Development of 12 sets of chromosome segment substitution lines that enhance allele mining in Asian cultivated rice.

Many agronomic traits that are important in rice breeding are controlled by multiple genes. The extensive time and effort devoted so far to identifying and selecting such genes are still not enough to target multiple agronomic traits in practical breeding in Japan because of a lack of suitable plant materials in which to efficiently detect and validate beneficial alleles from diverse genetic resources. To facilitate the comprehensive analysis of genetic variation in agronomic traits among Asian cultivated rice, we developed 12 sets of chromosome segment substitution lines (CSSLs) with the japonica background, 11 of them in the same genetic background, using donors representing the genetic diversity of Asian cultivated rice. Using these materials, we overviewed the chromosomal locations of 1079 putative QTLs for seven agronomic traits and their allelic distribution in Asian cultivated rice through multiple linear regression analysis. The CSSLs will allow the effects of putative QTLs in the highly homogeneous japonica background to be validated.

Transposable element finder (TEF): finding active transposable elements from next generation sequencing data.

BACKGROUND: Detection of newly transposed events by transposable elements (TEs) from next generation sequence (NGS) data is difficult, due to their multiple distribution sites over the genome containing older TEs. The previously reported Transposon Insertion Finder (TIF) detects TE transpositions on the reference genome from NGS short reads using end sequences of target TE. TIF requires the sequence of target TE and is not able to detect transpositions for TEs with an unknown sequence. RESULT: The new algorithm Transposable Element Finder (TEF) enables the detection of TE transpositions, even for TEs with an unknown sequence. TEF is a finding tool of transposed TEs, in contrast to TIF as a detection tool of transposed sites for TEs with a known sequence. The transposition event is often accompanied with a target site duplication (TSD). Focusing on TSD, two algorithms to detect both ends of TE, TSDs and target sites are reported here. One is based on the grouping with TSDs and direct comparison of k-mers from NGS without similarity search. The other is based on the junction mapping of TE end sequence candidates. Both methods succeed to detect both ends and TSDs of known active TEs in several tests with rice, Arabidopsis and Drosophila data and discover several new TEs in new locations. PCR confirmed the detected transpositions of TEs in several test cases in rice. CONCLUSIONS: TEF detects transposed TEs with TSDs as a result of TE transposition, sequences of both ends and their inserted positions of transposed TEs by direct comparison of NGS data between two samples. Genotypes of transpositions are verified by counting of junctions of head and tail, and non-insertion sequences in NGS reads. TEF is easy to run and independent of any TE library, which makes it useful to detect insertions from unknown TEs bypassed by common TE annotation pipelines.

Genetic effect of a new allele for the flowering time locus Ghd7 in rice.

The optimization of flowering time is a key aspect in maximizing grain productivity in rice. Allelic variations in genes for flowering time are major drivers in the wide adaptability of cultivated rice around the world. Here, we identified a novel allele of flowering time gene Grain number, plant height and heading date 7 (Ghd7). Loss-of-function ghd7, Ghd7-0a, is important for extremely early flowering time for adaptability to cultivation in Hokkaido, Japan. However, the rice variety Sorachi lacks a key functional nucleotide polymorphism of Ghd7, which results in a loss of function of the gene. Based on the sequence of Ghd7 allele in Sorachi, we identified the insertion of a transposon-like sequence at an upstream site of Ghd7. Segregation analysis using an F2 population derived from the cross between Hoshinoyume and Sorachi demonstrated that the Ghd7 locus contributed to extremely early flowering time in Sorachi. This Ghd7 allele in Sorachi showed a weak function in terms of delay of flowering time, compared with loss-of-function allele, and a distinct distribution in northern Japan.

Finding a novel QTL responsible for kernel cracking resistance from CSSLs of 'Itadaki' (O. sativa L.) × donor O. rufipogon.

To find new QTLs responsible for kernel cracking resistance, we screened 50 CSSLs derived from the moderately resistant cultivar 'Itadaki' (O. sativa L.) and the donor O. rufipogon. Two lines, IRSL 30 and IRSL 37, were selected as resistant. QTL analyses of the percentage of cracked kernels (PCK) in F4 individuals derived from "Itadaki/IRSL 30" and "Itadaki/IRSL 37" identified a major QTL, qCR (Cracking Resistance) 8-2, at the same position on chromosome 8 in both populations. 'IRSL 30' and 'IRSL 37' had a reduced PCK. These results show that qCR8-2 is likely to be an important QTL for kernel cracking resistance. Both lines had long awns, linked to qCR8-2, but the awnless line 'Chukei 19301' was also derived from "Itadaki/IRSL 37", so qCR8-2 is distinct from the gene for awn development. We consider that qCR8-2 will help in the breeding of new rice cultivars with high cracking resistance and in elucidating the physiological mechanism of kernel cracking.

Rapid DNA-genotyping system targeting ten loci for resistance to blast disease in rice.

The fungal pathogen Pyricularia oryzae causes blast, a severe disease of rice (Oryza sativa L.). Improving blast resistance is important in rice breeding programs. Inoculation tests have been used to select for resistance genotypes, with DNA marker-based selection becoming an efficient alternative. No comprehensive DNA marker system for race-specific resistance alleles in the Japanese rice breeding program has been developed because some loci contain multiple resistance alleles. Here, we used the Fluidigm SNP genotyping platform to determine a set of 96 single nucleotide polymorphism (SNP) markers for 10 loci with race-specific resistance. The markers were then used to evaluate the presence or absence of 24 resistance alleles in 369 cultivars; results were 93.5% consistent with reported inoculation test-based genotypes in japonica varieties. The evaluation system was successfully applied to high-yield varieties with indica genetic backgrounds. The system includes polymorphisms that distinguish the resistant alleles at the tightly linked Pita and Pita-2 loci, thereby confirming that all the tested cultivars with Pita-2 allele carry Pita allele. We also developed and validated insertion/deletion (InDel) markers for ten resistance loci. Combining SNP and InDel markers is an accurate and efficient strategy for selection for race-specific resistance to blast in breeding programs.

Switching genetic effects of the flowering time gene Hd1 in LD conditions by Ghd7 and OsPRR37 in rice.

Flowering time control in plants is a major limiting factor on the range of species. Day length, perceived via the photoperiodic pathway, is a critical factor for the induction of flowering. The module of GIGANTEA (GI)-CONSTANS (CO)-FLOWERING LOCUS T in the long day (LD) plant Arabidopsis is conserved in diverse plant species including the short day (SD) plant rice, where this module comprises OsGI-Heading date 1 (Hd1)-Heading date 3a. Hd1, the rice ortholog of Arabidopsis CO, has dual functions in the regulation of flowering time, promoting flowering in SD conditions and delaying it in LD conditions. We herein show genetic interactions among three LD repressor genes: Hd1, Grain number, plant height and heading date 7 (Ghd7), and Oryza sativa Pseudo-Response Regulator37 (OsPRR37). Genetic analyses, including segregation analyses, evaluations of near isogenic lines, and transformation for flowering time demonstrated that Hd1 promoted flowering time in inductive SD and non-inductive LD conditions in genetic condition of loss-of-function Ghd7 and OsPRR37 (ghd7osprr37) in rice. Functional Ghd7 or OsPRR37 may switch the genetic effects of Hd1 from the promotion to the delay of flowering times in LD conditions.

Genetic dissection of pre-harvest sprouting resistance in an upland rice cultivar.

Seed dormancy is important in rice breeding because it confers resistance to pre-harvest sprouting (PHS). To detect quantitative trait loci (QTLs) for pre-harvest sprouting resistance, we used chromosome segment substitution lines (CSSLs) derived from a cross between the Japanese upland rice cultivar 'Owarihatamochi' and the lowland rice cultivar 'Koshihikari'. In the CSSLs, several chromosomal regions were associated with PHS resistance. Among these, the chromosome 9 segment from 'Owarihatamochi' had the greatest association with increased PHS resistance. Further QTL analysis using an advanced backcross population (BC4F2) derived from a 'Koshihikari' × 'Owarihatamochi' cross revealed two putative QTLs, here designated qSDR9.1 (Seed dormancy 9.1) and qSDR9.2, on chromosome 9. The 'Owarihatamochi' alleles of the two QTLs reduced germination. Further fine mapping revealed that qSDR9.1 and qSDR9.2 were located within 4.1-Mb and 2.3-Mb intervals (based on the 'Nipponbare' reference genome sequence) defined by the simple sequence repeat marker loci RM24039 and RM24260 and Indel_2 and RM24540, respectively. We thus identified two QTLs for PHS resistance in 'Owarihatamochi', even though resistance levels are relatively low in this cultivar. This unexpected finding suggests the advantages of using CSSLs for QTL detection.

Fine Mapping of Carbon Assimilation Rate 8, a Quantitative Trait Locus for Flag Leaf Nitrogen Content, Stomatal Conductance and Photosynthesis in Rice.

Increasing the rate of leaf photosynthesis is one important approach for increasing grain yield in rice (Oryza sativa). Exploiting the natural variation in CO2 assimilation rate (A) between rice cultivars using quantitative genetics is one promising means to identify genes contributing to higher photosynthesis. In this study, we determined precise location of Carbon Assimilation Rate 8 (CAR8) by crossing a high-yielding indica cultivar with a Japanese commercial cultivar. Fine mapping suggested that CAR8 encodes a putative Heme Activator Protein 3 (OsHAP3) subunit of a CCAAT-box-binding transcription factor called OsHAP3H. Sequencing analysis revealed that the indica allele of CAR8 has a 1-bp deletion at 322 bp from the start codon, resulting in a truncated protein of 125 amino acids. In addition, CAR8 is identical to DTH8/Ghd8/LHD1, which was reported to control rice flowering date. The increase of A is largely due to an increase of RuBP regeneration rate via increased leaf nitrogen content, and partially explained by reduced stomatal limitation via increased stomatal conductance relative to A. This allele also increases hydraulic conductivity, which would promote higher stomatal conductance. This indicates that CAR8 affects multiple physiological aspects relating to photosynthesis. The detailed analysis of molecular functions of CAR8 would help to understand the association between photosynthesis and flowering and demonstrate specific genetic mechanisms that can be exploited to improve photosynthesis in rice and potentially other crops.

Hd18, Encoding Histone Acetylase Related to Arabidopsis FLOWERING LOCUS D, is Involved in the Control of Flowering Time in Rice.

Flowering time is one of the most important agronomic traits in rice (Oryza sativa L.), because it defines harvest seasons and cultivation areas, and affects yields. We used a map-based strategy to clone Heading date 18 (Hd18). The difference in flowering time between the Japanese rice cultivars Koshihikari and Hayamasari was due to a single nucleotide polymorphism within the Hd18 gene, which encodes an amine oxidase domain-containing protein and is homologous to Arabidopsis FLOWERING LOCUS D (FLD). The Hayamasari Hd18 allele and knockdown of Hd18 gene expression delayed the flowering time of rice plants regardless of the day-length condition. Structural modeling of the Hd18 protein suggested that the non-synonymous substitution changed protein stability and function due to differences in interdomain hydrogen bond formation. Compared with those in Koshihikari, the expression levels of the flowering-time genes Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and Rice flowering locus T1 (RFT1) were lower in a near-isogenic line with the Hayamasari Hd18 allele in a Koshihikari genetic background. We revealed that Hd18 acts as an accelerator in the rice flowering pathway under both short- and long-day conditions by elevating transcription levels of Ehd1 Gene expression analysis also suggested the involvement of MADS-box genes such as OsMADS50, OsMADS51 and OsMADS56 in the Hd18-associated regulation of Ehd1 These results suggest that, like FLD, its rice homolog accelerates flowering time but is involved in rice flowering pathways that differ from the autonomous pathways in Arabidopsis.

Natural Variation in the Flag Leaf Morphology of Rice Due to a Mutation of the NARROW LEAF 1 Gene in Oryza sativa L.

We investigated the natural variations in the flag leaf morphology of rice. We conducted a principal component analysis based on nine flag leaf morphology traits using 103 accessions from the National Institute of Agrobiological Sciences Core Collection. The first component explained 39% of total variance, and the variable with highest loading was the width of the flag leaf (WFL). A genome-wide association analysis of 102 diverse Japanese accessions revealed that marker RM6992 on chromosome 4 was highly associated with WFL. In analyses of progenies derived from a cross between Takanari and Akenohoshi, the most significant quantitative trait locus (QTL) for WFL was in a 10.3-kb region containing the NARROW LEAF 1 (NAL1) gene, located 0.4 Mb downstream of RM6992. Analyses of chromosomal segment substitution lines indicated that a mutation (G1509A single-nucleotide mutation, causing an R233H amino acid substitution in NAL1) was present at the QTL. This explained 13 and 20% of total variability in WFL and the distance between small vascular bundles, respectively. The mutation apparently occurred during rice domestication and spread into japonica, tropical japonica, and indica subgroups. Notably, one accession, Phulba, had a NAL1 allele encoding only the N-terminal, or one-fourth, of the wild-type peptide. Given that the Phulba allele and the histidine-type allele showed essentially the same phenotype, the histidine-type allele was regarded as malfunctional. The phenotypes of transgenic plants varied depending on the ratio of histidine-type alleles to arginine-type alleles, raising the possibility that H(233)-type products function differently from and compete with R(233)-type products.

Genetic architecture of variation in heading date among Asian rice accessions.

BACKGROUND: Heading date, a crucial factor determining regional and seasonal adaptation in rice (Oryza sativa L.), has been a major selection target in breeding programs. Although considerable progress has been made in our understanding of the molecular regulation of heading date in rice during last two decades, the previously isolated genes and identified quantitative trait loci (QTLs) cannot fully explain the natural variation for heading date in diverse rice accessions. RESULTS: To genetically dissect naturally occurring variation in rice heading date, we collected QTLs in advanced-backcross populations derived from multiple crosses of the japonica rice accession Koshihikari (as a common parental line) with 11 diverse rice accessions (5 indica, 3 aus, and 3 japonica) that originate from various regions of Asia. QTL analyses of over 14,000 backcrossed individuals revealed 255 QTLs distributed widely across the rice genome. Among the detected QTLs, 128 QTLs corresponded to genomic positions of heading date genes identified by previous studies, such as Hd1, Hd6, Hd3a, Ghd7, DTH8, and RFT1. The other 127 QTLs were detected in different chromosomal regions than heading date genes. CONCLUSIONS: Our results indicate that advanced-backcross progeny allowed us to detect and confirm QTLs with relatively small additive effects, and the natural variation in rice heading date could result from combinations of large- and small-effect QTLs. We also found differences in the genetic architecture of heading date (flowering time) among maize, Arabidopsis, and rice.

Gene pyramiding enhances durable blast disease resistance in rice.

Effective control of blast, a devastating fungal disease of rice, would increase and stabilize worldwide food production. Resistance mediated by quantitative trait loci (QTLs), which usually have smaller individual effects than R-genes but confer broad-spectrum or non-race-specific resistance, is a promising alternative to less durable race-specific resistance for crop improvement, yet evidence that validates the impact of QTL combinations (pyramids) on the durability of plant disease resistance has been lacking. Here, we developed near-isogenic experimental lines representing all possible combinations of four QTL alleles from a durably resistant cultivar. These lines enabled us to evaluate the QTLs singly and in combination in a homogeneous genetic background. We present evidence that pyramiding QTL alleles, each controlling a different response to M. oryzae, confers strong, non-race-specific, environmentally stable resistance to blast disease. Our results suggest that this robust defence system provides durable resistance, thus avoiding an evolutionary "arms race" between a crop and its pathogen.

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.

A natural variant of NAL1, selected in high-yield rice breeding programs, pleiotropically increases photosynthesis rate.

Improvement of leaf photosynthesis is an important strategy for greater crop productivity. Here we show that the quantitative trait locus GPS (GREEN FOR PHOTOSYNTHESIS) in rice (Oryza sativa L.) controls photosynthesis rate by regulating carboxylation efficiency. Map-based cloning revealed that GPS is identical to NAL1 (NARROW LEAF1), a gene previously reported to control lateral leaf growth. The high-photosynthesis allele of GPS was found to be a partial loss-of-function allele of NAL1. This allele increased mesophyll cell number between vascular bundles, which led to thickened leaves, and it pleiotropically enhanced photosynthesis rate without the detrimental side effects observed in previously identified nal1 mutants, such as dwarf plant stature. Furthermore, pedigree analysis suggested that rice breeders have repeatedly selected the high-photosynthesis allele in high-yield breeding programs. The identification and utilization of NAL1 (GPS) can enhance future high-yield breeding and provides a new strategy for increasing rice productivity.

Hd16, a gene for casein kinase I, is involved in the control of rice flowering time by modulating the day-length response.

The alteration of photoperiod sensitivity has let breeders diversify flowering time in Oryza sativa (rice) and develop cultivars adjusted to a range of growing season periods. Map-based cloning revealed that the rice flowering-time quantitative trait locus (QTL) Heading date 16 (Hd16) encodes a casein kinase-I protein. One non-synonymous substitution in Hd16 resulted in decreased photoperiod sensitivity in rice, and this substitution occurred naturally in an old rice cultivar. By using near-isogenic lines with functional or deficient alleles of several rice flowering-time genes, we observed significant digenetic interactions between Hd16 and four other flowering-time genes (Ghd7, Hd1, DTH8 and Hd2). In a near-isogenic line with the weak-photoperiod-sensitivity allele of Hd16, transcription levels of Ehd1, Hd3a, and RFT1 increased under long-day conditions, and transcription levels of Hd3a and RFT1 decreased under short-day conditions. Expression analysis under continuous light and dark conditions showed that Hd16 was not likely to be associated with circadian clock regulation. Biochemical characterization indicated that the functional Hd16 recombinant protein specifically phosphorylated Ghd7. These results demonstrate that Hd16 acts as an inhibitor in the rice flowering pathway by enhancing the photoperiod response as a result of the phosphorylation of Ghd7.

Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae.

Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map-based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST-OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower-related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild-type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blast-induced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus.

Roles of the Hd5 gene controlling heading date for adaptation to the northern limits of rice cultivation.

During the diversification of cultivated rice after domestication, rice was grown in diverse geographic regions using genetic variations attributed to the combination of alleles in loci for adaptability to various environmental conditions. To elucidate the key gene for adaptation in rice cultivars to the northern limit of rice cultivation, we conducted genetic analyses of heading date using extremely early-heading cultivars. The Hd5 gene controlling heading date (flowering time) generated variations in heading date among cultivars adapted to Hokkaido, where is the northernmost region of Japan and one of the northern limits of rice cultivation in the world. The association of the Hd5 genotype with heading date and genetical analysis clearly showed that the loss-of-function Hd5 has an important role in exhibiting earlier heading among a local population in Hokkaido. Distinct distribution of the loss-of-function Hd5 revealed that this mutation event of the 19-bp deletion occurred in a local landrace Bouzu and that this mutation may have been selected as an early-heading variety in rice breeding programs in Hokkaido in the early 1900s. The loss-of-function Hd5 was then introduced into the rice variety Fanny from France and contributed to its extremely early heading under the presence of functional Ghd7. These results demonstrated that Hd5 plays roles not only in generating early heading in variations of heading date among a local population in Hokkaido, but also in extremely early heading for adaptation to northern limits of rice cultivation.

Mapping and characterization of seed dormancy QTLs using chromosome segment substitution lines in rice.

Seed dormancy--the temporary failure of a viable seed to germinate under favorable conditions--is a complex characteristic influenced by many genes and environmental factors. To detect the genetic factors associated with seed dormancy in rice, we conducted a QTL analysis using chromosome segment substitution lines (CSSLs) derived from a cross between Nona Bokra (strong dormancy) and Koshihikari (weak dormancy). Comparison of the levels of seed dormancy of the CSSLs and their recurrent parent Koshihikari revealed that two chromosomal regions-on the short arms of chromosomes 1 and 6-were involved in the variation in seed dormancy. Further genetic analyses using an F(2) population derived from crosses between the CSSLs and Koshihikari confirmed the allelic differences and the chromosomal locations of three putative QTLs: Sdr6 on chromosome 1 and Sdr9 and Sdr10 on chromosome 6. The Nona Bokra alleles of the three QTLs were associated with decreased germination rate. We discuss the physiological features of the CSSLs and speculate on the possible mechanisms of dormancy in light of the newly detected QTLs.

Genetic interactions involved in the inhibition of heading by heading date QTL, Hd2 in rice under long-day conditions.

Heading date is the one of the most important traits in rice breeding, because it defines where rice can be cultivated and influences the expression of various agronomic traits. To examine the inhibition of heading by Heading date 2 (Hd2), previously detected on the distal end of chromosome 7's long arm by quantitative trait locus (QTL) analysis, we developed backcross inbred lines (BILs) from Koshihikari, a leading Japanese cultivar, and Hayamasari, an extremely early heading cultivar. The BILs were cultivated under natural field conditions in Tsukuba Japan, and under long-day (14.5 h), extremely long-day (18 h), and short-day (10 h) conditions. Combinations of several QTLs near Hd1, Hd2, Ghd7, Hd5, and Hd16 were detected under these four conditions. Analysis of advanced backcross progenies revealed genetic interactions between Hd2 and Hd16 and between Hd2 and Ghd7. In the homozygous Koshihikari genetic background at Hd16, inhibition of heading by the Koshihikari allele at Hd2 was smaller than that with the Hayamasari Hd16 allele. Similarly, in the homozygous Koshihikari genetic background at Ghd7, the difference in heading date caused by different alleles at Hd2 was smaller than in plants homozygous for the Hayamasari Ghd7 allele. Based on these results, we conclude that Hd2 and its genetic interactions play an important role in controlling heading under long-day conditions. In addition, QTLs near Hd2, Hd16, and Ghd7, which are involved in inhibition of heading under long-day conditions, function in the same pathway that controls heading date.

Ehd3, encoding a plant homeodomain finger-containing protein, is a critical promoter of rice flowering.

Oryza sativa (rice) flowers in response to photoperiod, and is a facultative short-day (SD) plant. Under SD conditions, flowering is promoted through the activation of FT-like genes (rice florigens) by Heading date 1 (Hd1, a rice CONSTANS homolog) and Early heading date 1 (Ehd1, with no ortholog in the Arabidopsis genome). On the other hand, under long-day (LD) conditions, flowering is delayed by the repressive function of Hd1 on FT-like genes and by downregulation of Ehd1 by the flowering repressor Ghd7 - a unique pathway in rice. We report here that an early heading date 3 (ehd3) mutant flowered later than wild-type plants, particularly under LD conditions, regardless of the Hd1-deficient background. Map-based cloning revealed that Ehd3 encodes a nuclear protein that contains a putative transcriptional regulator with two plant homeodomain (PHD) finger motifs. To identify the role of Ehd3 within the gene regulatory network for rice flowering, we compared the transcript levels of genes related to rice flowering in wild-type plants and ehd3 mutants. Increased transcription of Ghd7 under LD conditions and reduced transcription of downstream Ehd1 and FT-like genes in the ehd3 mutants suggested that Ehd3 normally functions as an LD downregulator of Ghd7 in floral induction. Furthermore, Ehd3 ghd7 plants flowered earlier and show higher Ehd1 transcript levels than ehd3 ghd7 plants, suggesting a Ghd7-independent role of Ehd3 in the upregulation of Ehd1. Our results demonstrate that the PHD-finger gene Ehd3 acts as a promoter in the unique genetic pathway responsible for photoperiodic flowering in rice.