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A complete mitochondrial genome sequence was determined by a hybrid approach with long- and short-read sequences in a japonica rice cultivar Taichung 65. The assembled mitochondrial genome was 401,060 bases in length and an overall GC content of 43.88% and predicted to harbor 57 protein-encoding, 18 transfer RNA, and 3 ribosomal RNA genes. Phylogenetic and sequence polymorphism analysis indicated that Taichung 65 is classified as typical japonica. The mitochondrial genome resource would serve as a fundamental tool to investigate cytoplasmic male sterility in rice.

Grain size is one of the most important traits that influence grain yield in rice. Here we report the isolation and characterization of the long grain1 (lin1) mutant, which exhibited long grains and increased grain weight. A field trial conducted over 2 years indicated that the grain weight increased 3.0–6.6% in the lin1 mutant compared with that of the wild-type Nipponbare. Whole-genome sequencing and genetic linkage analysis indicated that a 1-bp deletion within the coding sequence of Os06g0675200, which showed no homology to previously characterized genes, is responsible for the lin1 phenotype. Quantitative real-time PCR indicated that LIN1 transcripts were more abundant in young panicles than at advanced inflorescence developmental stages, which indicated that LIN1 controls grain length mainly at early stages of grain development. Sequence polymorphism analysis of LIN1 showed that all 15 temperate japonica cultivars tested as well as six out of the nine indica cultivars tested possessed the Nipponbare-type LIN1 allele, whereas the remaining three indica cultivars and one aus cultivar tested harbored an identical missense mutation in LIN1. These results revealed that the mutant allele of LIN1 has not been widely utilized in breeding temperate japonica cultivars currently in cultivation. Our findings indicate that the lin1 mutation may be useful to further improve grain length and presumably grain yield in temperate japonica and indica rice cultivars that harbor the Nipponbare-type LIN1 allele.

We isolated a cold sensitive virescent1 (csv1) mutant from a rice (Oryza sativa L.) population mutagenized by carbon ion irradiation. The mutant exhibited chlorotic leaves during the early growth stages, and produced normal green leaves as it grew. The growth of csv1 plants displayed sensitivity to low temperatures. In addition, the mutant plants that were transferred to low temperatures at the fifth leaf stage produced chlorotic leaves subsequently. Genetic and molecular analyses revealed translocation of a 13-kb genomic fragment that disrupted the causative gene (CSV1; LOC_Os05g34040). CSV1 encodes a plastid-targeted oxidoreductase-like protein conserved among land plants, green algae, and cyanobacteria. Furthermore, CSV1 transcripts were more abundant in immature than in mature leaves, and they did not markedly increase or decrease with temperature. Taken together, our results indicate that CSV1 supports chloroplast development under cold stress conditions, in both the early growth and tillering stages in rice. We isolated and characterized the rice virescent mutant csv1. The mutant showed low temperature sensitivity not only early growth stages but also the tillering stage.

Ion beams are recognized as alternative mutagens for plant and microbe breeding because they are thought to cause mutations by distinct mechanism from chemical mutagens or gamma rays. Valuable mutants have been successfully generated by ion beam irradiation not only in ornamental plants but also in crops such as rice. The characteristics of the ion-beam-induced mutations had been analyzed using specific marker genes; however, little is known about how many and what type of mutations are induced by ion beams in genomic level. Here, we conducted exome analysis of DNA samples of mutants identified in a carbon ion beam-irradiated rice population to understand the properties of mutations caused by ion beams at a genomic level. Rice seeds (Nipponbare) were irradiated with carbon ion beams (320 MeV 12C6+ at 40 Gy) accelerated by the cyclotron in QST Takasaki. Five mutant lines (2 dwarfs and 3 early heading date mutants) were selected and subjected to the exome analysis. As a result, we identified a total of 56 mutations including 24 single nucleotide variations and 28 insertions/deletions in the 5 lines. A large deletion (33.6k bp) and an inversion (534K bp) were also detected. In addition, we identified 6 mutations that potentially have high impact on protein function (frameshift or exon loss variants). Strong candidate genes that possibly link to the mutant phenotype were found in 4 lines out of 5 lines, suggesting that the combination of ion beam irradiation and genome analysis is helpful to narrow down a candidate gene responsible for a mutant phenotype., 16th International Symposium on Rice Functional Genomics

Genome size and complexity often present major challenges to genome-based approaches in crop plants and other agricultural species. For instance, repetitive sequences comprise 80% to 90% of the genome of hexaploid wheat, which has a haploid genome size of approximately 17 Gb. In this study, we developed an improved design and procedure for short-read library preparation that uses a modified adaptor and duplex-specific nuclease (DSN) for the efficient elimination of highly repeated sequence elements within genomes. The improved adapter, which has a hairpin-like form for stability, was constructed from truncated sequences adjacent to the original Illumina TruSeq adapter and can be converted to a full-length adapter structure during PCR amplification. Using the hairpin-structured adaptor, we prepared randomly sheared genomic libraries from rice and diploid, tetraploid, and hexaploid wheat cultivars and evaluated the efficiency of DSN for the enzymatic depletion of repetitive elements. According to real-time quantitative PCR analysis, the relative abundances of 18S and 25S ribosomal DNA decreased respectively to 1.15% and 3.54% in rice and 1.70%-1.95% and 14.71%-20.01% in the three wheat cultivars. Whole-genome sequencing analysis of a diploid wheat cultivar, KU104-1, indicated that DSN treatment with the designed hairpin-structured adapter dramatically reduced highly repetitive elements, such as Ty1-Copia and Ty3-Gypsy retrotransposons and DNA transposons, within the genome, while sequencing reads derived from low-copy genes and protein coding sequences increased more than 50%. Our new procedure should be useful not only for wheat genomes but also for other agricultural plant species with relatively large and complex genomes.

We irradiated Arabidopsis thaliana with several kinds of heavy-ion beams to investigate the linear energy transfer (LET)-dependent effects of heavy-ion beam irradiation. First, dry seeds were irradiated with C, N, Ne, Ar, or Fe ions at doses ranging from 5 to 400 Gy to compare the flowering and mutation rates among the ion species. The sensitivity of the flowering and mutation rates to irradiation differed markedly among the ion species. Of the ion species, N (30 keV mm � 1 ) was the most effective at inducing albino plants. Second, we examined the effects of LET on mutation induction. The LET of C, N, Ne, and Ar ion beams was controlled at 30-640 keV mm � 1 . Regardless of ion species, irradiation with the same LET value resulted in the same flowering rate and mutation rate. Thus, the LET of ion beams seems to be an important factor affecting mutagenesis. We found a 440-bp deletion in the hy (elongated hypocotyl) mutant that was isolated from M2 progeny. These fundamental data on LET-dependence can be used to develop advanced technologies for plant mutagenesis.

Ion-beam mutagenesis is a highly effective way to rapidly create new cultivars. To optimize conditions for heavy ion mutagenesis, we irradiated tobacco (Nicotiana tabacum L.) tissues at various developmental stages with heavy ion beams of various doses and examined the effects of irradiation by monitoring plant growth and mutation induction. The effects differed among irradiated tissues. Sensitivity to heavy ion-beam irradiation increased in the following order: dry seeds, imbibed seeds, and culture tissues. We isolated three white flower mutants. One, BWF1, was found to be a novel mutant, in which the synthesis of proanthocyanidin was up-regulated. The others may have a mutation in some regulatory genes involved in the flavonoid biosynthetic pathway. These results suggest that the developmental state of plant tissues is critical for efficient plant mutagenesis, and that the broad spectrum of mutations may be induced by heavy ion-beam irradiation at molecular level.

The biological effects of heavy-ion beam irradiation on cultured tissues of cyclamen were investigated by establishing an irradiation-mediated mutation breeding protocol for producing a new variety of cyclamen. Initially, a callus, a somatic embryo, and a plantlet were irradiated with 12 C 6� ion beam at doses of 10, 20, 40, 60, and 80 Gy. Distinct mutants were not obtained in plants regenerated from the irradiated cultured materials. Next, we used the tuber of cyclamen as a target for irradiation. Irradiation (8-16 Gy) of 8-15 mm diameter tubers produced male sterility, change in petal colour, and petal form. Mutation induction by heavy-ion beam irradiation to the tuber is useful for changing flower characteristics of cyclamen.

Mammalian promoters are categorized into TATA and CpG-related groups, and they have complementary roles associated with differentiated transcriptional characteristics. While the TATA box is also found in plant promoters, it is not known if CpG-type promoters exist in plants. Plant promoters contain Y Patches (pyrimidine patches) in the core promoter region, and the ubiquity of these beyond higher plants is not understood as well. Sets of promoter sequences were utilized for the analysis of local distribution of short sequences (LDSS), and approximately one thousand octamer sequences have been identified as promoter constituents from Arabidopsis, rice, human and mouse, respectively. Based on their localization profiles, the identified octamer sequences were classified into several major groups, REG (Regulatory Element Group), TATA box, Inr (Initiator), Kozak, CpG and Y Patch. Comparison of the four species has revealed three categories: (i) shared groups found in both plants and mammals (TATA box), (ii) common groups found in both kingdoms but the utilized sequence is differentiated (REG, Inr and Kozak) and (iii) specific groups found in either plants or mammals (CpG and Y Patch). Our comparative LDSS analysis has identified conservation and differentiation of promoter architectures between higher plants and mammals.

A fosmid library of Xanthomonas oryzae pathovar oryzae MAFF311018 (T7174), the causative agent of bacterial blight on rice, was constructed and characterized. The average fosmid library insert size was >34kb, and 967 clones were uniquely positioned on its sequenced genome. The entire Xoo MAFF311018 genome was covered by end-sequenced clones with at least 5kb of overlap. The fosmid vector contains both the single-copy Escherichia coli fertility factor origin, which enhances fosmid stability, and the multi-copy IncPα origin, allowing amplification of copy number upon induction with l-arabinose. Real-time quantitative PCR on 12 randomly picked fosmid library clones determined that fosmid copy number increased 8- to 58-fold after 5hour induction. This library provides a new resource for complementation experiments and systematic functional studies in Xoo and related species.

While heavy-ion beam irradiation is becoming popular technology for mutation breeding in Japan, the combination with genetic manipulation makes it more convenient to create greater variation in plant phenotypes. We have succeeded in producing over 200 varieties of transgenic torenia (Torenia fournieri Lind.) from over 2,400 regenerated plants by this procedure in only 2 years. Mutant phenotypes were observed mainly in flowers and showed wide variation in colour and shape. Higher mutation rates in the transgenics compared to those in wild type indicate the synergistic effect of genetic manipulation and heavy-ion beam irradiation, which might be advantageous to create greater variation in floral traits.

Background Plant promoter architecture is important for understanding regulation and evolution of the promoters, but our current knowledge about plant promoter structure, especially with respect to the core promoter, is insufficient. Several promoter elements including TATA box, and several types of transcriptional regulatory elements have been found to show local distribution within promoters, and this feature has been successfully utilized for extraction of promoter constituents from human genome. Results LDSS (Local Distribution of Short Sequences) profiles of short sequences along the plant promoter have been analyzed in silico, and hundreds of hexamer and octamer sequences have been identified as having localized distributions within promoters of Arabidopsis thaliana and rice. Based on their localization patterns, the identified sequences could be classified into three groups, pyrimidine patch (Y Patch), TATA box, and REG (Regulatory Element Group). Sequences of the TATA box group are consistent with the ones reported in previous studies. The REG group includes more than 200 sequences, and half of them correspond to known cis-elements. The other REG subgroups, together with about a hundred uncategorized sequences, are suggested to be novel cis-regulatory elements. Comparison of LDSS-positive sequences between Arabidopsis and rice has revealed moderate conservation of elements and common promoter architecture. In addition, a dimer motif named the YR Rule (C/T A/G) has been identified at the transcription start site (-1/+1). This rule also fits both Arabidopsis and rice promoters. Conclusion LDSS was successfully applied to plant genomes and hundreds of putative promoter elements have been extracted as LDSS-positive octamers. Identified promoter architecture of monocot and dicot are well conserved, but there are moderate variations in the utilized sequences.