Mutational effects of chronic gamma radiation throughout the life cycle of Arabidopsis thaliana: Insight into radiosensitivity in the reproductive stage.

Organisms living on Earth have always been exposed to natural sources of ionizing radiation, but following recent nuclear disasters, these background levels have often increased regionally due to the addition of man-made sources of radiation. To assess the mutational effects of ubiquitously present radiation on plants, we performed a whole-genome resequencing analysis of mutations induced by chronic irradiation throughout the life cycle of Arabidopsis thaliana grown under controlled conditions. We obtained resequencing data from 36 second generation post-mutagenesis (M2) progeny derived from 12 first generation (M1) lines grown under gamma-irradiation conditions, ranging from 0.0 to 2.0 Gray per day (Gy/day), to identify de novo mutations, including single base substitutions (SBSs) and small insertions/deletions (INDELs). The relationship between de novo mutation frequency and radiation dose rate from 0.0 to 2.0 Gy/day was assessed by statistical modeling. The increase in de novo mutations in response to irradiation dose fit the negative binomial model, which accounted for the high variability of mutation frequency observed. Among the different types of mutations, SBSs were more prevalent than INDELs, and deletions were more frequent than insertions. Furthermore, we observed that the mutational effects of chronic radiation were greater during the reproductive stage. These results will provide valuable insights into practical strategies for analyzing mutational effects in wild plants growing in environments with various mutagens.

Japanese "nameko" mushrooms (Pholiota microspora) produced via sawdust-based cultivation exhibit severe genetic bottleneck associated with a single founder.

Pholiota microspora ("nameko" in Japanese) is one of the most common edible mushrooms, especially in Japan, where sawdust-based cultivation is the most dominant method accounting for 99% of the production. The current strains for sawdust cultivation in Japan are considered to have been derived from a single wild strain collected from Fukushima, Japan, implying that commercial nameko mushrooms are derived from a severe genetic bottleneck. We tested this single founder hypothesis by developing 14 microsatellite markers for P. microspora to evaluate the genetic diversity of 50 cultivars and 73 wild strains isolated from across Japan. Microsatellite analysis demonstrated that sawdust-cultivated strains from Japan were significantly less genetically diverse than the wild strains, and the former displayed a significant bottleneck signature. Analyzing the genetic relationships among all genotypes also revealed that the sawdust-cultivated samples clustered into one monophyletic subgroup. Moreover, the sawdust-cultivated samples in Japan were more closely related than full-sibs. These results were consistent with the single founder hypothesis that suggests that all commercial nameko mushrooms produced in Japan are descendants of a single ancestor. Therefore, we conclude that cultivated P. microspora originated from a single domestication event that substantially reduced the diversity of commercial nameko mushrooms in Japan.

Genetic variation of the relict maple Acer miyabei: uncovering its history of disjunct occurrence and the role of mountain refugia in shaping genetic diversity.

PREMISE: Relict species provide valuable insights into the origin and formation of extant vegetation. Here, we aimed to elucidate the genetic structure and diversity of a riparian relic, Acer miyabei, in Japan. Once widely distributed, it now occurs in three isolated regions. The most northern regional group is located at low elevation on Hokkaido Island, whereas the southernmost group in central Honshu Island is at high elevation in a mountainous landscape. This contrastive distribution enables us to examine the effects of climate oscillations on genetic diversity in relation to topographic variation. METHODS: We collected 604 individuals of A. miyabei from 43 sites. Their genetic structure and diversity were analyzed using 12 microsatellite markers and cpDNA sequences. RESULTS: According to structure analyses, ∆K was lowest at K = 2; the clustering essentially separated many of the individuals in the most northern regional group from the others. In contrast, the two southern groups were not clearly differentiated from each other, despite their geographic discontinuity. The proportion of private alleles was high in populations from the mountain terrain in the southern group although the number of extant populations is limited. CONCLUSIONS: Genetic clustering of A. miyabei is not perfectly congruent with the current patterns of geographic distribution. We infer that disjunction of the two southern groups occurred more recently than that between these groups and the northern group. The mountainous landscape in the most southern region likely provided multiple refugia and contributed to the retention of distinctive genetic variation.

Development of microsatellite markers for a giant water bug, Appasus japonicus, distributed in East Asia.

We developed microsatellite markers for Appasus japonicus (Heteroptera: Belostomatidae). This belostomatid bug is distributed in East Asia (Japanese Archipelago, Korean Peninsula and mainland China) and often listed as an endangered species in the Red List or the Red Data Book at the national and local level in Japan. Here, we describe twenty novel polymorphic microsatellite loci developed for A. japonicus, and marker suitability was evaluated using 56 individuals from four A. japonicus populations (Nagano, Hiroshima and Yamaguchi prefectures, Japan, and Chungcheongnam-do, Korea). The number of alleles per locus ranged from 1 to 12 (mean = 2.5), and the average observed and expected heterozygosity and fixation index per locus were 0.270, 0.323 and 0.153, respectively. In addition, a population structure analysis was conducted using the software STRUCTURE, and its result suggested that the 20 markers described here will be useful for investigating the genetic structure of A. japonicus populations, which should contribute to population genetics studies of this species.

Experimental and Field Data Support Range Expansion in an Allopolyploid Arabidopsis Owing to Parental Legacy of Heavy Metal Hyperaccumulation.

Empirical evidence is limited on whether allopolyploid species combine or merge parental adaptations to broaden habitats. The allopolyploid Arabidopsis kamchatica is a hybrid of the two diploid parents Arabidopsis halleri and Arabidopsis lyrata. A. halleri is a facultative heavy metal hyperaccumulator, and may be found in cadmium (Cd) and zinc (Zn) contaminated environments, as well as non-contaminated environments. A. lyrata is considered non-tolerant to these metals, but can be found in serpentine habitats. Therefore, the parents have adaptation to different environments. Here, we measured heavy metals in soils from native populations of A. kamchatica. We found that soil Zn concentration of nearly half of the sampled 40 sites was higher than the critical toxicity level. Many of the sites were near human construction, suggesting adaptation of A. kamchatica to artificially contaminated soils. Over half of the A. kamchatica populations had >1,000 µg g-1 Zn in leaf tissues. Using hydroponic treatments, most genotypes accumulated >3,000 µg g-1 Zn, with high variability among them, indicating substantial genetic variation in heavy metal accumulation. Genes involved in heavy metal hyperaccumulation showed an expression bias in the A. halleri-derived homeolog in widely distributed plant genotypes. We also found that two populations were found growing on serpentine soils. These data suggest that A. kamchatica can inhabit a range of both natural and artificial soil environments with high levels of ions that either of the parents specializes and that it can accumulate varying amount of heavy metals. Our field and experimental data provide a compelling example of combining genetic toolkits for soil adaptations to expand the habitat of an allopolyploid species.

Seventeen-Armed Star Polystyrenes in Various Molecular Weights: Structural Details and Chain Characteristics.

Star-shaped polymers are very attractive because of their potential application ability in various technological areas due to their unique molecular topology. Thus, information on the molecular structure and chain characteristics of star polymers is essential for gaining insights into their properties and finding better applications. In this study, we report molecular structure details and chain characteristics of 17-armed polystyrenes in various molecular weights: 17-Arm(2k)-PS, 17-Arm(6k)-PS, 17-Arm(10k)-PS, and 17-Arm(20k)-PS. Quantitative X-ray scattering analysis using synchrotron radiation sources was conducted for this series of star polymers in two different solvents (cyclohexane and tetrahydrofuran), providing a comprehensive set of three-dimensional structure parameters, including radial density profiles and chain characteristics. Some of the structural parameters were crosschecked by qualitative scattering analysis and dynamic light scattering. They all were found to have ellipsoidal shapes consisting of a core and a fuzzy shell; such ellipse nature is originated from the dendritic core. In particular, the fraction of the fuzzy shell part enabling to store desired chemicals or agents was confirmed to be exceptionally high in cyclohexane, ranging from 74 to 81%; higher-molecular-weight star polymer gives a larger fraction of the fuzzy shell. The largest fraction (81%) of the fuzzy shell was significantly reduced to 52% in tetrahydrofuran; in contrast, the lowest fraction (19%) of core was increased to 48%. These selective shell contraction and core expansion can be useful as a key mechanism in various applications. Overall, the 17-armed polystyrenes of this study are suitable for applications in various technological fields including smart deliveries of drugs, genes, biomedical imaging agents, and other desired chemicals.

Draft Genome Sequence of Novel Metschnikowia sp. Strain JCM 33374, a Nectar Yeast Isolated from a Bumblebee.

Here, we report the draft genome sequence of Metschnikowia sp. strain JCM 33374, a nectar yeast isolated from a bumblebee (Bombus diversus). The genome of 20.1 Mb is a naturally heterozygous diploid. Phylogenetic analysis with related taxa demonstrated that the strain is very likely a novel species.

Ecotypic divergences of the alpine herb Potentilla matsumurae adapted to fellfield-snowbed habitats across a series of mountain sky islands.

PREMISE: Divergent selection due to environmental heterogeneity can lead to local adaptation. However, the ecological and evolutionary processes of local adaptation that occurs across multiple regions are often unknown. Our previous studies reported on the ecotypic divergence within a local area of variation of Potentilla matsumurae, an alpine herb adapted to the fellfield-snowbed environment. Here we investigated large-scale geographic patterns of ecotypic differentiation in this species to infer local adaptation and selective forces across multiple regions. METHODS: We compiled information on the overall distributions of fellfield and snowbed habitats on the mountains in Japan across the distribution of the species. Next, we conducted common garden experiments to test the adaptive divergence of the fellfield-snowbed plants derived from multiple regions. Finally, we evaluated phylogeographic structures based on cpDNA and allozyme variations and inferred the evolutionary history of ecotype differentiation. RESULTS: The mosaic distribution of the fellfield-snowbed ecotypes across isolated mountaintops constitutes indirect evidence for habitat-specific natural selection. The significant difference in survivorship between the ecotypes observed in a controlled snow environment provides more substantial evidence of local selection. Phylogeographic structures support the hypothesis that ecotypic divergence events from fellfield to snowbed populations occurred independently in at least two distinct regions. CONCLUSIONS: Ecotypic divergence of P. matsumurae has occurred across a series of mountain sky islands. Local selection in snowy environments is a driving force that maintains the divergent ecotypes across multiple mountain regions and can contribute to the diversification of plants in heavy-snow regions.

Precise Synthesis of Macromolecular Architectures by Novel Iterative Methodology Combining Living Anionic Polymerization with Specially Designed Linking Chemistry.

This article reviews the development of a novel all-around iterative methodology combining living anionic polymerization with specially designed linking chemistry for macromolecular architecture syntheses. The methodology is designed in such a way that the same reaction site is always regenerated after the polymer chain is introduced in each reaction sequence, and this "polymer chain introduction and regeneration of the same reaction site" sequence is repeatable. Accordingly, the polymer chain can be successively and, in principle, limitlessly introduced to construct macromolecular architectures. With this iterative methodology, a variety of synthetically difficult macromolecular architectures, i.e., multicomponent µ-star polymers, high generation dendrimer-like hyperbranched polymers, exactly defined graft polymers, and multiblock polymers having more than three blocks, were successfully synthesized.

Synthesis and Characterization of Multicomponent ABC- and ABCD-Type Miktoarm Star-Branched Polymers Containing a Poly(3-hexylthiophene) Segment.

The new series of ABC-type miktoarm star polymer (ABC star, A = polyisoprene (PI), B = polystyrene (PS), and C = poly(3-hexylthiophene) (P3HT)) and ABCD-type miktoarm star polymer (ABCD star, A = PI, B = PS, C = poly(α-methylstyrene) (PαMS), and D = P3HT) could be synthesized by the combination of the controlled KCTP, anionic linking reaction, and Click chemistry. By the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition click reaction of the azido-chain-end-functional P3HT (P3HT-N3) with the alkyne-in-chain-functional AB diblock copolymer (A = PI and B = PS) (AB-alkyne) or alkyne-core-functional ABC miktoarm star polymer (A = PI, B = PS, and C = PαMS) (ABC-alkyne), the target ABC star and ABCD star, respectively, were obtained, as confirmed by size exclusion chromatography (SEC) and proton nuclear magnetic resonance (1H NMR). The thermal and optical properties of these star polymers were examined by thermal gravimetric analysis (TGA) and UV-vis spectroscopy. The dynamic scattering calorimetry (DSC), atomic force micrograph (AFM) image, and grazing incidence small-angle X-ray scattering (GISAXS) results showed that the periodic P3HT fibril nanostructures rather than microphase separation occurred in the ABCD star film. In addition, it was found that highly crystalline P3HT domains aligned in the "edge-on" orientation, as supported by grazing incidence wide-angle X-ray scattering (GIWAXS).

Development and evaluation of microsatellite markers for Acer miyabei (Sapindaceae), a threatened maple species in East Asia.

PREMISE OF THE STUDY: Twelve microsatellite markers were developed and characterized in a threatened maple species, Acer miyabei (Sapindaceae), for use in population genetic analyses. METHODS AND RESULTS: Using Ion Personal Genome Machine (PGM) sequencing, we developed microsatellite markers with perfect di- and trinucleotide repeats. These markers were tested on a total of 44 individuals from two natural populations of A. miyabei subsp. miyabei f. miyabei in Hokkaido Island, Japan. The number of alleles per locus ranged from two to eight. The observed and expected heterozygosities per locus ranged from 0.05 to 0.75 and from 0.05 to 0.79, respectively. Some of the markers were successfully transferred to the closely related species A. campestre, A. platanoides, and A. pictum. CONCLUSIONS: The developed markers will be useful in characterizing the genetic structure and diversity of A. miyabei and will help to understand its spatial genetic variation, levels of inbreeding, and patterns of gene flow, thereby providing a basis for conservation.

Changes in pollinator fauna affect altitudinal variation of floral size in a bumblebee-pollinated herb.

Geographic trait variations are often caused by locally different selection regimes. As a steep environmental cline along altitude strongly influences adaptive traits, mountain ecosystems are ideal for exploring adaptive differentiation over short distances. We investigated altitudinal floral size variation of Campanula punctata var. hondoensis in 12 populations in three mountain regions of central Japan to test whether the altitudinal floral size variation was correlated with the size of the local bumblebee pollinator and to assess whether floral size was selected for by pollinator size. We found apparent geographic variations in pollinator assemblages along altitude, which consequently produced a geographic change in pollinator size. Similarly, we found altitudinal changes in floral size, which proved to be correlated with the local pollinator size, but not with altitude itself. Furthermore, pollen removal from flower styles onto bees (plant's male fitness) was strongly influenced by the size match between flower style length and pollinator mouthpart length. These results strongly suggest that C. punctata floral size is under pollinator-mediated selection and that a geographic mosaic of locally adapted C. punctata exists at fine spatial scale.

Development and evaluation of microsatellite markers for the gynodioecious shrub Daphne jezoensis (Thymelaeaceae).

PREMISE OF THE STUDY: Ten microsatellite markers were developed and characterized in a gynodioecious summer-deciduous shrub, Daphne jezoensis, to facilitate studies of the evolution of gynodioecy in the species. • METHODS AND RESULTS: We used a next-generation sequencing approach with the Ion Personal Genome Machine (PGM) system to identify and develop microsatellite markers with perfect di- and trinucleotide repeats. These markers were tested with 47 samples from two natural populations. The mean observed and expected heterozygosities per population ranged from 0.40 to 0.46 and 0.60 to 0.66, respectively. • CONCLUSIONS: The developed markers will be useful to study the mating system, gene flow, and population genetic structure of D. jezoensis.

Cylindrical Nanostructure of Rigid-Rod POSS-Containing Polymethacrylate from a Star-Branched Block Copolymer.

A nanostructure consisting of rectangular polyhedral oligomeric silsesquioxane (POSS) nanodomains packed into a hexagonal lattice was observed in POSS-containing A2B star-branched polymers. The A2B star-branched polymers, which comprised polystyrene (A) and bulky POSS-containing poly(methacrylate) (PMAPOSS) (B) units, were synthesized by anionic polymerization and addition reaction. The self-assembled structures of the A2B star-branched polymers were studied via transmission electron microscopy (TEM) and small- and wide-angle X-ray scattering (SAXS and WAXS, respectively). It was found that a rectangular nanostructure was packed into a hexagonal arrangement of nanodomains for a PMAPOSS volume fraction of 40 vol. % in the star-branched polymer. In addition, the cylinder-like nanostructure of bulky POSS observed, which is not observed in the case of PS-b-PMAPOSS diblock copolymers, was formed owing to the curvature effect, which is the result of the branched architecture of the copolymer.

Complex Self-Assembled Morphologies of Thin Films of an Asymmetric A3B3C3 Star Polymer.

An asymmetric nine-arm star polymer, (polystyrene)3-(poly(4-methoxystyrene))3-(polyisoprene)3 (PS3-PMOS3-PI3) was synthesized, and the details of the structures of its thin films were successfully investigated for the first time by using in situ grazing incidence X-ray scattering (GIXS) with a synchrotron radiation source. Our quantitative GIXS analysis showed that thin films of the star polymer molecules have very complex but highly ordered and preferentially in-plane oriented hexagonal (HEX) structures consisting of truncated PS cylinders and PMOS triangular prisms in a PI matrix. This HEX structure undergoes a partial rotational transformation process at temperatures above 190 °C that produces a 30°-rotated HEX structure; this structural isomer forms with a volume fraction of 23% during heating up to 220 °C and persists during subsequent cooling. These interesting and complex self-assembled nanostructures are discussed in terms of phase separation, arm number, volume ratio, and confinement effects.

A supramolecular approach on using poly(fluorenylstyrene)-block-poly(2-vinylpyridine):PCBM composite thin films for non-volatile memory device applications.

Supramolecular composite thin films of poly[4-(9,9-dihexylfloren-2-yl)styrene]-block-poly(2-vinylpyridine) (P(St-Fl)-b-P2VP):[6,6]-phenyl-C(61)-butyric acid methyl ester (PCBM) were prepared for write-once-read-many times (WORM) non-volatile memory devices. The optical absorption and photoluminescence results indicated the formation of charge transfer complexation between the P2VP block and PCBM, which led to the varied PCBM aggregated size and memory characteristics. The ITO/PCBM:(P(St-Fl)-b-P2VP)/Al device exhibited the WORM characteristic with low threshold voltage (-1.6 to -3.2 V) and high ON/OFF ratio (10(3) to 10(5)) by tuning the PCBM content. The switching behavior could be explained by the charge injection dominated thermionic emission in the OFF state and field-induced charge transfer in the ON state. The present study provides a novel approach system for tuning polymer memory device characteristics through the supramolecular materials approach.

Synchrotron X-ray scattering characterization of the molecular structures of star polystyrenes with varying numbers of arms.

We have synthesized well-defined multiarmed star polystyrenes, with 6, 9, 17, 33, and 57 arms, and studied their molecular shapes and structural characteristics in a good solvent (tetrahydrofuran at 25 degrees C) and in a theta (Theta) solvent (cyclohexane at 35 degrees C) by small-angle X-ray scattering (SAXS) using a synchrotron radiation source. Analysis of the SAXS data provided a detailed characterization of the molecular shapes, including the contributions of the blob morphology of the arms, the radius of gyration, the paired distance distribution, the radial electron density distribution, and the Zimm-Stockmayer and Roovers g-factor, for the multiarmed star polystyrenes. In particular, the molecular shapes of the star polystyrenes were found to change from a fuzzy ellipsoid, for the 6-armed polystyrene, to a fuzzy sphere, for the 57-armed polystyrene, with an increasing number of arms. The ellipsoidal character of the star polystyrenes with fewer arms may originate from the extended anisotropically branched architecture at the center of the molecule. The arms of the star polystyrenes were found to be more extended than those of the linear polystyrenes. Furthermore, the degree of chain extension in the arms increased with the number of arms.

Kinship between parents reduces offspring fitness in a natural population of Rhododendron brachycarpum.

BACKGROUND AND AIMS: A reduction in offspring fitness resulting from mating between neighbours is interpreted as biparental inbreeding depression. However, little is known about the relationship between the parents' genetic relatedness and biparental inbreeding depression in their progeny in natural populations. This study assesses the effect of kinship between parents on the fitness of their progeny and the extent of spatial genetic structure in a natural population of Rhododendron brachycarpum. METHODS: Kinship coefficients between 11,858 pairs of plants among a natural population of 154 R. brachycarpum plants were estimated a priori using six microsatellite markers. Plants were genotyped, and pairs were selected from among 60 plants to vary the kinship from full-sib to unrelated. After a hand-pollination experiment among the 60 plants, offspring fitness was measured at the stages of seed maturation (i.e. ripening) under natural conditions, and seed germination and seedling survival under greenhouse conditions. In addition, spatial autocorrelation was used to assess the population's genetic structure. KEY RESULTS: Offspring fitness decreased significantly with increasing kinship between parents. However, the magnitude and timing of this effect differed among the life-cycle stages. Measures of inbreeding depression were 0.891 at seed maturation, 0.122 (but not significant) at seed germination and 0.506 at seedling survival. The local population spatial structure was significant, and the physical distance between parents mediated the level of inbreeding between them. CONCLUSIONS: The level of inbreeding between individuals determines offspring fitness in R. brachycarpum, especially during seed maturation. Genetic relatedness between parents caused inbreeding depression in their progeny. Therefore, biparental inbreeding contributes little to reproduction and instead acts as a selection force that promotes outcrossing, as offspring of more distant (less related) parents survive better.

Combining living anionic polymerization with branching reactions in an iterative fashion to design branched polymers.

This paper reviews the precise synthesis of many-armed and multi-compositional star-branched polymers, exact graft (co)polymers, and structurally well-defined dendrimer-like star-branched polymers, which are synthetically difficult, by a commonly-featured iterative methodology combining living anionic polymerization with branched reactions to design branched polymers. The methodology basically involves only two synthetic steps; (a) preparation of a polymeric building block corresponding to each branched polymer and (b) connection of the resulting building unit to another unit. The synthetic steps were repeated in a stepwise fashion several times to successively synthesize a series of well-defined target branched polymers.

Morphological and genetic variations of Potentilla matsumurae (Rosaceae) between fellfield and snowbed populations.

Identifying ecological factors associated with local differentiation of populations is important for understanding microevolutionary processes. Alpine environments offer a unique opportunity to investigate the effects of habitat-specific selective forces and gene flow limitations among populations at a microscale on local adaptation because the heterogeneous snowmelt patterns in alpine ecosystems provide steep environmental changes. We investigated the variation in morphological traits and enzyme loci between fellfield and snowbed populations of Potentilla matsumurae, a common alpine herb with a wide distribution along snowmelt gradients in northern Japan. We found significant differences in morphological traits between fellfield and snowbed habitats in a northern distribution region. These differences were maintained when plants were grown under uniform conditions in a greenhouse. Allozyme variations among 15 populations from geographically separated regions with different historical backgrounds showed that the populations are more genetically differentiated between the fellfield and snowbed habitats within a region than between populations occupying the same habitat type in different regions. These results suggest that variation in snowmelt regimes could be a driving force creating local adaptation and genetic differentiation of alpine plant populations.