In order to obtain novel information about QTL-by-QTL, QTL-by-environment, and QTL-by-QTL-by-environment interactions (QQ, QE, and QQE, respectively), four loci controlling grain length in rice (Oryza sativa L.), that is, GW2, OsLG3b, and GS3, derived from a large-grain cultivar, BG 1, and LKF, from another cultivar, Fusayoshi, were targeted as a model system of quantitative trait. First, four single-QTL near-isogenic lines (NILs), each of which was a homozygote of a long-grain allele at one of the four loci in a common genetic background of cultivar Gimbozu, were developed. Then, pyramiding of these long-grain alleles was conducted through intercrossing between single-QTL NILs and selection of segregants of the longest grains, leading to another series of double-QTL NILs. All of these NILs, which were confirmed genotypes using molecular markers, were grown with their parents over eight years (environments). QQ, QE and QQE, as well as the additive effect and environmental effect, were estimated based on a linear model and analyzed using interaction principal component analysis (IPCA). All genetic terms and environmental effects showed highly significant variations. QQs showed all negative values and their magnitude tended to be smaller than those of the additive effects on average. The IPCA clearly showed that the additive effects and QQs responded to environments differentially and not randomly. In addition, a similar differential response was also observed between QQs including the long-grain allele at LKF and those excluding this allele. Such a demonstrated nature of these interactions may provide valuable suggestions on practical breeding.

Poor grain filling, characterized by incidence of partially filled grain, is one of the major problems to be improved for maximizing the yield potential in rice (Oryza sativa L.) cultivars with numerous spikelets in a panicle (extra-heavy panicle type [EHPT]). This study evaluated the grain-filling-related alleles at APS2 and APL2, the loci for the small and large subunits of ADP-glucose pyrophosphorylase (AGPase), respectively, and at SUT1, sucrose transporter, for their effects and interactions on grain filling and associated traits of EHPT cultivars. Two sets of derived lines were developed. Each of these lines was one of the eight possible homozygotes of alleles derived from rice cultivars Akenohoshi and Milyang 23 at the three loci in nearly common genetic backgrounds. They were examined for grain-filling traits in two different years. Results showed that APL2-2, and SUT1-2 in part, derived from Milyang 23 showing relatively good grain filling, significantly improved grain filling compared with APL2-1 and SUT1-1 from Akenohoshi showing poor filling. This improvement in grain filling may result from increased AGPase activity and expression of APL2 and SUT1 proteins, by means of APL2-2 and SUT1-2. However, the variations in AGPase activity, protein expression, and the degree of grain filling were not associated with the gene expression of the three loci, implying post-transcriptional regulation. These results strongly suggested that APL2-2 and SUT1-2 are associated with good grain filling.

A total of 145 accessions of the genus Citrus and related genera, maintained in the Conservation Garden for Citrus Germplasm at the Experimental Farm of Kindai University, Yuasa, Wakayama, Japan, were examined for their phylogenetic relationships. The present classification was conducted using an inter-retrotransposon amplified polymorphism (IRAP) method based on the insertion polymorphism of a retrotransposon, CIRE1, identified in C. sinensis. The objective of this study was to evaluate the applicability of the IRAP method for citrus classification. The constructed dendrogram showed that the 145 accessions and two outgroup species were successfully classified into five major clades. A large number of C. sinensis accessions were divided into three traditional groups, navel orange, sweet orange, and blood orange, almost corresponding to the sub-clades in the dendrogram. Several other accessions belonging to the same species, and also many hybrid cultivars from crossbreeding, were localized into the respective sub-clades or near positions in the dendrogram. Several unclassified accessions could also be located in the dendrogram, suggesting novel relationships with other accessions. It was concluded that the IRAP method based on CIRE1 insertion polymorphism was suitable for the classification of citrus from a molecular point of view.

The LKF locus, which regulates grain size in the rice cultivar ‘Fusayoshi’ showing large grain, has been mapped to the proximal part of the long arm of chromosome 3. An incomplete dominant allele, Lkf, caused large grain size of Fusayoshi. The structure and function of this locus, however, have not yet been determined. In a similar position to LKF on chromosome 3, two loci, Os03g0407400 (GS3) and LOC_Os03g44500, have been already reported as loci also regulating rice grain size. The objective of the present study was to determine the nucleotide sequences of both Os03g0407400 and LOC_Os03g44500 for different alleles at the LKF locus. Results showed that only one known single nucleotide polymorphism (SNP) in exon 10 of LOC_Os03g44500 was detected between a large-grain allele (Lkf) and a small-grain allele at the LKF locus, whereas no polymorphisms in Os03g0407400. This SNP, visualized using a dCAPS marker, clearly demonstrated nearly complete co-segregation with grain length in an F2 population segregating the Lkf at LKF. Other large-grain mutant lines with large-grain alleles at the LKF locus, which originated from another cultivar ‘Gimbozu’, also showed the same SNP in exon 10 of LOC_Os03g44500. It was concluded from these results that LKF is identical to LOC_Os03g44500, and the detected SNP in exon 10, at least, which is included in Kelch-like repeat motif, could be essential for expression of the large-grain phenotype.

gamma-Oryzanol is a main oleophilic component in rice bran oil and has been well recognized as a good dietary supplement for human health, as well as having uses in industrial materials. japonica-type rice cultivars generally showed significantly higher contents of total gamma-oryzanol in brown rice compared with indica-type cultivars, although within-group variation was significant. The objective of this study was to explore quantitative trait loci (QTLs) responsible for the difference in the gamma-oryzanol content between japonica-type and indica-type rice cultivars, using recombinant inbred lines (RILs), backcross inbred lines (BILs), and corresponding chromosome segment substitution lines (CSSLs) derived from crosses between japonica-type and indica-type. Results from RILs and BILs showed that eight QTLs were detected with R-2 from.09 to.16. Nine candidate regions for QTL were also suggested from corresponding CSSLs. These QTLs from RILs and BILs and the candidate regions from CSSLs were not overlapped, although one QTLs was mapped near the boundaries of the respective candidate region. At four QTLs and three candidate regions, alleles or segments from japonica-type caused higher contents than those from indica-type. On the other hand, at the other four QTLs and six candidate regions, alleles or segments from indica-type caused higher contents than those from japonica-type, which is a reverse result to the parental differences. This result strongly suggested that alleles with increasing effects on.-oryzanol content could be accumulated not only from japonica-type but also from indica-type, leading to a potential for increase in gamma-oryzanol content in future breeding programs.

A class II transposable element, mPing exists in the rice genome ubiquitously and can transpose even in ordinary cultivation conditions. A copy of mPing was identified at the long arm of chromosome 12. In reciprocal backcrossed F(1)s between a heterozygote and a homozygote without mPing, the male gametes with this mPing from heterozygotes were transmitted to the next generation at a lower frequency than those without mPing, resulting in distorted genetic segregation in self-fertilized progenies, as well as in F(1)s after backcrossing. Pollens with mPing tended to germinate on stigma less than those without mPing. These results, however, could not explain the lower transmission of male gametes with mPing. In addition, no excision of mPing was observed in a homozygote. Thus, it was suggested that male gametes with mPing were eliminated partly from pollination to fertilization by negative competition against male gametes without mPing. Less formation of microspores with mPing in meiosis could also be a cause for the distorted segregation, although this could not be examined. At least two ORFs, whose functions have not been identified, are located near this mPing. It is plausible that either of these ORFs or both are necessary for the normal functioning of male gametes.

Eight-carbon (C8) volatiles, such as 1-octen-3-ol, octan-3-one, and octan-3-ol, are ubiquitously found among fungi and bryophytes. In this study, it was found that the thalli of the common liverwort Marchantia polymorpha, a model plant species, emitted high amounts of C8 volatiles mainly consisting of (R)-1-octen-3-ol and octan-3-one upon mechanical wounding. The induction of emission took place within 40 min. In intact thalli, 1-octen-3-yl acetate was the predominant C8 volatile while tissue disruption resulted in conversion of the acetate to 1-octen-3-ol. This conversion was carried out by an esterase showing stereospecificity to (R)-1-octen-3-yl acetate. From the transgenic line of M. polymorpha (des6(KO)) lacking arachidonic acid and eicosapentaenoic acid, formation of C8 volatiles was only minimally observed, which indicated that arachidonic and/or eicosapentaenoic acids were essential to form C8 volatiles in M. polymorpha. When des6(KO) thalli were exposed to the vapor of 1-octen-3-ol, they absorbed the alcohol and converted it into 1-octen-3-yl acetate and octan-3-one. Therefore, this implied that 1-octen-3-ol was the primary C8 product formed from arachidonic acid, and further metabolism involving acetylation and oxidoreduction occurred to diversify the C8 products. Octan-3-one was only minimally formed from completely disrupted thalli, while it was formed as the most abundant product in partially disrupted thalli. Therefore, it is assumed that the remaining intact tissues were involved in the conversion of 1-octen-3-ol to octan-3-one in the partially disrupted thalli. The conversion was partly promoted by addition of NAD(P)H into the completely disrupted tissues, suggesting an NAD(P)H-dependent oxidoreductase was involved in the conversion. (C) 2014 Elsevier Ltd. All rights reserved.

Photoassimilates are accumulated as starch at rice stems before heading, are converted to sucrose, and are translocated to panicles after heading. Efficient translocation of photoassimilates to panicles should be a key to improve poor grain filling of rice cultivars with numerous spikelets per panicle. On the other hand, rice cultivars accumulating photoassimilates at stems should be utilized in the development of forage cultivars as whole crop silage. In this study, we investigated the starch content of the culms on the first node below frag leaf after heading for three years (in 2009, 2010 and 2012), using 9 rice recombinant inbred lines (RILs) (derived from Nakateshinsenbon/Milyang 23) which showed characteristic dynamics of non-structural carbohydrate contents after heading. Among these RILs, we selected three RILs showing stable dynamics in starch contents in culms among different years, and examined the expression of genes involved in starch metabolism in this organ. Results showed that one RIL, which increased the starch content at later stages of grain filling, tended to increase the expression levels of genes for soluble starch synthase (SSIIIB) and sucrose synthase (RSUS1), both of which are involved in starch synthesis pathway. However, other two RILs, which did not show such re-accumulation of the starch content, also showed no increase in the expression levels of these two genes. These results suggested that the expression of these two genes would contribute to the re-accumulation of starch content at the first node culm, and could be available to develop forage rice cultivars.

The utilization efficiency of solar energy through photosynthesis by crops is only a few percent at most. Therefore, most solar energy in the field (on-site) remains unused. Plants are, however, able to use certain types of monochromatic light as an environmental signal, and they exhibit various physiological responses to even small amounts of the light. In particular, the significant induction of secondary metabolite production by blue light is well documented. Here, we propose a novel cropping method where unused solar energy is captured on-site by solar cells and converted to monochromatic light by using LEDs. The crops are then irradiated to stimulate enhanced production of secondary metabolites, thus improving crop function without the need for an external energy source. In this study, the effect of blue light on the production of functional secondary metabolites was evaluated in perilla (Perilla frutescens var. crispa f. purpurea), a traditional medicinal crop in Japan. Perilla leaves contain several functional substances such as perillaldehyde, rosmarinic acid, and anthocyanins, which have notable stomachic, antipyretic, detoxification, sedation, and diuretic efficacy. Plants were grown under natural daylight supplemented with blue light irradiation (460 nm, approx. 10 mu mol/m(2)/s, 24 h,) for 108 days before harvest. Control plants were grown without supplemental lighting. The content ratio of perillaldehyde in plants grown with blue light was 60% higher at harvest than in the controls. Moreover, in the vegetative growth stage, the perillaldehyde content increased significantly after only three days of blue light irradiation. These findings suggest that the present approach will benefit agricultural practices in developing countries through more efficient usage of on-site solar energy. (C) 2014 The Authors. Published by Elsevier Ltd.

We found one line of Perilla herb with good smell, as a local genetic resource that may have been cultivated in the Kinokawa plain since ancient times. In this study, firstly, we revealed the morphological and aromatic characteristic of this line by comparison with that of four varieties of Perilla frutescens. Since the leaves of this plant were dark purplish-red color on both surfaces without crepe-like undulations, and this plant contained a notable amount of perillaldehyde and limonene, it was suggested that this line of perilla was one of the varieties belonging to the medicinal red perilla, P. frutescens var. crispa f. purpurea. Secondly, to grow up this resource as a local special product, we have begun to develop the cultivation method for year round production and open up new uses. Supplemental lighting by blue LEDs to the perilla plants which were grown under the natural daylight dreadfully inhibited the flower formation, and allowed the continuous differentiation of young leaves wealthy in perillaldehyde. This cultivation method is useful to inhibit flower formation during short-day period and to improve the functional component content in this perilla herb.

Miniature inverted-repeat transposable elements (MITEs) are widespread in both prokaryotic and eukaryotic genomes, where their copy numbers can attain several thousands. Little is known, however, about the genetic factor(s) affecting their transpositions. Here, we show that disruption of a gene encoding ubiquitin-like protein markedly enhances the transposition activity of a MITE mPing in intact rice plants without any exogenous stresses. We found that the transposition activity of mPing is far higher in the lines harboring a non-functional allele at the Rurm1 (Rice ubiquitin-related modifier-1) locus than in the wild-type line. Although the alteration of cytosine methylation pattern triggers the activation of transposable elements under exogenous stress conditions, the methylation degrees in the whole genome, the mPing-body region, and the mPing-flanking regions of the non-functional Rurm1 line were unchanged. This study provides experimental evidence for one of the models of genome shock theory that genetic accidents within cells enhance the transposition activities of transposable elements.© 2012 The Author.

The rice FRIZZY PANICLE (FZP) locus on chromosome 7, in which an ERF and acidic domain are present, is concerned with the regulation of spikelet meristem identity and the determination of panicle architecture. Many fzp mutants drastically alter panicle morphology with higher-order rachis-branches developing successively instead of the development of floral organs in these mutants. A new mutant showing the same fzp phenotype was induced by gamma-ray irradiation of seeds of a rice cultivar "Gimbozu". Examination of this fzp-like mutant for its allelism to a known allele of fzp-1, nucleotide sequence, and panicle and agronomic characteristics clearly indicated that the allele of this fzp-like mutant is located in FZP. Because there is a previously identified allele called fzp-9, we designated this new allele as fzp-10. fzp-10 has a single nucleotide (cytosine) deletion between the ERF domain and the acidic domain, which results in a frameshift mutation and a premature stop codon, thereby altering the C-terminus of the encoded protein. The degree of higher-order branching in the panicles was significantly reduced in the fzp-10 mutant compared with that of fzp-1. Moreover, the fzp-10 mutant showed highly depressed culm and panicle lengths and panicle number, as well as delayed heading dates compared with its wild type and also with fzp-1. fzp-10 has several new characteristics, its altered nucleotide position and severity of phenotype alteration, and, therefore, could be a new gene resource to examine the function of FZP and the determination of rice panicle architecture.

本研究は日本学術振興会科学研究費補助金22658077および近畿大学生物理工学部戦略的研究No.09-IV-1, 2010の助成を受けた.[要旨] 本研究は、機能性成分含有量の高い'紀州在来薬用紫蘇' の自然光併用型植物工場での高効率生産を目的とする。自然光下(ガラスハウス内)で栽培している'紀州在来薬用紫蘇'および'赤ちりめんしそ'に青色LEDの光を24時間連続照射し、草丈、主茎節数および機能性成分の一種であるペリルアルデヒドの含有量の変化を調査した。青色光補光による草丈の減少と主茎節数の増加が観察されたが、これらの変化は同時に観察された青色光補光による花成阻害に起因すると考えられた。一方、収穫された葉におけるペリルアルデヒド含有量は青色光照射によって有意に増加した。 [Abstract] Two cultivars of perilla, 'Kishu-zairai' (Perilla frutescens var. crispa f. purpurea, red s mooth 1 eat) and 'Aka-chirimen-shiso' (Perilla frutescens var. crispa f. crispa, red wrinkled leaf), were grown in a greenhouse and were lighted continuously by blue LEDs (460nm, approximately 10μmol/m^2/s). As a result, it was revealed that the blue supplemental-lighting reduced the plant height and increased the number of nodes on main stem in both kind of perilla. These two phenomena were thought to be associated with the inhibition of flower formation by the blue supplemental-lighting. While, the content of perillaldehyde (a kind of functional chemical component) in harvested leaves was increased by the blue supplemental-lighting.

Rice (Oryza sativa L.) genes AS2-2 and SUT1-2, which are alleles at OsAGPS2 (AS2) on chromosome 8 and OsSUT1 (SUT1) on chromosome 3, respectively, were suggested to improve grain filling in extra-heavy panicle rice cultivars. This study examined the distribution of these alleles among 315 rice cultivars. A discriminant function based on phenol reaction of hulls, apiculus hair length and KClO3 sensitivity in seedlings was used to classify cultivars into japonica (192 cultivars) and indica (123 cultivars). Genotyping for the AS2 and SUT1 alleles were performed with In/Del and SSR markers, respectively. In japonica cultivars, the frequencies of AS2-2 and SUT1-2 were very low (3.6% and 6.3%, respectively), whereas they were more common in indica cultivars (56.1% and 74.0%, respectively). In addition, in indica cultivars the genotypes of AS2-2 SUT1-2, as well as AS2-1 SUT1-1, were more frequent than expected from independent segregation of these alleles. It was suggested that artificial and/or natural selection contributed to this non-random distribution of alleles at AS2 and SUT1. The alleles for good grain filling should, thus, be explored in indica cultivars to improve the grain filling of extra-heavy panicle types of rice in future breeding programs.

Rice (Oryza sativa L.) cultivars of extra-heavy panicle type (EHPT), which have numerous spikelets in a panicle, often fail to realize their high-yield potential due to poor grain filling in general, while some EHPTs showed relatively better grain filling. This study first surveyed nucleotide polymorphisms in rice genes possibly related to grain filling, that is, genes for ADPglucose pyrophosphorylase (AGPase) subunits, OsAGPS2 and OsAGPL2, and a sucrose transporter gene, OsSUT1, to clarify the causes underlying such genetic variation. Many types of polymorphisms were detected in all three genes, but none of them affected amino acid sequences. OsAGPS2 and OsSUT1 had an identical series of polymorphisms in two EHPT cultivars, Milyang 23 and Nanjing 11, both of which have a high degree of grain filling, while another series for these genes was detected in other EHPTs showing a low grain filling. The former polymorphisms could constitute alleles for good grain filling in OsAGPS2 and OsSUT1, designated as AS2-2 and SUT1-2, respectively. Second, new molecular markers based on the polymorphisms for OsAGPS2 and OsSUT1 were developed. Finally, three EHPT cultivars with AS2-2 SUT1-2 genotype showed a higher degree of grain filling and higher activity of AGPase than three other EHPTs with another genotype at these loci. These results suggest that these AS2-2 and SUT1-2 alleles can contribute to the improvement of poor grain filling of EHPTs assisted with the developed markers.

The YABBY gene DROOPING LEAF (DL) regulates midrib formation in the leaves and carpel specification in the flowers of rice (Oryza sativa L). We found a new dl allele (dl-5) that caused a mild phenotype in the descendants of a mutable line, IM294. In plants homozygous for this allele, midrib structures were formed but their sizes were reduced. Molecular analysis revealed that a transposon, Ping, was inserted in the fourth intron of DL. Together with mPing and Pong, Ping is a member of a transposon family that was first identified as a group of active transposable elements in rice. Our finding of the Ping insertion in the DL gene is a first indication that Ping is active in planta, and that it can be transposed and integrated in a new locus. Ping seems to be still active because it was excised from intron 4 of DL at a relatively high frequency in rice calli. Real-time PCR analysis and in situ hybridization indicated that DL transcript levels were reduced in dl-5 without alterations in the spatial expression pattern of the DL gene. The reduction of DL expression may be due to inefficient splicing of the large intron caused by Ping insertion. By comparing the expression levels of DL and leaf phenotypes in the dl mutants with different severities, we confirmed our previous hypothesis that DL promotes cell proliferation in the central region of leaf primordia, and that this cell proliferation is critical for midrib formation in the mature leaves.

We developed a database system to support whole experiments which analyze correlation between genotypes and phenotypes by using transposons. We also evaluated the system by applying to our experiments with transposon mPing in rice. Our research group grow large-scale mutant series of rice by taking advantage of mPings, and study correlation between genotypes and phenotypes for predictions of gene functions. However, the analytical phase, in which we find mutation spots from waveform data, involves several problems from a viewpoint of labor amount, and it becomes one of bottlenecks in large-...

We are currently performing a functional genomics of rice (Oryza sativa L.) via insertion mutagenesis induced by mPing, an active inherent transposon of rice. Transposon display is an excellent method for exhaustive detection of numerous polymorphisms at mPing insertion sites in whole rice genome. In many cases, however, too many copies of mPing were present originally in rice genome, which makes difficult to identify truly polymorphic signals from such background involving many non-polymorphic amplified signals. In this study, we verified whether one or two selective bases added to an adap...

A mixture of artificial and natural light is indispensable for producing crops. This study develops a low-cost system for the short-time irradiation of plants in addition to natural daylight. Blue light (400-500nm) is employed by a highly autonomous production system. Using the proposed method, the total consumed energy is reduced by about 26%. The assistant light is supplied in the early morning to avoid energy generation conflicts.

This study describes a prototype TiNi shape memory alloy microgripper which could be used in an assembly process of a micro factory. The microgripper consists of two units, displacement direction-conversion unit and unit for the displacement drive, integrated with same material for miniaturization. Assuming to grip a pin of the about 0.5mm diameter, measuring the characteristics of the alloy on deformation and transformation, designing a microgripper using FEM, trial manufacturing and the characteristic evaluation of the gripper were carried out. As the results, the following were obtained:...

筆者らは、水稲の幼苗期耐冷性を、幼苗が急激に強度の低温に曝されたときに即座に発現する常発型耐冷性と、幼苗が徐々に強い低温にさらされていく過程でその発現が誘導される順化型耐冷性の2 つの側面に分けて考えている。本報では、この順化型耐冷性が、これを誘導する低温順化処理を行う幼苗のステージによって影響をうけるか否かを調査した。その結果、催芽種子の播種後3日から12日の幼苗に低温順化処理を行った場合、順化型耐冷性の発現が安定的に認められることが示された。

To construct the technical foundation of the new cropping system using short-time irradiation of blue light in addition to the natural daylight, we designed the irradiation equipment that consisted of blue fluorescence light and translucent solar cell. The additional irradiation of blue light to plantlets of pea (Pisum sativum L. cultivar Kisyu-Usui) for a half hour before daylight influenced the growth of the pea plants. The plant height, number of nodes on the main stem and fresh weight of the pea plants were significantly increased with the increase of relative intensity of irradiated bl...

イネ細粒遺伝子slg の内部に見出されたトランスポゾンmPing の転移活性は、slg 座の遺伝子型によって制御されているように見えた。本研究では、その原因を明らかにするため、slg がゲノム全体のシトシンのメチル化に及ぼす影響を調査した。その結果、slg は、ゲノム全体のシトシンのメチル化にも、また、CGメチラーゼおよびCNG メチラーゼの活性にも影響しないことが明らかになった。

The gene concerned with the formation of panicle neck of the rice plant is the gene which works at the conversion from the vegetative growth stage to the reproductive growth stage, therefore, the general and molecular genetic analysis of such genes is very important for breeding of rice (Oryza sativa L.). In this study, first of all, genetic analysis was done about two different types of mutant for knob formation on panicle neck, which are induced from a rice variety Gimbozu by gamma irradiation. As the result, it was clarified that this trait in both cases was controlled by single recessive gene. Next, the morphological observation of the knob on panicle neck was carried out using scanning electron microscopy. The result showed that this knob would be derived from hypertrophy of the palea leaf base and the panicle neck knot to support developing palea leaves. The gene related to this trait may regulate the timing of the growth termination of palea leaves.

Rice (Oryza sativa L.) is an important crop worldwide and, with the availability of the draft sequence(1,2), a useful model for analysing the genome structure of grasses(3),(4). To practice efficient rice breeding through genetic engineering techniques, it is important to identify the economically important genes in this crop. The use of mobile transposons as gene tags in intact plants is a powerful tool for functional analysis because transposon insertions often inactivate genes(5). Here we identify an active rice transposon named miniature Ping (mPing) through analysis of the mutability of a slender mutation of the glume(6)-the seed structure that encloses and determines the shape of the grain. The mPing transposon is inserted in the slender glume (slg) mutant allele but not in the wild-type allele. Search of the O. sativa variety Nipponbare genome identified 34 sequences with high nucleotide similarity to mPing, indicating that mPing constitutes a family of transposon elements. Excision of mPing from slg plants results in reversion to a wild-type phenotype. The mobility of the transposon mPing in intact rice plants represents a useful alternative tool for the functional analysis of rice genes.

水稲の幼苗期耐冷性には、幼苗が急激に強度の低温に遭遇すると即座に発現する常発型耐冷性と幼苗が徐々に強い低温にさらされていく過程で発現が誘導される順化型耐冷性の2 種が存在すると考えている。本報では、このうち順化型耐冷性に着目し、品種銀坊主の幼苗に休止期間を挟む2 回の低温処理、低温順化処理と耐冷性評価処理を行い、耐冷性の発現における低温順化処理の効果と誘導された順化型耐冷性の保持期間について検討した。その結果、5℃の低温順化処理によって順化型耐冷性が誘導されて耐冷性評価処理の障害を軽減すること、また、その誘導された順化型耐冷性は少なくとも8日間保持されることが明らかになった。

japonica 型水稲品種を用いて幼苗期耐冷性品種を育成する上で必要な遺伝学的知見を得るため、耐冷性弱の銀坊主と耐冷性強のはやゆきの交雑後代F_3 系統を供試し、生体重を評価指標として幼苗期における系統の耐冷性を調査した。その結果、F_3系統間で幼苗期耐冷性に関する大きな変異が認められ、また、両親品種の幼苗期耐冷性を超越するF_3 系統が出現した。このことから、幼苗期耐冷性は両親品種間で複数の遺伝子座について異なっており､交雑後代で幼苗期耐冷性遺伝子を集積できることが推察された。

水稲品種銀坊主種子のγ 線照射後代に見いだされた突然変異誘発遺伝子mutator を効率良く育種に利用するには，mutator によって誘発される変異のスペクトラムを明らかにする必要がある．本研究では，この観点から，葉から抽出した脂質の脂肪酸組成に関する変異の検出を試みた．種子に含まれる全脂質の脂肪酸組成に変異が認められたmutator 由来の突然変異固定型系統12系統について，葉から抽出した全脂質を８脂質分子種に分画した上で，各脂質分子種における脂肪酸組成を調査したところ，いずれの分子種についても，脂肪酸含有比率が原品種銀坊主と明瞭に異なる系統が認められ，mutator によって脂肪酸組成に関する変異が誘発されることが示された．

A mutagenic factor, mutator, that was detected in the progeny from a γ-ray irradiated M_1 seed of a japonica rice variety Gimbozu, generated novel mutations at many different loci, when activated by the reversion of a mutable slender-glume gene slg, which was induced by γrays in the above identical M_1 seed. For the effective utilization of the mutator in plant breeding, the reversion mutability of slg was analyzed for its genetic behavior and relationship with the mutator activity. First, changes in the reverse mutation frequency of slg(RMF)were traced among several generations, using 39 slender-glume lines which originated from a single slender-glume mutant but were more or less different from each other in background genotype. The results revealed that the mutability of slg was essentially an inherited character, but that it often increased or decreased. Secondly, eleven of the 39 slender-glume lines were each estimated for the mutator activity by examining the frequency of mutator-induced heading-date mutations that appeared in the porgenies of non-slender-glume plants from the slender-glume lines, using 424 sublines derived from the 11 lines. As a result, it was found that the mutability of slg did not influence the mutagenic activity of the mutator.

継続後誌:近畿大学先端技術総合研究所紀要 = Memoirs of Institute of Advanced Technology, Kinki University水稲品種銀坊主種子のγ線照射後代に見いだされた突然変異誘発造伝子mutatorは、上記γ線照射で誘発された1細粒遺伝子slgの正常粒遺伝子(＋)への復帰突然変異に伴って活性化し、形態的あるいは生理的形質に種々の変異を誘発する。しかし、植物体構成物質の成分についてはまだ突然変異誘発の有無が明らかにされていない。そこで、本研究では、生体膜を形成する重要な成分であり、すでに一部の代謝反応系が解明されている脂質に着目し、mutatorによって誘発された形態的あるいは生理的形質に関する突然変異固定型48系統を供試して、脂質代謝経路におけるmutator誘発突然変異の有無を探索した。まず、供試全系統について全脂質の脂肪酸組成を調査したところ、組成脂肪酸の含有比率が正規分布から有意に外れる9系統が見いだされた。ついで、これら9系統のうち解析に十分な量の種子が得られた6系統および上記9系統についで大きな変異を示した8系統計14系統について、種子中に多く含まれる3脂質分子種フォスファチジルセリン、フォスファチジルエタノールアミンおよびフォスファチジルコリンの脂肪酸組成を調査したところ、いずれの分子種についても、脂肪酸含有比率が原品種銀坊主と有意に異なる系統が相当数認められた、このことから,mutatorは植物体構成物質の成分に関する変異も誘発すると考えられた。 （英文） The mutator that was found out first in the M_3 progeny derived from a γ-ray irradiated M_1 seed of a japonica rice variety Gimbozu, causes novel mutations in many different loci for morphological and/or physiological traits, when activated by the reversion of a mutable slender-glume gene slg., which was induced by γ rays in the above identical M_1 seed. However, it has not yet been confirmed if the mutator also mutate the structure material of plants. Lipid is one of the most important components of biomembrane, and has partly been elucidated for metabolic pathway. In view of this situation, the mutations that might have been induced by the mutator on lipid metabolic pathway was screened through the analysis of the fatty acid composition of lipid contained in seeds, using 48 mutant lines with some mutator-induced morphological and/or physiological mutations. First, the 48 lines were analyzed for their respective fatty acid composition in total lipid, and nine of these lines each proved to be significantly apart from the population mean in at least one of the percent contents of 5 sorts of fatty acid. Then, 14 lines the percent contents of which largely deviated from the population mean, inclusive of 6 from the above 9 lines, were selected and analyzed for fatty acid composition of 3 lipid molecular classes, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine. As a result, it was observed in all the classes that a considerable number of lines were significantly different from the original variety in the percent content of fatty acid. This may strongly suggest that the mutator induces mutations also in the components of structure material of plants.

The genetics of leaf senescence, one of the most important yield-determinant characters in cereals, has been a great concern in plant breeding. From this point of view, an early leaf-senescence (ELS) mutant induced by tissue culture of a rice variety, Nipponbare, and detected first in the next generation (R_1) of a regenerated plant (R_o) was analyzed for the gene concerning the relevant mutation. First, the R_2 progeny lines derived from the normal-type plants which belonged to the same R_1 line as the ELS mutant did were examined for their genetic segregation with early leaf-senescence. The result revealed that ELS was controlled by a single recessive gene. Secondly, using 10 normal-type plants and 15 mutants of R_3 eneration, chlorophyll content in several leaves per plant was measured every 3 days for 2 months around heading time. The result suggested that the gene responsible for ELS might advance the time of beginning of senescence in lower leaves (8-12 th leaves), while retard the progress of senescence in each leaf.

イネの稈におけるデンプン含量およびデンプン代謝関連遺伝子の発現量の推移を，組換え近交系を用いて解析し，稲体のデンプン含量の制御に関する育種の基礎としての知見を得ることを試みた．その結果，登熟後期で稈のデンプン含量が再増加するものとしないものを比較すると，デンプン合成酵素，ショ糖合成酵素に関連する遺伝子の発現量が前者では後者よりも増加していることがあきらかになった．

植物工場の経済性モデルを考案し、人工光源を用いた植物工場は電力コストにより栽培できる作物は、レタスなどの葉もの野菜に限定されることを示した。この経済性モデルは、エネルギー源として期待される芋などの燃料作物の栽培は、太陽光を分散利用しない限り経済性がマイナスとなるため、屋外の多層棚栽培が唯一の実現手段であることを示す。

植物工場の経済性モデルを考案し、人工光源を用いた植物工場は電力コストにより栽培できる作物は、レタスなどの葉もの野菜に限定されることを示した。この経済性モデルは、エネルギー源として期待される芋などの燃料作物の栽培は、太陽光を分散利用しない限り経済性がマイナスとなるため、屋外の多層棚栽培システムの開発を提唱した。

イネ極穂重型の登熟程度の向上を目的とし，登熟程度に変異を示す品種間のOsAGPS2およびOsSUT1にける塩基配列多型から，登熟を向上させることが示唆される対立遺伝子を見いだした．これらを識別する分子マーカーを開発し，この対立遺伝子は発育胚乳中のADPグルコースピロホスホリラーゼ活性を増加させ，登熟程度を向上させることが確認された（英文）．

極穂重型のイネの低登熟性を改良する遺伝資源として，ADPグルコースピロホスホリラーゼ遺伝子(OsAGPS2)およびショ糖トランスポーター遺伝子(OsSUT1)において良登熟型対立遺伝子を検出し，それらの登熟に及ぼす効果を組換え型自殖系統を用いて検証した（英文）．

イネに内在するトランスポゾンmPingの活発な転移を利用すれば、農業形質に関する有用遺伝子のノックアウトおよびタギングを効果的に行うことが可能である。本報では、アンカーPCRによるトランスポゾンディスプレイと、イネトランスポゾン解析支援システムによるデータ解析を紹介し、有用形質に関する変異と共分離するmPingコピーの同定を試みた結果を報告する。

イネ紋枯病に対する抵抗性遺伝資源を、我国内外のイネ50品種を供試して探索した。我国の品種群では抵抗性をほとんど示さないか、比較的弱い抵抗性を示すのみであったが、外国イネ品種の中には、極めて高い抵抗性を示すものが認められた。

イネの低温耐性および病害抵抗性に関する研究の成果を報告する。まず、イネの耐冷性に関しては、分子生物学発展の成果を取り入れやすくするために、発芽後の極めて初期の耐冷性にスポットをあてて研究を始めた。その中で筆者らは、耐冷性の程度が苗の生育ステージによって変化すること、および、比較的程度の緩い低温によって誘導される順化型耐冷性が亜熱帯起源のイネにも存在することを見出した。一方、病害抵抗性に関しては、イネの主要病害である紋枯病について研究を進めており、イネの抗菌性防御物質の一つが紋枯病菌の生長を抑制することを突き止めた。

mPingの転移におけるRurm1の役割を解明するために、Rurm1のコードするタンパク質RURM1の活性化因子についてデータベースを用いた検索を行った。酵母における活性化因子であるUba4pのアミノ酸配列と相同性の高い配列をデータベース中から検出することを試みたところ、Uba4pと相同なタンパク質をコードする遺伝子を見出すことができた。

耐冷性遺伝子の集積による高度耐冷性水稲品種の育成を目的として、japonica型イネ2品種（中生新千本およびアケノホシ）の幼苗期耐冷性を支配するQTLの連鎖分析を行った。一般に、近縁品種間では連鎖分析に必要な分子マーカーが十分に得られないため、ここでは、indica型品種密陽23号を共通親とする2組の分離集団を作成し、両集団内で個別にQTL解析を行ってその結果を総合する、比較QTL解析を試みた。実験に用いたjaponica型2品種の幼苗期耐冷性はindica型品種密陽23号に比べて極めて強いものである。両分離集団の各々について各個体からDNAを抽出し、SSRマーカーを用いて連鎖地図を作成した。また、上記各個体の個体別次代系統に対して低温障害程度を指標とした耐冷性調査を行い、その平均値を各個体の耐冷性評価値とした。連鎖分析の結果、幼苗期耐冷性に関連すると見られるいくつかの遺伝子座を見出した。

極穂重型イネ品種間でみられる登熟能力の変異をもたらす一要因として，発育胚乳中のショ糖代謝関連酵素に注目した．これら酵素中，発育胚乳でのショ糖合成酵素とADPグルコースピロホスホリラーゼ活性は，登熟良好な品種の方が登熟不良な品種に比べて高いこと，後者の酵素遺伝子に関しては登熟程度の異なる品種間で塩基配列の多型が認められること，等が示された．これによって，登熟能力を遺伝的に改良していく可能性が示唆された（英文）．

登熟能力に関して変異を呈するような極穂重型イネ品種間の発育胚乳中で発現するショ糖代謝関連酵素遺伝子，特にADPグルコースピロホスホリラーゼ遺伝子の5'側上流域について塩基配列の多型を解析した．その結果，登熟が比較的良好な品種南京１１号のみが他の品種とは異なる配列を数カ所において示すことが明らかとなった（英文）．

イネのトランスポゾンmPingの生物学的特性について概述した後、プロジェクトにおける研究の目的と内容ならびに最近の研究成果について紹介した。本プロジェクトでは、経済作物であると同時に優れたモデル植物でもあるイネを対象として、まずトランスポゾンmPingの転移による遺伝子破壊系統群の作出し、破壊された遺伝子の同定を行う。ついで、特定の有用経済形質を支配する遺伝子群の構造および機能に関する情報をコンピュータ内に集積して、個々の遺伝子の発現から形質発現に至る過程の遺伝子間相互作用ならびに遺伝子ネットワークの解析を行う。

イネのRurm1座に挿入されたトランスポゾンmPingは活発に転移することが知られているが、他のゲノムサイトに挿入されているmPingの転移については十分な解析がなされていない。そこで本研究では、トランスポゾンディスプレイ法によりRurm1座以外のサイトにあるmPingの可動性を検証した。その結果、細粒系統では、Rurm1座以外のサイトにあるmPingも自然栽培条件下で頻繁に転移することが示された。

イネのトランスポゾンmPingの活発な転移を利用したイネの遺伝子機能解析技術を確立するため、mPingの新規挿入を検出する方法およびmPingによってタグされた遺伝子の同定法の開発を行った。その結果、mPingによって新たにタグされた染色体8上の遺伝子を同定することができた。

イネの易変性細粒遺伝子slg の内部に見出されたトランスポゾンmPing について、自然栽培条件下の植物体内における可動性を示した。またイネ品種群におけるmPing の分布から、可動性を維持しているシステムに関する考察を行った。

イネのトランスポゾンmPing の転移を全ゲノムに渡ってモニタリングするためのトランスポゾンディスプレイの実施条件について最適化を行った。その結果、この方法を用いれば、供試サンプル間で複数コピーのmPing の転移を検出できることが明らかになった。

イネのトランスポゾンmPing の端部にあるGGCC 配列中のシトシンのメチル化の有無についてPCR による調査を行った。その結果、葉組織では、この配列がメチル化されており、このことがmPing の可動性を制御する要因の一つではないかと考えられた。

細粒突然変異系統IM294のmPing挿入サイトのうち9サイトについて、細粒個体4790個体におけるmPingの切り出しを調査した結果、2サイトでのみ切り出しが確認された。このことは、mPingが挿入された部位によって切り出し頻度が異なる可能性を示唆している。

イネの易変性細粒遺伝子 s/g と Rurm1 座内の MITE 様転移因子 Sairyu の挿入との対立性検定を行った。 その結果、 両者は互いに対立関係にあって、 s/g の易変性は Sairyu の転移によって発現することが明らかになった。

A mutagenic factor, mutator, that was detected in the progeny from a γ-ray irradiated M_1 seed of a japonica rice variety Gimbozu, generated novel mutations at many different loci, when activated by the reversion of a mutable slender-glume gene slg, which was induced by γrays in the above identical M_1 seed. For the effective utilization of the mutator in plant breeding, the reversion mutability of slg was analyzed for its genetic behavior and relationship with the mutator activity. First, changes in the reverse mutation frequency of slg(RMF)were traced among several generations, using 39 s...

2021年度は、研究室に保存されている品種、および、トランスポゾンmPingの転移によって生じた突然変異遺伝子を保有する系統について、浸水処理にともなうGABAの増加量を調査した。これまでGABAの定量にはGABAseを利用した酵素法を用いてきたが、2段階の酵素反応を利用しているため、特にGABA濃度が低い場合に著しく定量性が低下することが課題となっいた。このため、HPLC装置を用いた遊離アミノ酸定量法の適用も検討したが、当研究室が保有している装置では定量することができなかった。そこでさらに、Enzyme Sensor Co. Ltd.から発売されたGABAseとは異なる酵素を利用した酵素法を用いたGABA定量キットの適用を検討している。定量の精度について現在検証をすすめつつあるが、実験操作は極めて容易であり所要時間を大幅に短縮できるこメリットがある。定量性の検証が終われば、この方法に採用して研究を進める予定である。 これに並行して、供試する品種および突然変異系統について、十分な量の玄米サンプルを得るための種子増殖をはかった。紀の川市内の実験圃場に、100品種および1000突然変異系統を栽植し、それぞれから籾を収穫し乾燥させた。 また、水浸過程ではGABAの増加と同時に、胚から誘導されるアミラーゼによるデンプンの部分分解も始まる。このため催芽した玄米に由来する白米では、デンプンの物理特性が変化して食味が変化する可能性がある。そこで、デンプンの物理特性を評価するための装置を準備して、GABAを増加させるための処理が食味に与える影響を評価する準備を進めている。 これまでに得られた研究成果については、2021年6月に近畿作物・育種研究会において口頭発表を行い、同内容で投稿準備中である。

In this study, we described one line of Perilla herb, as a local genetic resource in the Kinokawa plain. First, we observed morphological and aromatic characteristics of the line and evaluated the genetic variation for perillaldehyde content. As a result, this line has been fixed genetically for these characteristics. And then, we have begun to develop the cultivation method for year round production and for enrichment of the functionality of the perilla. Supplemental lighting by blue LEDs significantly inhibited the flower formation of perilla, and allowed the continuous differentiation of young leaves related to higher content of perillaldehyde.

本研究は、透光性太陽電池によって得られた電力で単色光蛍光灯やLEDを点灯し、その特定波長の光を花卉に照射することによって、経済的な特性に有用な変化を誘導しようとするものである。これまでの研究で、スプレーギクのつぼみへの紫外線照射が花色の変化を誘導することや、ビオラへの青色光の照射が着花数の増大をもたらすことが明らかになった。そこで本年度は、まず、スプレーギクについて、花色の変化を誘導するのに適した紫外線の照射時期を検討したところ、開花の10日前、すなわち出荷の数日前に紫外線を照射すると顕著に花色が変化することが明らかになった。したがって、通常の出荷時期よりも少し早く採花して、低音貯蔵中に紫外線を照射すれば極めて低コストで花色を改善することか可能と考えられる。ただし、一部の花に紫外線によると見られる障害が認められたため、紫外線照射強度や用いる紫外線の波長に関してさらに条件を検討する必要がある。また着花数については、ビオラを含む数種の花卉苗に青色光または赤色光を照射し、これらの処理が着花数に及ぼす効果を検討した。その結果、単色光照射によって着花数は顕著に増加したが、照射光の波長に対する反応特性は、種によって、また一部では品種によっても異なっていた。このように、反応に一意性が認められないため、現状では処理の効果を予測するのは困難である。単色光を用いて着花数を制御するためには、さらに...

Transposable activity of an active-transposon mPing in a rice mutant line, IM294 is much higher than in the original variety Gimbozu. Because IM294 is considered harboring a recessive mutation allele at Rurm1 locus, it is very likely that the destruction of the normal Rurm1 gene participates in high mPing transposable activity of IM294. We analyzed a change to occur by function loss of the Rurm1 gene to elucidate a novel rice protein conjugation system relating to the RURM1 protein (Rurm1 system), and this study was going to get the due to clarify the induction mechanism for the transpositi...

In a mutant line IM294 (slender glume mutant) which harbors Rurm1 (Rice Ubiquitine Related Modifier-1) allele inactivated by mPing insertion shows high excision frequency (ca.1%) of mPing from mutant Rurm1 allele (Rurm1^m). Expression levels of two ORFs of Ping were also high in IM294 comparing to the original variety Gimbozu. To clarify the relation between the function of Rurm1 and Ping activity under the same genetic background, the mutant allele of Rurm1 was introduced into Gimbozu background through recurrent backcrossing. Also, a pair of non-slender glume line (Rurm1^+/Rrum1^+) and sl...

本研究は、イネのMITE型トランスポゾンmPingの活発な転移を利用したノックアウト遺伝子の効率的クローニング法の開発を目指している。同様の目的で開発された遺伝子タギング法はこれまでにも存在するが、本法の特色は一般に量的形質であり通常の分離分析を行うことが困難な農業形質関連遺伝子のクローニングを目的とする点である。この目的を達成するため、突然変異分離集団の大規模なトランスポゾンディスプレイを行い、QTL解析に準じた統計処理によって対象形質の変異に密接に関連するmPing挿入多型を見出そうと考えた。平成15年度には、細粒形質に関するキメラ個体をモデルとして、トランスポゾンディスプレイから突然変異遺伝子のタギングに到る一連の技術的基盤を完成させた。平成16年度には、タグライン作成用細粒系統の大規模圃場展開を行って、mPing挿入突然変異体を収集するとともに、分離系統の一部についてトランスポゾンディスプレイを行って脱粒性と密接に関連する117bpの増幅断片を生じるmPing挿入多型を見出した。平成17年度は、前年に得られたmPing挿入突然変異体の個体別次代系統を圃場に展開し、農業形質に関する突然変異の有無を調査すると同時に、農業形質に関する分離が認められた2系統の全個体について個体別にトランスポゾンディスプレイを行い、共分離するmPing挿入多型の検出を試みた。その結果、稈長につ...

A mutable slender mutant (line : IM294) was induced with gamma-ray irradiation to seeds of the rice variety Gimbozu. This mutant character is governed by a recessive mutant gene and has never been fixed in spite of repeated self-propagation. Our previous study suggested that the mutability is caused by the precise excision of a MITE (Miniature Invereted-repeat Transposable Element mPing) that was inserted in the wild type allele at the Rurm1(Rice ubiquitin related modifier 1) locus. The present study confirmed this point, and also indicated that mPing, which lacks coding region, is a deleti...

A mutable slender glume gene slg was induced by gamma-ray irradiation to seeds of the rice variety Gimbozu. Occasionally reverting to its wild-type, slg is assumed to involve an active transposable element in it. To identify the molecular characteristics of this element, we attempted to clone slg with a positional cloning method. slg has already been mapped on the overlapping region of two YAC clones, Y1774 and Y3356. We first selected 209 candidate clones from the cDNA clone library of the rice variety Nipponbare using a YAC clone Y1774 as a probe, and then partially sequenced 54 clones. B...

イネゲノム内の他の遺伝子座に新たな変異を誘発するmutatorを突然変異育種上の新たな変異原として効果的に利用するには、その発現契機である細粒遺伝子(slg)の復帰突然変異が安定的に高頻度で生じる必要がある。しかしながら、多数のslgを持つ細粒系統の中には、slgの復帰突然変異頻度(RMF)が著しく少ない系統が少なからず認められる。本研究は、組織培養によってこれら低RMF系統のRMFを増加させ、mutatorの有する突然変異誘発活性の有効な利用法を確立しようとするものである。平成10年、育成経過の異なる40の細粒系統からRMFの比較的低い5系統(IM21、IM54、IM136、IM157およびIM294)を選び、系統ごとに、胚盤由来のカルスから得られた再分化植物(R_0細粒系統)と組織培養を経ていない細粒個体の自殖によって得られた細粒系統(NC細粒系統)との間でRMFを比較した。その結果、5系統の全てにおいてRMFの増加が認められた。平成11年は、上記再分化植物中に見いだされた細粒固体の個体別自殖次代R_1細粒系統とNC細粒系統との間でRMFを比較し、組織培養がRMFに及ぼす遺伝的な効果を観察した。その結果、大部分のR_1細粒系統では組織培養によるRMFの有意な増加が認められなかったものの、少数のR_1細粒系統ではNC細粒系統に比べてRMFが有意に増加していた。以上のように、...

耐冷性評価の確立を目的に、これまで茎葉部の低温反応を指標とする耐冷性の評価を行ってきたが、本年度はこれに加えて、種子根の発育状態や冠根の発生程度など、根部の低温反応を指標とする耐冷性の評価を試みた。その結果、根部に示される耐冷性は、茎葉部に示される耐冷性と弱いながらも有意な相関を示し、初期の耐冷性イネの選抜は、観察容易な茎葉部の耐冷性について行えばよいと考えられた。また、根部においても茎葉部と同様、幼苗の発育ステ-ジに伴う耐冷性発現の変化が認められた(日本育種学会第88回講演会において発表)。つぎに、茎葉部の耐冷性に基づいて、日本国内で育成されたジャポニカ型水稲70余品種の耐冷性を評価したところ、北海道地域および東北地域で育成された品種では、耐冷性が多様で極強の品種が多く認められたが、北陸以西の地域で育成された品種では多様性に乏しく、極強の品種はみられなかった(日本育種学会第89回講演会において発表予定)。また、mutator(突然変異誘発遺伝子)によって誘発された固定型突然変異80余系統の耐冷性を評価したところ、耐冷性に系統間差が認められ、mutatorを用いても耐冷性の強化が可能と考えられた。一方、耐冷性獲得機構の生化学的解析を進めるために、カラムクロマトグラフ、薄層クロマトグラフおよびガスクロマトグラフを用いて、少量試料(種子、根および葉)からリン脂質および糖脂質を抽出...