De novo genome assembly of the partial homozygous dihaploid potato identified PVY resistance gene (Rychc) derived from Solanum chacoense.

The isolation of disease resistance genes introduced from wild or related cultivated species is essential for understanding their mechanisms, spectrum and risk of breakdown. To identify target genes not included in reference genomes, genomic sequences with the target locus must be reconstructed. However, de novo assembly approaches of the entire genome, such as those used for constructing reference genomes, are complicated in higher plants. Moreover, in the autotetraploid potato, the heterozygous regions and repetitive structures located around disease resistance gene clusters fragment the genomes into short contigs, making it challenging to identify resistance genes. In this study, we report that a de novo assembly approach of a target gene-specific homozygous dihaploid developed through haploid induction was suitable for gene isolation in potatoes using the potato virus Y resistance gene Rychc as a model. The assembled contig containing Rychc-linked markers was 3.3 Mb in length and could be joined with gene location information from the fine mapping analysis. Rychc was successfully identified in a repeated island located on the distal end of the long arm of chromosome 9 as a Toll/interleukin-1 receptor-nucleotide-binding site-leucine rich repeat (TIR-NBS-LRR) type resistance gene. This approach will be practical for other gene isolation projects in potatoes.

Adding collagen to adipose tissue transplant increases engraftment by promoting cell proliferation, neovascularisation and macrophage activity in a rat model.

To clarify the effect of collagen addition to transplanted adipose tissue on angiogenesis, cell proliferation and tissue remodelling process and reveal whether collagen addition contributes to improving transplanted adipose tissue engraftment in rats. Adipose tissue was harvested from the inguinal and injected into the back of the rat, in addition to collagen. Engraftment tissue was harvested, semi-quantitatively evaluated and underwent haematoxylin and eosin or Perilipin staining. Moreover, we evaluated viable adipocyte counts and neovascularisation. Macrophages were evaluated using flow cytometry, and the adiponectin or vascular endothelial growth factor (VEGF) mRNA was detected using real-time polymerase chain reaction. By collagen addition to transplanted adipose tissue, higher engraftment rate semi-quantitatively and a greater number of new blood vessels histologically were identified. Perilipin staining revealed a higher adipocyte number. The total cell, M1 macrophage and M2 macrophage count were higher. There was increased adiponectin mRNA significantly at week 4 compared to that at week 1 after transplantation. Note that the expression levels of VEGF mRNA increased. In rats, adding collagen enhanced cell proliferation, induced M2 macrophages, which are involved in wound healing, and promoted adipocytes and neovascularisation. Therefore, collagen addition to transplanted adipose tissue could increase the engraftment rate of adipose tissue.

A pubescence color gene enhances tolerance to cold-induced seed cracking in yellow soybean.

In yellow soybean, severe cold weather causes seed cracking on the dorsal side. Yellow soybeans carry the I or ii allele of the I locus and have a yellow (I) or pigmented (ii ) hilum. We previously isolated an additional allele, designated as Ic, of the I locus, and reported that yellow soybeans with the IcIc genotype may be tolerant to cold-induced seed cracking. The Ic allele by itself, however, does not confer high tolerance. The association of a pubescence color gene (T) with suppression of low-temperature-induced seed coat deterioration has been previously reported. In the present study, we tested whether T is effective for the suppression of cold-induced seed cracking using two pairs of near-isogenic lines for the T locus in the iiii or IcIc background. In both backgrounds, the cracked seed rate of the near-isogenic line with the TT genotype was significantly lower than that with the tt genotype, which indicates that T has an inhibitory effect on cold-induced seed cracking. Furthermore, we also showed that gene pyramiding of Ic and T can improve tolerance to cold-induced seed cracking. Our findings should aid the development of highly SC-tolerant cultivars in soybean breeding programs.

[Determination of Inorganic Arsenic for Pet Food by LC-ICP-MS].

We have developed a quantitative determination method of the concentration of inorganic arsenic in pet foods using a liquid chromatograph-inductively coupled plasma-mass spectrometer (LC-ICP-MS). After adding 2 w/v% TMAH solution to a sample, inorganic arsenic was extracted by heating and the extract was collected by water. The pH of the solution was adjusted, and injected into a LC-ICP-MS to determine the concentration of inorganic arsenic. LC separation was carried out on an ODS column with 10 mmol/L sodium 1-butanesulfonate, 4 mmol/L malonic acid, 4 mmol/L TMAH and 0.05% methanol solution as a mobile phase. A collaborative study was conducted by nine laboratories using dry and wet-type pet foods, formed jerky, dried jerky and biscuit. Dry-type pet food and dried jerky was added with 2 mg/kg of As (III). Wet-type pet food was added with 0.5 mg/kg of As (III). Formed jerky was added with 1 mg/kg of As (III). Biscuit was added with 0.2 mg/kg of As (III). The mean recoveries, repeatabilities and reproducibilities in the form of relative standard deviation (RSDr and RSDR), and HorRat, were 95.4% to 98.3%, less than 2.9%, less than 9.1%, and 0.22 to 0.51, respectively.

Ecological, physiological, and biomechanical differences between gametophytes and sporophytes of Chondrus ocellatus (Gigartinales, Rhodophyta)1.

Although variation among habitats in the ratio of gametophytes to sporophytes has been reported in various gigartinacean species, factors controlling the phase ratio remain poorly understood. Over 18 months, we examined the phase ratio of Chondrus ocellatus at three sites: a sheltered intertidal site, Hiruga A; an exposed intertidal site, Hiruga B; and a subtidal site, Shikimi. The mean proportion of gametophytes at Hiruga A (73.1%) was significantly higher than that at Shikimi (51.2%) and Hiruga B (44.7%). Due to a significantly higher water retention ability of the gametophytes, it was expected that the gametophytes would exhibit higher desiccation tolerance. After dehydration treatments, however, neither the photosynthetic rate of vegetative blades nor the survival rate of spores was significantly different between the phases. Measurements of blade strength indicated that the sporophytic blades were less stiff and more flexible, and a culture experiment revealed that the sporophytic germlings showed a significantly higher growth rate. Flexible blades and fast-growing germlings are considered advantageous for colonizing wave-swept intertidal habitats, so these properties may have caused the different fluctuation pattern of phase ratio among the sites. The present data demonstrate that biomechanical and physiological differences between the two phases of C. ocellatus make one phase advantageous in certain environmental conditions, and that these differences likely cause an unequal ratio of isomorphic phases.

Mapping soybean rhg2 locus, which confers resistance to soybean cyst nematode race 1 in combination with rhg1 and Rhg4 derived from PI 84751.

The soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is a devastating pest of soybean (Glycine max (L.) Merr.) in the world. Three soybean QTLs for resistance to SCN race 1 were detected through QTL analyses using recombinant inbred lines (RILs) derived from a cross between 'Tokei 758' (susceptible) and 'To-8E' (resistant to races 1 and 3, derived from 'PI 84751' and 'Gedenshirazu'). Two of the three QTLs appear to be rhg1 and Rhg4 from their locations on the linkage map. The third QTL, detected around Satt359 on chromosome 11, was tentatively identified as rhg2. All RILs resistant to race 1 had all three QTLs. We developed lines carrying the three loci in various combinations, including all and none, from descendants of a cross between 'NIL-SCN' (with resistance derived from 'PI 84751' in the 'Natto-shoryu' background) and 'Natto-shoryu'. Evaluating these lines in a race 1-infected field in Mito, Ibaraki, showed that resistance to race 1 required all three loci. Through field evaluation of 10 recombinant fixed pairs that we developed, we located the rhg2 locus to an 821 kb-region between SSR markers Sat_123 (=WGSP11_0140) and BARCSOYSSR11_1420 on chromosome 11.

Assessing change of environmental dynamics by legislation in Japan, using red tide occurrence in Ise Bay as an indicator.

Tokyo Bay, Ise Bay, and the Seto Inland Sea are the total pollutant load control target areas in Japan. A significant correlation between the incidence of red tides and water quality has been observed in the Seto Inland Sea (Honjo, 1991). However, while red tides also occur in Ise Bay and Tokyo Bay, similar correlations have not been observed. Hence, it is necessary to understand what factors cause red tides to effectively manage these semi-closed systems. This study aims to investigate the relationship between the dynamics of the Red Tide Index and nitrogen regulation as well as phosphorus regulation, even in Ise Bay where, unlike Tokyo Bay, there are few observation items, by selecting a suitable objective variable. The introduction of a new technique that uses the Red Tide Index has revealed a possibility that the total pollution load control has influenced the dynamics of red tide blooms in Ise Bay.

Waste reduction and recycling initiatives in Japanese cities: lessons from Yokohama and Kamakura.

Waste reduction and recycling at the city level will acquire greater significance in the near future due to rising global volumes of waste. This paper seeks to identify policy-relevant drivers for successful promotion of waste reduction and recycling. Factors influencing the success of waste reduction and recycling campaigns are identified. Two case study cities in Japan which depict the successful use of the 3Rs (reduce, reuse and recycle) at the municipal level are presented. In these cases, the existence of incinerators, which are generally considered as disincentives for recycling, was not functioning as a disincentive but rather as an incentive for waste reduction. Owing to the high cost of incineration facilities, the movement to close incinerators has become a strong incentive for waste reduction and recycling in these two cities. The study suggests that careful consideration is necessary when making decisions concerning high-cost waste treatment facilities with high installation, maintenance and renewal outlays. In addition, intensive source separation and other municipal recycling initiatives have a high potential for producing positive results.

Method for selection of soybeans tolerant to seed cracking under chilling temperatures.

In Hokkaido, northern Japan, soybean [Glycine max (L.) Merr.] crops are damaged by cold weather. Chilling temperatures result in the appearance of cracking seeds (CS) in soybean crops, especially those grown in eastern and northern Hokkaido. Seed coats of CS are severely split on the dorsal side, and the cotyledons are exposed and frequently separated. CS occurrence causes unstable production because these seeds have no commodity value. However, little is known about the CS phenomenon. The aims of this study were to identify the cold-sensitive stage associated with CS occurrence and to develop a method to select CS-tolerant lines. First, we examined the relationship between chilling temperatures after flowering and CS occurrence in field tests. The average temperature 14 to 21 days after flowering was negatively correlated with the rate of CS. Second, we evaluated differences in CS tolerance among soybean cultivars and breeding lines in field tests. 'Toyohomare' and 'Toiku-238' were more CS-tolerant than 'Yukihomare' and 'Toyomusume'. Third, we developed a selection method in which plants were subjected to 21-day chilling-temperature treatment from 10 days after flowering in a phytotron. This enabled comparisons of CS tolerance among cultivars. This selection method will be useful for breeding CS-tolerant soybeans.

Decrease in fungal biodiversity along an available phosphorous gradient in arable Andosol soils in Japan.

Andosols comprise one of the most important soil groups for agricultural activities in Japan because they cover about 46.5% of arable upland fields. In this soil group, available phosphorus (P) is accumulated by application of excessive fertilizer, but little is known about the influence of increasing P availability on microbial community diversity at large scales. We collected soil samples from 9 agro-geographical sites with Andosol soils across an available P gradient (2048.1-59.1 mg P2O5·kg(-1)) to examine the influence of P availability on the fungal community diversity. We used polymerase chain reaction - denaturing gradient gel electrophoresis to analyze the fungal communities based on 18S rRNA genes. Statistical analyses revealed a high negative correlation between available P and fungal diversity (H'). Fungal diversity across all sites exhibited a significant hump-shaped relationship with available P (R(2) = 0.38, P < 0.001). In addition, the composition of the fungal community was strongly correlated with the available P gradient. The ribotype F6, which was positively correlated with available P, was closely related to Mortierella. The results show that both the diversity and the composition of the fungal community were influenced by available P concentrations in Andosols, at a large scale. This represents an important step toward understanding the processes responsible for the maintenance of fungal diversity in Andosolic soils.

Genetic relationships of soybean cyst nematode resistance originated in Gedenshirazu and PI84751 on Rhg1 and Rhg4 loci.

Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is one of the most damaging pests of soybean (Glycine max (L.) Merr.). Host plant resistance has been the most effective control method. Because of the spread of multiple SCN races in Hokkaido, the Tokachi Agricultural Experiment Station has bred soybeans for SCN resistance since 1953 by using 2 main resistance resources PI84751 (resistant to races 1 and 3) and Gedenshirazu (resistant to race 3). In this study, we investigated the genetic relationships of SCN resistance originating from major SCN resistance genes in Gedenshirazu and PI84751 by using SSR markers. We confirmed that race 1 resistance in PI84751 was independently controlled by 4 genes, 2 of which were rhg1 and Rhg4. We classified the PI84751- type allele of Rhg1 as rhg1-s and the Gedenshirazu-type allele of Rhg1 as rhg1-g. In the cross of the Gedenshirazu-derived race 3-resistant lines and the PI84751-derived races 1- and 3-resistant lines, the presence of rhg1-s and Rhg4 was responsible for race 1-resistance. These results indicated that it was possible to select race 1 resistant plants by using marker-assisted selection for the rhg1-s and Rhg4 alleles through a PI84751 origin × Gedenshirazu origin cross.

Combined analyses of bacterial, fungal and nematode communities in andosolic agricultural soils in Japan.

We simultaneously examined the bacteria, fungi and nematode communities in Andosols from four agro-geographical sites in Japan using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and statistical analyses to test the effects of environmental factors including soil properties on these communities depending on geographical sites. Statistical analyses such as Principal component analysis (PCA) and Redundancy analysis (RDA) revealed that the compositions of the three soil biota communities were strongly affected by geographical sites, which were in turn strongly associated with soil characteristics such as total C (TC), total N (TN), C/N ratio and annual mean soil temperature (ST). In particular, the TC, TN and C/N ratio had stronger effects on bacterial and fungal communities than on the nematode community. Additionally, two-way cluster analysis using the combined DGGE profile also indicated that all soil samples were classified into four clusters corresponding to the four sites, showing high site specificity of soil samples, and all DNA bands were classified into four clusters, showing the coexistence of specific DGGE bands of bacteria, fungi and nematodes in Andosol fields. The results of this study suggest that geography relative to soil properties has a simultaneous impact on soil microbial and nematode community compositions. This is the first combined profile analysis of bacteria, fungi and nematodes at different sites with agricultural Andosols.

Effect of soil type and fertilizer management on archaeal community in upland field soils.

The effects of soil and fertilizer types on archaeal communities were evaluated by real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene of total DNA directly extracted from upland field soils. Twelve experimental upland field plots containing four different soil types, i.e., Cumulic Andosol, Low-humic Andosol, Yellow Soil and Gray Lowland Soil, were maintained under three different fertilizer management systems for 8 years (chemical fertilizer, rice husks and cow manure, and pig manure, respectively). Two-way ANOVA and RDA analyses showed that the copy number and PCR-DGGE profile of archaeal 16S rRNA gene were affected mainly by soil type, especially between Andosol and non-Andosol, but were also influenced by fertilizer type. Among several soil chemical properties, total N content showed a significant correlation to archaeal community. Sequence analyses showed that most of the major DGGE bands corresponded to uncultured Crenarchaeota of Group I.1b that contained ammonia-oxidizing archaea (AOA). These sequences were separated into two clusters in the phylogenetic tree and each lineage showed a different response to total N content.