Spatiotemporal transcriptomic atlas of rhizome formation in Oryza longistaminata.

Rhizomes are modified stems that grow underground and produce new individuals genetically identical to the mother plant. Recently, a breakthrough has been made in efforts to convert annual grains into perennial ones by utilizing wild rhizomatous species as donors, yet the developmental biology of this organ is rarely studied. Oryza longistaminata, a wild rice species featuring strong rhizomes, provides a valuable model for exploration of rhizome development. Here, we first assembled a double-haplotype genome of O. longistaminata, which displays a 48-fold improvement in contiguity compared to the previously published assembly. Furthermore, spatiotemporal transcriptomics was performed to obtain the expression profiles of different tissues in O. longistaminata rhizomes and tillers. Two spatially reciprocal cell clusters, the vascular bundle 2 cluster and the parenchyma 2 cluster, were determined to be the primary distinctions between the rhizomes and tillers. We also captured meristem initiation cells in the sunken area of parenchyma located at the base of internodes, which is the starting point for rhizome initiation. Trajectory analysis further indicated that the rhizome is regenerated through de novo generation. Collectively, these analyses revealed a spatiotemporal transcriptional transition underlying the rhizome initiation, providing a valuable resource for future perennial crop breeding.

Solving the mystery of Obake rice in Africa: population structure analyses of Oryza longistaminata reveal three genetic groups and evidence of both recent and ancient introgression with O. sativa.

The undomesticated rice relative Oryza longistaminata is a valuable genetic resource for the improvement of the domesticated Asian rice, Oryza sativa. To facilitate the conservation, management, and use of O. longistaminata germplasm, we sought to quantify the population structure and diversity of this species across its geographic range, which includes most of sub-Saharan Africa, and to determine phylogenetic relationships to other AA-genome species of rice present in Africa, including the prevalence of interspecific hybridization between O. longistaminata and O. sativa. Though past plant breeding efforts to introgress genes from O. longistaminata have improved biotic stress resistance, ratooning ability, and yield in O. sativa, progress has been limited by substantial breeding barriers. Nevertheless, despite the strong breeding barriers observed by plant breeders who have attempted this interspecific cross, there have been multiple reports of spontaneous hybrids of O. sativa and O. longistaminata (aka "Obake") obtained from natural populations in Africa. However, the frequency and extent of such natural introgressions and their effect on the evolution of O. longistaminata had not been previously investigated. We studied 190 O. longistaminata accessions, primarily from the International Rice Research Institute genebank collection, along with 309 O. sativa, 25 Oryza barthii, and 83 Oryza glaberrima control outgroups, and 17 control interspecific O. sativa/O. longistaminata hybrids. We analyzed the materials using 178,651 single-nucleotide polymorphisms (SNPs) and seven plastid microsatellite markers. This study identified three genetic subpopulations of O. longistaminata, which correspond geographically to Northwestern Africa, Pan-Africa, and Southern Africa. We confirmed that O. longistaminata is, perhaps counterintuitively, more closely related to the Asian species, O. sativa, than the African species O. barthii and O. glaberrima. We identified 19 recent spontaneous interspecific hybrid individuals between O. sativa and O. longistaminata in the germplasm sampled. Notably, the recent introgression between O. sativa and O. longistaminata has been bidirectional. Moreover, low levels of O. sativa alleles admixed in many predominantly O. longistaminata accessions suggest that introgression also occurred in the distant past, but only in Southern Africa.

The discovery and utilization of favorable genes in Oryza longistaminata.

Asian cultivated rice has been domesticated from ancestors of the wild rice species Oryza rufipogon. During this process, important changes have occurred in many agronomic traits, such as plant height, grain shattering, and panicle shape, and the yield has also greatly increased. However, many favored traits (e.g., stress resistance) have been lost. The genome of O. longistaminata is of the same AA type as O. sativa, harboring many genes conferring resistance to biotic and abiotic stresses, and it is considered as a potential gene pool for genetic improvement of O. sativa. In this review, we summarize the basic research on O. longistaminata, including its resistance to biotic and abiotic stresses, its rhizome traits, and other traits that are of potential application value, such as bacterial blight resistance, drought resistance, heat tolerance, self-incompatibility, nitrogen efficiency, and high yield. Furthermore, we present the current applied research progress on perennial rice breeding based on the rhizome trait of O. longistaminata. Lastly, the possibility of de novo domestication of O. longistaminata is discussed. We expect this article to provide information to enhance the basic research of O. longistaminata and accelerate the genetic improvement of cultivated rice.

An adaptive joint CCA-ICA method for ocular artifact removal and its application to emotion classification.

BACKGROUND: The quality of Electroencephalogram (EEG) signals is critical for revealing the neural mechanism of emotions. However, ocular artifacts decreased the signal to noise ratio (SNR) and covered the inherent cognitive component of EEGs, which pose a great challenge in neuroscience research. NEW METHOD: We proposed a novel unsupervised learning algorithm to adaptively remove the ocular artifacts by combining canonical correlation analysis (CCA), independent component analysis (ICA), higher-order statistics, empirical mode decomposition (EMD), and wavelet denoising techniques. Specifically, the combination of CCA and ICA aimed to improve the quality of source separation, while the higher-order statistics further located the source of ocular artifacts. Subsequently, these noised sources were further corrected by EMD and wavelet denoising to improve SNR of EEG signals. RESULTS: We evaluated the performance of our proposed method with simulation studies and real EEG applications. The results of simulation study showed our proposed method could significantly improve the quality of signals under almost all noise conditions compared to four state-of-art methods. Consistently, the experiments of real EEG applications showed that the proposed methods could efficiently restrict the components of ocular artifacts and preserve the inherent information of cognition processing to improve the reliability of related analysis such as power spectral density (PSD) and emotion recognition. COMPARISON WITH EXISTING METHODS: Our proposed model outperforms the comparative methods in EEG recovery, which further improve the application performance such as PSD analysis and emotion recognition. CONCLUSIONS: The superior performance of our proposed method suggests that it is promising for removing ocular artifacts from EEG signals, which offers an efficient EEG preprocessing technology for the development of brain computer interface such as emotion recognition.

Regeneration pattern and genome-wide transcription profile of rhizome axillary buds after perennial rice harvest.

Perennial rice is a new type of rice that allows the harvest of rice for multiple years without growing new seedlings annually. This technology represents a green and sustainable agricultural production mode with many advantages for balancing agricultural ecology and food security. However, the differences in regeneration patterns between perennial and annual rice and the gene regulatory pathways of the apical dominance in axillary bud growth after harvest in perennial rice are still unclear. In this study, perennial rice (PR23) and annual rice (Chugeng28) were used to investigate axillary bud growth patterns before and after apical spike removal. After elimination of apical dominance at different development stages, perennial rice rhizome axillary buds at the compression nodes germinated more rapidly than others and developed into new seedlings. The axillary buds at the high-position nodes in annual rice grew faster than those at other nodes. Furthermore, the global gene expression patterns of PR23 rhizome buds at compression nodes grown for 1, 3, 4, and 5 days after apical spike removal were analyzed by transcriptome sequencing. Compared with the control buds without apical removal, 264, 3,484, 2,095, and 3,398 genes were up-regulated, and 674, 3,484, 1,594, and 1,824 genes were down-regulated in the buds grown for 1, 3, 4, and 5 days after apical spike removal, respectively. Trend analysis of the expressed genes at different time points was performed and co-expression network was constructed to identify key genes in rhizome axillary bud regrowth. The results showed that 85 hub genes involved in 12 co-regulatory networks were mainly enriched in the light system, photosynthesis-antenna protein, plant hormone signal transduction, ABC transporter and metabolic pathways, which suggested that hormone and photosynthetic signals might play important roles in the regulation of rhizome axillary bud regeneration in perennial rice. Overall, this study clarified the differences in the regeneration patterns of axillary buds between perennial and annual rice and provided insight into the complex regulatory networks during the regeneration of rhizome axillary buds in perennial rice.

A Genetic Network Underlying Rhizome Development in Oryza longistaminata.

The rhizome is an important organ through which many perennial plants are able to propagate vegetatively. Its ecological role has been thoroughly studied on many grass species while the underlying genetic basis is mainly investigated using a rhizomatous wild rice species-Oryza longistaminata. Previous studies have revealed that the rhizome trait in O. longistaminata is jointly controlled by multiple loci, yet how these loci interact with each other remains elusive. Here, an F2 population derived from Oryza sativa (RD23) and O. longistaminata was used to map loci that affect rhizome-related traits. We identified 13 major-effect loci that may jointly control rhizomatousness in O. longistaminata and a total of 51 quantitative trait loci (QTLs) were identified to affect rhizome abundance. Notably, some of these loci were found to have effects on more than one rhizome-related trait. For each trait, a genetic network was constructed according to the genetic expectations of the identified loci. Furthermore, to gain an overview of the genetic regulation on rhizome development, a comprehensive network integrating all these individual networks was assembled. This network consists of three subnetworks that control different aspects of rhizome expression. Judging from the nodes' role in the network and their corresponding traits, we speculated that qRHZ-3-1, qRHZ-4, qRHI-2, and qRHI-5 are the key loci for rhizome development. Functional verification using rhizome-free recombinant inbred lines (RILs) suggested that qRHI-2 and qRHI-5, two multi-trait controlling loci that appeared to be critical in our network analyses, are likely both needed for rhizome formation. Our results provide more insights into the genetic basis of rhizome development and may facilitate identification of key rhizome-related genes.

Extramural vascular invasion nomogram before radical resection of rectal cancer based on magnetic resonance imaging.

Purpose: This study verified the value of magnetic resonance imaging (MRI) to construct a nomogram to preoperatively predict extramural vascular invasion (EMVI) in rectal cancer using MRI characteristics. Materials and methods: There were 55 rectal cancer patients with EMVI and 49 without EMVI in the internal training group. The external validation group consisted of 54 rectal cancer patients with EMVI and 55 without EMVI. High-resolution rectal T2WI, pelvic diffusion-weighted imaging (DWI) sequences, and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) were used. We collected the following data: distance between the lower tumor margin and the anal margin, distance between the lower tumor margin and the anorectal ring, tumor proportion of intestinal wall, mrT stage, maximum tumor diameter, circumferential resection margin, superior rectal vein width, apparent diffusion coefficient (ADC), T2WI EMVI score, DWI and DCE-MRI EMVI scores, demographic information, and preoperative serum tumor marker data. Logistic regression analyses were used to identify independent risk factors of EMVI. A nomogram prediction model was constructed. Receiver operating characteristic curve analysis verified the predictive ability of the nomogram. P < 0.05 was considered significant. Result: Tumor proportion of intestinal wall, superior rectal vein width, T2WI EMVI score, and carbohydrate antigen 19-9 were significant independent predictors of EMVI in rectal cancer and were used to create the model. The areas under the receiver operating characteristic curve, sensitivities, and specificities of the nomogram were 0.746, 65.45%, and 83.67% for the internal training group, respectively, and 0.780, 77.1%, and 71.3% for the external validation group, respectively. Data conclusion: A nomogram including MRI characteristics can predict EMVI in rectal cancer preoperatively and provides a valuable reference to formulate individualized treatment plans and predict prognosis.

The Elite Alleles of OsSPL4 Regulate Grain Size and Increase Grain Yield in Rice.

Grain weight and grain number, the two important yield traits, are mainly determined by grain size and panicle architecture in rice. Herein, we report the identification and functional analysis of OsSPL4 in panicle and grain development of rice. Using CRISPR/Cas9 system, two elite alleles of OsSPL4 were obtained, which exhibited an increasing number of grains per panicle and grain size, resulting in increase of rice yield. Cytological analysis showed that OsSPL4 could regulate spikelet development by promoting cell division. The results of RNA-seq and qRT-PCR validations also demonstrated that several MADS-box and cell-cycle genes were up-regulated in the mutation lines. Co-expression network revealed that many yield-related genes were involved in the regulation network of OsSPL4. In addition, OsSPL4 could be cleaved by the osa-miR156 in vivo, and the OsmiR156-OsSPL4 module might regulate the grain size in rice. Further analysis indicated that the large-grain allele of OsSPL4 in indica rice might introgress from aus varieties under artificial selection. Taken together, our findings suggested that OsSPL4 could be as a key regulator of grain size by acting on cell division control and provided a strategy for panicle architecture and grain size modification for yield improvement in rice.

Confirmation of a Gametophytic Self-Incompatibility in Oryza longistaminata.

Oryza longistaminata, a wild species of African origin, has been reported to exhibit self-incompatibility (SI). However, the genetic pattern of its SI remained unknown. In this study, we conducted self-pollination and reciprocal cross-pollination experiments to verify that O. longistaminata is a strictly self-incompatible species. The staining of pollen with aniline blue following self-pollination revealed that although pollen could germinate on the stigma, the pollen tube was unable to enter the style to complete pollination, thereby resulting in gametophytic self-incompatibility (GSI). LpSDUF247, a S-locus male determinant in the gametophytic SI system of perennial ryegrass, is predicted to encode a DUF247 protein. On the basic of chromosome alignment with LpSDUF247, we identified OlSS1 and OlSS2 as Self-Incompatibility Stamen candidate genes in O. longistaminata. Chromosome segment analysis revealed that the Self-Incompatibility Pistil candidate gene of O. longistaminata (OlSP) is a polymorphic gene located in a region flanking OlSS1. OlSS1 was expressed mainly in the stamens, whereas OlSS2 was expressed in both the stamens and pistils. OlSP was specifically highly expressed in the pistils, as revealed by RT-PCR and qRT-PCR analyses. Collectively, our observations indicate the occurrence of GSI in O. longistaminata and that this process is potentially controlled by OlSS1, OlSS2, and OlSP. These findings provide further insights into the genetic mechanisms underlying self-compatibility in plants.

Comparison of the two up-to-date sequencing technologies for genome assembly: HiFi reads of Pacific Biosciences Sequel II system and ultralong reads of Oxford Nanopore.

BACKGROUND: The availability of reference genomes has revolutionized the study of biology. Multiple competing technologies have been developed to improve the quality and robustness of genome assemblies during the past decade. The 2 widely used long-read sequencing providers-Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)-have recently updated their platforms: PacBio enables high-throughput HiFi reads with base-level resolution of >99%, and ONT generated reads as long as 2 Mb. We applied the 2 up-to-date platforms to a single rice individual and then compared the 2 assemblies to investigate the advantages and limitations of each. RESULTS: The results showed that ONT ultralong reads delivered higher contiguity, producing a total of 18 contigs of which 10 were assembled into a single chromosome compared to 394 contigs and 3 chromosome-level contigs for the PacBio assembly. The ONT ultralong reads also prevented assembly errors caused by long repetitive regions, for which we observed a total of 44 genes of false redundancies and 10 genes of false losses in the PacBio assembly, leading to over- or underestimation of the gene families in those long repetitive regions. We also noted that the PacBio HiFi reads generated assemblies with considerably fewer errors at the level of single nucleotides and small insertions and deletions than those of the ONT assembly, which generated an average 1.06 errors per kb and finally engendered 1,475 incorrect gene annotations via altered or truncated protein predictions. CONCLUSIONS: It shows that both PacBio HiFi reads and ONT ultralong reads had their own merits. Further genome reference constructions could leverage both techniques to lessen the impact of assembly errors and subsequent annotation mistakes rooted in each.

Identification of Key Genes for the Ultrahigh Yield of Rice Using Dynamic Cross-tissue Network Analysis.

Significantly increasing crop yield is a major and worldwide challenge for food supply and security. It is well-known that rice cultivated at Taoyuan in Yunnan of China can produce the highest yield worldwide. Yet, the gene regulatory mechanism underpinning this ultrahigh yield has been a mystery. Here, we systematically collected the transcriptome data for seven key tissues at different developmental stages using rice cultivated both at Taoyuan as the case group and at another regular rice planting place Jinghong as the control group. We identified the top 24 candidate high-yield genes with their network modules from these well-designed datasets by developing a novel computational systems biology method, i.e., dynamic cross-tissue (DCT) network analysis. We used one of the candidate genes, OsSPL4, whose function was previously unknown, for gene editing experimental validation of the high yield, and confirmed that OsSPL4 significantly affects panicle branching and increases the rice yield. This study, which included extensive field phenotyping, cross-tissue systems biology analyses, and functional validation, uncovered the key genes and gene regulatory networks underpinning the ultrahigh yield of rice. The DCT method could be applied to other plant or animal systems if different phenotypes under various environments with the common genome sequences of the examined sample. DCT can be downloaded from https://github.com/ztpub/DCT.

Evolution of Plant Architecture in Oryza Driven by the PROG1 Locus.

The genetic control of plant architecture in crops is critical for agriculture and understanding morphological evolution. This study showed that an open reading frame (ORF) of the rice domestication gene PROG1 appeared 3.4-3.9 million years ago (Mya). Subsequently, it acquired a novel protein-coding gene function in the genome of O. rufipogon (~0.3-0.4 Mya). This extremely young gene and its paralogous C2H2 genes located nearby define the prostrate architecture of O. rufipogon and, thus, are of adaptive significance for wild rice in swamp and water areas. However, selection for dense planting and high yield during rice domestication silenced the PROG1 gene and caused the loss of the RPAD locus containing functional C2H2 paralogs; hence, domesticated lines exhibit an erect plant architecture. Analysis of the stepwise origination process of PROG1 and its evolutionary genetics revealed that this zinc-finger coding gene may have rapidly evolved under positive selection and promoted the transition from non- or semi-prostrate growth to prostrate growth. A transgenic assay showed that PROG1 from O. rufipogon exerts a stronger function compared with PROG1 sequences from other Oryza species. However, the analysis of the expression levels of PROG1 in different Oryza species suggests that the transcriptional regulation of PROG1 has played an important role in its evolution. This study provides the first strong case showing how a fundamental morphological trait evolved in Oryza species driven by a gene locus.

Neo-functionalization of a Teosinte branched 1 homologue mediates adaptations of upland rice.

The rice orthologue of maize domestication gene Teosinte branched 1 (Tb1) affects tillering. But, unlike maize Tb1 gene, it was not selected during domestication. Here, we report that an OsTb1 duplicate gene (OsTb2) has been artificially selected during upland rice adaptation and that natural variation in OsTb2 is associated with tiller number. Interestingly, transgenic rice overexpressing this gene shows increased rather than decreased tillering, suggesting that OsTb2 gains a regulatory effect opposite to that of OsTb1 following duplication. Functional analyses suggest that the OsTb2 protein positively regulates tillering by interacting with the homologous OsTb1 protein and counteracts the inhibitory effect of OsTb1 on tillering. We further characterize two functional variations within OsTb2 that regulate protein function and gene expression, respectively. These results not only present an example of neo-functionalization that generates an opposite function following duplication but also suggest that the Tb1 homologue has been selected in upland rice.

Decrease of gene expression diversity during domestication of animals and plants.

BACKGROUND: The genetic mechanisms underlying the domestication of animals and plants have been of great interest to biologists since Darwin. To date, little is known about the global pattern of gene expression changes during domestication. RESULTS: We generated and collected transcriptome data for seven pairs of domestic animals and plants including dog, silkworm, chicken, rice, cotton, soybean and maize and their wild progenitors and compared the expression profiles between the domestic and wild species. Intriguingly, although the number of expressed genes varied little, the domestic species generally exhibited lower gene expression diversity than did the wild species, and this lower diversity was observed for both domestic plants and different kinds of domestic animals including insect, bird and mammal in the whole-genome gene set (WGGS), candidate selected gene set (CSGS) and non-CSGS, with CSGS exhibiting a higher degree of decreased expression diversity. Moreover, different from previous reports which found 2 to 4% of genes were selected by human, we identified 6892 candidate selected genes accounting for 7.57% of the whole-genome genes in rice and revealed that fewer than 8% of the whole-genome genes had been affected by domestication. CONCLUSIONS: Our results showed that domestication affected the pattern of variation in gene expression throughout the genome and generally decreased the expression diversity across species, and this decrease may have been associated with decreased genetic diversity. This pattern might have profound effects on the phenotypic and physiological changes of domestic animals and plants and provide insights into the genetic mechanisms at the transcriptome level other than decreased genetic diversity and increased linkage disequilibrium underpinning artificial selection.

Differential transcriptome profiling of chilling stress response between shoots and rhizomes of Oryza longistaminata using RNA sequencing.

Rice (Oryza sativa) is very sensitive to chilling stress at seedling and reproductive stages, whereas wild rice, O. longistaminata, tolerates non-freezing cold temperatures and has overwintering ability. Elucidating the molecular mechanisms of chilling tolerance (CT) in O. longistaminata should thus provide a basis for rice CT improvement through molecular breeding. In this study, high-throughput RNA sequencing was performed to profile global transcriptome alterations and crucial genes involved in response to long-term low temperature in O. longistaminata shoots and rhizomes subjected to 7 days of chilling stress. A total of 605 and 403 genes were respectively identified as up- and down-regulated in O. longistaminata under 7 days of chilling stress, with 354 and 371 differentially expressed genes (DEGs) found exclusively in shoots and rhizomes, respectively. GO enrichment and KEGG pathway analyses revealed that multiple transcriptional regulatory pathways were enriched in commonly induced genes in both tissues; in contrast, only the photosynthesis pathway was prevalent in genes uniquely induced in shoots, whereas several key metabolic pathways and the programmed cell death process were enriched in genes induced only in rhizomes. Further analysis of these tissue-specific DEGs showed that the CBF/DREB1 regulon and other transcription factors (TFs), including AP2/EREBPs, MYBs, and WRKYs, were synergistically involved in transcriptional regulation of chilling stress response in shoots. Different sets of TFs, such as OsERF922, OsNAC9, OsWRKY25, and WRKY74, and eight genes encoding antioxidant enzymes were exclusively activated in rhizomes under long-term low-temperature treatment. Furthermore, several cis-regulatory elements, including the ICE1-binding site, the GATA element for phytochrome regulation, and the W-box for WRKY binding, were highly abundant in both tissues, confirming the involvement of multiple regulatory genes and complex networks in the transcriptional regulation of CT in O. longistaminata. Finally, most chilling-induced genes with alternative splicing exclusive to shoots were associated with photosynthesis and regulation of gene expression, while those enriched in rhizomes were primarily related to stress signal transduction; this indicates that tissue-specific transcriptional and post-transcriptional regulation mechanisms synergistically contribute to O. longistaminata long-term CT. Our findings provide an overview of the complex regulatory networks of CT in O. longistaminata.

A genomic perspective on the important genetic mechanisms of upland adaptation of rice.

BACKGROUND: Cultivated rice consists of two important ecotypes, upland and irrigated, that have respectively adapted to either dry land or irrigated cultivation. Upland rice, widely adopted in rainfed upland areas in virtue of its little water requirement, contains abundant untapped genetic resources, such as genes for drought adaptation. With water shortage exacerbated and population expanding, the need for breeding crop varieties with drought adaptation becomes more and more urgent. However, a previous oversight in upland rice research reveals little information regarding its genetic mechanisms for upland adaption, greatly hindering progress in harnessing its genetic resources for breeding and cultivation. RESULTS: In this study, we selected 84 upland and 82 irrigated accessions from all over the world, phenotyped them under both irrigated and dry land environments, and investigated the phylogenetic relations and population structure of the upland ecotype using whole genome variation data. Further comparative analysis yields a list of differentiated genes that may account for the phenotypic and physiological differences between upland and irrigated rice. CONCLUSIONS: This study represents the first genomic investigation in a large sample of upland rice, providing valuable gene list for understanding upland rice adaptation, especially drought-related adaptation, and its subsequent utilization in modern agriculture.

Analysis of elite variety tag SNPs reveals an important allele in upland rice.

Elite crop varieties usually fix alleles that occur at low frequencies within non-elite gene pools. Dissecting these alleles for desirable agronomic traits can be accomplished by comparing the genomes of elite varieties with those from non-elite populations. Here we deep-sequence six elite rice varieties and use two large control panels to identify elite variety tag single-nucleotide polymorphism alleles (ETASs). Guided by this preliminary analysis, we comprehensively characterize one protein-altering ETAS in the 9-cis-epoxycarotenoid dioxygenase gene of the IRAT104 upland rice variety. This allele displays a drastic frequency difference between upland and irrigated rice, and a selective sweep is observed around this allele. Functional analysis indicates that in upland rice, this allele is associated with significantly higher abscisic acid levels and denser lateral roots, suggesting its association with upland rice suitability. This report provides a potential strategy to mine rare, agronomically important alleles.

Single-base resolution maps of cultivated and wild rice methylomes and regulatory roles of DNA methylation in plant gene expression.

BACKGROUND: DNA methylation plays important biological roles in plants and animals. To examine the rice genomic methylation landscape and assess its functional significance, we generated single-base resolution DNA methylome maps for Asian cultivated rice Oryza sativa ssp. japonica, indica and their wild relatives, Oryza rufipogon and Oryza nivara. RESULTS: The overall methylation level of rice genomes is four times higher than that of Arabidopsis. Consistent with the results reported for Arabidopsis, methylation in promoters represses gene expression while gene-body methylation generally appears to be positively associated with gene expression. Interestingly, we discovered that methylation in gene transcriptional termination regions (TTRs) can significantly repress gene expression, and the effect is even stronger than that of promoter methylation. Through integrated analysis of genomic, DNA methylomic and transcriptomic differences between cultivated and wild rice, we found that primary DNA sequence divergence is the major determinant of methylational differences at the whole genome level, but DNA methylational difference alone can only account for limited gene expression variation between the cultivated and wild rice. Furthermore, we identified a number of genes with significant difference in methylation level between the wild and cultivated rice. CONCLUSIONS: The single-base resolution methylomes of rice obtained in this study have not only broadened our understanding of the mechanism and function of DNA methylation in plant genomes, but also provided valuable data for future studies of rice epigenetics and the epigenetic differentiation between wild and cultivated rice.