DNA methylation remodeling and the functional implication during male gametogenesis in rice.

BACKGROUND: Epigenetic marks are reprogrammed during sexual reproduction. In flowering plants, DNA methylation is only partially remodeled in the gametes and the zygote. However, the timing and functional significance of the remodeling during plant gametogenesis remain obscure. RESULTS: Here we show that DNA methylation remodeling starts after male meiosis in rice, with non-CG methylation, particularly at CHG sites, being first enhanced in the microspore and subsequently decreased in sperm. Functional analysis of rice CHG methyltransferase genes CMT3a and CMT3b indicates that CMT3a functions as the major CHG methyltransferase in rice meiocyte, while CMT3b is responsible for the increase of CHG methylation in microspore. The function of the two histone demethylases JMJ706 and JMJ707 that remove H3K9me2 may contribute to the decreased CHG methylation in sperm. During male gametogenesis CMT3a mainly silences TE and TE-related genes while CMT3b is required for repression of genes encoding factors involved in transcriptional and translational activities. In addition, CMT3b functions to repress zygotic gene expression in egg and participates in establishing the zygotic epigenome upon fertilization. CONCLUSION: Collectively, the results indicate that DNA methylation is dynamically remodeled during male gametogenesis, distinguish the function of CMT3a and CMT3b in sex cells, and underpin the functional significance of DNA methylation remodeling during rice reproduction.

Transcriptome-wide association analyses reveal the impact of regulatory variants on rice panicle architecture and causal gene regulatory networks.

Panicle architecture is a key determinant of rice grain yield and is mainly determined at the 1-2 mm young panicle stage. Here, we investigated the transcriptome of the 1-2 mm young panicles from 275 rice varieties and identified thousands of genes whose expression levels were associated with panicle traits. Multimodel association studies suggested that many small-effect genetic loci determine spikelet per panicle (SPP) by regulating the expression of genes associated with panicle traits. We found that alleles at cis-expression quantitative trait loci of SPP-associated genes underwent positive selection, with a strong preference for alleles increasing SPP. We further developed a method that integrates the associations of cis- and trans-expression components of genes with traits to identify causal genes at even small-effect loci and construct regulatory networks. We identified 36 putative causal genes of SPP, including SDT (MIR156j) and OsMADS17, and inferred that OsMADS17 regulates SDT expression, which was experimentally validated. Our study reveals the impact of regulatory variants on rice panicle architecture and provides new insights into the gene regulatory networks of panicle traits.

OsbHLH067, OsbHLH068, and OsbHLH069 redundantly regulate inflorescence axillary meristem formation in rice.

Rice axillary meristems (AMs) are essential to the formation of tillers and panicle branches in rice, and therefore play a determining role in rice yield. However, the regulation of inflorescence AM development in rice remains elusive. In this study, we identified no spikelet 1-Dominant (nsp1-D), a sparse spikelet mutant, with obvious reduction of panicle branches and spikelets. Inflorescence AM deficiency in nsp1-D could be ascribed to the overexpression of OsbHLH069. OsbHLH069 functions redundantly with OsbHLH067 and OsbHLH068 in panicle AM formation. The Osbhlh067 Osbhlh068 Osbhlh069 triple mutant had smaller panicles and fewer branches and spikelets. OsbHLH067, OsbHLH068, and OsbHLH069 were preferentially expressed in the developing inflorescence AMs and their proteins could physically interact with LAX1. Both nsp1-D and lax1 showed sparse panicles. Transcriptomic data indicated that OsbHLH067/068/069 may be involved in the metabolic pathway during panicle AM formation. Quantitative RT-PCR results demonstrated that the expression of genes involved in meristem development and starch/sucrose metabolism was down-regulated in the triple mutant. Collectively, our study demonstrates that OsbHLH067, OsbHLH068, and OsbHLH069 have redundant functions in regulating the formation of inflorescence AMs during panicle development in rice.

Genome-wide binding analysis of transcription factor Rice Indeterminate 1 reveals a complex network controlling rice floral transition.

RICE INDETERMINATE 1 (RID1) plays a critical role in controlling floral transition in rice (Oryza sativa). However, the molecular basis for this effect, particularly the target genes and regulatory specificity, remains largely unclear. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) in young leaves at the pre-floral-transition stage to identify the target genes of RID1, identifying 2,680 genes associated with RID1 binding sites genome-wide. RID1 binding peaks were highly enriched for TTTGTC, the direct binding motif of the INDETERMINATE DOMAIN protein family that includes RID1. Interestingly, CACGTG and GTGGGCCC, two previously uncharacterized indirect binding motifs, were enriched through the interactions of RID1 with the novel flowering-promoting proteins OsPIL12 and OsTCP11, respectively. Moreover, the ChIP-seq data demonstrated that RID1 bound to numerous rice heading-date genes, such as HEADING DATE 1 (HD1) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1). Notably, transcriptome sequencing (RNA-seq) analysis revealed roles of RID1 in diverse developmental pathways. Genetic analysis combined with genome-wide ChIP-seq and RNA-seq results showed that RID1 directly binds to the promoter of OsERF#136 (a repressor of rice flowering) and negatively regulates its expression. Overall, our findings provide new insights into the molecular and genetic mechanisms underlying rice floral transition and characterize OsERF#136 as a previously unrecognized direct target of RID1.

miR395-regulated sulfate metabolism exploits pathogen sensitivity to sulfate to boost immunity in rice.

MicroRNAs (miRNAs) play important roles in plant physiological activities. However, their roles and molecular mechanisms in boosting plant immunity, especially through the modulation of macronutrient metabolism in response to pathogens, are largely unknown. Here, we report that an evolutionarily conserved miRNA, miR395, promotes resistance to Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), two destructive bacterial pathogens, by regulating sulfate accumulation and distribution in rice. Specifically, miR395 targets and suppresses the expression of the ATP sulfurylase gene OsAPS1, which functions in sulfate assimilation, and two sulfate transporter genes, OsSULTR2;1 and OsSULTR2;2, which function in sulfate translocation, to promote sulfate accumulation, resulting in broad-spectrum resistance to bacterial pathogens in miR395-overexpressing plants. Genetic analysis revealed that miR395-triggered resistance is involved in both pathogen-associated molecular pattern-triggered immunity and R gene-mediated resistance. Moreover, we found that accumulated sulfate but not S-metabolites inhibits proliferation of pathogenic bacteria, revealing a sulfate-mediated antibacterial defense mechanism that differs from sulfur-induced resistance. Furthermore, compared with other bacteria, Xoo and Xoc, which lack the sulfate transporter CysZ, are sensitive to high levels of extracellular sulfate. Accordingly, miR395-regulated sulfate accumulation impaired the virulence of Xoo and Xoc by decreasing extracellular polysaccharide production and biofilm formation. Taken together, these results suggest that rice miR395 modulates sulfate metabolism to exploit pathogen sensitivity to sulfate and thereby promotes broad-spectrum resistance.

RID1 sets rice heading date by balancing its binding with SLR1 and SDG722.

Rice (Oryza sativa) is a major crop that feeds billions of people, and its yield is strongly influenced by flowering time (heading date). Loss of RICE INDETERMINATE1 (RID1) function causes plants not to flower; thus, RID1 is considered a master switch among flowering-related genes. However, it remains unclear whether other proteins function together with RID1 to regulate rice floral transition. Here, we revealed that the chromatin accessibility and H3K9ac, H3K4me3, and H3K36me3 levels at Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) loci were significantly reduced in rid1 mutants. Notably, RID1 interacted with SET DOMAIN GROUP PROTEIN 722 (SDG722), a methyltransferase. We determined that SDG722 affects the global level of H3K4me2/3 and H3K36me2/3, and promotes flowering primarily through the Early heading date1-Hd3a/RFT1 pathway. We further established that rice DELLA protein SLENDER RICE1 (SLR1) interacted with RID1 to inhibit its transactivation activity, that SLR1 suppresses rice flowering, and that messenger RNA and protein levels of SLR1 gradually decrease with plant growth. Furthermore, SLR1 competed with SDG722 for interaction with RID1. Overall, our results establish that interplay between RID1, SLR1, and SDG722 feeds into rice flowering-time control.

VPB1 Encoding BELL-like Homeodomain Protein Is Involved in Rice Panicle Architecture.

Inflorescence architecture in rice (Oryza sativa) is mainly determined by spikelets and the branch arrangement. Primary branches initiate from inflorescence meristem in a spiral phyllotaxic manner, and further develop into the panicle branches. The branching patterns contribute largely to rice production. In this study, we characterized a rice verticillate primary branch 1(vpb1) mutant, which exhibited a clustered primary branches phenotype. Gene isolation revealed that VPB1 was a allele of RI, that it encoded a BELL-like homeodomain (BLH) protein. VPB1 gene preferentially expressed in the inflorescence and branch meristems. The arrangement of primary branch meristems was disturbed in the vpb1 mutant. Transcriptome analysis further revealed that VPB1 affected the expression of some genes involved in inflorescence meristem identity and hormone signaling pathways. In addition, the differentially expressed gene (DEG) promoter analysis showed that OsBOPs involved in boundary organ initiation were potential target genes of VPB1 protein. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter system further verified that VPB1 protein bound to the promoter of OsBOP1 gene. Overall, our findings demonstrate that VPB1 controls inflorescence architecture by regulating the expression of genes involved in meristem maintenance and hormone pathways and by interacting with OsBOP genes.

OsMLH1 interacts with OsMLH3 to regulate synapsis and interference-sensitive crossover formation during meiosis in rice.

Meiotic recombination is essential for reciprocal exchange of genetic information between homologous chromosomes and their subsequent proper segregation in sexually reproducing organisms. MLH1 and MLH3 belong to meiosis-specific members of the MutL-homolog family, which are required for normal level of crossovers (COs) in some eukaryotes. However, their functions in plants need to be further elucidated. Here, we report the identification of OsMLH1 and reveal its functions during meiosis in rice. Using CRISPR-Cas9 approach, two independent mutants, Osmlh1-1 and Osmlh1-2, are generated and exhibited significantly reduced male fertility. In Osmlh1-1, the clearance of PAIR2 is delayed and partial ZEP1 proteins are not loaded into the chromosomes, which might be due to the deficient in resolution of interlocks at late zygotene. Thus, OsMLH1 is required for the assembly of synapsis complex. In Osmlh1-1, CO number is dropped by ~53% and the distribution of residual COs is consistent with predicted Poisson distribution, indicating that OsMLH1 is essential for the formation of interference-sensitive COs (class I COs). OsMLH1 interacts with OsMLH3 through their C-terminal domains. Mutation in OsMLH3 also affects the pollen fertility. Thus, our experiments reveal that the conserved heterodimer MutLγ (OsMLH1-OsMLH3) is essential for the formation of class I COs in rice.

Phosphorylation of OsFD1 by OsCIPK3 promotes the formation of RFT1-containing florigen activation complex for long-day flowering in rice.

Heading date is a critical trait that determines the regional adaptability and grain productivity of many crops. Although rice is a facultative short-day plant, its domestication led to the Ghd7-Ehd1-Hd3a/RFT1 pathway for adaptation to long-day conditions (LDs). The formation of the "florigen activation complex" (FAC) containing florigen Hd3a has been characterized. However, the molecular composition of the FAC that contains RFT1 for long-day flowering is unclear. We show here that RFT1 forms a ternary FAC with 14-3-3 proteins and OsFD1 to promote flowering under LDs. We identified a calcineurin B-like-interacting protein kinase, OsCIPK3, which directly interacts with and phosphorylates OsFD1, thereby facilitating the localization of the FAC to the nucleus. Mutation in OsCIPK3 results in a late heading date under LDs but a normal heading date under short-day conditions. Collectively, our results suggest that OsCIPK3 phosphorylates OsFD1 to promote RFT1-containing FAC formation and consequently induce flowering in rice under LDs.

Rice and Arabidopsis homologs of yeast CHROMOSOME TRANSMISSION FIDELITY PROTEIN 4 commonly interact with Polycomb complexes but exert divergent regulatory functions.

Both genetic and epigenetic information must be transferred from mother to daughter cells during cell division. The mechanisms through which information about chromatin states and epigenetic marks like histone 3 lysine 27 trimethylation (H3K27me3) are transferred have been characterized in animals; these processes are less well understood in plants. Here, based on characterization of a dwarf rice (Oryza sativa) mutant (dwarf-related wd40 protein 1, drw1) deficient for yeast CTF4 (CHROMOSOME TRANSMISSION FIDELITY PROTEIN 4), we discovered that CTF4 orthologs in plants use common cellular machinery yet accomplish divergent functional outcomes. Specifically, drw1 exhibited no flowering-related phenotypes (as in the putatively orthologous Arabidopsis thaliana eol1 mutant), but displayed cell cycle arrest and DNA damage responses. Mechanistically, we demonstrate that DRW1 sustains normal cell cycle progression by modulating the expression of cell cycle inhibitors KIP-RELATED PROTEIN 1 (KRP1) and KRP5, and show that these effects are mediated by DRW1 binding their promoters and increasing H3K27me3 levels. Thus, although CTF4 orthologs ENHANCER OF LHP1 1 (EOL1) in Arabidopsis and DRW1 in rice are both expressed uniquely in dividing cells, commonly interact with several Polycomb complex subunits, and promote H3K27me3 deposition, we now know that their regulatory functions diverged substantially during plant evolution. Moreover, our work experimentally illustrates specific targets of CTF4/EOL1/DRW1, their protein-proteininteraction partners, and their chromatin/epigenetic effects in plants.

Rice RBH1 Encoding A Pectate Lyase is Critical for Apical Panicle Development.

Panicle morphology is one of the main determinants of the rice yield. Panicle abortion, a typical panicle morphological defect results in yield reduction due to defective spikelet development. To further elucidate the molecular mechanism of panicle abortion in rice, a rice panicle bald head 1 (rbh1) mutant with transfer DNA (T-DNA) insertion showing severely aborted apical spikelets during panicle development was identified and characterized. The rbh1-1 mutant showed obviously altered cell morphology and structure in the degenerated spikelet. Molecular genetic studies revealed that RBH1 encodes a pectate lyase protein. Pectate lyase-specific activity of Rice panicle Bald Head 1 (RBH1) protein assay using polygalacturonic acid (PGA) as substrates illustrated that the enzyme retained a significant capacity to degrade PGA. In addition, immunohistochemical analysis showed that the degradation of pectin is inhibited in the rbh1-1 mutant. Further analysis revealed that a significant increase in reactive oxygen species (ROS) level was found in degenerated rbh1-1 spikelets. Taken together, our findings suggest that RBH1 is required for the formation of panicle and for preventing panicle abortion.

Structural Insight into DNA Recognition by CCT/NF-YB/YC Complexes in Plant Photoperiodic Flowering.

CONSTANS, CONSTANS-LIKE, and TIMING OF CAB EXPRESSION1 (CCT) domain-containing proteins are a large family unique to plants. They transcriptionally regulate photoperiodic flowering, circadian rhythms, vernalization, and other related processes. Through their CCT domains, CONSTANS and HEADING DATE1 (HD1) coordinate with the NUCLEAR FACTOR Y (NF-Y) B/C dimer to specifically target a conserved 'CCACA' motif within the promoters of their target genes. However, the mechanism underlying DNA recognition by the CCT domain remains unclear. Here we determined the crystal structures of the rice (Oryza sativa) NF-YB/YC dimer and the florigen gene Heading date 3a (Hd3a)-bound HD1CCT/NF-YB/YC trimer with resolutions of 2.0 Å and 2.55 Å, respectively. The CCT domain of HD1 displays an elongated structure containing two α-helices and two loops, tethering Hd3a to the NF-YB/YC dimer. Helix α2 and loop 2 are anchored into the minor groove of the 'CCACA' motif, which determines the specific base recognition. Our structures reveal the interaction mechanism among the CCT domain, NF-YB/YC dimer, and the target DNA. These results not only provide insight into the network between the CCT proteins and NF-Y subunits, but also offer potential approaches for improving productivity and global adaptability of crops by manipulating florigen expression.

Friction and Wear Properties of Ni3Si Alloy with Ti Addition at High Temperatures.

The tribological properties of Ni3Si alloy were studied at high temperatures. The effect of the addition of Ti was also analyzed. The surface composition was analyzed by Raman spectroscopy. The results showed that the friction coefficient decreased with the increasing temperature, and the wear rate changed slightly from 25 to 400 °C. However, the wear resistance of the alloys decreased sharply at 600 °C, and this was due to the decrease of the high-temperature strength and the severe oxidation of the alloys. Although the oxidation resistance of Ni3Si alloy decreased with Ti addition, the tribological property was improved by the addition of Ti. The Ni3Si alloy with 5% Ti addition had the best wear resistance at high temperatures as compared to pure Ni3Si alloy and with 10% Ti addition, and the wear rates of the alloys were in the order of magnitude of 10-5 mm3/Nm. With the increase of temperature, the wear mechanism of pure Ni3Si alloy transformed from abrasive wear to oxidation wear. As the Ti content increased, the wear mechanisms of the alloys changed from abrasive wear to fatigue wear at low temperature, and oxidation wear and fatigue wear at high temperature.

OsmiR167a-targeted auxin response factors modulate tiller angle via fine-tuning auxin distribution in rice.

Rice tiller angle determines plant growth density and further contributes grain production. Although a few genes have been characterized to regulate tiller angle in rice, the molecular mechanism underlying the control of tiller angle via microRNA is poorly understood. Here, we report that rice tiller angle is controlled by OsmiR167a-targeted auxin response factors OsARF12, OsARF17 and OsARF25. In the overexpression of OsMIR167a plants, the expression of OsARF12, OsARF17 and OsARF25 was severely repressed and displayed larger tiller angle as well as the osarf12/osarf17 and osarf12/ osarf25 plants. In addition, those plants showed compromised abnormal auxin distribution and less sensitive to gravity. We also demonstrate that OsARF12, OsARF17 and OsARF25 function redundantly and might be involved in HSFA2D and LAZY1-dependent asymmetric auxin distribution pathway to control rice tiller angle. Our results reveal that OsmiR167a represses its targets, OsARF12, OsARF17 and OsARF25, to control rice tiller angle by fine-tuning auxin asymmetric distribution in shoots.

Panicle Morphology Mutant 1 (PMM1) determines the inflorescence architecture of rice by controlling brassinosteroid biosynthesis.

BACKGROUND: Panicle architecture is one of the main important agronomical traits that determine branch number and grain number in rice. Although a large number of genes involved in panicle development have been identified in recent years, the complex processes of inflorescence patterning need to be further characterized in rice. Brassinosteroids (BRs) are a class of steroid phytohormones. A great understanding of how BRs contribute to plant height and leaf erectness have been reported, however, the molecular and genetic mechanisms of panicle architecture influenced by BRs remain unclear. RESULTS: Here, we identified PMM1, encoding a cytochrome P450 protein involved in BRs biosynthesis, and characterized its role in panicle architecture in rice. Three alleles of pmm1 were identified from our T-DNA insertional mutant library. Map-based cloning revealed that a large fragment deletion from the 2nd to 9th exons of PMM1 was responsible for the clustered primary branch morphology in pmm1-1. PMM1 is a new allele of DWARF11 (D11) PMM1 transcripts are preferentially expressed in young panicles, particularly expressed in the primordia of branches and spikelets during inflorescence development. Furthermore, overexpression of OsDWARF4 (D4), another gene encoding cytochrome P450, completely rescued the abnormal panicle phenotype of pmm1-1. Overall, it can be concluded that PMM1 is an important gene involved in BRs biosynthesis and affecting the differentiation of spikelet primordia and patterns of panicle branches in rice. CONCLUSIONS: PMM1 is a new allele of D11, which encodes a cytochrome P450 protein involved in BRs biosynthesis pathway. Overexpression of D4 could successfully rescue the abnormal panicle architecture of pmm1 plants, indicating that PMM1/D11 and D4 function redundantly in BRs biosynthesis. Thus, our results demonstrated that PMM1 determines the inflorescence architecture by controlling brassinosteroid biosynthesis in rice.

The E3 Ubiquitin Ligase HAF1 Modulates Circadian Accumulation of EARLY FLOWERING3 to Control Heading Date in Rice under Long-Day Conditions.

The ubiquitin 26S proteasome system (UPS) is critical for enabling plants to alter their proteomes to integrate internal and external signals for the photoperiodic induction of flowering. We previously demonstrated that HAF1, a C3HC4 RING domain-containing E3 ubiquitin ligase, is essential to precisely modulate the timing of Heading Date1 accumulation and to ensure appropriate photoperiodic responses under short-day conditions in rice (Oryza sativa). However, how HAF1 mediates flowering under long-day conditions remains unknown. In this study, we show that OsELF3 (EARLY FLOWERING3) is the direct substrate of HAF1 for ubiquitination in vitro and in vivo. HAF1 is required for maintaining the circadian rhythm of OsELF3 accumulation during photoperiodic responses in rice. In addition, the haf1 oself3 double mutant headed as late as oself3 plants under long-day conditions. An amino acid variation (L558S) within the interaction domain of OsELF3 with HAF1 greatly contributes to the variation in heading date among japonica rice accessions. The japonica accessions carrying the OsELF3(L)-type allele are found at higher latitudes, while varieties carrying the OsELF3(S)-type allele are found at lower latitudes. Taken together, our findings suggest that HAF1 precisely modulates the diurnal rhythm of OsELF3 accumulation to ensure the appropriate heading date in rice.

Rice Functional Genomics Research: Past Decade and Future.

Rice (Oryza sativa) is a major staple food crop for more than 3.5 billion people worldwide. Understanding the regulatory mechanisms of complex agronomic traits in rice is critical for global food security. Rice is also a model plant for genomics research of monocotyledons. Thanks to the rapid development of functional genomic technologies, over 2000 genes controlling important agronomic traits have been cloned, and their molecular biological mechanisms have also been partially characterized. Here, we briefly review the advances in rice functional genomics research during the past 10 years, including a summary of functional genomics platforms, genes and molecular regulatory networks that regulate important agronomic traits, and newly developed tools for gene identification. These achievements made in functional genomics research will greatly facilitate the development of green super rice. We also discuss future challenges and prospects of rice functional genomics research.

Suppressor of rid1 (SID1) shares common targets with RID1 on florigen genes to initiate floral transition in rice.

The transition from vegetative to reproductive growth is a critical process in the life cycle of higher plants. Previously, we cloned Rice Indeterminate 1 (RID1), which acts as the master switch for the transition from the vegetative to reproductive phase in rice. Although the photoperiod pathway of RID1 inducing expression of the florigen genes Hd3a and RFT1 via Ehd1 has been established, the alternative pathways for the essential flowering transition need to be further examined. Here, we identified a Suppressor of rid1 (SID1), which rescues the never-flowering phenotype of rid1. SID1 encodes an INDETERMINATE DOMAIN (IDD) transcription factor. Mutation in SID1 showed the delayed flowering phenotype. Gain-of-function of SID1, OsIDD1, or OsIDD6 could restore the rid1 to flowering. Further analyses showed SID1 and RID1 directly target the promoter regions of Hd3a and RFT1, two florigen genes in rice. Taken together, our results reveal an autonomous flowering pathway might be mediated by RID1, thereby controlling the phase transition from vegetative to reproductive development in rice.

Building two indica rice reference genomes with PacBio long-read and Illumina paired-end sequencing data.

Over the past 30 years, we have performed many fundamental studies on two Oryza sativa subsp. indica varieties, Zhenshan 97 (ZS97) and Minghui 63 (MH63). To improve the resolution of many of these investigations, we generated two reference-quality reference genome assemblies using the most advanced sequencing technologies. Using PacBio SMRT technology, we produced over 108 (ZS97) and 174 (MH63) Gb of raw sequence data from 166 (ZS97) and 209 (MH63) pools of BAC clones, and generated ~97 (ZS97) and ~74 (MH63) Gb of paired-end whole-genome shotgun (WGS) sequence data with Illumina sequencing technology. With these data, we successfully assembled two platinum standard reference genomes that have been publicly released. Here we provide the full sets of raw data used to generate these two reference genome assemblies. These data sets can be used to test new programs for better genome assembly and annotation, aid in the discovery of new insights into genome structure, function, and evolution, and help to provide essential support to biological research in general.

Extensive sequence divergence between the reference genomes of two elite indica rice varieties Zhenshan 97 and Minghui 63.

Asian cultivated rice consists of two subspecies: Oryza sativa subsp. indica and O. sativa subsp. japonica Despite the fact that indica rice accounts for over 70% of total rice production worldwide and is genetically much more diverse, a high-quality reference genome for indica rice has yet to be published. We conducted map-based sequencing of two indica rice lines, Zhenshan 97 (ZS97) and Minghui 63 (MH63), which represent the two major varietal groups of the indica subspecies and are the parents of an elite Chinese hybrid. The genome sequences were assembled into 237 (ZS97) and 181 (MH63) contigs, with an accuracy >99.99%, and covered 90.6% and 93.2% of their estimated genome sizes. Comparative analyses of these two indica genomes uncovered surprising structural differences, especially with respect to inversions, translocations, presence/absence variations, and segmental duplications. Approximately 42% of nontransposable element related genes were identical between the two genomes. Transcriptome analysis of three tissues showed that 1,059-2,217 more genes were expressed in the hybrid than in the parents and that the expressed genes in the hybrid were much more diverse due to their divergence between the parental genomes. The public availability of two high-quality reference genomes for the indica subspecies of rice will have large-ranging implications for plant biology and crop genetic improvement.