Development of microsatellite markers and evaluation of genetic diversity of the Amazonian ornamental fish Pterophyllum scalare

The objective of the present work was to develop species-specific microsatellite markers for P. scalare and to analyze the diversity and genetic structure of a wild population, from the Amazon River, and three commercial stocks (common, marble, and clown morphological variants), from farmers in Vieras-Minas Gerais. Through microsatellite-enriched genetic libraries, 11 microsatellite markers with adequate amplification patterns were characterized. Population genetic analysis identified eight polymorphic loci that generated 66 alleles ranging from two alleles (PSCA1B3) to nine (PSCA2H1). The polymorphic information content ranged from 0.031 to 0.827. High genetic differentiation was observed between the wild population and the stocks, and moderate differentiation between the three stocks. Deviation in the Hardy-Weinberg equilibrium was observed in one locus in the wild population, in five loci in the common morphological variant, in two in the marble, and in two in the clown morphological variant. Bayesian analysis of genetic structure revealed the existence of two clusters, one represented by the natural population and the other by the stocks. The developed microsatellite markers serve as a tool for the analysis of diversity and genetic structure and conservation studies of P. scalare.

     

Effect of nitrite accumulation on nitrous oxide emission and total nitrogen loss during swine manure composting

The present study investigated the nitrogen balance in swine manure composting to evaluate the effect of nitrite (     ) accumulation, which induces nitrogenous emissions, such as N₂O, during compost maturation. During active composting, most N losses result from NH₃ emission, which was 9.5% of the initial total nitrogen (TN_initial), after which,     began to accumulate as only ammonia-oxidizing bacteria proliferated. After active composting, the addition of mature swine compost (MSC), including nitrite-oxidizing bacteria (NOB), could prevent     accumulation and reduce N₂O emission by 70% compared with the control in which     accumulated as a result of delayed growth of indigenous NOB. Total N₂O emissions in the control and in the treatment of MSC addition (MA) were 9.3% and 3.0% of TN_initial, respectively, whereas N losses as the sum total of NH₃ and N₂O over the whole period were 19.0% (control) and 12.8% (MA) of TN_initial, respectively. However, the difference in total N losses was markedly greater than that measured as NH₃ and N₂O, which were 27.8% (control) and 13.3% (MA) of TN_initial, respectively. These results demonstrated that the magnitude of nitrogen losses induced by     accumulation is too large to ignore in the composting of swine manure.     

The National BioResource Project (NBRP) Lotus and Glycine in Japan

The objective of the National BioResource Project (NBRP) in Japan is to collect, conserve and distribute biological materials for life sciences research. The project consists of twenty-eight bioresources, including animal, plant, microorganism and DNA resources. NBRP Lotus and Glycine aims to support the development of legume research through the collection, conservation, and distribution of these bioresources. Lotus japonicus is a perennial legume that grows naturally throughout Japan and is widely used as a model plant for legumes because of such advantages as its small genome size and short life cycle. Soybean (Glycine max) has been cultivated as an important crop since ancient times, and numerous research programs have generated a large amount of basic research information and valuable bioresources for this crop. We have also developed a “LegumeBase” a specialized database for the genera Lotus and Glycine, and are maintaining this database as a part of the NBRP. In this paper we will provide an overview of the resources available from the NBRP Lotus and Glycine database site, called “LegumeBase”.     

Population structure of rice blast isolates resistant to scytalone dehydratase inhibitors in Mie Prefecture and implications for their origin

In Mie Prefecture in Japan, rice blast isolates resistant to melanin biosynthesis inhibitors targeting scytalone dehydratase (SDH) were first observed in 2005. To analyze the distribution of the resistant isolates, 527 isolates were collected from wide areas in this prefecture during 2006 and 2007. Almost half of the isolates collected (233 of 527 isolates) carried a point mutation in the SDH gene conferring the resistant phenotype. To compare population structures of resistant and sensitive isolates, we analyzed the isolates with repetitive-element-based PCR DNA fingerprinting using a single primer complementary to a sequence in the terminal inverted repeat of transposable element Pot2. A majority of the resistant isolates were classified into a single DNA fingerprint haplotype, Mie1. Despite its prevalence in the resistant isolates, Mie1 was not found in the sensitive isolates. Furthermore, in a dendrogram constructed from the DNA fingerprint data, Mie1 and six other haplotypes formed a cluster composed of resistant isolates alone. These results suggest that the resistant isolates that belonged to the Mie1 haplotype had migrated from regions outside Mie Prefecture and selectively propagated in a short period in this prefecture.     

Detection and analysis of genetic variations in GOB locus of Plasmopara viticola by DNA sequence analysis

The population genetic structure of Plasmopara viticola in Japan was analyzed using grapevine downy mildew specimens collected from two islands, Honshu and Hokkaido. By simple sequence repeat analysis with the GOB microsatellite marker and DNA sequencing, an accurate copy number of the (CT) _n (CTAT) _n repeat was determined. Consequently, we found a large number of genetic variations in (CT) _n and (CTAT) _n repeats. Also, a single nucleotide polymorphism in the (CTAT) _n repeat of the GOB locus was detected. These genetic variations may serve as a valuable tool to understand the population structure of P. viticola.     

The genetic mechanisms of warfarin resistance in Rattus rattus found in the wild in Japan

Warfarin is commonly used worldwide as a rodenticide. It inhibits blood coagulation by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity leading to hemorrhage. However, it has been reported that repeated or long-term treatment with warfarin results in resistance emerging in wild rodents. Such resistance may explain why it is difficult to control rodents in many regions in Japan. In this report, we studied mutations in the VKOR gene (including the VKOR complex subunit 1 (VKORC1)), while also analyzing VKOR and clotting factor activity in black rats (Rattus rattus) in order to understand better the mechanism of warfarin resistance in this species.We sequenced the VKORC1 gene from 275 rats living in the wild in Japan. We found several types of novel base substitutions, some of which conferred warfarin resistance.There was no difference in coagulation times between warfarin-sensitive and resistant rats measured under physiological conditions. However, after warfarin administration, no effect was noted in warfarin-resistant rats, although a prolonged coagulation time was noted in warfarin-sensitive rats.We also determined the kinetic differences in hepatic microsomal VKOR-dependent activity between warfarin-resistant and sensitive rats. Warfarin-resistant rats showed 2-3-fold lower V_max/K_m values than did sensitive rats. In addition, we report that resistant rats found in the Tokyo area had a VKOR activity which was poorly inhibited by warfarin.Finally, we conclude that reduced VKOR activity and warfarin resistance in the Japanese black rat might be due to mutations in the VKORC1 gene. However, further study is needed to clarify how such rats can maintain adequate vitamin K-dependent clotting factor levels, while simultaneously exhibiting low VKOR activity and warfarin resistance.     

Stimulation of Adrenal Chromaffin Cell Proliferation by Hypercalcemia Induced by Intravenous Infusion of Calcium Gluconate in Rats

Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.     

A case of congenital hyperostosis in a newborn piglet

One female newborn piglet showed prominent thickening of both forelimbs and died soon after birth. Histopathologically, thin and woven trabeculae of bone was extending out from the edge of cortical bone in the affected forelimbs, and diagnosed as congenital hyperostosis. The extent of radially proliferated trabeculae was most prominent in radioulna. Many round- to spindle-shaped cells were observed in periosteum, which were considered to be osteoblasts. Around the periosteum, the mesenchymal proliferation was extensive with abundant mucus, and cartilaginous metaplastic changes were observed mainly around the radioulna and humerus. Dilatation of vessels with fibrin deposition in vessel walls was often observed, which were considered to reflect the localized circulatory disturbance.     

High-grade myofibroblastic sarcoma of inguinal region in a dog

A subcutaneous tumor in the left inguinal region was present in an 11-year-old female bloodhound. Histopathologically, the tumor showed invasive growth and extensive necroses, and it was composed of spindle-shaped, elongated, and stellate neoplastic cells accompanied by occasional giant cells arranged in fascicular, herringbone, or irregular storiform patterns with abundant production of collagen fibers. The cytoplasm of most tumor cells was positive for vimentin, alpha-smooth muscle actin, and calponin, but was negative for desmin, smoothelin, and S-100. Furthermore, most of the tumor cells were negative for Iba1 while some tumor cells were weakly positive. Thus, this tumor was diagnosed as a high-grade myofibroblastic sarcoma according to the diagnostic criteria for human myofibroblastic sarcomas.