Elevated centromeric crossovers in <i>met1–3.</i>

<p>(A) Schematic diagram illustrating generation of wild type and <i>met1–3^−/−</i> recombinant male backcross populations from Col and Ler homozygous parents. (B) Chromosome physical maps with overlaid cM/Mb (red) and DNA methylation (blue) plots; black vertical lines indicate the position of polymorphic Col/Ler markers tested for segregation frequency. Vertical magenta lines indicate centromeres. (C) Segregation data and centromeric CO measurements in wild type and <i>met1–3^−/−</i> male backcross populations.</p

Elevated crossover hotspot <i>3a1</i> activity in <i>met1–3</i>.

<p>(A) CO frequency distributions (cM/Mb, blue) within <i>420</i> map interval 8 measured by dCAPs PCR marker segregation (white bars represent genes, with triangles indicating strand). (B) Plots of cM/Mb for the <i>3a</i> CO hotspot shown for wild type and <i>met1–3^−/−.</i> Vertical black lines indicate the position of the inner PCR primers used to amplify <i>3a</i>. Epigenomic annotation of the <i>3a</i> region with plots displaying low nucleosome density, histone H3K4m (black), H3K4m2 (red) H3K4m3 (green) and DNA methylation densities. (C) Table summarizing quantification of <i>3a</i> parental and CO molecule amplifications from pollen genomic DNA and calculation of cM, cM/Mb and associated standard deviations (S.D.). (D) RNA-seq RPKM (total counts mapping to gene/length of gene×total mapped reads, multiplied by 10⁶) for <i>3a</i> associated genes in wild type (Col) and <i>met1–3</i>. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s012" target="_blank">Table S10</a>.</p

Decreased pericentromeric crossovers in <i>met1–3</i>.

<p>(A) Physical map of chromosome 3 with overlaid genes/Mb (green), cM/Mb (red) and DNA methylation (blue) plots. The dotted, horizontal red line indicates the cM/Mb weighted mean. Outer vertical black lines indicate the position of FTL transgene insertions that define <i>CEN3</i>. Inner vertical black lines indicate the position of centromeric markers analyzed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen-1002844-g002" target="_blank">Figure 2</a>. The vertical magenta line indicates the centromere. (B) Chromosomes heterozygous for <i>trans-</i>linked <i>FTL332</i> (eYFP) and <i>FTL2536</i> (DsRed) transgenes, which flank the centromere (black circle) segregating through meiosis-I and –II in the absence (left) or presence (right) of a CO within <i>CEN3</i>. (C) Fluorescence micrographs of <i>qrt1–2</i> pollen showing patterns of inheritance associated with (tetratype) or without (parental ditype) a CO within <i>CEN3</i>. BF shows bight field illumination and R and G indicate red and green UV fluorescence. (D) <i>CEN3</i> genetic map lengths for naïve wild type (Col), <i>MET1</i>, <i>met1–3^+/−</i>, <i>met1–3^−/−</i> segregants and self-fertilized <i>met1–3^−/−</i> measured by <i>qrt1–2^−/−</i> tetrad counting. (E) Southern blotting and hybridization analysis of <i>CEN180</i> following digestion of genomic DNA using DNA methylation sensitive <i>HpaII</i>. DNA was prepared from <i>CEN3 qrt1–2^−/−</i> individuals whose measured genetic distance in cM is indicated above the blot in blue in addition to their <i>met1–3</i> genotype. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s005" target="_blank">Table S3</a>.</p

Elevated euchromatic recombination topology in wild type and <i>met1–3.</i>

<p>(A) Physical map of chromosome 3 with overlaid gene/Mb (green), DNA methylation (blue) and cM/Mb (red) plots. Black vertical lines indicate the positions of <i>napA</i> transgene insertions that define the <i>420</i> interval and the vertical magenta line indicates the centromere. (B) Fluorescence micrographs of seed expressing different combinations of <i>napA</i> transgenes. (C) Segregation diagram showing <i>cis-</i>heterozygous arrangement of <i>420 napA</i> lines. (D) <i>420</i> genetic distance measured in naïve wild type (Col) and <i>met1–3^+/−</i> segregants. (E) Black lines indicate recombination frequency (cM/Mb) maps of the <i>420</i> interval in wild type or <i>met1–3^−/−</i> with horizontal dotted lines indicating weighted means. Red lines represent merged map recombination frequency data for the <i>420</i> interval. The red star indicates the interval containing the <i>3a</i> CO hotspot. (F) Plots showing cumulative recombination value (cM/Mb) of ranked <i>420</i> mapping intervals in wild type (black) and <i>met1–3^−/−</i> (red). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s009" target="_blank">Tables S7</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s010" target="_blank">S8</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s011" target="_blank">S9</a>.</p

Total crossover numbers are similar between wild type and <i>met1–3.</i>

<p>(A) Physical maps of chromosomes (vertical black lines) with KASPar marker (horizontal black lines) and centromere (horizontal red lines) positions indicated. Histograms showing the frequency of total CO numbers identified in male backcross individuals from either Col/Ler F₁ (wild type) or <i>met1–3^−/−</i> Col/Ler F₁ (<i>met1–3</i>) parents. (B) Micrographs of DAPI-stained anther meiocytes showing the labeled stage of meiosis in Col and <i>met1–3^−/−</i>. (C) Micrographs of diplotene and diakineses stage male meiocytes stained with DAPI (white) and immunostained for MLH1 (green). (D) Micrographs showing co-localisation of dense-DAPI staining and <i>in situ</i> hybridization with the <i>CEN180</i> satellite repeat (red). (E) Micrographs of male meiocytes stained with DAPI (white) and immunostained with MLH1 (green) and the axis component ASY1 (red). (F) The upper table lists mean MLH1 foci numbers in wild type and <i>met1–3^−/−</i> at diplotene or diakinesis with standard deviation (+/−). The lower table lists the relative proportions (%) of MLH1 foci localizing to chromosome arm regions (arms) vs densely-DAPI staining regions (DAPI-dense). All scale bars represent 10 µM. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s006" target="_blank">Table S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s007" target="_blank">S5</a>.</p

Epigenomic organisation and CO frequency in the <i>A. thaliana</i> genome.

<p>(A) Physical maps of <i>A. thaliana</i> chromosomes showing genes/Mb (olive green), repeats/Mb (black), cM/Mb (red), H3K3me3/Mb (light green), LND/Mb (dark green) and DNA methylation density (blue). Dotted horizontal lines indicate the means weighted by intermarker distance. Vertical magenta lines indicate the centromeres. Grey shaded areas indicate the pericentromeres. (B) Pairwise correlations between cM/Mb, genes/Mb, H3K4me3/Mb, LND/Mb and DNA methylation in either chromosome arms or pericentromeres. Pearson's correlation coefficients (r) and associated p-values (p) are shown and regression lines are plotted in red. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s003" target="_blank">Tables S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002844#pgen.1002844.s004" target="_blank">S2</a>.</p

Meiotic recombination hotspots and coldspots within <i>MAJOR RESISTANCE CLUSTER1</i> and <i>MAJOR RESISTANCE CLUSTER5</i>.

<p><b>(A)</b> Crossing scheme used to double-select <i>MRC</i> crossovers. <i>KAN</i> (SGT) Ler chromosomes are shown in red and <i>BAR</i> (SAIL) Col chromosomes are shown in green. Triangles represent insertions and are labeled with the resistance they confer (<i>KAN</i> = kanamycin, <i>BAR</i> = glufosinate). <b>(B)</b> Diagram illustrating <i>MRC</i> genotype expectations in double-resistant backcross progeny, depending on crossover location. A representative ethidium-stained genotyping gel for a Col/Ler single nucleotide polymorphism (SNP) dCAPs marker is shown beneath [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.ref070" target="_blank">70</a>]. Colored stars above the genotyped samples match the chromosome diagrams above. <b>(C-D)</b> Phylogenetic trees generated using NBS domain amino acid sequences are plotted, with branch tips connected to the corresponding gene physical location. <b>(E-F)</b> Physical maps of chromosomes 1 and 5 with red dots indicating NBS-LRR gene positions (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.s007" target="_blank">S1 Table</a>). The selected <i>MRC</i> regions are indicated by black vertical lines and <i>KAN</i> and <i>BAR</i> symbols. <b>(G-H)</b> Recombination rate (cM/Mb) throughout the genotyped <i>MRC1</i> and <i>MRC5</i> regions (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.s008" target="_blank">S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.s009" target="_blank">S3</a> Tables). Red x-axis ticks indicate NBS-LRR gene positions (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.s007" target="_blank">S1 Table</a>). The dotted horizontal line indicates the male Col/Ler chromosome average recombination rate [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.ref071" target="_blank">71</a>]. The <i>RPP8</i> and <i>RPS6</i> coldspot locations are indicated beneath the <i>MRC5</i> map. <b>(I)</b> Crossover frequency within the <i>HRG1</i> hotspot interval (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.s010" target="_blank">S4 Table</a>). Arrows represent genes, with the <i>R</i> gene highlighted in red. <b>(J)</b> As for (I) but for <i>HRG2/HRG3</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.s011" target="_blank">S5 Table</a>). <b>(K-L)</b> Historical recombination rates across <i>HRG1</i> and <i>HRG2/HRG3</i>. Crossover frequency (cM/Mb) estimates (LDhat) from 80 Eurasian and 180 Swedish accessions are shown in blue and red respectively [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.ref072" target="_blank">72</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.ref074" target="_blank">74</a>]. X-axis ticks indicate SNP positions used for analysis. Horizontal dotted lines indicate chromosome mean historical recombination values. Arrows represent genes, with the NBS-LRR genes highlighted in red.</p

Intragenic crossover hotspots within the <i>RAC1</i> resistance gene.

<p><b>(A)</b> Diagram illustrating pollen-typing methods used to fine-map plant crossover hotspots. Pollen is collected from Col/Ler F₁ plants and genomic DNA is used for two rounds of nested allele-specific PCR amplifications with either crossover (ASO-Ler+ASO-Col) or parental (ASO-Col+ASO-Col) allele-specific oligonucleotide (ASO) primer combinations. An ethidium-bromide stained agarose gel is shown with crossover-specific amplifications from Col/Ler F₁ pollen, which are not observed when using Col/Ler F₁ leaf (somatic) DNA as a template. Crossover positions are identified using either Sanger sequencing from single crossover molecule amplifications, or paired-end sequencing of crossover molecule libraries. <b>(B)</b> Historical crossover frequency over <i>RAC1</i> estimated by LDhat analysis of polymorphisms from 80 Eurasian (blue) and 180 Swedish (red) accessions [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.ref072" target="_blank">72</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.ref073" target="_blank">73</a>]. X-axis ticks indicate SNP positions used for analysis. Horizontal dotted lines indicate chromosome mean values. Black arrows represent genes and green horizontal lines indicate transposable elements. Black vertical lines indicate <i>RAC1</i> TSS and TTS positions. <b>(C)</b> Crossover frequency within the <i>RAC1</i> amplicon measured by crossover and parental molecule titration, followed by single crossover molecule Sanger sequencing. The positions of <i>RAC1</i> and the adjacent gene (At1g31550) are indicated by horizontal black arrows above the plot. Exons are indicated by horizontal black lines, with sequences encoding RAC1 TIR (green), NB-ARC (red) and LRR (blue) domains highlighted as coloured lines. For the NB-ARC domain, the position of the Walker A, Walker B, ARC1 and ARC2 motifs are shown. Red x-axis ticks show Col/Ler SNP positions. Blue vertical lines indicate the positions of gene TSS and TTS from TAIR10 representative gene models.</p

<i>RAC1</i> crossovers measured by high-throughput sequencing, nucleosome occupancy and CTT-repeat motifs.

<p><b>(A)</b> Normalized crossover read pair values are plotted relative to the TAIR10 reference sequence, with the position of Col/Ler polymorphisms indicated by red x-axis ticks. Labelling is as for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.g003" target="_blank">Fig 3C</a>. <b>(B)</b> The sequence of the <i>RAC1</i> CTT-repeat motif is printed and connected to its physical location in (A) with dashed lines. Note the sequence is the reverse complement relative to the orientation of plotting in (A). The presence of CTT-motifs in <i>RAC1</i>-related paralogs in the Col-0 accession are shown in a multiple sequence alignment, where gene start codons (ATG) are in bold type. The asterisks beneath the alignment indicate identical positions. <b>(C)</b> Normalized nucleosome occupancy values (MNase-seq) plotted as for (A).</p

NBS-LRR cluster recombination rate is modified by natural genetic variation.

<p><b>(A)</b> Physical map of chromosome 5 with NBS-LRR gene positions indicated by red dots. The positions of FTL T-DNAs that define <i>I5a</i> are indicated by vertical blue lines, with triangles indicating the colour (red or yellow) of fluorescent protein encoded. Beneath is a diagram illustrating the generation of FTL hemizygous F₁ plants and scoring of crossover recombination in pollen. Crossover within <i>I5a</i> leads to pollen with red or yellow fluorescence alone, whereas the parental classes are either without fluorescence or show both red and yellow. <b>(B)</b> Fluorescence micrograph of pollen from <i>I5a/++</i> hemizygotes, showing the presence of four fluorescent classes. <b>(C)</b> A representative flow cytometry plot of pollen from <i>I5a/++</i> hemizygotes. Points represent individual pollen grains measured for yellow (FL1 YFP) and red (FL2 dsRed2) fluorescence. <b>(D)</b> <i>I5a</i> genetic distance (cM) measured from individual F₁ plants (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006179#pgen.1006179.s024" target="_blank">S18 Table</a>). Mean values are shown in red. The accession crossed to is indicated on the x-axis. The Col/Col homozygous sample is labelled and indicated with an arrow.</p