Article Figures & Data
Figures
- Figure 1.
Flow-Chart for Analyses of Endosperm RNA-Seq Data.
The number of trimmed reads is shown for each of the sequenced samples. These reads were aligned to the AGPv2_FGS5b gene models. The data from the inbreds were used to confirm SNPs and to evaluate differential expression in B73 relative to Mo17. The graph shows the RPKM values for the two genotypes, and the black data points indicate genes with significantly (q < 0.05) different expression in the two genotypes. The RNA-seq data from the hybrids were used for allele-specific expression analyses to identify imprinted genes.
- Figure 2.
Identification of Imprinted Genes in Maize Endosperm.
(A) The relative proportion of transcripts derived from the B73 allele was determined for both reciprocal hybrids (limited to only genes with at least 10 reads assigned to one allele in both hybrids). Genes indicated with a color are those that exhibit a significant (χ2 < 0.01) bias from the expected 2:1 ratio in both hybrids. Red coloration indicates genes that are biased toward higher expression of the B73 allele, while blue indicates genes that are biased toward higher expression of the Mo17 allele. The yellow and black spots represent genes with maternal or paternal bias, respectively. The triangles represent the 46 genes that have at least 90% of the transcripts derived from the paternal allele, and the star symbols represent the 72 genes with at least 90% of the transcripts derived from the maternal allele.
(B) The proportion of maternal (blue) and paternal (red) transcripts in both hybrids are shown for three genes previously identified as imprinted genes. The total number of allele-specific reads for each gene in each sample is indicated above the bar.
(C) Chromosomal distribution of imprinted genes. The chromosomal positions are shown for each of the 100 imprinted genes (red spots indicate maternal expression and blue spots indicate paternal expression). The black arrows indicate two loci where adjacent genes are both expressed from the maternal allele.
- Figure 3.
Persistent Imprinting throughout Endosperm Development.
For several genes, we developed CAPS assays to assess allele-specific expression at multiple stages of endosperm development including 10, 12, 18, and 22 DAP. The outer lanes provide the expected patterns for the B73 and Mo17 alleles (data are from 14-DAP B73 and Mo17 tissue). The inner lanes show the digests of products derived from reciprocal hybrids. The BxM samples represent hybrids in which B73 was the maternal parent and the MxB samples had Mo17 as the maternal parent.
- Figure 4.
Developmental Expression Pattern of Imprinted Genes.
Expression atlas data (Sekhon et al., 2011) were available for 71 of the 100 imprinted genes. The expression atlas data were normalized across all tissues. These patterns of expression were then assessed by hierarchical clustering using Ward’s method. The resulting heat map illustrates expression of each gene across the different tissues, with black indicating low expression and yellow indicating higher levels of expression. The three columns to the right of the dendrogram illustrate features of each imprinted gene. The first column indicates whether the gene is a MEG (pink) or a PEG (blue). The second column indicates which genes were classified as having endosperm-preferred expression (black). The final column indicates the genes that had a DMR in purple.
- Figure 5.
Identification of DMRs at Imprinted Genes.
(A) The panels display array-based methylation profiling data for three genes. The y axis represents the relative differences in methylation levels in endosperm and leaf tissue (negative values indicate lower methylation in endosperm). The x axis indicates the base pair distance of the probe from the transcription start site (TSS). Each data point represents a single probe, and the values are determined from the average of three replicates of B73 and Mo17 tissues. The red lines indicate regions that were identified as hypomethylated in endosperm.
(B) For each of these three genes, we identified SNPs within the hypomethylated region (listed under traces in [B73/Mo17] format). The SNP-containing regions were amplified and sequence in both reciprocal hybrids. The sequencing plots from the undigested DNA from the reciprocal hybrids (first and last traces) reveal presence of both alleles. However, following digestion with the methylation-dependent restriction enzyme FspEI (middle traces), only the maternal allele is detected confirming specific hypomethylation of the maternal allele.
Tables
GO_ID Description Genes P Valuea PEGsb 3677 DNA binding 11 0.01 9908 Flower development 4 0.01 3676 Nucleic acid binding 14 0.02 16301 Kinase activity 7 0.02 5488 Binding 24 0.02 MEGsb 7275 Multicellular organismal development 11 0.00 32501 Multicellular organismal process 11 0.00 50896 Response to stimulus 13 0.02 50789 Regulation of biological process 11 0.04 Maize Gene ID Chromosome Maternal Read No. Paternal Read No. Maize Pattern Rice Gene ID Rice Description Luo et al., 2011 Arabidopsis Gene ID AC191534.3_FG003 chr7 23 2397 PEG Os04g22240 C3HC4-type zinc-finger PEG AT1G57820 GRMZM2G000404 chr7 0 160 PEG Os09g28940 Ubiquitin-specific protease PEG GRMZM2G028366 chr5 15 13763 PEG Os02g12840 DEAD-box ATP-dependent RNA helicase PEG AT1G54270 GRMZM2G073700 chr6 15858 592 MEG Os06g11730 RNA binding (RRM/RBD/RNP motifs) MEG AT1G78260 GRMZM2G110306 chr1 0 33 PEG Os08g27240 ARID/BRIGHT DNA binding domain PEG AT3G43240 GRMZM2G118205 chr4 9320 17 MEG Os08g04290 OsFIE MEG AT3G20740 GRMZM2G170099 chr3 2891 20 MEG Os01g18810 Hypothetical protein MEG GRMZM2G365731 chr1 2 396 PEG Os10g30944 ARID/BRIGHT DNA binding domain PEG AT4G11400 GRMZM2G379898 chr1 72 0 MEG Os03g01320 Protease inhibitor/seed storage/LTP MEG AT1G62500 GRMZM2G447406 chr8 100 3155 PEG Os01g70060 Hypothetical protein PEG GRMZM5G845175 chr10 24 708 PEG Os04g32880 CBS domain-membrane protein PEG AT1G09020 Maize Gene ID Chromosome Maternal Read No. Paternal Read No. Maize Pattern Arabidopsis Gene ID Arabidopsis Description Gehring et al. (2011)a Hsieh et al. (2011)a Wolff et al. (2011)a AC191534.3_FG003 chr7 23 2397 PEG-DMR AT1G57820 VARIANT IN METHYLATION1 (VIM1) PEG-DMRb MEGfil GRMZM2G064905 chr8 3 62 PEG AT5G53150 DNAJ heat shock domain PEG MEGfil GRMZM2G127160 chr6 1354 84630 PEG AT1G70560 Trp aminotransferase of Arabidopsis1 (TAA1) PEG-DMR PEG GRMZM2G365731 chr1 2 396 PEG AT4G11400 ARID/BRIGHT DNA binding domain DMR PEG GRMZM2G014119 chr6 6869 10 MEG-DMR AT5G03240 Polyubiquitin3 (UBQ3) MEG MEGfil MEGfil GRMZM2G044440 chr8 2795 9 MEG-DMR AT5G03240 Polyubiquitin3 (UBQ3) MEG MEGfil MEGfil GRMZM2G103247 chr9 98 2 MEG-DMR AT2G28890 Poltergeist like4 (PLL4) MEG-DMR MEGfil GRMZM2G112925 chr1 492 49 MEG AT2G28890 Poltergeist like4 (PLL4) MEG-DMR MEGfil GRMZM2G370991 chr5 15027 22 MEG-DMR AT5G03280 ETHYLENE INSENSITIVE2 (EIN2) MEG-DMR MEG MEGfil GRMZM2G088020 chr8 106 0 MEG AT5G53870 Early nodulin-like protein1 (ENODL1) MEGfil MEG MEGfil ↵a The genes listed as MEG or PEG passed all filters in the cited reference. Genes listed as MEGfil were filtered due to potential seed coat expression or because they did not pass stricter statistical cutoffs for imprinting in these studies but may actually be imprinted.
↵b The presence of a DMR from Gehring et al. (2009) is indicated.
Additional Files
Author profile
Amanda J. Waters
Current Position: Graduate student in the Department of Plant Biological Sciences at the University of Minnesota
Education: B.S. (2010) in Biology at the University of Minnesota
Non-scientific Interests: Ceramics, biking, and swimming.
During my time at the University of Minnesota I realized that my interest lies in understanding the effects of epigenetic factors on gene expression. I found fascinating the idea that factors other than sequence differences can account for phenotypic variation. I was introduced to this research while working as an undergraduate in Nathan Springer�s lab, which does research to learn more about the prevalence and mechanisms of epigenetic variation in maize. I worked with a post-doc, Ruth Swanson-Wagner to profile the patterns of methylation between maize inbred lines and was also introduced to the epigenetic phenomena of imprinting. Following my graduation with a B.S. in biology, I continued to work as a technician in the Springer lab. My work included the project that identified 100 imprinted genes in two maize inbred lines, as described in this paper. I entered the Plant Biological Sciences graduate program in the fall of 2010 and plan to continue working on maize imprinting in the Springer lab. As a graduate student, my research focuses on discovering and understanding the variability of imprinted genes among diverse maize inbred lines. My goal is to understand how epigenetic factors affect the biased expression of imprinted alleles and to investigate possible phenotypic effects of these biases.
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