Characterization of imprinted genes in rice reveals ... - Plant Physiology

Plant Physiology Preview. Published on June 18, 2018, as DOI:10.1104/pp.17.01621

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Characterization of imprinted genes in rice reveals conservation of regulation and

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imprinting with other plant species

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Chen Chena,1,2, Tingting Lib,1, Shan Zhuc,1, Zehou Liud, Zhenyuan Shia, Xiaoming Zhenge,

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Rui Chenf, Jianfeng Huangg, Yi Shenh, Shiyou Luoc, Lei Wangb, Qiao-Quan Liua, and Zhiguo

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Eb,2

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a

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Modern Production Technology of Grain Crops, Key Laboratory of Plant Functional

Jiangsu Key Laboratory of Crop Genetics and Physiology, Co-Innovation Center for

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Genomics of the Ministry of Education, Yangzhou University, Yangzhou, China

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b

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c

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d

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e

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Facilities for Crop Gene Resources and Genetic Improvement, Beijing, China

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f

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Academy of Agricultural Sciences, Tianjin, China

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g

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h

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1

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China National Rice Research Institute, Hangzhou, China

Rice Research Institute, Jiangxi Academy of Agricultural Sciences, Nanchang, China Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China

Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, National Key

Tianjin Institute of Agricultural Quality Standard and Testing Technology, Tianjin

Shanghai Biotechnology Corporation, Shanghai, China Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, China

These authors contributed equally to this work Address correspondence to [email protected], [email protected]

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Running title: Genomic Imprinting of Rice

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One-sentence summary

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Compared with other species, rice imprinted genes are less associated with transposable

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elements, and the epigenetic regulation of imprinting occurs both pre- and 1

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Copyright 2018 by the American Society of Plant Biologists

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post-fertilization in rice.

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Author Contributions

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C.C. conceived the original screening and research plans; C.C., Z.E., and Q.Q.L. designed

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the research; C.C., T.L., S.Z., Z.L., Z.S., and S.L. performed the experiments; X.Z., R.C., J.H.,

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Y.S., and L.W. analyzed the data. C.C. wrote the article with contributions of all the

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authors.

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Abstract

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Genomic imprinting is an epigenetic phenomenon by which certain genes display

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differential expression in a parent-of-origin-dependent manner. Hundreds of imprinted

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genes have been identified from several plant species. Here we identified, with a high

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level of confidence, 208 imprinted gene candidates from rice (Oryza sativa). Imprinted

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genes of rice showed limited association to the transposable elements, which contrasts

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with findings from Arabidopsis thaliana. Generally, imprinting in rice is conserved within

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a species, but intraspecific variation was also detected. The imprinted rice genes do not

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show signatures of selection, which suggests that domestication has had a limited

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evolutionary consequence on genomic imprinting. Though conservation of imprinting in

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plants is limited, we show that some loci are imprinted in several different species.

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Moreover, our results suggest that different types of epigenetic regulation can be

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established either before or after fertilization. Imprinted 24-nt small RNAs and their

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neighboring genes tend to express alleles from different parents. This association was

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not observed between 21-nt small RNAs and their neighboring genes. Together, our

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findings suggest that regulation of imprinting can be diverse, and genomic imprinting

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has evolutionary and biological significance.

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Introduction

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Imprinted genes are expressed in only one of the parental alleles in a

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parent-of-origin-dependent manner (Köhler et al., 2012; Gehring, 2013). Genes that

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exclusively or preferentially express the maternal or paternal alleles are termed

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maternally expressed genes (MEGs) or paternally expressed genes (PEGs), respectively.

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Genomic imprinting has been observed in many species, from mammals to flowering

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plants (Pires and Grossniklaus, 2014), and hundreds of putative imprinted loci, including

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protein-coding genes and non-coding RNAs, have been discovered in plants (Gehring et

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al., 2011; Hsieh et al., 2011; Luo et al., 2011; Waters et al., 2011; Wolff et al., 2011;

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Zhang et al., 2011; Waters et al., 2013; Xin et al., 2013; Pignatta et al., 2014; Xu et al.,

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2014; Florez-Rueda et al., 2016; Hatorangan et al., 2016; Klosinska et al., 2016; Zhang et

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al., 2016). However, there is little conservation of imprinting among plant species

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(Waters et al., 2013; Hatorangan et al., 2016), which suggests that the evolution of

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genomic imprinting in plants is very rapid.

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Genomic imprinting is regulated epigenetically by either DNA methylation or histone

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modification, and in some circumstances, both mechanisms are involved (Huh et al.,

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2008; Köhler and Weinhofer-Molisch, 2010). In plants, imprinting is predominantly

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expressed in the endosperm (Luo et al., 2011; Raissig et al., 2013; Klosinska et al., 2016),

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which is hypomethylated relative to the embryo and vegetative tissues (Gehring et al.,

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2009; Zemach et al., 2010; Rodrigues et al., 2013; Xing et al., 2015; Klosinska et al., 2016).

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The DNA glycosylase DEMETER (DME), which is responsible for DNA demethylation, is

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expressed predominantly in the central cell prior to fertilization (Choi et al., 2004).

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Imprinting disorders can be found in dme mutants (Hsieh et al., 2011; Wolff et al., 2011;

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Vu et al., 2013), indicating that proper DNA methylation status is required for the

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establishment of genetic imprinting in the endosperm. Several studies have revealed

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that imprinted genes usually neighbor transposable elements (TEs) (Wolff et al., 2011;

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Pignatta et al., 2014; Hatorangan et al., 2016). High expression of DME in the central cell 4


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promotes demethylation of TEs and their adjacent imprinted genes (Gehring et al., 2009).

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Therefore, maternal alleles of imprinted genes are activated in the central cell, but the

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paternal alleles are hypermethylated and remain silenced in the sperm cell, which

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eventually leads to expression of only the maternal allele after fertilization. Polycomb

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Repressive Complex 2 (PRC2)-mediated Histone H3 Lysine 27 Trimethylation (H3K27me3)

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is required for proper genomic imprinting (Huh et al., 2008; Köhler and

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Weinhofer-Molisch, 2010). Mutation of fertilization-independent endosperm (FIE), a

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member of the Fertilization Independent Seed (FIS)-PRC2 family, may result in disruption

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of imprinting at some loci (Hsieh et al., 2011; Wolff et al., 2011).

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Genetic evidence has revealed that several MEGs are likely important for seed and/or

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endosperm development in Arabidopsis thaliana (Chaudhury et al., 1997; Luo et al.,

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2000; Tiwari et al., 2008; Gerald et al., 2009; Costa et al., 2012; Liu et al., 2014). However,

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many mutants of paternally expressed imprinted genes show no altered phenotype

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(Köhler et al., 2005; Shirzadi et al., 2011; Bratzel et al., 2012; Vu et al., 2013; Wolff et al.,

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2015). In addition, conservation of imprinting between closely related species is limited

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(Waters et al., 2013; Hatorangan et al., 2016). These observations cast doubt on the

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importance of genomic imprinting in plants. The kinship theory (Haig and Westoby, 1991)

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hypothesizes that imprinting arose as a consequence of conflict between male and

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female gametes. Specifically, this theory suggests that males gain fitness benefits from

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greater transmission of resources from the mother to the offspring, but females benefit

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by suppression of growth-related demands from the offspring that are driven by

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paternally active genes. Numerous observations support the kinship hypothesis (Köhler

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et al., 2012; Pires and Grossniklaus, 2014; Rodrigues and Zilberman, 2015). However, in

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plants, this theory has been challenged (Rodrigues and Zilberman, 2015). Recent

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findings in Arabidopsis revealed that some PEGs may be involved in hybrid

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incompatibility (Wolff et al., 2015), implying that genomic imprinting may play a role in

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speciation. However, the evolutionary significance of genomic imprinting requires 5


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further investigation in different plant species.

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Rice (Oryza sativa) is a model monocot and a vital agricultural crop. Consistent with

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results from dicots, studies in maize (Zea mays) (another monocot) have revealed that

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differential DNA methylation and H3K27me3 modification between parental genomes

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are essential for imprinting of monocots (Waters et al., 2013; Zhang et al., 2014a).

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However, our understanding of the epigenetic regulation of genomic imprinting in rice

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remains limited. Through a genome-wide survey of imprinted genes from reciprocal

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intraspecific crosses, Luo et al. (2011) and Yuan et al. (2017) identified more than three

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hundred imprinted candidates in rice. However, there was little overlap in the identity of

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these candidates between the studies (Yuan et al., 2017). The ability to identify

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imprinted genes is dependent largely on the availability of informative single-nucleotide

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polymorphisms (SNPs). Therefore, discovery of imprinted genes from distinct

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intraspecific crosses is necessary to obtain greater certainty for identifying imprinted

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candidates. Moreover, due to its rapid evolution, variation in imprinting has been found

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among accessions in both Arabidopsis and maize (Pignatta et al., 2014; Waters et al.,

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2013). In this study, we explored imprinted genes in rice by making two independent

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reciprocal crosses between indica and japonica cultivars, as well as crosses between

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cultivated and wild rice. Our results showed that the regulatory mechanisms of genomic

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imprinting in rice are diverse. The findings will help us to better understand the

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regulation and evolution of imprinting in plants.

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Results

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Identification of imprinted genes in cultivated rice

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To explore the imprinted genes in rice, we used three-line hybrid rice strains to construct

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reciprocal crosses. A cytoplasmic male sterile (CMS) line carries a sterility gene encoded

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in the cytoplasmic genome, such that the pollen produced is sterile. A “maintainer” line

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shares the same nuclear genome as its corresponding CMS line but has a distinct

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cytoplasmic genome. Due to the absence of the cytoplasmic sterility gene, the

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maintainer line is fertile. Using rice CMS and maintainer lines for crossing greatly

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increases the efficiency of hybridization and enables us to rigorously control the timing

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of fertilization and to avoid false hybridization events. Here, we used the japonica CMS

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lines (Liuqianxin-A and Yu6-A) and their maintainer lines (Liuqianxin-B and Yu6-B), and

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the indica CMS lines (Rongfeng-A and Wufeng-A) with their corresponding maintainer

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lines (Rongfeng-B and Wufeng-B) to make two distinct reciprocal-cross sets, which we

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refer to as Liuqianxin-A/Rongfeng-B (LR), Rongfeng-A/Liuqianxin-B (RL), Yu6-A/Wufeng-B

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(YW), and Wufeng-A/Yu6-B (WY). The LR and RL reciprocal crosses, and YW and WY

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reciprocal crosses are indicated as LR-RL and YW-WY hereafter for convenience.

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Meanwhile, we made crosses of Wufeng-A/Wufeng-B (WW), Rongfeng-A/Rongfeng-B

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(RR), Liuqianxin-A/Liuqianxin-B (LL), and Yu6-A/Yu6-B (YY), which resembled selfing of

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inbred lines as controls to validate imprinting.

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Morphological observations suggested that seeds produced by LR, RL, YW, and WY

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developed normally (Figure 1A and B). The endosperm from these reciprocal crosses

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was collected at five days after fertilization (DAF) for RNA-sequencing (RNA-seq). By

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comparing LR-RL transcriptome data to whole genomic resequencing data from

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Liuqianxin-A and Rongfeng-B, we identified 62,102 SNPs from 12,299 endosperm

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expressed genes in the LR-RL cross that could be used to distinguish parental alleles.

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Similarly, by comparing the RNA-seq data from YW-WY to genomic resequencing of

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Yu6-A and Wufeng-B, we identified 59,107 SNPs from 11,044 genes. In total, parental 7


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expression of 4,946 genes in LR-RL and 6,308 genes in YW-WY deviated from the

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expected 2:1 maternal-to-paternal ratio [χ2 test; p < 0.05, false discovery rate (FDR) < 8


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0.05].

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Next, we surveyed parent-of-origin effects of these genes by examining expression levels

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that were greater than 2-fold (>4:1 for moderate MEGs and 10:1 for strong MEGs and 5-fold bias) shared between LR-RL and YW-WY in the following

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studies (Figure 1F).

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Using Reverse Transcription PCR and sequencing, we confirmed that all 14 of the tested

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candidates were imprinted genes (Supplemental Figure 1A). We also performed

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single-colony sequencing and pyro-sequencing (Supplemental Figure 1B to D) to confirm

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the imprinting status of two quantitative trait loci, Grain Weight 2 (GW2) and protein

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phosphatase with Kelch-like repeat domain 2 (OsPPKL2), for rice seed development

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(Song et al., 2007; Zhang et al., 2012b), suggesting that genome imprinting may function

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directly for seed development in rice.

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For MEGs, Gene Ontology (GO) analysis showed enrichment for the term “DNA binding”

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(p = 0.00031, FDR = 0.017), whereas for PEGs, the terms “transferase activity” (p =

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6.3e-05, FDR = 0.0014), “kinase activity” (p = 6.3e-05, FDR = 0.0014) and “protein

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binding” (p = 0.001, FDR = 0.015) were overrepresented. This result suggested that PEGs

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and MEGs might have different functions. Interestingly, we noticed that several of the 10


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imprinted genes were involved in epigenetic regulation (Supplemental Table 2). A

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previous study reported that OsFIE1 was the only imprinted PRC2 gene in rice (Luo et al.,

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2009). Here we found that rice Embryonic Flower 2a (OsEMF2a), a homolog of the

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Su(z)12 family, also was imprinted, which was validated by different approaches

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(Supplemental Figure 2).

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Comparison to previously identified imprinted genes in rice

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Luo et al. (2011) conducted a genome-wide survey of imprinted genes in rice using the

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japonica cultivar Nipponbare and the indica cultivar 9311 as the parents (N9-9N).

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Approximately 74% (53/72) of the PEGs and 53% (49/93) of the MEGs identified

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overlapped with the ones identified in the present study (Figure 1F). Similarly, about 80%

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(76/97) of the PEGs and 44% (72/162) of the MEGs discovered from L0-0L (Yuan et al.,

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2017) were included in our list of candidates (Figure 1F). However, there were fewer

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overlaps between N9-9N and L0-0L (Yuan et al., 2017). In total, 300 MEGs and 289 PEGs

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have been identified from rice (Figure 1F and Supplemental Table 1), with PEGs being

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more conserved than MEGs. About 54.3% of the PEGs (157/289) could be found in at

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least two sets of the four reciprocal crosses (RL-LR, WY-YW, N9-9N and L0-0L), while only

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39% of the MEGs (117/300) coincided among the different combinations.

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Most of the imprinted genes identified in one cross also tended to be imprinted in

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other reciprocal crosses (Figure 2A to C and Supplemental Figure 3). The ones found in

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only one set of reciprocals usually lacked informative SNPs or sufficient reads to identify

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if they were imprinted in other crosses (Supplemental Figure 3). For example, among

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the 156 imprinted genes identified in the other two studies but not in the present study

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(Figure 1F), 62 (40%) lacked polymorphisms or expression in WY-YW and LR-RL.

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Allele-specific imprinting has been found in maize and Arabidopsis (Waters et al.,

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2013; Pignatta et al., 2014). To test for allele-specific imprinting in rice, we used a cut-off

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criterion. If a strong imprinted gene identified in one reciprocal-cross set failed to show 11


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moderate imprinting (