Flowering Date1, a major photoperiod sensitivity gene in adzuki bean, is a soybean floral repressor E1 ortholog.

Adzuki bean is an important legume crop originating in temperate regions, with photoperiod in sensitivity being a key factor in its latitudinal adaptation. The Flowering Date1 (FD1) gene has a large effect on the photoperiodic response of flowering time, but the molecular basis for the effect of this locus is undetermined. The present study delimited the FD1 locus to a 17.1 kb sequence, containing a single gene, an E1 ortholog (VaE1). A comparison between Vigna angularis 'Shumari' (photoperiod insensitive) and 'Acc2265' (photoperiod sensitive) identified 29 insertions/deletions and 178 SNPs upstream of VaE1 in the FD1 locus. VaE1 expression in 'Acc2265' was greater under long-day than short-day conditions, whereas VaE1 expression in 'Shumari' was lower regardless of day length. These findings suggested that responsible gene of FD1 is a VaE1, which acts as a floral repressor by being upregulated in response to long-day conditions. The inability to upregulate VaE1 under long-day conditions was linked to its ability to flower under these conditions. These results provide greater understanding of the molecular control of a flowering date and clues enabling the breeding of adzuki bean at higher latitudes.

Mapping of QTLs controlling epicotyl length in adzuki bean (Vigna angularis).

Epicotyl length (ECL) of adzuki bean (Vigna angularis) affects the efficiency of mechanized weeding and harvest. The present study investigated the genetic factors controlling ECL. An F2 population derived from a cross between the breeding line 'Tokei1121' (T1121, long epicotyls) and the cultivar 'Erimo167' (common epicotyls) was phenotyped for ECL and genotyped using simple sequence repeats (SSRs) and single-nucleotide polymorphism (SNP) markers. A molecular linkage map was generated and fifty-two segregating markers, including 27 SSRs and 25 SNPs, were located on seven linkage groups (LGs) at a LOD threshold value of 3.0. Four quantitative trait loci (QTLs) for ECL, with LOD scores of 4.0, 3.4, 4.8 and 6.4, were identified on LGs 2, 4, 7 and 10, respectively; together, these four QTLs accounted for 49.3% of the phenotypic variance. The segregation patterns observed in F5 residual heterozygous lines at qECL10 revealed that a single recessive gene derived from T1121 contributed to the longer ECL phenotype. Using five insertion and deletion markers, this gene was fine mapped to a ~255 kb region near the end of LG10. These findings will facilitate marker-assisted selection for breeding in the adzuki bean and contribute to an understanding of the mechanisms associated with epicotyl elongation.

The temperature sensitive hybrid breakdown 1 induces low temperature-dependent intrasubspecific hybrid breakdown in rice.

Hybrid breakdown (HB) is an important type of post-zygotic reproductive barrier that inhibits hybrid production during the process of cross-breeding. A novel low temperature-dependent HB was identified in a chromosomal segment substitution line (CSSL) library derived from a cross of two rice (Oryza sativa L. japonica) cultivars, Yukihikari and Kirara397. A set of weakness symptoms in a target CSSL was observed at 23°C, but was rescued at 27°C and/or 30°C. Genetic analysis of HB using an F2:3 population of a cross between a target CSSL and Kirara397 found that a recessive temperature sensitive hybrid breakdown1 (thb1) gene from Yukihikari caused HB in the genetic background of Kirara397. Molecular mapping showed that thb1 was located within a 199-kb fragment on chromosome 6. A genetic study of F2 populations of reciprocal crosses between Yukihikari and Kirara397 confirmed that this HB was induced by the interaction of two recessive genes. These results provide important clues to further dissect the mechanism of generation of a novel temperature sensitive HB in rice intrasubspecific crosses and suggest that these linked markers will useful in rice breeding.

Development and characterization of chromosome segment substitution lines derived from backcross between japonica donor rice cultivar Yukihikari and japonica recipient cultivar Kirara397.

Grain yield-related traits and grain quality-related traits are important for rice cultivars. The quantitative trait loci (QTLs) involved in controlling the natural variation in these traits among closely related cultivars are still unclear. The present study describes the development of a novel chromosome segment substitution line (CSSL) population derived from a cross between the temperate japonica cultivars Yukihikari and Kirara397, which are grown in Hokkaido, the northernmost limit for rice cultivation. Days to heading, culm length, panicle length, panicle number, brown grain weight per plant, thousand brown grain weight, brown grain length, brown grain width, brown grain thickness, apparent amylose content, and protein content were evaluated. Panicle length, brown grain length and amylose content differed significantly in the parental cultivars. Thirty-five significant changes in the evaluated traits were identified in the CSSLs. A total of 28 QTLs were located on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 10, 11 and 12. These findings could be useful for breeding rice cultivars in the northernmost limit for rice cultivation.

Identification of quantitative trait loci for rice grain quality and yield-related traits in two closely related Oryza sativa L. subsp. japonica cultivars grown near the northernmost limit for rice paddy cultivation.

Quantitative trait loci (QTLs) associated with eating quality, grain appearance quality and yield-related traits were mapped in recombinant inbred lines (RILs) derived from closely related rice (Oryza sativa L. subsp. japonica) cultivars, Yukihikari (good eating quality) and Joiku462 (superior eating quality and high grain appearance quality). Apparent amylose content (AAC), protein content (PC), brown grain length (BGL), brown grain width (BGWI), brown grain thickness (BGT), brown grain weight per plant (BGW) and nine yield-related traits were evaluated in 133 RILs grown in four different environments in Hokkaido, near the northernmost limit for rice paddy cultivation. Using 178 molecular markers, a total of 72 QTLs were detected, including three for AAC, eight for PC, two for BGL, four for BGWI, seven for BGT, and six for BGW, on chromosomes 1, 2, 3, 4, 6, 7, 8, 9, 11 and 12. Fifteen intervals were found to harbor multiple QTLs affecting these different traits, with most of these QTL clusters located on chromosomes 4, 6, 8, 9 and 12. These QTL findings should facilitate gene isolation and breeding application for improvement of eating quality, grain appearance quality and yield of rice cultivars.

Ethylene-dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize.

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen-deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene-dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1-aminocyclopropane-1-carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen-deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis.

Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure.

Development of genome-wide PCR-based markers from insertion, deletion and single nucleotide polymorphisms for closely related Japanese rice cultivars and identification of QTLs for the appearance of cooked rice and polished rice.

Appearance of rice grain is an important property, affecting its acceptance by consumers. Moreover, appearance is a complex characteristic involving many components, including glossiness and whiteness. The genetic bases for the glossiness of cooked rice and the whiteness of polished rice (WPR) were determined using 133 recombinant inbred lines (RILs) derived from a cross between two closely related cultivars from Hokkaido, Joiku462, with high glossiness and whiteness, and Yukihikari, an ancestor of Joiku462 with low glossiness and whiteness. Analyses identified 167 genome-wide InDel markers, five cleaved amplified polymorphic sequences (CAPS) and eight derived CAPS markers differentiating the parental lines. The glossiness area (GLA) and glossiness strength (GLS) of cooked rice and WPR were determined for RILs in two locations, Pippu and Sapporo, Hokkaido. Four QTLs were detected. qGLA10 and qGLS9 were detected on chromosomes 10 and 9, respectively, with both being significant at both geographic locations. qWPR1 on chromosome 1 was significant at Pippu, and qWPR4 on chromosome 4 was significant at Sapporo. The Joiku462 alleles at all QTLs increased each trait. The PCR-based markers flanking these four QTLs may be useful for improvement of GLA, GLS and WPR.

Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots.

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.

The rice RCN11 gene encodes ß1,2-xylosyltransferase and is required for plant responses to abiotic stresses and phytohormones.

Seed germination rates and plant development and growth under abiotic stress are important aspects of crop productivity. Here, our characterization of the rice (Oryza sativa L.) mutant reduced culm number11 (rcn11) showed that RCN11 controls growth of plants exposed to abnormal temperature, salinity and drought conditions. RCN11 also mediates root aerenchyma formation under oxygen-deficient conditions and ABA sensitivity during seed germination. Molecular studies showed that the rcn11 mutation resulted from a 966-bp deletion that caused loss of function of ß1,2-xylosyltransferase (OsXylT). This enzyme is located in the Golgi apparatus where it catalyzes the transfer of xylose from UDP-xylose to the core ß-linked mannose of N-glycans. RCN11/OsXylT promoter activity was observed in the basal part of the shoot containing the shoot and axillary meristems and in the base of crown roots. The level of RCN11/OsXylT expression was regulated by multiple phytohormones and various abiotic stresses suggesting that plant specific N-glycosylation is regulated by multiple signals in rice plants. The present study is the first to demonstrate that rice ß1,2-linked xylose residues on N-glycans are critical for seed germination and plant development and growth under conditions of abiotic stress.

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.

Rice RCN1/OsABCG5 mutation alters accumulation of essential and nonessential minerals and causes a high Na/K ratio, resulting in a salt-sensitive phenotype.

Mineral balance and salt stress are major factors affecting plant growth and yield. Here, we characterized the effects of rice (Oryza sativa L.) reduced culm number1 (rcn1), encoding a G subfamily ABC transporter (OsABCG5) involved in accumulation of essential and nonessential minerals, the Na/K ratio, and salt tolerance. Reduced potassium and elevated sodium in field-grown plants were evident in rcn1 compared to original line 'Shiokari' and four independent rcn mutants, rcn2, rcn4, rcn5 and rcn6. A high Na/K ratio was evident in the shoots and roots of rcn1 under K starvation and salt stress in hydroponically cultured plants. Downregulation of SKC1/OsHKT1;5 in rcn1 shoots under salt stress demonstrated that normal function of RCN1/OsABCG5 is essential for upregulation of SKC1/OsHKT1;5 under salt stress. The accumulation of various minerals in shoots and roots was also altered in the rcn1 mutant compared to 'Shiokari' under control conditions, potassium starvation, and salt and d-sorbitol treatments. The rcn1 mutation resulted in a salt-sensitive phenotype. We concluded that RCN1/OsABCG5 is a salt tolerance factor that acts via Na/K homeostasis, at least partly by regulation of SKC1/OsHKT1;5 in shoots.

The rice REDUCED CULM NUMBER11 gene controls vegetative growth under low-temperature conditions in paddy fields independent of RCN1/OsABCG5.

Low temperature tolerance during vegetative growth is an important objective in rice (Oryza sativa L.) breeding programs. We isolated a novel reduced culm number mutant, designated reduced culm number11 (rcn11), by screening under low-temperature condition in a paddy fields. Since the shoot architecture of the rcn11 was very similar to that of the rcn1, we examined whether RCN11 is involved in RCN1/OsABCG5-associated vegetative growth control. The rcn11 mutant has no mutation in the RCN1/OsABCG5 gene and rcn11 has no effect on RCN1/OsABCG5 gene expression. In the rcn1 mutant, RCN1/OsABCG5 was upregulated showing that RCN1/OsABCG5 is controlled by negative feedback regulation. Absence of an effect of rcn11 on RCN1/OsABCG5 feedback regulation supported that RCN11 is not involved in the RCN1/OsABCG5-associated transport system. A genetic allelism test and molecular mapping study showed that rcn11 is independent of rcn1 on rice chromosome 3 and located on chromosome 8. The rcn1 rcn11 phenotype suggests that RCN11 acts on vegetative growth independent of RCN1/OsABCG5. A root development comparison between rcn1 and rcn11 in young seedlings represented that rcn11 reduced crown root number and elongation, whereas rcn1 reduced lateral root density and elongation. Thus, rcn11 will shed new light on vegetative growth control under low temperature.

Genome-wide analysis and expression profiling of half-size ABC protein subgroup G in rice in response to abiotic stress and phytohormone treatments.

The roles of the proteins encoded by half-size adenosine triphosphate-binding cassette transporter subgroup G (ABCG) genes in abiotic stress responses are starting to be established in the dicot model Arabidopsis thaliana. In the monocot model rice, the functions of most half-size ABCG proteins in abiotic stress responses are unknown. Rcn1/OsABCG5 is an essential transporter for growth and development under abiotic stress, but its molecular function remains largely unclear. Here, we present a comprehensive overview of all 30 half-size ABCG genes in rice, including their gene structures, phylogeny, chromosome locations, and conserved motifs. Phylogenetic analysis revealed that the half-size OsABCG proteins were divided to four classes. All seven rice intronless genes, including Rcn1/OsABCG5, were in Class III, like the 12 intronless ABCG genes of Arabidopsis. The EST and FL-cDNA databases provided expression information for 25 OsABCG genes. Semi-quantitative and quantitative RT-PCR analyses demonstrated that seven OsABCG genes were up-regulated in seedlings, shoots or roots following treatments with abiotic stresses (6, 17, 42 °C, NaCl, or mannitol) and abscisic acid. Another 15 OsABCG genes were up-regulated under at least one of the abiotic stress conditions and other phytohormones besides abscisic acid. Hierarchical clustering analysis of gene expression profiles showed that expression of the OsABCG genes could be classified into four clusters. The Rcn1/OsABCG5 cluster was up-regulated by abscisic acid and included OsABCG2, 3, 13, and 27. The present study will provide a useful reference for further functional analysis of the ABCGs in monocots.

The genetically unstable dwarf locus in azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi).

We characterized a spontaneous dwarf mutant showing extremely short internodes and dark green leaves originating from azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi) cultivar "Erimo-shouzu." F(1) plants of 3 cross combinations between the dwarf mutant and several representative wild-type plants, Erimo-shouzu, V. angularis accession Acc2265 and wild relative V. riukiuensis accession Acc2482, supported the dwarf genotype being recessive. In a total of 3328 F(2) progeny of these 3 crosses, 65 dwarfs (2.0%) and 5 chimeric dwarfs (0.2%) segregated and the remainder were wild-type plants (97.8%). In F(3) progeny derived from self-pollinated dwarf F(2) plants, we observed wild type (54.3%), dwarf (39.1%), and chimeric dwarf (6.5%) plants. Two types of chimeric plants were observed: dwarf branches on the axils of wild-type plant stems and wild-type branches on the axils of dwarf stems. In 21 dwarf F(2) plants, the dwarf trait cosegregated with simple sequence repeat marker CEDG154 on chromosome 4. Conversely, homozygote F(2) plants at this chromosomal segment from the dwarf mutant frequently (>90%) expressed the wild-type phenotype. We concluded that the dwarf phenotype is mitotically and meiotically inheritable and controlled by a single genetically unstable locus, designated Azuki Dwarf1 (AD1), which converts between 2 phenotypic states bidirectionally.

Genomic diversity of germinating scutellum specific gene P23k in barley and wheat.

P23k is a 23 kDa protein involved in sugar translocation in the scutellum of germinating barley seeds. The present study was carried out to provide the genomic characterization for P23k gene in terms of copy number, chromosome mapping, genetic mapping and expression analysis in germinating sculletum in two major Triticeae crops, barley and wheat, and their relatives. Southern blotting showed that a variable copy number with different restriction fragment sizes was found among 15 Hordeum accessions, while low copy number were found to be conserved in 23 Triticum and 3 Aegilops accessions. Genetic and physical mapping study identified that the P23k gene is duplicated in wild and cultivated barley on chromosomes 1H, 2H, and 3H, and further tandem duplication on chromosomes 1H and 3H. In contrast, the wheat P23k is located on chromosome 3A of durum wheat and at the distal portion of the long arms of 3A and 3D chromosomes of bread wheat. Northern blotting showed remarkably high accumulation of P23k transcript in the germinating scutellum in cultivated and wild barley, whereas very few or no accumulation was detected in diploid, tetraploid, and hexaploid wheat accessions. The present study suggests a simple scenario where the ancestral P23k is encoded on the distal portion of an ancestral chromosome of homoeologous chromosome 3. Beside of polyploidy, dispersed and tandem duplications could trigger generation of the P23k family in the Hordeum lineage, while an ancestral P23k has been conserved in homoeologous 3A and 3D chromosomes in the wheat lineage.

The male sterility locus ms3 is present in a fertility controlling gene cluster in soybean.

Soybean [Glycine max (L.) Merr.] is self-pollinated. To produce large quantities of hybrid seed, insect-mediated cross-pollination is necessary. An efficient nuclear male-sterile system for hybrid seed production would benefit from molecular and/or phenotypic markers linked to male fertility/sterility loci to facilitate early identification of phenotypes. Nuclear male-sterile, female-fertile ms3 mutant is a single recessive gene and displays high outcrossed seed set with pollinators. Our objective was to map the ms3 locus. A segregating population of 150 F(2) plants from Minsoy (PI 27890) x T284H, Ms3ms3 (A00-68), was screened with 231 simple sequence repeat markers. The ms3 locus mapped to molecular linkage group (MLG) D1b (Gm02) and is flanked by markers Satt157 and Satt542, with a distance of 3.7 and 12.3 cM, respectively. Female-partial sterile-1 (Fsp1) and the Midwest Oilseed male-sterile (msMOS) mutants previously were located on MLG D1b. msMOS and Fsp1 are independent genes located very close to each other. All 3 genes are located in close proximity of Satt157. We believe that this is the first report of clustering of fertility-related genes in plants. Characterization of these closely linked genes may help in understanding the evolutionary relationship among them.

Rice shoot branching requires an ATP-binding cassette subfamily G protein.

* Shoot branching is important for the establishment of plant architecture and productivity. * Here, characterization of rice (Oryza sativa) reduced culm number 1 (rcn1) mutants revealed that Rcn1 positively controls shoot branching by promoting the outgrowth of lateral shoots. Molecular studies revealed that Rcn1 encodes a novel member of ATP-binding cassette protein subfamily G (ABCG subfamily), also known as the white-brown complex (WBC) subfamily, and is designated OsABCG5. * Rcn1 is expressed in leaf primordia of main and axillary shoots, and in the vascular cells and leaf epidermis of older leaves. In addition, Rcn1 is expressed in the crown root primordia, endodermis, pericycle and stele in the root. No effect on Rcn1 expression in shoots or roots was seen when the roots were treated with auxins. Phenotypic analyses of rcn1 and tillering dwarf 3 (d3) double mutants at the seedling stage clarified that Rcn1 works independently of D3 in the branching inhibitor pathway. * Rcn1 is the first functionally defined plant ABCG protein gene that controls shoot branching and could thus be significant in future breeding for high-yielding rice.

Genetic interaction between 2 tillering genes, reduced culm number 1 (rcn1) and tillering dwarf gene d3, in rice.

Mutant genes, reduced culm number 1 (rcn1) and bunketsuwaito tillering dwarf (d3), affect tiller number in rice (Oryza sativa L.) in opposite directions. The d3 mutant was reported to increase tiller number and reduce plant stature. Our objective was to compare the phenotype of the d3rcn1 double mutant with each single mutant and parental rice cultivar "Shiokari" and to clarify whether the Rcn1 gene interacted with the D3 gene. We recovered a new rcn1 mutant from Shiokari and developed d3rcn1 double mutant with Shiokari genetic background. A new rcn1 mutant, designated as "S-97-61" exhibited a reduction in tiller number and plant stature to about the same level as the previously reported original rcn1 mutant. Three near-isogenic lines, rcn1 mutant, d3 mutant, and d3rcn1 double mutant, were grown together with the parental Shiokari. The reduction in tillering by the rcn1 mutation was independent of the d3 genotype, and tillering number of d3rcn1 double mutant was between those of the d3 and rcn1 mutants. These results demonstrated that the Rcn1 gene was not involved in the D3-associated pathway in tillering control.