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The DNA binding with one finger (Dof) proteins are plant-specific transcription factors that contain a particular class of zinc-finger DNA-binding domain. Thirty-different Dof genes are predicted to exist in the rice genome based on in silico analysis. A member of the Dof transcription factor family, Dof1 of rice (Oryza sativa), was identified from a dissociation (Ds) insertion mutant population. The Ds-tagged rice mutant line osdof1-Ds was identified from a population of rice tagged with promoterless β-glucuronidase (GUS) in T-DNA, and showed wound-responsive GUS expression. Genomic Southern analysis revealed that this mutant line contained a single copy of the Ds gene trap. Four wound-response-like cis-acting elements (PI-II, EIRE, W box, and G box-like elements) were found in the OsDof1 promoter region. To analyze the cis-acting elements, we constructed fusion genes between the OsDof1 promoter and the GUS reporter gene and transformed Arabidopsis and rice plants with these constructs. GUS gene expression was induced in leaf, seed, culm, and flower tissues of the osdof1-Ds mutant after wounding. Furthermore, histochemical analysis of the OsDof1 promoter::GUS fusion transgenic plants showed an early response to wounding. In addition, OsDof1 mRNA was induced by pathogen treatment. These results suggest that OsDof1 is a transcriptional activator that regulates gene expression in response to wounding.

Two genetic linkage maps, constructed by DH and RILs populations derived from the same parents, were carried out for the identification and comparison of QTLs controlling yield traits across different years in rice ( Oryza sativa L.). A total of 194 SSR and STS markers were used in two maps, of which 114 markers were same. The distribution of Samgang allele was higher in RILs population than it in DH population. Comparing with DH population, RILs population has more lines with higher yield and wider phenotypic transgressive segression for yield traits. Although most of QTLs for the same trait were different in two populations across different years, 8 QTLs (including gwp11.1, spp5.1, spp10.1, spp11.2, ssr1.1, ssr11.1, tgw9.1 and tgw11.1) were detected over 2 yr. It is important to note that ppp10.1, spp10.1 and tgw9.1 were identified in two populations, while spp10.1 and tgw9.1 were simultaneity observed across different years. Epistatic effects were more important than additive effects for PPP, SPP, yield in DH population and TGW, yield in RILs population. Epistatic effects of DH and RILs populations were different on the same genetic background in the present study, which illuminated the QE interaction played an important role on epistatic effect. Identification and comparison of QTLs for yield traits in DH and RILs populations should provide various and more precise information. The QTLs identified in present study would be valuable in marker-assisted selection program for improving rice yield.

Two genetic linkage maps based on doubled haploid (DH) and recombinant inbred lines (RILs) populations, derived from the same indica-japonica cross ‘Samgang × Nagdong’, were constructed to analyze the quantitative trait loci (QTLs) affecting agronomic traits in rice. The segregations of agronomic traits in RILs population showed larger variations than those in DH population. A total of 10 and 12 QTLs were identified on six chromosomes using DH population and seven chromosomes using RILs population, respectively. Three stable QTLs including pl9.1, ph1.1, and gwp11.1 were detected through different years. The percentages of phenotypic variation explained by individual QTLs ranged from 8 to 18% in the DH population and 9 to 33% in the RILs population. Twenty-three epistatic QTLs were identified in the DH population, while 21 epistatic QTLs were detected in the RILs population. Epistatic interactions played an important role in controlling the agronomic traits genetically. Four significant main-effect QTLs were involved in the digenic interactions. Significant interactions between QTLs and environments (QE) were identified in two populations. The QTLs affecting grain weight per panicle (GWP) were more sensitive to the environmental changes. The comparison and QTLs analysis between two populations across different years should help rice breeders to comprehend the genetic mechanisms of quantitative traits and improve breeding programs in marker-assisted selection (MAS).

Rice is the staple food of more than 50% of the worlds population. Cultivated rice has the AA genome (diploid, 2n=24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized transposable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos (Hirochika. 1997) have been used to generate gene-tagged populations. To date T-DNA and maize transposable element systems has been utilized as main insertional mutagens in rice. A main drawback of a T-DNA scheme is that Agrobacteria- mediated transformation in rice requires extensive facilities, time, and labor. In contrast, the Ac/Ds system offers the advantage of generating new mutants by secondary transposition from a single tagged gene. Revertants can be utilized to correlate phenotype with genotype. To enhance the efficiency of gene detection, advanced gene-tagging systems i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of insertionally mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been launched by collaborative works from 2001 in Korea.

With 3 figures Abstract Male sterility is an important trait for rice breeding programmes based on heterosis. To generate male-sterile plants, we utilized RNAi technology with the promoters for two anther-specific genes, Osc4 and Osg6b, and one for rice allene oxide synthase (OsAOS). AOS catalyzes the biosynthesis of jasmonic acid and plays an important role in anther development. The promoters isolated from Osc4 and Osg6b were introduced into pK7gwiwgL, a promoter-less RNAi Gateway binary vector. Partial DNA fragments of OsAOS1 and OsAOS2 were combined into the Osc4::RNAi and Osg6b::RNAi cassettes by LR recombination. The resulting four RNAi constructs were transformed into rice calli by Agrobacterium. During the reproductive growth stage, 28% of OsAOS1 and 48% of OsAOS2 transgenic plants under the control of the Osg6b promoter exhibited a complete- or severe-sterility phenotype compared with the normal wild type. Here, we report that RNA interference of OsAOS2 with the Osg6b promoter is the most effective system for obtaining engineered genic male sterility in rice.

Seed shattering is an important factor causing loss of grain yield before and during rice harvest. In the present study, the quantitative trait loci regarding shattering scale, breaking tensile strength (BTS) and abscission layer (AL), the parameters evaluating seed shattering habit by hand gripping, a digital force gauge and observation on AL, respectively, were identified by using an doubled haploid line (DHL) population from a cross between a loose-shattering type Tongil variety, ‘Samgang’, and a moderately difficult shattering japonica variety, ‘Nagdong’. Eight QTLs consisted in four QTLs for shattering scale, two QTLs for AL, each one QTL for pulling and bending strength were detected on six chromosomes, respectively. Among them, Qss1 with flanking markers RM6696 and RM476 explained 31% of phenotype variation in shattering scale. Furthermore, two new QTLs controlling shattering habit, Qss5-2 and Qal5-1, were located on chromosome 5 at the interval 5028–5037 and 5021-RM289. They explained 10% and 12% of phenotype variations, respectively. A total of eleven digenic epistatic loci were identified for four parameters. The identification of QTLs affecting seed shattering habits is favorable to thoroughly dissect the genetic mechanism of the shattering habit and to apply for marker-assisted selection in rice breeding system of specific regions.

Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h, from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F(2) plants and five F(3) lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3' splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 (OsCPL1). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development.

Quantitative trait loci (QTL) associated with six physical traits of cooked rice and seven chemical properties of rice grain were identified using a recombinant inbred (RI) population of rice evaluated over 3 years at the National Honam Agricultural Research Institute in Korea. The RI population consisted of 164 lines derived from a cross between Milyang23 and Gihobyeo, and the genetic map consisted of 414 molecular markers. A total of 49 QTL were identified for the 13 physico-chemical properties using composite interval mapping. Of these, 13 QTL were identified for 2 or more years, while 36 were detected in only 1 year. Five QTL were identified over all 3 years and will be useful for marker-assisted improvement of rice grain quality in Korea. The two QTL with the highest LOD scores, adhesiveness1.2 and potassium content7.1, provide a valuable starting point for positional cloning of genes underlying these QTL.

본 연구는 동진벼 유래의 Ac/Ds 삽입변이집단의 GUS 분석을 통하여 뿌리 및 미숙종자에서 강하게 GUS가 발현한 개체를 선발하여 FST(flanking sequence tag) 분석 한 결과 Ds 전이 인자가 3번 염색체 zinc finger RING-H2 관련 Oszinc626 유전자의 첫 번째 exon 부위에 single copy로 삽입되어 있었으며, 선발변이체는 뿌리 및 종자 발달이 정상인 동진벼에 비해 매우 낮은 것으로 나타났다. Oszinc626 유전자는 RING-H2 type(C3-H2-C3)으로 $Cys-X_2-Cys-X_{28}-Cys-X-His-X_2-His-X_2-Cys-X_{14}-Cys-X_2-Cys$ 배열이 C-terminus 가장 말단에 위치하며, 49 kDa의 분자량을 가지고 있다. 또한 Southern blot 분석에서 Oszinc626 유전자는 벼 게놈상에 single copy로 존재하였다. RT-PCR을 통한 돌연변이 유전자의 발현분석 결과 250 mM의 염과, $4^{\circ}C$ 저온등과 같은abiotic stress에 의해 발현이 증가함을 보였고, 호르몬처리에 있어서 ABA와 IAA의 식물호르몬을 처리했을 경우 24시간까지 계속해서 발현양이 증가하는 것을 보이는 반면, 2,4-D 처리의 경우 30분 후에 발현이 일시적으로 증가되었으나 이후 발현이 급속히 감소한 것을 보였다. 벼의 조직 별 발현 검정에서 미성숙한 종자, 뿌리 분열조직 및 신초 등 주로 생장점 부위에서 강하게 발현되는 것을 보임에 따라 Oszinc626 유전자의 경우 식물의 생장에 관여하는 주동 유전자의 하나로 판단된다. 【Ac/Ds mutant lines of this study were transgenic rice plants, each of which harbored the maize transposable element Ds together with a GUS coding sequence under the control of a promoterless(Ds-GUS). We selected the mutants that were GUS expressed lines, because the GUS positive lines will be useful for identifying gene function in rice. One of these mutants was identified knock-out at Oszinc626(NP_001049991) gene, encoding a RING-H2 zinc-finger protein, by Ds insertion. In this mutant, while primary root development is normal, secondary root development from lateral root was very poor and seed development was incomplete compare with normal plant. RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, a few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. The consensus of the RING-H2(C3-H2-C3 type) domain for this group of protein is $Cys-X_2-Cys-X_{28}-Cys-X-His-X_2-His-X_2-Cys-X_{14}-Cys-X_2-Cys$ . Oszinc626 encodes a predicted protein product of 445 amino acids residues with a molecular mass of 49 kDa, with a RING-zinc-finger motif located at the extreme end of the C-terminus. RT-PCR analysis indicated that the expression of Oszinc626 gene was induced by IAA, cold, dehydration, high-salinity and abscisic acid, but not by 2,4-D, and the transcription of Oszinc626 gene accumulated primarily in rice immature seeds, root meristem and shoots. The gene accumulation patterns were corresponded with GUS expression.】

The Korean Rice Ds-tagging lines Database (KRDD) is designed to provide information about Ac/Ds insertion lines and activation tagging lines using japonica rice. This database has provided information on 18,158 Ds lines, which includes the ID, description, photo image, sequence information, and gene characteristics. The KRDD is visualized using a web-based graphical view, and anonymous users can query and browse the data using the search function. It has four major menus of web pages: (i) a Blast Search menu of a mutant line; Blast from rice Ds-tagging mutant lines; (ii) a primer de-sign tool to identify genotypes of Ds insertion lines; (iii) a Phenotype menu for Ds lines, searching by identi-fication name and phenotype characteristics; and (iv) a Management menu for Ds lines. Availability: The KRDD can be accessed at the web site of the National Institute of Agricultural Biotechnology (http://www. niab.go.kr/RDS/) Keywords: Ac/Ds database, Ds phenotype, Ds-tagging line, insertion line, KRDD

A population of 164 recombinant inbred lines (RILs) of rice (Oryza sativa L.) derived from a cross between Milyang23 and Gihobyeo was evaluated for nine phenotypic characters over three years and two regions in Korea. The population had been previously mapped using 414 molecular markers. Genotype x environment (G x E) interaction was analyzed for six grain yield-related traits and three agronomic traits across years and locations using the AMMI model. The quantitative trait loci (QTLs) were detected by interval mapping and composite interval mapping. A total of 75 QTLs were identified for the nine traits across five environments and they were categorized as (i) 29 QTLs with main effect, (ii) 18 QTLs with minor effect, (iii) 13 QTLs with G x E interaction effect, (iv) six QTLs with both main effect and G x E interaction effect, and (v) nine potential QTLs with low log of the odds (LOD) scores. The AMMI model explained from 68.6% to 84.7% of the interaction effect and 19 QTLs were significantly associated with G x E interaction. Culm length had the least G x E, while the maximum G x E interaction was exhibited for spikelets per panicle (39.7%) and percent ripened grain (35.3%). Markers closely linked to main effect QTLs will be most useful for marker-assisted breeding.

OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a "blade to sheath transformation" phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::DeltaOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles.

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes.

A phosphate starvation-induced acid phosphatase cDNA was cloned from the rice, Oryza sativa. The cDNA encoding O. sativa acid phosphatase (OsACP1) has 1100 bp with an open reading frame of 274 amino acid residues. The deduced amino acid sequence of OsACP1 cDNA showed 53% identity to tomato acid phosphatase and 46-50% identity to several other plant phosphatases. OsACP1 expression was up-regulated in the rice plant and in cell culture in the absence of phosphate (Pi). The induced expression of OsACP1 was a specific response to Pi starvation, and was not affected by the deprivation of other nutrients. OsACP1 expression was responsive to the level of Pi supply, with transcripts of OsACP1 being abundant in Pi-deprived root. The OsACP1 cDNA was expressed as a 30 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsACP1 gene was introduced into Arabidopsis via Agrobacterium-mediated transformation. Functional expression of the OsACP1 gene in the transgenic Arabidopsis lines was confirmed by Northern blot and Western blot analyses, as well as phosphatase activity assays. These results suggest that the OsACP1 gene can be used to develop new transgenic dicotyledonous plants able to adapt to Pi-deficient conditions.

Using a population of recombinant inbred lines of the 164 genotypes derived from a cross between ‘Milyang 23’ (indica) and ‘Gihobyeo’ (japonica) in rice (Oryza sativa L.), salt tolerance was evaluated at a young seedling stage in concentrations of 0.5% and 0.7% NaCl. Mapping quantitative trait loci (QTLs) related to salt tolerance was carried out by interval mapping using Qgene 3.0. Two QTLs (qST1 and qST3) conferring salt tolerance at young seedling stage were mapped on chromosome 1 and 3, respectively, and explained 35.5–36.9% of the total phenotypic variation in 0.5% and 0.7% NaCl. The favourable allele of qST1 was contributed by ‘Gihobyeo’, and that of qST3 by ‘Milyang 23’. The results obtained in 0.5% and 0.7% NaCl for 2 years were similar in flanked markers and phenotypic variation.

Easy shattering reduces yield due to grain loss during harvest in cereals. Shattering is also a hindrance in breeding programs that use wild accessions because the shattering habit is often linked to desirable traits. We characterized a shattering mutant line of rice, Hsh, which was derived from a nonshattering japonica variety, Hwacheong, by N-methyl-N-nitrosourea (MNU) treatment. The breaking tensile strength (BTS) of the grain pedicel was measured using a digital force gauge to evaluate the degree of shattering of rice varieties at 5, 10, 15, 20, 25, 30, 35, and 40 days after heading (DAH). The BTS of Hwacheong did not decrease with increasing DAH, maintaining a level of 180–240 gf, while that of Hsh decreased greatly during 10–20 DAH and finally stabilized at 50 gf. Optical microscopy revealed that Hsh had a well-developed abscission layer similar to the wild rice Oryza nivara (accession IRGC105706), while Hwacheong did not produce an abscission layer, indicating that the shattering of Hsh was caused by differentiation of the abscission layer. On the basis of the BTS value and morphology of the abscission layer of F1 plants and segregation data in F2 populations, it was concluded that the easy shattering of Hsh was controlled by the single recessive gene sh-h. The gene sh-h was determined to be located on rice chromosome 7 by bulked segregant analysis. Using 14 SSR markers on rice chromosome 7, the gene sh-h was mapped between the flanking markers RM8262 and RM7161 at distances of 1.6 and 2.0 cM, respectively. An SSR marker Rc17 cosegregated with the gene sh-h. The locus sh-h for shattering was tightly linked to the Rc locus conferring red pericarp, as well as a QTL qSDs-7-1 for seed dormancy, implying that this region might represent a domestication block in the evolutionary pathway of rice.

The spikelet identity gene "fzp" (frizzy panicle) is required for transformation of the floral meristems to inflorescent shoots. In fzp mutants, spikelets are replaced by branches and spikelet meristems produce massive numbers of branch meristems. We have isolated and characterized a new fzp mutant derived from anther culture lines in rice and designated as fzp-9(t). The fzp-9(t) mutant showed retarded growth habit and developed fewer tillers than those of the wild-type plant. The primary and secondary rachis branches of fzp-9(t) appeared to be normal, but higher-order branches formed continuous bract-like structures without developing spikelets. The genetic segregation of fzp-9(t) showed a good fit to the expected ratio of 3: 1. The sequence analysis of fzp-9(t) revealed that there is a single nucleotide base change upstream of the ERF (ethylene-responsive element-binding factor) domain compare to wild-type plant. The mutation point of fzp-9(t) (W66G) was one of the six amino acids of the ERF domain that contributed to GCC box-specific binding. The premature formation of a stop codon at the beginning of the ERF domain might cause a non-functional product.

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.

Anther culturability of rice is a quantitative trait controlled by nuclear-encoded genes. The identification of quantitative trait loci (QTL) and associated marker selection for anther culturability is important for increasing the efficiency of green plant regeneration from microspores. QTL associated with the capacity for green plant regeneration in anther culture of rice were mapped on chromosomes 3 and 10 using 164 recombinant inbred (RI) lines from a cross between ‘Milyang 23’ and ‘Gihobyeo’. The quantitative trait locus located on chromosome 10 was detected repeatedly when three anther culture methods were applied and was tightly linked to the markers, RG323, RG241 and RZ400. Associations between these markers and the efficacy of green plant regeneration in 43 rice cultivars and two F2 populations, ‘MG RI036’/‘Milyang 23’, and ‘MG RI036’;/‘IR 36’ were analysed. One of these markers, RZ400, was able to identify effectively genotypes with good (> 10.0%) and poor (< 3.0%) regenerability, based on the marker genotypes in the cultivars and two F2 populations. This marker enables the screening of rice germplasm for anther culturability and introgression into elite lines in breeding programmes.

Two cDNA clones, pOS-ACO2 and pOS-ACO3, encoding 1-aminocyclopropane-1-carboxylate (ACC) oxidase were isolated from rice seedling cDNA library. pOS-ACO3 is a 1,299 bp full-length clone encoding 321 amino acids (Mr=35.9 kDa), while pOS-ACO2 is 1,072 bp long and is a partial cDNA clone encoding 314 amino acids. These two deduced amino acid sequences share 70% identity, and display a high degree of sequence identity (72-92%) with previously isolated pOS-ACO1 of deepwater rice. The chromosomal location studies show that OS-ACO2 is positioned on the long arm of chromosome 9, while OS-ACO3 on the long arm of chromosome 2 of rice genome. A marked increase in the level of OS-ACO2 transcript was observed in IAA-treated etiolated rice seedlings, whereas the OS-ACO3 mRNA was greatly accumulated by ethylene treatment. Results of ethylene inhibitor studies indicated that auxin promotion of the OS-ACO2 transcription was not mediated through the action of auxin-induced ethylene. Thus, it appears that there are two groups of ACC oxidase transcripts in rice plants, either auxin-induced or ethylene-induced. The auxin-induced OS-ACO2 expression was partially inhibited by ethylene, while ethylene induction of OS-ACO3 transcription was completely blocked by auxin. These results indicate that the expression of ACC oxidase genes is regulated by complex hormonal networks in a gene specific manner in rice seedlings. Okadaic acid, a potent inhibitor of protein phosphatase, effectively suppressed the IAA induction of OS-ACO2 expression, suggesting that protein dephosphorylation plays a role in the induction of ACC oxidase by auxin. A scheme of the multiple regulatory pathways for the expression of ACC oxidase gene family by auxin, ethylene and protein phosphatase is presented.

We have previously transformed rice (Oyrza sativa L.) with the maize ribosome-inactivating protein b-32 gene (Zmcrip3a) and the phosphinothricin resistance gene (bar). In the present study, Southern blot hybridization analysis of 56 primary fertile transformants resulted in distinct band patterns, indicating that all the transformants had been generated by independent integration events and 30% of them contained a single copy of the transgene. Segregation analysis of 15 R0 plants revealed that transgene was stably transmitted to their progenies and Southern blot band patterns of R1 progenies remained the same as the corresponding parents, suggesting that all the loci of multiple integration events are genetically linked. Also, in most of the lines, physical presence of the b-32 transgene co-segregated with the phosphinothricin- resistant phenotype, confirming that the transgene is behaving as a normal locus in the genome. However, some of R1 seedlings that contained multiple copies of the transgene became sensitive to phosphinothricin, indicating that its expression was silenced. Immunoblot analysis demonstrated that b-32 protein was produced and the levels of expression differed in different lines, estimated to be 0.5–1% of total soluble protein in the leaf tissues. In addition, the transgene-encoded protein was preferentially processed in germinating seeds and young leaves of R2 transgenic plants in a way similar to that in maize kernels, suggesting that the processing mechanism is conserved in the germination stage between rice and maize.

To mutagenize rice genomes, a two-element system is utilized. This system comprises an immobile Ac element driven by the CaMV 35S promoter, and a gene trap Ds carrying a partial intron with alternative splice acceptors fused to the GUS coding region. Rapid, large-scale generation of a Ds transposant population was achieved using a plant regeneration procedure involving the tissue culture of seed-derived calli carrying Ac and Ds elements. During tissue cultures, Ds mobility accompanies changes in methylation patterns of a terminal region of Ds, where over 70% of plants contained independent Ds insertions. In the transposon population, around 12% of plants expressed GUS at the early seedling stage. A flanking-sequence-tag (FST) database has been established by cloning over 19,968 Ds insertion sites and the Ds map shows relatively uniform distribution across the rice chromosomes.

To mutagenize rice genomes, a two-element system is utilized. This system comprises an immobile Ac element driven by the CaMV 35S promoter, and a gene trap Ds carrying a partial intron with alternative splice acceptors fused to the GUS coding region. Rapid, large-scale generation of a Ds transposant population was achieved using a plant regeneration procedure involving the tissue culture of seed-derived calli carrying Ac and Ds elements. During tissue cultures, Ds mobility accompanies changes in methylation patterns of a terminal region of Ds, where over 70% of plants contained independent Ds insertions. In the transposon population, around 12% of plants expressed GUS at the early seedling stage. A flanking-sequence-tag (FST) database has been established by cloning over 19,968 Ds insertion sites and the Ds map shows relatively uniform distribution across the rice chromosomes.

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.

During brown planthopper (BPH) feeding on rice plants, we employed a modified representational difference analysis (RDA) method to detect rare transcripts among those differentially expressed in SNBC61, a BPH resistant near-isogenic line (NIL) carrying the Bph1 resistance gene. This identified 3 RDA clones: OsBphi237, OsBphi252 and OsBphi262. DNA gel-blot analysis revealed that the loci of the RDA clones in SNBC61 corresponded to the alleles of the BPH resistant donor Samgangbyeo. Expression analysis indicated that the RDA genes were up-regulated in SNBC61 during BPH feeding. Interestingly, analysis of 64 SNBC NILs, derived from backcrosses of Samgangbyeo with a BPH susceptible Nagdongbyeo, using a cleaved amplified polymorphic sequence (CAPS) marker indicated that OsBphi252, which encodes a putative lipoxygenase (LOX), co-segregates with BPH resistance. Our results suggest that OsBphi252 is tightly linked to Bph1, and may be useful in marker-assisted selection (MAS) for resistance to BPH.

With the completion of rice genome sequencing, the new challenge for the rice community is to unravel the biological functions of approximately 40,000 rice genes. To achieve this goal, a wide range of functional genomics tools, such as microarray, serial analysis of gene expression (SAGE), RNA interference (RNAi), insertional mutagenesis, and bioinformatics, have been established and employed. Insertional (T-DNA, transposon or retrotransposon) mutagenesis has proven to be one of the most efficient methodologies, because studies of mutants with detectable phenotypes have given us the greatest insight into the mechanisms underlying a wide range of biological processes in plants. Compared with T-DNA insertional mutagenesis, transposon insertional mutagenesis (or transposon tagging) has distinct advantages. Large-scale transposon mutagenized populations can be produced using a relatively small number of starter lines, as many independent insertions can be generated among the progeny of a single line. The tagged gene can be confirmed by revertants resulting from excision of the transposon.

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.

The spikelet identity gene "fzp" (frizzy panicle) is required for transformation of the floral meristems to inflorescent shoots. In fzp mutants, spikelets are replaced by branches and spikelet meristems produce massive numbers of branch meristems. We have isolated and characterized a new fzp mutant derived from anther culture lines in rice and designated as fzp-9(t). The fzp-9(t) mutant showed retarded growth habit and developed fewer tillers than those of the wild-type plant. The primary and secondary rachis branches of fzp-9(t) appeared to be normal, but higher-order branches formed continuous bract-like structures without developing spikelets. The genetic segregation of fzp-9(t) showed a good fit to the expected ratio of 3: 1. The sequence analysis of fzp-9(t) revealed that there is a single nucleotide base change upstream of the ERF (ethylene-responsive element-binding factor) domain compare to wild-type plant. The mutation point of fzp-9(t) (W66G) was one of the six amino acids of the ERF domain that contributed to GCC box-specific binding. The premature formation of a stop codon at the beginning of the ERF domain might cause a non-functional product.

Even though Ac/Ds gene-tagging systems have been established in many higher plants, maize is the only major plant in which short-distance transposition of Ac/Ds has been utilized to probe gene function. This study was performed to evaluate the efficiency of obtaining new alleles and functional revertants from Ds insertion loci in rice. By analyzing 1,580 plants and the progeny of selected lines, the insertion sites and orientations of Ds elements within 16 new heritable alleles of three rice loci were identified and characterized. Intragenic transposition was detected in both directions from the original insertion sites. The closest interval was 35 bp. Three of the alleles had two Ds elements in cis configuration in the same transcription units. We also analyzed the excision footprints of intragenic and extragenic transpositions in Ds-inserted alleles at 5 loci. The 134 footprints obtained from different plants revealed predominant patterns. Ds excision at each locus left a predominant footprint at frequencies of 30-75%. Overall, 66% of the footprints were 7-bp additions. In addition, 16% of the excisions left 0-, 3-, 6-, and 9-bp additions with the potential of conserving reading frame.

Salt tolerance was evaluated at the young seedling stage of rice (Oryza sativa L.) using recombinant inbred lines (MG RILs) from a cross between Milyang 23 (japonica/indica) and Gihobyeo (japonica). 22 of 164 MG RILs were classified as tolerant with visual scores of 3.5-5.0 in 0.7% NaCl. Interval mapping of QTLs related to salt tolerance was conducted on the basis of the visual scores at the young seedling stage. Two QTLs, qST1 and qST3, conferring salt tolerance, were detected on chromosome 1 and 3, respectively, and the total phenotypic variance explained by the two QTLs was 36.9% in the MG RIL population. qST1 was the major QTL explaining 27.8% of the total phenotypic variation. qST1 was flanked by Est12-RZ569A, and qST3 was flanked by RG179-RZ596. The detection of new QTLs associated with salt tolerance will provide important information for the functional analysis of rice salt tolerance.

We describe a rapid and simple procedure for homogenizing leaf samples suitable for mini/midi‐scale DNA preparation in rice. The methods used tungsten carbide beads and general vortexer for homogenizing leaf samples. In general, two samples can be ground completely within 11.3±1.5 sec at one time. Up to 20 samples can be ground at a time using a vortexer attachment. The yields of the DNA ranged from 2.2 to 7.6 µg from 25–150 mg of young fresh leaf tissue. The quality and quantity of DNA was compatible for most of PCR work and RFLP analysis.

A cDNA library was constructed using mRNA extracted from rice leaves infected with Xanthomonas oryzae pv. oryzae (Xoo), a bacterial leaf blight pathogen, to isolate rice genes induced by Xoo infection. Subtractive hybridization and differential screening of the cDNA library led to the isolation of many induced genes including a nucleotide diphosphate kinase 1 (OsNDPK1) and a pathogenesis-related protein 1 (OsPR1) cDNA. Nucleoside diphosphate kinases (NDPKs) are key metabolic enzymes that maintain the balance between cellular ATP and other nucleoside triphosphates (NTPs). Three other OsNDPK genes (NP922751, OsNDPK2 and OsNDPK3) found in databases were obtained by RT-PCR. Three different programs for predicting subcellular targeting indicated that OsNDPK1 and NP922751 were non-organellar, OsNDPK2 plastidic, and OsNDPK3 mitochondrial. Only transcripts of OsNDPK1 accumulated strongly after infection with Xoo. When rice plants were infected with Burkholderia glumae, a bacterial grain/seedling rot pathogen, the pattern of expression of the rice NDPK genes was similar to that following infection with Xoo. OsNDPK1 gene expression was also strongly induced in response to exposure to salicylic acid, jasmonic acid, and abscisic acid, although the level of transcripts and their pattern of expression depended on the inducer.

With the completion of genomic sequencing of rice, rice has been firmly established as a model organism for both basic and applied research. The next challenge is to uncover the functions of genes predicted by sequence analysis. Considering the amount of effort and the diversity of disciplines required for functional analyses, extensive international collaboration is needed for this next goal. The aims of this review are to summarize the current status of rice mutant resources, key tools for functional analysis of genes, and our perspectives on how to accelerate rice gene discovery through collaboration.

The resistance of rice to ozone (O3) is a quantitative trait controlled by nuclear genes. The identification of quantitative trait loci (QTL) and analysis of molecular markers of O3 resistance is important for increasing the resistance of rice to O3 stress. QTL associated with the O3 resistance of rice were mapped on chromosomes 1, 7 and 11 using 164 recombinant inbred (RI) lines from a cross between 'Milyang 23' and 'Gihobyeo'. The quantitative trait loci were tightly linked to the markers RG109, C507 and RG1094 and were detected in each of three replications. The association between these markers and O3 resistance in 26 rice cultivars and doubled haploid (DH) populations was analysed. The markers permit the screening of rice germplasm for O3 resistance and the introduction of resistance into elite lines in breeding programs.

Many aspects of epigenetic phenomena have been elucidated via studies of transposable elements. An active transposable element frequently loses its ability to mobilize and goes into an inactive state during development. In this study, we describe the cyclic activity of a maize transposable element dissociation (Ds) in rice. In rice genome, Ds undergoes the spontaneous loss of mobility. However, an inactive state of Ds can be changed into an active state during tissue culture. The recovery of mobility accompanies not only changes in the methylation patterns of the terminal region of Ds, but also alteration in the steady state level of the activator (Ac) mRNA that is expressed by a constitutive CaMV 35S promoter. Furthermore, the Ds-reactivation process is not random, but stage-specific during plantlet regeneration. Our findings have expanded previous observations on Ac reactivation in the tissue culture of maize.

A marker-assisted selection (MAS) breeding program was used to improve the plant regenerability of indica rice. A significant quantitative trait loci (QTL) that is associated with the capacity for green plant regeneration in the anther culture of rice was mapped on chromosome 10 using recombinant inbred (RI) population from Milyang 23/Gihobyeo. The marker that was chosen to follow the QTL region was used in MAS. This marker co-segregated with the regeneration ability in F2 individuals that were derived from MGRI 079/IR 36. In order to clarify the relationship between this marker and plant regenerability, the backcross population was screened with a RFLP marker. The capacity of plant regeneration of the backcross population was clearly distinguished by the marker genotype. The development of near isogenic line (NILs) with high regenerability through MAS will save time, labor, and cost in indica rice breeding.

Twenty primers of 20 mer referred to universal rice primer (URP) were developed from a repetitive sequence of rice genome. URP-PCR protocol employed stringent PCR with high annealing temperature throughout the thermo-cycling reaction, giving high reproducibility. Under the PCR condition, each single URP primer produced characteristic fingerprints from diverse genomes containing 14 plants, 7 animals and 6 microbes, indicating its universal applicability. The generality of URP-PCR was demonstrated by applying it to 15 cultivars from five rice species, 23 isolates in four Alternaria species producing host-specific toxins on different host plants and 12 bacterial strains including Escherichia coli, Salmonella spp., and Blucella abortus. PCR approach using URP primers will be useful for studying DNA diversity of most eukaryotic or prokaryotic genomes, especially at inter- and intraspecies levels.

The rice species Oryza sativa is considered to be a model plant because of its small genome size, extensive genetic map, relative ease of transformation and synteny with other cereal crops. Here we report the essentially complete sequence of chromosome 1, the longest chromosome in the rice genome. We summarize characteristics of the chromosome structure and the biological insight gained from the sequence. The analysis of 43.3 megabases (Mb) of non-overlapping sequence reveals 6,756 protein coding genes, of which 3,161 show homology to proteins of Arabidopsis thaliana, another model plant. About 30% (2,073) of the genes have been functionally categorized. Rice chromosome 1 is (G + C)-rich, especially in its coding regions, and is characterized by several gene families that are dispersed or arranged in tandem repeats. Comparison with a draft sequence indicates the importance of a high-quality finished sequence.

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.