Figure 1.
Epigenomic organisation and CO frequency in the A. thaliana genome.
(A) Physical maps of A. thaliana 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 Tables S1 and S2.
Figure 2.
Elevated centromeric crossovers in met1–3.
(A) Schematic diagram illustrating generation of wild type and met1–3−/− 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 met1–3−/− male backcross populations.
Figure 3.
Decreased pericentromeric crossovers in met1–3.
(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 CEN3. Inner vertical black lines indicate the position of centromeric markers analyzed in Figure 2. The vertical magenta line indicates the centromere. (B) Chromosomes heterozygous for trans-linked FTL332 (eYFP) and FTL2536 (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 CEN3. (C) Fluorescence micrographs of qrt1–2 pollen showing patterns of inheritance associated with (tetratype) or without (parental ditype) a CO within CEN3. BF shows bight field illumination and R and G indicate red and green UV fluorescence. (D) CEN3 genetic map lengths for naïve wild type (Col), MET1, met1–3+/−, met1–3−/− segregants and self-fertilized met1–3−/− measured by qrt1–2−/− tetrad counting. (E) Southern blotting and hybridization analysis of CEN180 following digestion of genomic DNA using DNA methylation sensitive HpaII. DNA was prepared from CEN3 qrt1–2−/− individuals whose measured genetic distance in cM is indicated above the blot in blue in addition to their met1–3 genotype. See also Table S3.
Figure 4.
Total crossover numbers are similar between wild type and met1–3.
(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 met1–3−/− Col/Ler F1 (met1–3) parents. (B) Micrographs of DAPI-stained anther meiocytes showing the labeled stage of meiosis in Col and met1–3−/−. (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 in situ hybridization with the CEN180 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 met1–3−/− 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 Table S4 and S5.
Figure 5.
Elevated euchromatic crossovers in met1–3.
(A) Physical map of chromosome 1 with overlaid gene/Mb (green), DNA methylation (blue) and cM/Mb (red) plots. Black vertical lines indicate the I1b transgene insertions and the magenta vertical line indicates the centromere. (B) Schematic diagram showing homologous chromosomes (black lines) heterozygous for cis-linked FTL567 (eYFP) and FTL1262 (RFP) transgenes segregating through meiosis in the absence or presence of a CO. (C) Fluorescence micrographs showing qrt1–2−/− or QRT1 pollen from I1b cis-heterozygotes. (D) I1b genetic map length for naïve wild type (Col), MET1, met1–3+/− and met1–3−/− segregants and self-fertilized met1–3−/− (met1-self) measured by qrt1–2−/− tetrad counting. (E) I1b genetic map length for naïve wild type (Col) and MET1, met1–3+/− and met1–3−/− segregants measured by flow cytometry of individual pollen grains. A representative flow cytometry histogram from an I1b cis-heterozygote together with gate quadrant R6 counts, adjusted total pollen counts and cM. See also Table S6.
Figure 6.
Elevated euchromatic recombination topology in wild type and met1–3.
(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 napA transgene insertions that define the 420 interval and the vertical magenta line indicates the centromere. (B) Fluorescence micrographs of seed expressing different combinations of napA transgenes. (C) Segregation diagram showing cis-heterozygous arrangement of 420 napA lines. (D) 420 genetic distance measured in naïve wild type (Col) and met1–3+/− segregants. (E) Black lines indicate recombination frequency (cM/Mb) maps of the 420 interval in wild type or met1–3−/− with horizontal dotted lines indicating weighted means. Red lines represent merged map recombination frequency data for the 420 interval. The red star indicates the interval containing the 3a CO hotspot. (F) Plots showing cumulative recombination value (cM/Mb) of ranked 420 mapping intervals in wild type (black) and met1–3−/− (red). See also Tables S7, S8 and S9.
Figure 7.
Elevated crossover hotspot 3a1 activity in met1–3.
(A) CO frequency distributions (cM/Mb, blue) within 420 map interval 8 measured by dCAPs PCR marker segregation (white bars represent genes, with triangles indicating strand). (B) Plots of cM/Mb for the 3a CO hotspot shown for wild type and met1–3−/−. Vertical black lines indicate the position of the inner PCR primers used to amplify 3a. Epigenomic annotation of the 3a region with plots displaying low nucleosome density, histone H3K4m (black), H3K4m2 (red) H3K4m3 (green) and DNA methylation densities. (C) Table summarizing quantification of 3a 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 3a associated genes in wild type (Col) and met1–3. See also Table S10.