Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana
- Swiss Federal Institute of Technology, Switzerland
- Swedish University of Agricultural Sciences, Sweden
Figures
Figure 1 with 6 supplements
Impact of PEG function on diploid seed development.
(A) Percentage of normal, unfertilized, and aborted seeds from self-fertilized wild-type and homozygous peg mutant plants. A minimum of 300 seeds was analyzed for each genotype. (B) Size measurements of mature seeds derived from crosses of maternal Col plants pollinated with heterozygous peg pollen (black line) were normalized, plotted on a histogram and distribution was compared with wild type control crosses (grey line). A minimum of 400 seeds was analyzed for each cross.
Figure 1—figure supplement 1
Imprinting of PEGs in Col/Ler.
Allele-specific expression analysis of indicated PEGs in siliques derived from crosses of Col x Ler and Ler x Col. Siliques were harvested at 4 DAP and allele-specific expression was tested by PCR and subsequent DNA sequencing.
Figure 1—figure supplement 2
T-DNA insertions in peg mutants.
Schematic presentation of T-DNA insertions (red triangle) in peg mutants that were analyzed in this study and not previously published. Dark grey bars indicate exons, light grey bars indicate UTRs, black lines indicate introns and promoter regions are indicated by a dotted line.
Figure 1—figure supplement 3
PEG expression in peg mutants.
Expression of PEG genes in peg mutants and wild type in whole siliques at 4 DAP, measured by qRT-PCR. Expression of PEG9 and SUVH7 was analyzed in peg9-1 and suvh7-1. Error bars indicate SEM.
Figure 1—figure supplement 4
Transmission analysis of peg mutant alleles through the male germ line.
Frequency of peg mutants among the progeny of crosses derived from wild-type plants pollinated with heterozygous peg pollen. 24 seedlings were genotyped for each cross.
Figure 1—figure supplement 5
Effect of stress on seed set in Col x peg/+ crosses.
Number of seeds per silique in crosses of unstressed and stressed Col plants pollinated with Col pollen or heterozygous peg pollen. A minimum of 8 siliques was analyzed for each cross. Error bars indicate SEM.
Figure 1—figure supplement 6
Seed size analysis under stress conditions.
Size measurements of mature seeds derived from crosses of stressed Col plants pollinated with heterozygous peg pollen (black line) were normalized, plotted on a histogram and distribution was compared with wild-type control crosses (grey line). A minimum of 400 seeds was analyzed for each cross.
Figure 2 with 2 supplements
PEGs establish interploidy hybridization barriers.
(A) Percentages of non-collapsed and germinated seeds of wild-type plants pollinated with osd1 and peg osd1 pollen. Numbers on top of bars correspond to number of analyzed seeds. (B) Triploid seedlings 14 days after germination. Scale bar, 1 cm. (C) Percentages of non-collapsed and germinated seeds of peg mutant plants pollinated with osd1 and peg osd1 pollen. Numbers on top of bars correspond to number of analyzed seeds. (D) PEG2 and SUVH7 remain imprinted in diploid and triploid seeds. Siliques of crosses of Ler plants pollinated with Col or osd1 pollen were harvested at 4 DAP and imprinted expression was tested by PCR and subsequent DNA sequencing. Siliques of Col and Ler plants pollinated with Col and Ler pollen were used as controls. (E) Sections of seeds derived from crosses of Col plants pollinated with Col, osd1, peg2 osd1, peg9-1 osd1 and suvh7-1 osd1 pollen at 8 DAP. Scale bar, 0.1 mm.
Figure 2—figure supplement 1
PEG expression in triploid seeds.
Log2 fold changes of PEG expression in triploid seeds compared to PEG expression in diploid seeds at 6 DAP, measured by whole genome deep sequencing transcriptome analysis.
Figure 2—figure supplement 2
Analysis of independent mutant alleles for suvh7 and peg9 and genomic complementation of peg2.
Percentages of non-collapsed seeds of wild-type plants pollinated with osd1, suvh7-2 osd1, peg9-2 osd1 or peg2 osd1;PEG2::PEG2 pollen. Numbers on top of bars correspond to number of analyzed seeds.
Figure 3 with 1 supplement
Transcriptome analysis of AGL genes and PEGs in triploid adm-2, suvh7-1 and peg2 seeds.
(A) Log2 fold change expression of AGLs in triploid adm-2, suvh7-1 and peg2 mutants compared to triploid wild-type seeds. Only AGLs were tested that were up-regulated in triploid wild-type seeds. (B) Log2 fold change expression of PEGs in triploid adm-2, suvh7-1 and peg2 mutants compared to triploid wild-type seeds. Only PEGs were tested that were up-regulated in triploid wild-type seeds. (C) Left panel: Venn diagram showing overlap of genes being up-regulated in seeds derived from wild type x osd1 crosses (signal log ratio [SLR] > 1, p < 0.05) and down-regulated in wild type x adm-2 osd1 (SLR < −1, p < 0.05), wild type x suvh7-1 osd1 (SLR < −1, p < 0.05), and wild type x peg2 osd1 (SLR < −1, p < 0.05) Hypergeometric testing was used to test for significance of overlap; p = 5.853e-09. Right panel: Venn diagram showing overlap of genes being down-regulated in seeds derived from wild type x osd1 crosses (SLR < −1, p < 0.05) and up-regulated in wild type x adm-2 osd1 (SLR >1, p < 0.05), wild type x suvh7-1 osd1 (SLR >1, p < 0.05), and wild type x peg2 osd1 (SLR >1, p < 0.05). Hypergeometric testing was used to test for significance of overlap; p = 4.622 e−16.
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Figure 3—source data 1
List of genes deregulated in seeds derived from interploidy crosses.
- https://doi.org/10.7554/eLife.10074.014
Figure 3—figure supplement 1
Gene ontology analysis.
GO analysis of genes commonly up- and down-regulated in triploid adm-2, suvh7-1 and peg2 mutants compared to triploid wild-type seeds. Only GO categories with a p-value <0.05 are shown.
Figure 4 with 1 supplement
Analysis of pectin biosynthesis and degradation genes in diploid and triploid seeds.
(A) Venn diagram showing overlap of pectin degradation genes and genes being upregulated in seeds derived from wild type x osd1 crosses (signal log ratio [SLR] > 1, p < 0.05) and genes being downregulated in either wild type x adm-2 osd1 (SLR < −1, p < 0.05), wild type x suvh7-1 osd1 (SLR < −1, p < 0.05), or wild type x peg2 osd1 (SLR < −1, p < 0.05). Hypergeometric testing was used to test for significance of overlap, p = 4.048 e−10. (B) Venn diagram showing overlap of pectin biosynthesis genes and genes being down-regulated in seeds derived from wild type x osd1 crosses (SLR <1, p < 0.05). (C) Cluster analysis of pectin degradation genes that are upregulated in triploid wild-type seeds, based on their expression in endosperm during different stages of diploid seed development (Belmonte et al., 2013). Each row represents a gene, and each column represents a tissue type. Tissue types are: micropylar (MPE), peripheral (PE), chalazal (CZE) and cellularized endosperm (CES) derived from seeds containing embryos of the preglobular stage to the mature stage. Red or green indicate tissues in which a particular gene is highly expressed or repressed, respectively. (D) Log2 fold change expression of pectin degradation genes in triploid wild-type seeds (compared to diploid wild-type seeds) and triploid adm-2, suvh7-1 and peg2 mutants (compared to triploid wild-type seeds). Genes marked by an asterisk are not included in the seed transcriptome dataset (Belmonte et al., 2013) and are therefore not included in panel (C). (E) Ruthenium red staining of sections of seeds derived from crosses of Col plants pollinated with Col and osd1 at 4 DAP and 6 DAP. Red color marks the presence of demethylesterified pectin, blue color is derived from the counterstain with toluidine blue. A minimum of 100 seeds was analyzed for each cross. Scale bar, 0.1 mm.
Figure 4—figure supplement 1
Additional ruthenium red staining of seeds.
Ruthenium red staining of sections of additional seeds derived from crosses of Col plants pollinated with Col and osd1 at 4 DAP and 6 DAP. A minimum of 100 seeds was analyzed for each cross. Scale bar, 0.1 mm.
Additional files
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Supplementary file 1
Primers used in this study.
- https://doi.org/10.7554/eLife.10074.018
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Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana
eLife 4:e10074.
https://doi.org/10.7554/eLife.10074
