<i>mea</i> seed abortion can be paternally suppressed.

<p>(A) Mature F1 seeds derived from crosses between Ler or homozygous <i>mea-1</i>/<i>mea-1</i> and pollen from Ler, C24, and Cvi-0. The scale bar represents 500μm. (B) Percentage of viable plump seeds derived from crosses between heterozygous <i>mea-2</i>/<i>MEA</i> introgressed into C24 and Cvi-0 backgrounds and pollen from Ler, Cvi-0, and C24. Three independent backcross lineages were generated for each accession, and one maternal individual per lineage was assayed. (C) Percentage of viable plump seeds derived from crosses between heterozygous <i>mea-2</i>/<i>MEA</i>, <i>fis2</i>/<i>FIS2</i> and <i>fie</i>/<i>FIE</i> and pollen from Ler, Cvi-0, and C24. The numbers in the bars indicate the number of seeds sampled; the error bars denote 95% binomial confidence intervals.</p

Bulk-Seq analysis to map parent-of-origin effects.

<p>(A) Schematic representation of the procedure for Bulk-Seq. (B) Relative proportion of Cvi-0 reads in the <i>mea</i> (red) and WT (blue) pools. The dashed line represents the expected 25% average proportion of Cvi-0 reads in the WT pool in the absence of segregation distortion. (C) Enrichment of Cvi-0 reads in the <i>mea</i> pool relative to the WT pool. The dotted lines indicate the position of the main peaks; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005806#pgen.1005806.t002" target="_blank">Table 2</a>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005806#pgen.1005806.s004" target="_blank">S4 Fig</a> for plots of the three independent replicates.</p

QTL analysis demonstrates that six loci contribute independently to <i>mea</i> seed rescue.

<p>(A) Percentage of viable plump seeds derived from crosses between homozygous <i>mea-1</i>/<i>mea-1</i> and pollen from a Ler/Cvi RIL population. Error bars indicate 95% binomial confidence intervals. The inset is a histogram representation of the same data. (B) Standard QTL interval mapping. The dotted line represents the 1% significance LOD threshold. (C) Bayesian estimates for the localization of each of the six QTLs from a multiple QTL model. The values above the peaks represent the best estimate for the localization of the QTL. (D) Effect of individual QTLs. In each subpanel the percentage of plump seeds of all the 80 RILs as in panel A are shown, but sorted according to the genotype (Cvi-0 or Ler) at the respective QTL. The black horizontal line and error bars represent the mean and standard errors. (E) Cumulative effects of Cvi-0 QTLs. The percentage of plump seeds of all the 80 RILs is shown, but sorted according to the total number of Cvi-0 alleles in the six QTLs. The red horizontal lines represent the mean and standard errors. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005806#pgen.1005806.t001" target="_blank">Table 1</a>.</p

A Cvi/Ler NIL population confirms the polygenic architecture of <i>mea</i> rescue.

<p>(A) Percentage of viable plump seeds in crosses between homozygous <i>mea-1</i>/<i>mea-1</i> and pollen from 33 Cvi/Ler NILs. The numbers under the bars represent the number of seeds sampled. Error bars denote 95% binomial confidence intervals. The asterisks indicate that the proportions are significantly different from the crosses with Ler pollen (Bonferroni-corrected one-sided binomial tests, * p < 0.01, ** p < 0.001). (B) Genetic map of the Cvi-0 introgressions in each of the 33 NILs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005806#pgen.1005806.ref040" target="_blank">40</a>]. The solid boxes correspond to the Cvi-0 segments. The thin grey lines indicate the position of Ler segments. The colour scale represents the percentage of viable plump seeds as in panel A. The blue lines indicate the position of the six QTLs identified using the RIL population.</p

Cryptic genetic variation for parent-of-origin effects during <i>Arabidopsis</i> seed development.

<p>(A-C) Percentage of viable plump seeds in the F2 progeny of <i>mea-2</i> with 164 <i>Arabidopsis</i> accessions. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005806#pgen.1005806.s006" target="_blank">S2 Table</a>. Error bars indicate 95% binomial confidence intervals. (D) Manhattan plots indicating the significance of associations between variation at SNPs across the genome and <i>mea</i> rescue. The black triangles indicate the position of the peaks from the Bulk-Seq analysis. The dashed line indicates the 5% significance threshold with Bonferroni correction.</p

<i>TUN(RNAi)</i> lines show <i>fer</i>-like vegetative dwarf phenotype.

<p>(A–D) Plant size of 30-d-old seedlings of wild-type (A), <i>fer-2/fer-2</i> (B), <i>TUN(RNAi)</i> (C), and <i>EVN(RNAi)</i> lines (D). Asterisks indicate <i>TUN(RNAi)</i> seedlings, that accumulate athocyanins and degenerate without further growth. (E–H) Plant size of adult wild-type (E), <i>fer-2/fer-2</i> (F), <i>TUN(RNAi)</i> (G), and <i>EVN(RNAi)</i> (H) plants. (F) Left plant is at the same developmental stage as wild-type, <i>TUN(RNAi)</i>, and <i>EVN(RNAi)</i> individuals. Scale bars: 1 cm. All lines are in the Col-0 background.</p

Overview of phenotypes in <i>tun</i> and <i>evn</i> mutant plants.

<p>^a<i>pTUN::TUN-GFP</i> is homozygous</p><p>^b<i>anx1/2</i>-like pollen burst phenotype was assessed</p><p>^c<i>evn</i>-dependent pollen degeneration was assessed</p><p>NA, not applicable</p><p>Overview of phenotypes in <i>tun</i> and <i>evn</i> mutant plants.</p

<i>tun</i> and <i>evn</i> ovules display pollen tube overgrowth and increased callose accumulation at the filiform apparatus.

<p>(A–C) Aniline Blue staining of callose in PT cell walls 2 d after pollination (DAP). (A) PT reception in a wild-type FG. Arrowhead indicates site of PT growth arrest. (B–C) PT overgrowth in <i>tun-1</i> (B) and <i>evn-1</i> mutant FGs (C). Asterisks indicate PT overgrowth. (D–F) β-glucuronidase (GUS) staining of synergid marker ET2634 2 d after emasculation (DAE) in wild-type (D), <i>tun-1</i> (E), and <i>evn-1</i> mutant FGs (F). Arrow indicates abnormal structure at the FA. (G–I) Chloral hydrate clearings of ovules 2 DAE in wild-type (G), <i>tun-1</i> (H), and <i>evn-1</i> mutants (I). Arrows indicate abnormal structure at the FA. (J–L) Aniline Blue staining of callose in 6 μm sections of wild-type (J), <i>tun-1</i> (K), and <i>evn-1</i> ovules 2 DAE (L). Boxes represent close-ups of indicated regions, whereby mutant close-ups in (K) and (L) were captured with reduced exposure time compared to the wild type (J). Scale bars in A–F and J–L = 20 μm; scale bars in G–I = 10 μm.</p

NTA, FER and LRE show proper localization in <i>tun</i> and <i>evn</i> mutant embryo sacs.

<p>(A–I) Confocal microscope analysis of fluorescently labeled proteins. (A–C) Vesicle-associated NTA-GFP localization in the cytoplasm of a wild-type (A), <i>tun-2</i> (B), and <i>evn-3</i> FG (C). (D–F) FER-GFP at the FA and in membranes of sporophytic tissue of a wild-type (D), <i>tun-2</i> (E), and <i>evn-3</i> FG (F). (G–I) Extracellular localization of LRE-Citrine in a wild-type (G), <i>tun-2</i> (H), and <i>evn-3</i> FG (I). Scale bars: 20 μm.</p

ANX1-YFP fluorescence is not detectable in <i>tun</i> mutant pollen grains.

<p>(A–D) Confocal microscope analysis of <i>ANX1-YFP</i> expression under a pollen-specific promoter. (A) <i>ANX1-YFP</i> expression in <i>TUN/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP</i> (wild-type segregants homozygous for the reporter gene). (B) <i>ANX1-YFP</i> expression in <i>TUN/TUN;qrt/qrt;ANX1-YFP/-</i> (wild-type segregants hemizygous for the reporter gene). (C) <i>ANX1-YFP</i> expression in <i>tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP</i> mutant tetrads. Arrowheads indicate missing fluorescence in <i>tun</i> pollen grains. (D) <i>ANX1-YFP</i> expression in <i>tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP</i> mutant tetrads after Kifunensine (Kif) treatment. (E) <i>ANX1-YFP</i> expression in <i>tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP</i> mutant tetrads after mock treatment for fluorescence intensity decrease comparison. (F–H) <i>ANX1-YFP</i> expression in <i>tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP</i> mutant tetrads after Eeyarestatin I (EerI) treatment. (F) 10 μm EerI recovers ANX1-YFP fluorescence in <i>tun</i> pollen grains. (G) Higher concentrations of EerI can lead to cytosolic inclusions (asterisk) or pollen grain burst (arrow). (H) ANX1-YFP fluorescence recovery in several <i>tun-2/TUN;qrt/qrt;ANX1-YFP/ANX1-YFP</i> mutant tetrads after EerI treatment. Residual fluorescence signal from the pollen coat is autofluorescence. Scale bars: 20 μm.</p