Microsatellite data of core and edge populations of Coenagrion scitulum in Western Europe

Genotype data of Coenagrion scitulum individuals from five core and five edge populations. Samples were collected in the field and the extracted DNA was genotyped for twelve microsatellite loci

<i>De novo</i> sperm crossover activity in the distal region.

(A) Sperm CO profiles for each of the two ePAR-carrying men analysed. A total of 158 recombinants were recovered from 76,800 sperm from man 53 using reciprocal crossover assays (i.e. Ab plus Ba COs, where AB and ab are the parental haplotypes) compared with 42 from a total of 92,000 sperm from man 20. Recombination activity expressed in cM/Mb along the assayed intervals is shown in the central graphs with the crossover activity of man 53 shown by the dark grey histogram and that of man 20 by the light grey histogram. The combined least-squares best-fit normal distribution for both men is shown by the black curve. The recovered CO structures together with their frequencies are shown above (man 53) and below (man 20) with heterozygous SNP locations represented by circles. SNPs marked with an asterisk were exploited for recombinant recovery (see Methods and S6 Table). The pink panel spans the interval in which male meiotic DSBs were previously mapped by DMC1-SSDS [19] and coincides with the peak CO activity determined from de novo sperm events in this study. (B) Transmission frequencies of SNP alleles into reciprocal COs with 95% credible intervals determined by Bayesian analysis. Transmission of the ‘strong’ allele (C or G) is shown for transition polymorphisms and transmission of the purine allele (A or G) is shown for the transversion polymorphisms. The upper panel shows the transmission data from man 53, the lower panel those for man 20. All markers with the exception of rs1970797 in man 53 were consistent with the expected 50:50 transmission of the two alleles into reciprocal events. This polymorphism lies 126 bp proximal to the predicted hotspot centre and 210 bp distal of the closest hotspot motif [73], as indicated at the top of the upper panel. CO asymmetry has previously been noted at hotspots that do not contain obvious matches to this motif, yet are nonetheless specifically activated by PRDM9 A [38,42]. Note that our failure to observe asymmetry for man 20 may simply be a consequence of the small number of COs detected for this donor.</p

Choosing target regions within ePAR to assay for male germline <i>de novo</i> recombinants.

(A) Distribution and intensity of DSB clusters that fall within the X-derived portion of the ePAR. Clusters were defined [19] by anti-DMC1 SSDS using testis biopsies from five men and designated as being induced by PRDM9 A (dark green) or PRDM9 C (light green); DSB strength is shown as the mean across relevant individuals using the arbitrary values reported in the original work [19]. (B) Frequency of heterozygous SNP markers per 1-kb interval identified in each of the two ePAR-positive sperm donors (man 20, man 53) as determined by Ion Torrent sequencing. (C) Linkage disequilibrium (LD) heat map derived from the 50 CEU females from the 1000 Genomes Project [34] (more intense orange equates to stronger LD). Data are based on |D´| values determined from SNPs with minor allele frequency >0.2 that passed tests for Hardy-Weinberg equilibrium specifically derived for markers on the X chromosome [72]; these stringent criteria meant that LD corresponding to the most proximal portion of the ePAR interval could not be examined. Scaling is shown with respect to GRCh38/hg38, and the two chosen assay intervals, distal and proximal, are indicated by the dashed boxes and arrows. See also S2 Fig.</p

Simple interpretation of the I2a ePARs.

(A) Schematic of consensus X-derived portion of ePAR carried by individuals 6889_01 and man 20. Green boxes with black outlines represent the shared haplotypes at each of the nine blocks of SNP markers whilst the intervening black boxes coincide with mapped PRDM9 A and C DSB clusters [19]; widths of all boxes are proportional to their length. The black triangle to the left points towards the canonical 2.7-Mb PAR1 and ultimately to the Yp telomere; the start of Y-specific I2a sequence is shown to the right. The frequency of phase-known X haplotypes among the 95 CEU+GBR males that match the modal ePAR haplotype for each block of SNPs are shown in green; the frequencies of singleton haplotypes amongst the same are shown in purple. (B) The remaining eight I2a ePARs, assuming they are the result of a single crossover between the consensus and an incoming X-linked haplotype depicted by yellow boxes (crossover interval shown with blue cross). Boxes with red outlines show haplotype blocks that differ from the consensus with the number of SNP changes shown in red; asterisks identify three haplotype blocks that differ from the consensus by the same single base pair change. Black numbers beneath boxes indicate the observed frequency of the non-consensus haplotype amongst the 95 phase-known X haplotypes from the CEU+GBR males. Total SNP counts per block are shown in italics at the bottom.</p

Schematic representation of formation and organization of the ePAR.

(A) Normal pairing of the short arms of the X and Y chromosomes in male meiosis is limited to PAR1 (purple) such that homologous recombination can occur up to the canonical boundary as marked. However, mispairing of the PAR1 LTR6B element (yellow box) carried on a Y chromosome (blue) with one located more proximally on an X chromosome (pink) can result in non-allelic homologous recombination and the generation of gametes containing either an ePAR-carrying Y chromosome, or the reciprocal deleted X chromosome. Schematics not to scale. (B) Pairing and therefore homologous exchange between an ePAR Y chromosome and a normal X chromosome is predicted to extend proximally to a new boundary. The first three exons of the XG blood group gene fall within PAR1, while the remaining exons are carried on the X chromosome; men carrying the ePAR thus have two full-length XG genes like females. Similarly, whilst most men are hemizygous for the GYG2 gene, ePAR carriers have two copies of this gene.</p

Comparison of Y chromosome SNP/CN probe intensities between a male, a female and a male with an isodicentric Y chromosome.

<p>The Y axis denotes the log ratio of the average signal intensities for each 20 consecutive SNP/CN probes, while the X axis denotes the genomic position on the Y chromosome, in million base pairs. The signals for a control male are represented in black, female signals are in blue and the signals for the male with an isodicentric Y chromosome are in yellow. Only probes corresponding to the male specific Y region are included.</p

Comparison of inferred ePAR haplotypes with phase-known haplotypes from the corresponding region of the X chromosome.

(A) SNP haplotypes from each of the eleven independently sampled ePARs (ten from the haplogroup I2a Y chromosome lineage and one from the R1b Y lineage) are shown in rows, clustered according to the distal haplotype block. Individual 6889_01 is shown at the top with all his alleles colour-coded blue; yellow denotes the alternative SNP alleles not carried by this man. Black vertical lines correspond to the relative locations of mapped PRDM9 A and C DSB clusters; in two instances, marked by asterisks, an A and C cluster lie in very close proximity. Arrows indicate the distal and proximal sperm recombination assay regions and the red box indicates a second ePAR that is identical to that of 6889_01. In total, nine of the ten ePAR haplotypes are unique to this dataset. (B) Phased X haplotypes from the 1000 Genomes Project [34] for the 49 CEU males and 46 GBR males. One haplotype is shared between the two sample sets as indicated. In addition, three pairs of identical X haplotypes were noted among the CEU and one X haplotype was found to be carried by three different GBR men (red boxes). In total, 42 of the 46 CEU haplotypes and 43 of the 44 GBR haplotypes are unique. None of these X haplotypes matches any of the ePAR haplotypes. (C) Relative scaling of the regions depicted together with summary count of the number of SNPs, number of ePAR haplotypes and the corresponding total number of haplotypes seen amongst the ePAR, CEU and GBR datasets per block of SNPs.</p

<i>De novo</i> recombination in the proximal region.

(A) Sperm CO profiles in relation to the proximal DSB cluster as determined by DMC1-SSDS [19] (pink panel) for man 20 (light grey histogram) and man 53 (dark grey histogram), with the combined least-squares best-fit normal distribution shown by the black curve. As for Fig 3, data from reciprocal assays have been pooled and the recovered structures and their frequencies for each man are shown above and below the histograms with informative SNPs represented by circles. In these assays, ASPs were designed against the SNPs marked with asterisks and were used in conjunction with universal primers (triangles) to selectively amplify each parental haplotype; recombinants were then detected by probing for the alleles of the opposite haplotype represented here by white circles (see Methods, S6 and S7 Tables). Note that CO events involving only the terminal marker closest to the universal primers are indistinguishable from NCO events in this assay, so we arbitrarily designated half of such events as COs in these cases (numbers given in italics) but indicate with dashed boxes in the graph how the profiles would appear should all such events actually be COs. In the latter case, the hotspot width would be reduced by 250 bp, the centre point would be shifted proximally by 116 bp, and the peak activity would be ~ 830 cM/Mb. (B) Testing for GC bias amongst NCOs. Of the four informative SNPs for man 53 that carry a ‘weak’ and ‘strong’ allele, SNP 97.4 shows over-transmission into NCOs of the ‘strong’ allele G relative to the ‘weak’ allele A (P = 0.011, one-tailed binomial exact test). This SNP lies 97 bp proximal to the hotspot centre as shown by the black curve in (A). Whilst we cannot be sure of the number of NCOs involving the terminal marker SNP 97.8, both alleles at this SNP base pair with three hydrogen bonds (i.e. are ‘strong’) and there is no evidence of disparity between the orientations assuming at least half the terminal recombinants are NCOs (i.e. 9). For man 20, terminal marker SNP 96.0 recombinants were recovered in the two orientations with similar frequencies, again suggesting an absence of TD. Note a further two NCOs each affecting a different single site (SNP 99.8 or SNP 100.1) were also recovered for this man but are not depicted in this figure.</p

Principal component analysis of Y chromosome specific SNP variation between haplogroups.

<p>A. The figure shows the graphical representation of the first two eigenvectors after PCA analysis. Y-axis corresponds to the first vector explaining 24.1% of the variation and X-axis explains 13.4% of the remaining variation. Each dot represents the results from one individual and the colour represent each HG as denoted by letters in the figure. The plus symbols in black denote individuals for which HG determination was ambiguous. The black triangles denote individuals for which no HG could be assigned. B. The results from the individuals carrying the blue-grey dupl are represented in blue, while the results from individuals carrying “blue-grey like dupl.” are in red. All cases are included within the NO-M214(xM175) haplogroup. In total 11 individuals carry these variants (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137223#pone.0137223.t001" target="_blank">Table 1</a>) but they are superimposed in the figure, due to high similarity between their HG.</p

qPCR validation of the newly discovered P6 duplication.

<p>Amplification plots for a female (green), a control male (purple) and a male with P6 dupl. (blue) are shown for markers RH38681 (A), sY1081 (B) and sY933 (C). D. Intensity signal plot (Log 2 ratio) for an individual with P6 dupl. showing that markers sY1081 and sY933 are positioned within the duplicated region, while RH38681 is located outside.</p