a HOM region does not depend on having a HET-type CO interference process.
Figure 8: In the larger/left interval, where canonical reciprocal effects are observed: HET reduces COs in HOM: HOM/HOM 16cM; HOM/HET 11cM; and for interference, HOM/HET looks like HOM/HOM. HOM increases COs in HET: HET/HOM 18cM; HET/HET 16cM and for interference, HET/HOM looks like HET/HET, and the same is seen in Figure 6D, E, F. This figure compared a HOM/HOM situation with a HET/HOM and found them to have the same (HOM) interference. And the “canonical” reciprocal effects are observed.
7) The last item is the interplay between fancm and CO interference, keeping in mind that the information does not tell us anything about what is going on in FANCM+ (and thus the general cases of heterozygosity seen in nature).
One expectation is that if more COs are coming from the fancm-revealed process, there will be less interference. This expectation is met in Figure 8. More or less, there is plenty of interference in all four strains in FANCM+. And more or less, in both HOM/HET and HOM/HOM, where the entire test region is homozygous and the fancm mutation is having its full effect, there is a big reduction in interference. Whereas, in both HET/HOM and HET/HET, where the entire test region is heterozygous and there are fewer extra COs in the fancm mutant, interference is less reduced. And the zip4 mutation, in the fancm background, has the expected effects in all cases, reducing interference because it decreases the fraction of “interfering” COs. These results provide more support for the nature of the fancm interplay with heterozygosity (discussed in 2 above).
The authors also ask about variations in the “interplay” of fancm with HET/HOM differences. The underlying idea (which I finally realized after many hours) is that in fancm zip4 there are still COs but they do not show interference, so one can ask if the variations in 420 according to what is adjacent are still seen. If no, then interference is required.
The data for 420 in Figure 7 are:
HOM/HOM 35cM HET/HET 12cm HET/HOM 10cM HOM/HET 35cm. Most importantly, since HOM/HET = HOM/HOM, there is no transmission of information from HET to HOM if interference is absent, which implicates interference in the process (in accord with the suggestions above). The reciprocal case: HET/HET 12cm HET/HOM 10cM seems to run against all rules, with HET increasing COs in the adjacent HET, but again, maybe this is a small effect and/or can be ignored. The fact that HET/HET << HOM/HOM is explained by the fact that there are fewer COs from the fancm pathway in heterozygous regions (above).
Similarly in Figure 8: for left and right regions
HOM/HOM 25, 12cM; HET/HET 8, 3.5; HET/HOM 7.5, 2.5cM HOM/HET 22, 12cm. These data are clear and very useful in showing that interference is relevant.
8) A final point on statistics: p-values are given everywhere as Chi-square p-values, but it seems the authors are comparing means, not comparing a result against an expectation. Is this a typo? Is it a different kind of test, or did they really use a Chi-square? (If the latter, they need to justify and clarify what the expectation was, otherwise give p-values for something that compares distributions, e.g. a T-test).
There is no question that the manuscript of Ziolkowski et al. is of interest as it is challenging current views of crossover control, not only in plants.
I also read the thoughtful comment and valuable suggestions of both reviewers with great interest. I think that many of the points raised by the reviewers the authors were able to address in their response, especially by the inclusion of new experimental data.
I have to say that the main conclusion that interfering crossovers are enhanced in heterozygous regions is for me as for the other reviewers counterintuitive. Also, the model the authors bring forward at the end, that recombination intermediates are stabilized by mismatch recognition is quite special. Therefore it is of course important to take other explanations into account.
There is one thing that worries me a bit and that is quality of the sequence data available for the different Arabidopsis cultivars. Do heterozygous region really attract crossovers from homozygous regions in hybrids? As the authors state themselves, these sequences were aligned by short read technology and so the occurrence of inversions and of duplication in the range from hundreds to hundred-thousands of bps might be dramatically underestimated. Thus, changes above the nucleotide level were not taken into account by the authors. Nevertheless these changes might drastically influence recombination patterns.
Nevertheless, all in all, the authors supply us in the revised version with enough hard data that in my opinion strengthen their hypothesis to the point that it should be considered (and challenged) by the community. Also, it might well be that such a mechanism is exceptional and restricted to self-fertilizing species like Arabidopsis. But this would be still interesting.https://doi.org/10.7554/eLife.03708.044
- Ian R Henderson
- Ian R Henderson
- Gregory P Copenhaver
- Piotr A Ziolkowski
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Research was supported by a Royal Society University Research Fellowship and Gatsby Charitable Foundation Grant 2962 to IRH, and United States National Science Foundation grant MCB-1121563 to GPC. We thank Raphaël Mercier for providing fancm and zip4 mutations and genotyping information and Avi Levy for the 420 line. PAZ was supported by Polish Mobility Plus Fellowship 605/MOB/2011/0. We thank the editor and reviewers for insightful comments.
- Detlef Weigel, Reviewing Editor, Max Planck Institute for Developmental Biology, Germany
© 2015, Ziolkowski et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.