PGENETICS-D-26-00438

High-resolution global recombination mapping in C. elegans reveals sexual dimorphisms shaped by meiotic chromosomal features and structures

PLOS Genetics

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Reviewers' comments:

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Reviewer #1: The manuscript by Bush et al., presents the genome-wide crossover landscape of C. elegans oocytes and spermatocytes. Leveraging WGS, the investigators show that there are significant differences in the crossover landscape on individual chromosomes and in the sexes. Analyzing a synaptonemal complex (SC) mutant heterozygote, the authors find that the SC plays a differential role in the crossover landscape on individual chromosomes and in the sexes. Chromatin environment also appears to influence the crossover landscape in a sex-specific manner. Overall, this is a well-done study that provides an important dataset to facilitate future hypotheses on crossover regulation. The following should be addressed:

Figure 1D, RAD-51 Cut + Run: It is important to state in the results that this was just done on hermaphrodites – presumably adults only undergoing oogenesis (but not in males). Given that previous published cytological and CHiP-seq data revealed enrichment of RAD-51 on chromosome arms, the authors need to discuss why their data shows a random pattern. In the figure legend (line 1345) it states that the histogram shows global distribution of crossovers in oocytes versus oocytes – I am assuming this is supposed to be oocytes vs. spermatocytes.

Figure 3, DCOs in syp-2/+: It appears that there is no statistical difference in DCOs genome-wide in syp-2/+ oocytes and spermatocytes (Figure 3C). On individual chromosomes it appears that there are fewer DCOs on chromosome IV in oocytes and no change in spermatocytes, while an increase of DCOs on the other chromosomes. Are any of these statistically different? In the results section, please indicate that on chromosome IV, there is a reduction of DCOs in the syp-2/+ oocytes and consider the possibility that syp-2/+ alters the distribution of COs to individual chromosomes but not necessarily the number, particularly in oocytes?

Analysis of H3K36me3/H3K27me3: The difference between crossover enrichment in H3K36me3 in spermatocytes and H3K27me3 in oocytes is very interesting. However, it was unclear why only these two marks were examined. Ideally, additional euchromatin and heterochromatin marks should be examined. In the absence of this, please provide a justification for only looking at these two chromatin marks.

Discussion: I would really like to see a discussion of the sex-specific differences observed in this study in relation to what has been observed in other systems as indicated in the intro (lines 101-14) and perhaps what is says more globally about crossover landscapes.

Typos:

Line 157: remove the first “the”

Line 244: remove “the” equal

Line 522: add “be” to “may be one”

Line 687: remove “for”

Reviewer #2: In this manuscript, the authors use genomic and bioinformatic analysis to analyze sexual dimorphism in crossover distribution and number during C. elegans meiosis. They show that oocytes and spermatocytes show different distributions of crossovers at large scales and finer scales, different numbers of double crossovers, and investigate whether these differences correlate with chromosomal features, such as Pairing Centers, sequence level annotations, germline gene expression and chromatin states. Moreover, they evaluate the contribution of gene dosage of a synaptonemal complex component and find that reducing syp-2 gene dosage in oocytes results in crossover distribution patterns that more closely resemble crossover distribution patterns in spermatogenesis. In general, this paper is an important contribution to the field and requires some minor revisions.

Figure 1C: I agree that oocytes show enrichment of crossovers on the PC end of Chromosomes I and III but I don’t see the enrichment of crossovers on the PC end of Chromosome II. I would also include the enrichment of crossovers on the PC end of Chromosome IV but understand why they don’t, given the lack of sexual dimorphism. Similarly, I agree that there are more crossovers in the central part of Chromosome V but it does not seem as dramatic as what is observed on the X chromosomes. In general. I think this section could be slightly expanded to include finer details of their analysis depicted in Figure 1.

I appreciated the inclusion of the RAD-51 CUT&RUN data to reinforce the points about lack of hotspots.

Could the authors mention number of syp-2/+ genomes in results section (300 oocyte-derived and 300 spermatogenesis-derived) that make up 1292 recombinant chromosomes?

Can the authors explain the lower sequencing coverage in syp-2/+ animal sequencing and if this affects their analysis?

For those not well-versed in these approaches to assess crossover number and distribution: Is there any concern about bias in what fraction of chromosomes are reported for crossover location and number?

Could the authors specify in their analysis of chromatin states and crossover distributions that the histone modification data comes from analysis of germ cells?

In their section of the discussion, “Chromosome synapsis and sexually dimorphic crossover distributions,” the authors discuss synapsis defects as a possible source of the altered crossover distributions in syp-2/+ oocytes. Another possibility the authors should consider discussing is that SYP-2 dosage in syp-2/+ oocytes approximates SYP-2 dosage in wildtype spermatogenesis. For example, Cahoon et al show that SYP-2 fluorescence on meiotic chromosomes is reduced at early stages of meiosis during spermatogenesis, compared to oogenesis. Given their analysis that syp-2 gene dosage affects crossover distributions and number in both sexes, it’s possible that the amount of SYP-2 in the synaptonemal complex contributes to control of these events. Similarly, syp-2 gene dosage could have consequences on the amount of other synaptonemal complex components in this structure, affecting crossover distribution aside from affecting the process of synapsis itself.

In lines 521-523, the be is missing: the prolonged duration of pairing in wild-type C. elegans oogenesis may *be* one factor that contributes to the broad-scale differences in the crossover distribution on the pairing center “arms” of these chromosomes.

Reviewer #3: Bush et al. used whole-genome sequencing to map crossovers and non-crossover gene conversion events in C. elegans males and females. Intensive analysis of the sequencing data provides support for several conclusions. Although some of these may seem to merely buttress results from genetic experiments, these have typically been restricted to part of a single chromosome pair. The genome-wide analyses in this manuscript provide robust data along each chromosome, among chromosomes, and between the sexes. This and the accompanying analyses (e.g., the SC mutant) make a significant contribution to quantifying differences in crossover distribution with respect to several metrics. Although most of the manuscript focuses on crossover positions, mapping of non-crossovers and of RAD-51 binding bring additional important insights.

The analysis is thorough and broad. I don’t believe there are additional experiments that need to be done, but the points below should be addressed in revision.

1. RAD-51 binding sites are found across every chromosome, including in the central region of each, so one might expect total recombination frequency (NCO + CO) to mirror this. The lower SNP density in the central regions means that if there are COs there they will be mapped with low resolution, but still detected. As noted, NCOs in these regions will be missed because of low SNP density. The regions of low SNP density seem to be a third to a half of each chromosome. This makes it puzzling that the total NCOs observed is approximately the number predicted through other studies (lines 245-257). Can the authors reconcile these observations?

2. The heatmaps in Figs 3 and 4 can be difficult to parse. There are certainly some features that must be analyzed on a per chromosome basis, but is there any reason to expect that association with chromatin or gene expression states would vary between chromosomes (or perhaps between groups of chromosomes that seem to be similar, i.e. [I, II, & III], [IV, V], [X])?

3. Tables 1 and 2 should include number of chromosomes sequenced, as well as an indication of what is statistically significant (most informative might be oocyte versus spermatocyte).

4. In several places, the authors use logistic regression analyses, but there are no details provided. For example, is the model being used binomial (e.g., parental versus recombinant) or multinomial (parental, single crossover, double crossover)?

Typos and minor corrections:

• line 81: phenomenon is the singular

• line 151: “near sites where homologous chromosomes pair” should instead say where pairing is initiated, since they eventually are paired along their entire lengths

• lines 191-194: The wording is confusing. Does “…our crossover mapping pipeline was able to successfully call crossovers in 297/300 oocyte samples and 300/310 spermatocyte samples” mean the pipeline failed for 3 oocyte and 10 spermatocyte samples or that there were just no crossovers detected in those samples? The numbers are about what would be expected if there is an average of one crossover per bivalent and recovery of nonrecombinant versus recombinant chromatids is independent between bivalents. Also, since the first clause is “In the oocyte data,” the last clause “and 738…” implies that the spermatocyte data are from the oocyte data.

• lines 198-203: This paragraph concludes by saying that the whole-genome sequence method is reliable, but it’s unclear how the preceding two sentences support this conclusion. Rather, it appears that the intent is to say that despite some samples having insufficient read coverage (as is to be expected) and there being genetic incompatibilities), this approach is still informative. Is the argument that because there are distinctive patterns detected across multiple analyses (examples should be given or something like “described below” included) this lends validity to the approach?

• lines 210-213: The sex-specific bias is noted for I, II, and III, but later IV is lumped in as being different from V and X.

• lines 214-215: What does “seemingly elevated” mean? Is there a statistically significant elevation?

• line 260-262: It’s not really “base pair resolution” obtained with CUT&RUN and 300 bp windows. Also, the analyses cannot reveal that there are “no statistically enriched binding sites for RAD-51,” only that none were detected with the parameters used.

• line 265: The suggestion of peaks of RAD-51 about every 10 kb is interesting. Does this correspond to any known or hypothesized structural feature of meiotic chromosomes?

• line 290: “each” should be “one”

• lines 291-301: The apparent sexually dimorphic windows may be interesting, but the discussion lacks some statistical framework. For the analysis, what is the interval between sliding windows, and how many such windows were compared in total? Figure 2B shows the sex-specific differences clearly, but the numbers seen here appear to be far lower than stated in the text (e.g., it doesn’t look like there are 31 adjacent differences for chromosome I). Is this because the figure shows only one set of adjacent 200 kb intervals, with no sliding?

• line 352-353: “is sexually dimorphic…in oocytes versus spermatocytes” is redundant

• lines 353-355: “…and some of this sexual dimorphism can be attributed to crossover formation relative to their distance from the chromosome pairing centers.” The phrase “can be attributed to” suggest that the distance is the cause of the dimorphism. It may be, but the data presented only show that this is an observation. The observation is that a greater proportion of oocyte crossovers are within or near the PCs.

• line 616: “during in”

• The term “arm” is sometimes in quotation marks and sometimes not. For consistency, I suggest that early in the manuscript the authors clearly define what they are going to refer to as an arm (e.g., a percentage of physical or genetic length or the region with high SNP density) and then drop the quotation marks.

• All of the intervals are defined by SNPs. One presumes there are also indels. Were these not used because of mapping complications?

• line 719: Does accuracy refer to call v. not called or precision of the location? Even at 0.1x coverage it seems like very crossover should be called, just with less precision on the position.

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Reviewer #1: Yes

Reviewer #2: No: Illumina data availability is currently in progress and I didn't see numerical data for the graphs.

Reviewer #3: Yes

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Dear Dr Libuda,

We are pleased to inform you that your manuscript entitled "High-resolution global recombination mapping in C. elegans reveals sexual dimorphisms shaped by meiotic chromosomal features and structures" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Comments from the reviewers (if applicable):

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