Characterization of Imprinted Genes in Rice Reveals Conservation of Regulation and Imprinting with Other Plant Species

Abstract

Genomic imprinting is an epigenetic phenomenon by which certain genes display differential expression in a parent-of-origin-dependent manner. Hundreds of imprinted genes have been identified from several plant species. Here, we identified, with a high level of confidence, 208 imprinted gene candidates from rice (Oryza sativa). Imprinted genes of rice showed limited association with the transposable elements, which contrasts with findings from Arabidopsis (Arabidopsis thaliana). Generally, imprinting in rice is conserved within a species, but intraspecific variation also was detected. The imprinted rice genes do not show signatures of selection, which suggests that domestication has had a limited evolutionary consequence on genomic imprinting. Although conservation of imprinting in plants is limited, we show that some loci are imprinted in several different species. Moreover, our results suggest that different types of epigenetic regulation can be established either before or after fertilization. Imprinted 24-nucleotide small RNAs and their neighboring genes tend to express alleles from different parents. This association was not observed between 21-nucleotide small RNAs and their neighboring genes. Together, our findings suggest that the regulation of imprinting can be diverse, and genomic imprinting has evolutionary and biological significance.

Figure 1.

Identification of imprinted genes in rice. A, Seed morphology of Wufeng-A × Wufeng-B (WW), Wufeng-A × Yu6-B (WY), Yu6-A × Yu6-B (YY), and Yu6-A × Wufeng-B (YW) at 1 to 7 d after fertilization (DAF). B, Seed morphology of Rongfeng-A × Rongfeng-B (RR), Rongfeng-A × Liuqianxin-B (RL), Liuqianxin-A × Liuqianxin-B (LL), and Liuqianxin-A × Rongfeng-B (LR) from 1 to 7 DAF. C and D, Allele-specific expression analysis in WY-YW (C) and RL-LR (D). The highlighted areas indicate moderately (2-fold higher than expected; blue) and strongly (5-fold higher than expected; red) imprinted genes. Red lines denote the 0.67 expected value. The proportion of maternal alleles is calculated by fragments per kilobase million (FPKM)_maternal/(FPKM_maternal + FPKM_paternal). E, Distribution of the proportion of maternal alleles at each locus with informative SNPs in WY, YW, RL, LR, Longtepu × 02428 (L0), and 0L. The dashed line indicates the 0.67 expected value. F, Venn diagrams of imprinted genes identified from MY-YM, RL-LR, L0-0L, and Nipponbare and 9311 (N9-9N) reciprocal crosses.

Figure 2.

Intraspecific imprinting variation in rice. A to C, Proportion of maternal alleles [FPKM_maternal/(FPKM_maternal + FPKM_paternal)] in the reciprocal crosses. A, Proportion of maternal alleles in the imprinted genes identified from RL-LR in WY-YW and L0-0L. B, Proportion of maternal alleles in the imprinted genes identified from WY-YW in RL-LR and L0-0L. C, Proportion of maternal alleles of imprinted genes identified from L0-0L in RL-LR and WY-YW. Most of the imprinted genes identified from one reciprocal cross set are imprinted in other sets. D, Examples of allele-specific imprinting in rice. The bar charts show the proportion of parental alleles in different crosses; the sequencing diagrams show the validation of intraspecific imprinting variation using RT-PCR sequencing. Dashed lines indicate the 0.67 expected value, and the informative SNPs are boxed.

Figure 3.

TEs in rice do not enrich with PEGs or MEGs. A, Frequency distribution showing the distance of rice imprinted genes to their nearest TE. There is no TE enrichment in the 2-kb region flanking MEGs or PEGs. PEGs-all and MEGs-all indicate the combined PEGs and MEGs found in RL-LR, WY-YW, N9-9N, and L0-0L. PEGs-conserved and MEGs-conserved indicate the common PEGs and MEGs shared among RL-LR, WY-YW, N9-9N, and L0-0L. B and C, The combined PEGs of rice are found farther upstream (B) and downstream (C) of TEs compared with the combined set of MEGs; the overall distances of combined MEGs to their upstream (B) and downstream (C) TE vicinities do not differ from all genes encoded in the rice genome. A Kruskal-Wallis one-way analysis was used for statistical analysis. Error bars indicate sd.

Figure 4.

Expression profiles of imprinted genes in rice. A, Expression analysis of the common MEGs (top) and PEGs (bottom) of RL-LR and WY-YW by Genevestigator. Red and black arrowheads indicate sperm cell and endosperm, respectively. B, Dynamic expression patterns of the MEGs (left) and PEGs (right) in developing Nipponbare seed.

Figure 5.

DNA methylation of imprinted genes in rice. A to F, Average CG (A and B), CHG (C and D), and CHH (E and F) methylation profiles of the MEGs and their most similar nonimprinted homologs (A, C, and E) and the PEGs and their most similar nonimprinted homologs (B, D, and F). em, Embryo; en, endosperm. G, Heat maps of the expression changes of MEGs (top) and PEGs (bottom) in response to AZA, a DNA methylation inhibitor. −AZA, AZA-free seedlings; +AZA, AZA-treated seedlings.

Figure 6.

The genic neighbors of imprinted 24-nucleotide sRNAs show opposite parental expression bias and high CHH methylation in endosperm. A, Distribution frequency of the proportion of maternal alleles (FPKM_maternal/FPKM_maternal + FPKM_paternal) of the genes that group with a paternally expressed 24-nucleotide sRNA (PESR) and all genes encoded by the genome in different crosses. B, Distribution frequency of the proportion of maternal alleles of genes that group with a maternally expressed 24-nucleotide sRNA (MESR) and all genes encoded by the genome in different crosses. C, CHH methylation of the imprinted genes that colocalize with 24-nucleotide sRNAs and the imprinted genes that colocalized with 21-nucleotide sRNAs in embryo and endosperm. D, Examples of CHH methylation increased imprinted genes that grouped with 24-nucleotide sRNAs.

Figure 7.

Detection of gene imprinting status in cultivated rice × wild rice reciprocal crosses. A and B, Imprinting status of the PEGs (A) and MEGs (B) identified from RL-LR and WY-YW in cultivated rice × wild rice reciprocal crosses. Polymorphic sites are shaded in gray. W, R, and D indicate Wufeng, Rongfeng, and Dongxiang wild rice, respectively. The gene that does not display a parent-of-origin expression pattern is boxed. C and D, Differences in π between W1-type wild rice and indica (C) and W2-type wild rice and japonica (D). A Mann-Whitney rank sum test was used for statistical analyses. **, Significant difference at P < 0.01. The bars indicate 15th/75th percentiles, and the dots indicate 5th/95th percentiles. All genes encoded by the rice genome are used as the control.

Figure 8.

Some loci exhibit conserved imprinting in plants. A, Venn diagram analysis of imprinting conservation in plants. Only a few of the most similar rice homologs (top one hit) of the imprinted genes found in maize, sorghum, and Arabidopsis show imprinting in rice. B, Many of the conserved imprinting loci are imprinted in barley. Conserved imprinted genes refer to those identified from at least two species of rice, maize, sorghum, and Arabidopsis. The top three BLAST hits (e < 10e-10) are used to evaluate imprinting conservation. C, Validation of the imprinting status of 11 conserved imprinted loci in barley. M and B indicate Morex and Bowman, respectively, and the superscripts indicate the polymorphic nucleotides of each allele. Polymorphic sites are highlighted in gray.