Volume 8, Issue 12, Pages 1766-1775 (December 2015) An Epigenetic Role for Disrupted Paternal Gene Expression in Postzygotic Seed Abortion in Arabidopsis Interspecific Hybrids  Ryan C. Kirkbride, Helen Hong Yu, Gyoungju Nah, Changqing Zhang, Xiaoli Shi, Z. Jeffrey Chen  Molecular Plant  Volume 8, Issue 12, Pages 1766-1775 (December 2015) DOI: 10.1016/j.molp.2015.09.009 Copyright © 2015 The Author Terms and Conditions

Figure 1 Interspecific Seed Abortion Phenotypes. Seeds in siliques (mature stage) of three F1 interspecific hybrids or neo-allotetraploids between A. arenosa (Aa) and A. thaliana Columbia-0 (Col), Landsberg erecta, (Ler), or C24 (C24) to illustrate abortive seed phenotype. Molecular Plant 2015 8, 1766-1775DOI: (10.1016/j.molp.2015.09.009) Copyright © 2015 The Author Terms and Conditions

Figure 2 Identification of Transcripts Showing Paternally and Maternally Biased Expression in Seeds. (A) Scatter plot showing the T-allele (A. thaliana, maternal parent) and A-allele (A. arenosa, paternal parent) by mapping reads from ColXAa to the SNP dataset. Red and green lines represent the paternal and maternal filtering thresholds, respectively. Red, green, and blue show the paternally expressed genes (PEGs), maternally biased reads, and maternally biased reads with seed-biased expression, respectively. Black dots lie between maternal and paternal expression thresholds. (B) Heatmap showing seed subregion mRNA abundance (Belmonte et al., 2013) for PEGs (top) primarily in the embryo and endosperm, and maternally expressed genes (MEGs) (bottom) primarily in the seed coat. Molecular Plant 2015 8, 1766-1775DOI: (10.1016/j.molp.2015.09.009) Copyright © 2015 The Author Terms and Conditions

Figure 3 Analysis of MEGs and PEGs Using Maternal-to-Paternal Ratio (4:1 for MEG and 1:2 for PEG) of Expression Values (q < 0.05). (A) Pairwise comparison showed coordinated maternal-biased (II) and paternal-biased (IV) expression of genes in three F1 crosses or maternal-biased in one F1 but paternal-biased in another cross (I, III). T and A refer to the signal from A. thaliana alleles and the A. arenosa allele, respectively. (B) MEGs and PEGs in three F1 interspecific crosses were classified into the shared group (SG, in all three crosses), partially shared group (PSG, in two crosses), and unshared group (USG, only in one cross). Molecular Plant 2015 8, 1766-1775DOI: (10.1016/j.molp.2015.09.009) Copyright © 2015 The Author Terms and Conditions

Figure 4 Altered Allelic Expression in Three F1 Interspecific Crosses. MEGs and PEGs in three F1 crosses were compared with those in the reciprocal intraspecific crosses ColXLer and LerXCol. Those MEGs in the intraspecific crosses that were converted to PEGs are shown in blue, MEGs remaining as MEG in red, and PEGs remaining as PEGs in orange. No intraspecific PEGs were observed converting to MEGs in the interspecific crosses. Gray boxes indicate no match between intraspecific and interspecific MEGs or PEGs. Molecular Plant 2015 8, 1766-1775DOI: (10.1016/j.molp.2015.09.009) Copyright © 2015 The Author Terms and Conditions

Figure 5 Comparison of PEG and MEG RNA Levels in Three F1 Interspecific Hybrids with Those in Interploidy Cross (Diploid × Hexaploid, 2X6 or Hexaploid × Diploid, 6X2) Relative to the Diploid Cross (2X2). (A) Expression correlation coefficients of PEGs with those in the 2X6 (left) or 6X2 (right) cross. (B) Expression correlation coefficients of MEGs with those in the 2X6 (left) or 6X2 (right) cross. (C) Distribution of PEGs in SG (blue), PSG (green), and USG (red) relative to those in the 2X6 (left) or 6X2 (right) cross. Correlation coefficient (ρ) values are shown (Spearman test, P < 0.001). Molecular Plant 2015 8, 1766-1775DOI: (10.1016/j.molp.2015.09.009) Copyright © 2015 The Author Terms and Conditions

Figure 6 Expression Correlations of PEGs from ColXAa Cross in the met1XWT or in the Interploidy Cross (Diploid × Hexaploid, 2X6 or Hexaploid × Diploid, 6X2). Expression ratios to the wild-type (WT) or control (2X2) were used for correlation tests (Spearman test, P < 0.05). Correlation coefficient (ρ) values are shown (Kankel et al., 2003). Molecular Plant 2015 8, 1766-1775DOI: (10.1016/j.molp.2015.09.009) Copyright © 2015 The Author Terms and Conditions

Figure 7 Comparative Analysis of MEGs and PEGs with Upregulated Genes in meaXWT. Proportions of MEGs (left) and PEGs (right) in three F1 interspecific hybrids matched upregulated genes in meaXWT. mea, medea; WT, wild-type. Molecular Plant 2015 8, 1766-1775DOI: (10.1016/j.molp.2015.09.009) Copyright © 2015 The Author Terms and Conditions

Figure 8 Validation of Allelic Expression of Candidate MEGs and PEGs in Arabidopsis Interspecific Hybrids or Neo-Allotetraploids. Sequencing products of qRT–PCR with an equal mix of A. thaliana (At) and A. arenosa (Aa) cDNAs were used as controls, in which both SNP-containing alleles were amplified at similar levels, except for At3g47350 (At/Aa = 2.5). SNPs of A/T in At2g46960, and C/T in At3g18780 were used for the validation of A. thaliana-allelic expression of MEGs. SNPs of T/G in At1g05260, A/C in At1g70270, and T/A in At3g47350 were used for the validation of A. arenosa-allelic expression of PEGs. Expression ratios of At/Aa alleles in the F1 interspecific hybrids are shown below the sequencing chromatography (upper panel: qRT–PCR sequencing; lower panel: RNA-seq data). One of three qRT–PCR sequencing replicates is shown. Molecular Plant 2015 8, 1766-1775DOI: (10.1016/j.molp.2015.09.009) Copyright © 2015 The Author Terms and Conditions