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Tricholoma matsutake and its related species are ectomycorrhizal Agaricomycetes that produce prized mushrooms collectively called “ matsutake”. These mushrooms are mainly distributed in the Northern Hemisphere. In this review, we describe both the pioneering work and recent advances in our understanding of the distribution and evolution of matsutake, with a special focus on genomic studies. Deoxyribonucleic acid (DNA) sequence analyses revealed that Tricholoma caligatum was the earliest species within this group, followed by Tricholoma fulvocastaneum, the cluster with Tricholoma ilkkae, Tricholoma dulciolens, and Tricholoma bakamatsutake, and finally the cluster with Tricholoma murrillianum, Tricholoma mesoamericanum, Tricholoma anatolicum, and Tricholoma matsutake. Although analyses based on mobile DNAs and whole-genome sequences revealed a similar clustering pattern, there are distinct differences in the distribution of mobile DNAs and genomic structure of Tricholoma bakamatsutake and Tricholoma matsutake. Furthermore, repetitive DNA can be used as markers to distinguish among strains and populations of Tricholoma matsutake from different geographical regions, including identifying dispersals of basidiospores. “Telomere-to-telomere genome sequencing” analyses unearthed that both Tricholoma matsutake and Tricholoma bakamatsutake underwent explosive multiplications of retrotransposons within their genomes but with different mobile DNA elements expanded between them. Contrastingly, the structure of the mating loci between Tricholoma matsutake and Tricholoma bakamatsutake is highly conserved.

SummaryTricholoma bakamatsutake, which is an edible ectomycorrhizal fungus associated with Fagaceae trees, may have diverged before the other species inTricholomasectionCaligata. We generated a highly contiguous whole-genome sequence forT. bakamatsutakeSF-Tf05 isolated in an oak (Quercus salicina) forest in Japan. The assembly of high-fidelity long reads, with a median read length of 12.3 kb, resulted in 13 chromosome-sized contigs comprising 142,068,211 bases with an average GC content of 43.94%. The 13 chromosomes were predicted to encode 11,060 genes. A contig (122,566 bases) presumably containing the whole circular mitochondrial genome was also recovered. The chromosome-wide comparison ofT. bakamatsutakeandT. matsutake(TMA_r1.0) indicated that the basic number of chromosomes (13) was conserved, but the structures of the corresponding chromosomes diverged, with multiple inversions and translocations. Gene conservation and cluster analyses revealed at least three groups inTricholoma. Specifically, allT. bakamatsutakestrains belonged to the “bakamatsutake” clade, which is most proximal to the “caligatum” clade consisting ofT. caligatumandT. fulvocastaneum. The constructed highly contiguous telomere-to-telomere genome sequence of aT. bakamatsutakeisolate will serve as a fundamental resource for future research on the evolution and differentiation ofTricholomaspecies.

Enzymes of the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase superfamilies are involved in the reduction of compounds containing a ketone group. In most cases, multiple isoforms appear to be involved in the reduction of a compound, and the enzyme(s) that are responsible for the reaction in the human liver have not been elucidated. The purpose of this study was to quantitatively evaluate the contribution of each isoform to reduction reactions in the human liver. Recombinant cytosolic isoforms were constructed, i.e., AKR1C1, AKR1C2, AKR1C3, AKR1C4, and carbonyl reductase 1 (CBR1), and a microsomal isoform, 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1), and their contributions to the reduction of 10 compounds were examined by extrapolating the relative expression of each reductase protein in human liver preparations to recombinant systems quantified by liquid chromatography-mass spectrometry (MS)/MS. The reductase activities for acetohexamide, doxorubicin, haloperidol, loxoprofen, naloxone, oxcarbazepine, and pentoxifylline were predominantly catalyzed by cytosolic isoforms, and the sum of the contributions of individual cytosolic reductases was almost 100%. Interestingly, AKR1C3 showed the highest contribution to acetohexamide and loxoprofen reduction, although previous studies have revealed that CBR1 mainly metabolizes them. The reductase activities of bupropion, ketoprofen, and tolperisone were catalyzed by microsomal isoform(s), and the contributions of HSD11B1 were calculated to be 41%, 32%, and 104%, respectively. To our knowledge, this is the first study to quantitatively evaluate the contribution of each reductase to the reduction of drugs in the human liver.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized matsutake mushroom in conifer forests. Here, we isolated mutants generated by the argon-ion beam irradiation of T. matsutake NBRC 33136, designated as Ar 5002 and Ar 5012, which are defective in β-1,4-endoglucanase activity, that promoted the above- and below-ground growth of the symbiotic partner Pinus densiflora in vitro, compared with the control. Pine seedlings associated with Ar 5002, compared with NBRC 33136, had significantly higher above-ground dry mass and significantly greater plant growth, while those associated with Ar 5012 had significantly higher above- and below-ground dry mass. There were no significant differences between the numbers of mycorrhizal root-tips synthesized by the wild-type and mutants, indicating that β-1,4-endoglucanase did not influence the mycorrhization of T. matsutake. In seedlings inoculated with Ar 5002- and Ar 5012, the Hartig nets resembled those of seedlings inoculated with NBRC 33136. The data suggest that T. matsutake lacking β-1,4-endoglucanase activity confer beneficial effects on the growth of P. densiflora. Further studies are necessary to determine whether the symbiotic relationship could be strengthened by such a mutated fungal trait.

The aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies are responsible for the reduction in compounds containing the aldehyde, ketone, and quinone groups. In humans, 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 6 SDR isoforms (CBR1, CBR3, CBR4, HSD11B1, DHRS4, and DCXR) have been found to catalyze the reduction in xenobiotics, but their hepatic expression levels are unclear. The purpose of this study is to determine the absolute mRNA expression levels of these 18 isoforms in the human liver. In 22 human livers, all isoforms, except for AKR1B15, are expressed, and AKR1C2 (on average 1.6 × 106 copy/μg total RNA), AKR1C3 (1.3 × 106), AKR1C1 (1.3 × 106), CBR1 (9.7 × 105), and HSD11B1 (1.1 × 106) are abundant, representing 67% of the total expression of reductases in the liver. The expression levels of AKR1C2, AKR1C3, AKR1C1, CBR1, and HSD11B1 are significantly correlated with each other, except between AKR1C2 and CBR1, suggesting that they might be regulated by common factor(s). In conclusion, this study comprehensively determined the absolute expression of mRNA expression of each AKR and SDR isoform in the human liver.

Chalky grains of rice are increased due to high temperature (HT) during a ripening period. However, the underlying mechanisms of the chalkiness are not well known, seemingly due to the obtuse response of wild type and lack of effective mutants. In this study, we isolated and characterized the flo11-2 mutant, which showed higher number of chalky grains than wild type under HT but quite small number of chalkiness under cool temperature as well as similar growth, development, and yield to that of the wild type. Using this high sensitivity of the flo11-2 mutant, we identified the most critical meteorological factor and developmental stage affecting chalkiness with 5 days HT treatments over 4 consecutive years. The results demonstrated that daily maximum temperature was more causative than daily mean or minimum temperatures which have been regarded as important factors before this. Besides, the developmental stage around 20days after flowering (DAF) was most sensitive to HT rather than the early stage up to 15 DAF. In addition, we found that the flo11-2 mutant with a high chalky ratio was vulnerable to preharvest sprouting, which has never been reported before for chalky grains, but could cause significant yield and quality loss after extremely hot and dry summers followed by rainy cool autumns. The flo11-2 mutant is, therefore, a useful material for chalky grain research. Abbreviations: Tmax: Daily maximum temperature; Tmean: Daily mean temperature; Tmin: Daily minimum temperature; DAF: Days after flowering; DAH: Days after heading; Ext30: Excess temperature above 30 ºC; HDT: High day temperature; HNDT: High night and high day temperature; HNT: High night temperature; HT; High temperature; HTR: High temperature resistant; HTS: High temperature susceptible; BW: Basal white; MW: Milky white; WB: White back.

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature-sensitive floury endosperm11-2 (flo11-2) mutant was isolated from ion beam-irradiated rice of 1116 lines. The flo11-2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole-exome sequencing of the flo11-2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid-localized 70-kDa heat shock protein 2 (cpHSP70-2). The cpHSP70-2 of the flo11-2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11-2 mutants that express the wild-type cpHSP70-2 showed significantly lower chalkiness than the flo11-2 mutant. Moreover, the accumulation level of cpHSP70-2 was negatively correlated with the chalky ratio, indicating that cpHSP70-2 is a causal gene for the chalkiness of the flo11-2 mutant. The intrinsic ATPase activity of recombinant cpHSP70-2 was lower by 23% at Vmax for the flo11-2 mutant than for the wild type. The growth of DnaK-defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild-type DnaK. The results indicate that the lowered cpHSP70-2 function is involved with the chalkiness of the flo11-2 mutant.

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.

The Sanriku district is one of the largest Undaria pinnatifida (Wakame) cultivation areas in Japan. However, the production has steadily declined in recent years due to the high retirement rate among fishers. Extending the cultivation period is a potential way to improve productivity by decentralizing the workforce through the production process. We aimed to investigate the phenotypic differentiation between regional strains of U. pinnatifida collected from Matsushima Bay (MAT) and Hirota Bay (HRT) in the Sanriku district through a cultivation trial to verify the application for the purpose of extending the cultivation period. The growth of MAT was better than that of HRT when the cultivation started earlier (i.e., 9 and 19 October 2014); in contrast, HRT outperformed MAT when the cultivation started later (6 November and 12 December 2014). The yield of MAT reached over the standard amount in the Sanriku district in February. On the other hand, the yield of HRT reached over this value in April. Furthermore, the photosynthetic performance and nutrient uptake rates differed between MAT and HRT, indicating that the differences may result in maturation characteristics. According to these results, the combined use of MAT and HRT would be a valuable strategy by which to extend the cultivation period.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces matsutake mushrooms in association with conifers. Here, we isolated a pleiotropic mutant, designated as G1, following γ-ray irradiation of T. matsutake NBRC 33136. In addition to exhibiting increased cellulose- and amylose-degrading activities and altered mycelial morphology, G1 degenerated lateral roots and caused stunting or fatal wilting of seedlings of its symbiotic partner, Pinus densiflora, in vitro. A mutant, designated Ar 59, previously isolated after argon-ion beam irradiation, exhibited the same phenotypes as G1, but without its detrimental effects. Ar 59, like NBRC 33136, developed a Hartig net around healthy cortical cells that was composed of uniseriate hyphae. In G1-inoculated seedlings, the Hartig net was composed of heavily bundled hyphae penetrating the intercellular space, and it was accompanied by somewhat unusual forms of plant cortical cells. Sequences of rRNA gene-related markers, including four single nucleotide polymorphisms present within each strain, were 100% identical among NBRC 33136, G1, and Ar 59, indicating that they are mutants of the wild-type. Thus, γ-ray irradiation can convert the fungus from a beneficial to a harmful agent. These findings suggest the presence of an unknown mechanism in the fungal genome that can transform mutualism into parasitism.

Summary Heavy‐ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost‐efficient whole‐exome sequencing procedure in rice, and its application to characterize an unselected population of heavy‐ion beam‐induced mutations. The bioinformatics pipeline identified single‐nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy‐ion beams at the population level. In total, 165 individual M2 lines derived from six irradiation conditions as well as eight pools from non‐irradiated ‘Nipponbare’ controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon‐ion beam‐induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M2 lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing‐based determination of the optimal irradiation condition for induction of mutations., Significance Statement We developed a multiplexed and cost‐efficient whole‐exome sequencing procedure and bioinformatics filtering pipeline for rice, and demonstrated the rapid identification of authentic mutations from a large dataset of candidate mutations. We applied this technique for the characterization of mutation spectra by heavy‐ion beams in the absence of the mutant selection, and showed that 9.06 ± 0.37 (average ± SE) mutations were induced within the target exon regions by 150 Gy irradiation of dry seeds in rice.

Tobacco (Nicotiana tabacum L.) is a complex allotetraploid species with a large 4.5-Gb genome that carries duplicated gene copies. In this study, we describe the development of a whole-exome sequencing (WES) procedure in tobacco and its application to characterize a test population of ethyl methanesulfonate (EMS)-induced mutations. A probe set covering 50.3-Mb protein coding regions was designed from a reference tobacco genome. The EMS-induced mutations in 19 individual M2 lines were analyzed using our mutation analysis pipeline optimized to minimize false positives/negatives. In the target regions, the on-target rate of WES was approximately 75%, and 61,146 mutations were detected in the 19 M2 lines. Most of the mutations (98.8%) were single nucleotide variants, and 95.6% of them were C/G to T/A transitions. The number of mutations detected in the target coding sequences by WES was 93.5% of the mutations detected by whole-genome sequencing (WGS). The amount of sequencing data necessary for efficient mutation detection was significantly lower in WES (11.2 Gb), which is only 6.2% of the required amount in WGS (180 Gb). Thus, WES was almost comparable to WGS in performance but is more cost effective. Therefore, the developed target exome sequencing, which could become a fundamental tool in high-throughput mutation identification, renders the genome-wide analysis of tobacco highly efficient.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

Aim Abiraterone acetate for metastatic castration-resistant prostate cancer is an acetylated prodrug to be hydrolyzed to abiraterone. Abiraterone acetate is known to be hydrolyzed by pancreatic cholesterol esterase secreted into the intestinal lumen. This study aimed to investigate the possibility that arylacetamide deacetylase (AADAC) expressed in enterocytes contributes to the hydrolysis of abiraterone acetate based on its substrate preference. Materials and methods Abiraterone acetate hydrolase activity was measured using human intestinal (HIM) and liver microsomes (HLM) as well as recombinant AADAC. Correlation analysis between activity and AADAC expression was performed in 14 individual HIMs. The in vivo pharmacokinetics of abiraterone acetate was examined using wild-type and Aadac knockout mice administered abiraterone acetate with or without orlistat, a pancreatic cholesterol esterase inhibitor. Key findings Recombinant AADAC showed abiraterone acetate hydrolase activity with similar Km value to HIM and HLM. The positive correlation between activity and AADAC levels in individual HIMs supported the responsibility of AADAC for abiraterone acetate hydrolysis. The area under the plasma concentration-time curve (AUC) of abiraterone after oral administration of abiraterone acetate in Aadac knockout mice was 38% lower than that in wild-type mice. The involvement of pancreatic cholesterol esterase in abiraterone formation was revealed by the decreased AUC of abiraterone by coadministration of orlistat. Orlistat potently inhibited AADAC, implying its potential as a perpetrator of drug-drug interactions. Significance AADAC is responsible for the hydrolysis of abiraterone acetate in the intestine and liver, suggesting that concomitant use of abiraterone acetate and drugs potently inhibiting AADAC should be avoided.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom "matsutake" in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

Undaria pinnatifida is grown for food and industrial materials worldwide; therefore, advanced breeding is needed to meet quality and productivity requirements. In this study, we examined regional lines of U. pinnatifida from five cultivation sites in Japan with different environmental conditions: Oga (OGA, the northern Sea of Japan coast), Hirota Bay (HRT, the northeastern Pacific coast), Matsushima Bay (MAT, the northeastern Pacific coast), Naruto (the Seto Inland Sea coast) and Shimonoseki (SIM, the southern Sea of Japan coast). The sporophytes of these lines were cultured in a tank culture system under controlled environmental conditions, and their morphological characteristics, nutrient uptake kinetics (V max, K s and V max/K s ), and carbon, nitrogen and phosphorus contents were determined. Sporophytes from MAT grew faster, whereas those from SIM were smaller than those from the other sites. Although the blade thickness of sporophytes cultivated in the sea significantly differs among cultivation sites in the previous study, there was no significant difference in blade thickness among the regional lines cultivated in the tank. Sporophytes from OGA had the greatest V max/K s values and significantly greater nitrogen contents than the other lines. Therefore, the morphological characteristics of MAT and SIM sporophytes, and the nutrient uptake kinetics of OGA sporophytes may have a genetic origin. This indicates that these lines may represent useful resources for selective breeding, with MAT sporophytes providing faster growth and OGA sporophytes being well-adapted to low-nutrient conditions.

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.

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.

Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a distinct mechanism from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10-7per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.

Identifying functional elements in promoter sequences is a major goal in computational and experimental genome biology. Here, we describe an algorithm, Local Distribution of Short Sequences for Prokaryotes (LDSS-P), to identify conserved short motifs located at specific positions in the promoters of co-expressed prokaryotic genes. As a test case, we applied this algorithm to a symbiotic nitrogen-fixing bacterium, Sinorhizobium meliloti. The LDSS-P profiles that overlap with the 5′ section of the extracytoplasmic function RNA polymerase sigma factor RpoE2 consensus sequences displayed a sharp peak between -34 and -32 from TSS positions. The corresponding genes overlap significantly with RpoE2 targets identified from previous experiments. We further identified several groups of genes that are co-regulated with characterized marker genes. Our data indicate that in S. meliloti, and possibly in other Rhizobiaceae species, the master cell cycle regulator CtrA may recognize an expanded motif (AACCAT), which is positionally shifted from the previously reported CtrA consensus sequence in Caulobacter crescentus. Bacterial one-hybrid experiments showed that base substitution in the expanded motif either increase or decrease the binding by CtrA. These results show the effectiveness of LDSS-P as a method to delineate functional promoter elements.

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.

In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of ‘4 bases—a spacer with a flexible length—4 bases’. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein–DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly.

CcrM is one of the solitary bacterial DNA methyltransferases which does not have corresponding restriction enzymes. We established a stable ccrM-overexpressing mutant of Mesorhizobium loti, MlccrM-OX, and performed molecular and phenotypic characterization of this strain. In the M. loti MlccrM-OX infected plants, nodulation was apparently delayed at 7 days after inoculation (dai), however, the nodules that eventually formed on the MlccrM-OX roots showed nitrogen fixing ability by at least 21 dai. These results suggest that the initial morphogenic events were affected by ccrM-overexpression and that the correct pattern of DNA methylation of the bacterial genome is not essential for plant-microbe symbiosis, but are required for efficient nodulation.

A positive selection system for gene disruption using a sucrose-sensitive transgenic rhizobium was established and used for the molecular characterization of mutations induced by ion beam irradiations. Single nucleotide substitutions, insertions, and deletions were found to occur in the sucrose sensitivity gene, sacB, when the reporter line was irradiated with highly accelerated carbon and iron ion beams. In all of the insertion lines, fragments of essentially the same sequence and of approximately 1188bp in size were identified in the sacB regions. In the deletion lines, iron ions showed a tendency to induce larger deletions than carbon ions, suggesting that higher LET beams cause larger deletions. We found also that ion beams, particularly "heavier" ion beams, can produce single gene disruptions and may present an effective alternative to transgenic approaches.

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.

Biological samples are irradiated with heavy-ion beams at a heavy-ion beam irradiation facility in RIKEN to perform mutation breeding. The energies of the heavy-ion beams are sufficiently high to irradiate biological samples of macroscopic thickness in air. A uniform dose distribution is a key to a systematic study of the effect of the heavy-ion beams, and thus to the improvement of mutation efficiency. We selected a sufficiently high beam energy to avoid the Bragg peak to realize a uniform dose distribution along the beam path. The outline of the beam line is presented. The linear energy transfers (LETs) of the heavy-ion beams are selected using a range shifter and an energy adjuster to investigate the LET dependence of the irradiation effect. More than five hundred samples are automatically sent to the beam position using an automatic sample changer, which put the heavy-ion beam induced mutation breeding to practical use. The structure and function of the automatic irradiation system are also presented.

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.

The DNA adenine methylation status on specific 5′-GANTC-3′ sites and its change during the establishment of plant–microbe interactions was demonstrated in several species of α-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.

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.

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.

アクリルアミド, N-イソプロピルアクリルアミドおよびN-アクリロイルピペリジンを用いて表面疎水性度の異なるN-イソプロピルアクリルアミド共重合体ゲルを合成した.これらのゲルは, 熱刺激により水和・脱水和する.これらのゲルの表面特性とゲルの分子構造ならびに熱刺激の効果を安息香酸とフタル酸の吸着実験によって明らかにした.安息香酸やフタル酸は, 相転移温度 (Tp) 以上の高温では吸着し, 相転移温度 (Tp) 以下の低温では脱着する.これは, ゲルの表面状態 (親・疎水のバランス) に依存している.相転移温度以下の293Kおよび相転移温度以上の323Kにおいてこれらの吸着等温線はヘンリー型を示した.相転移温度 (Tp) は, 分子設計の段階で制御可能であり, 親水性のモノマー (アクリルアミド) の添加によりTpは上昇し, 疎水性のモノマー (N-アクリロイルピペリジン) の添加によりTpは低下する.温度を293Kと323Kの間で段階的に変化させることにより, 吸着質の吸脱着は速やかにかつ可逆的に行われることが解った.

We have developed a restriction landmark genome scanning (RLGS) system in silico, involving two-dimensional electrophoretic analysis of DNA by computer simulation that is based on the availability of whole-genome sequences for specific organisms. We applied the technique to the analysis of the Xanthomonas oryzae pathovar oryzae (Xoo) MAFF 311018, which causes bacterial blight in rice. The coverage that was found to be achievable using RLGS in silico, as a percentage of the genomic regions that could be detected, ranged from 44.5% to 72.7% per image. However, this reached a value of 96.7% using four images that were obtained with different combinations of landmark restriction enzymes. Interestingly, the signal intensity of some of the specific spots obtained was significantly lower than that of other surrounding spots when MboI, which cleaves unmethylated 5′-GATC-3′ sites, was used. DNA gel blot analysis with both DNA adenine methylase (Dam)-sensitive and -insensitive isoschizomers (MboI and Sau3AI) revealed that Dam-mediated DNA adenine methylation had indeed occurred at these particular sites. These results suggest that a significant portion of the 5′-GATC-3′ sites within the Xoo genome is stably methylated by Dam.

The DNA adenine methylation status on specific 5'-GANTC-3' sites and its change during the establishment of plant-microbe interactions was demonstrated in several species of alpha-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.

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.