A simple and efficient method for extraction of PCR-amplifiable DNA from chicken eggshells

Recently, we reported a method for discriminating a Japanese brand of chicken, the Hinai-jidori. As an application of this method for discriminating Hinai-jidori eggs, we here report an efficient method for extracting maternal DNA from eggshells. Eggshell powder was completely decalcified with EDTA solution, and then DNA was isolated by conventional phenol-chloroform extraction and ethanol precipitation. The efficiency of DNA recovery from eggshells was 50-fold higher than that of a previously reported method. The recovered DNA could be used for PCR, and 10 markers for identifying the Hinai-jidori chicken were detected. The genotypes of the Hinai-jidori exactly matched those of the Hinai-dori breed. Using this method, Hinai-jidori and Hinai-dori eggs could be distinguished from the eggs of Rhode Island Reds. This is the first report of a technique that can be used to discriminate the eggs of Hinai-jidori from those of other chickens, and it can also be utilized to validate the labeling of Hinai-jidori eggs in the market.     

Use of fluorescent proteins to visualize interactions between the Bakanae disease pathogen <Emphasis Type="Italic">Gibberella fujikuroi</Emphasis> and the biocontrol agent <Emphasis Type="Italic">Talaromyces</Emphasis> sp. KNB-422

Talaromyces sp. isolate KNB-422, isolated from a rice seedling, is a biofungicidal agent effective against several seedborne pathogens of rice including Gibberella fujikuroi, which causes Bakanae disease. Because the fungal mode of action (MOA) has not yet been clarified, we used the fluorescent protein markers GFP and RFP to visualize cell–cell interactions between the biocontrol agent and the pathogen G. fujikuroi. In slide culture, the hyphal cell wall of G. fujikuroi collapsed, and fluorescence of its cytoplasm disappeared 3 days after contact with hyphae of Talaromyces sp. On inoculated rice plants, both fungi occupied the same regions of coleoptiles and roots, where the biocontrol effect of Talaromyces sp. must be exerted. Our observations suggest that the MOA of Talaromyces sp. is mycoparasitic.     

Twenty-eight new microsatellite loci in chicken and their cross-species amplification in Japanese quail and helmeted guinea fowl

Twenty‐eight original chicken microsatellite markers were isolated and characterized to determine their utility as cross‐reactive markers for comparative genetic mapping in the order Galliformes. Primer pairs were typed in 12 unrelated chickens and also tested on Japanese quail and helmeted guinea fowl deoxyribonucleic acid (DNA). Polymorphism was observed in 23 (82.1%) of the markers and the average number of alleles per locus was 2.9 while the mean heterozygosity was 0.19. Eleven (39.3%) of the chicken markers cross‐reacted with Japanese quail DNA and 2 (7.1%) with helmeted guinea fowl DNA. The cross‐reactive markers described would serve as useful resources for comparative genetic mapping in poultry species belonging to the order Galliformes.     

Intrinsic Biodegradation of Heavy Oil from Nakhodka and the Effect of Exogenous Fertilization at a Coastal Area of the Sea of Japan

We performed a field experiment in thebiodegradation of heavy oil spilled from the Russian tankerNakhodka on a beach in the Sea of Japan. We collectedoil-contaminated cobbles and treated half with nitrogen andphosphorus slow-release fertilizers to stimulate microbialdegradation of the oil; the other half acted as unfertilizedcontrols. The cobbles were placed in porous acrylic vessels andsubmerged. We monitored changes in the oils, macronutrients,microbial community structure and amount of chlorophyll a. There were no significant differences in these criteriabetween the fertilized and unfertilized vessels, apart from anincrease in chlorophyll a in the fertilized vessels.However, there was a major intrinsic degradation of semi-volatile oil compounds in the unfertilized vessels; this occurred at a rate similar to that in the fertilized vessels, despite the low concentration of macronutrients in the seawater at the site.     

Chemical Characteristics in Stemflow of Japanese Cedar in Japan

To clarify the characteristics in stemflow of Japanese cedar (Cryptomeria japonica), we conducted the annual and extensive observation. We examined the chemistry of bulk and wet deposition, throughfall and stemflow at 26 forested sites in June and September 1998. The each sampling site was broadly distributed in all over the Japanese archipelago. The stemflow pH of Japanese cedar was significantly lower (p<0.01) than precipitation and stemflow of broad-leaved species in both months. There were significant anion deficits in stemflow of Japanese cedar, suggesting that organic anions derived from plant sources play an important role in the stemflow acidity. Our results suggested that the strong stemflow acidity in Japanese cedar was derived from an internal biological characteristic rather than influences of external acidic deposition.     

SIZE FRACTIONATION, DISSOLUTION ANALYSIS, AND INFRA-RED SPECTROSCOPY OF HUMUS COMPLEXES IN ANDO SOILS

Eight samples of Ando soil A₁ and buried A₁ horizons of different ages were fractionated into clay-, silt-, and sand-size separates, and the yields and carbon contents of these separates were determined. The clay-size separates were subjected to two series of successive extractions, 1) 0.1 M Na₄ P₂ O₇/2% Na₂ CO₃/0.5 M NaOH and 2) dithionite-citrate/2% Na₂ CO₃/0.5 M NaOH; and to difference infra-red spectroscopy for identification of the extracted constituents. The result indicated that humus evolves from forms with a very low complexing ability for Al and Fe into forms that complex Al and Fe in the A₁ horizon, and that humus further interacts with allophane-like constituents, allophane and imogolite in the buried A₁ horizon. These reactions between humus and inorganic constituents result in formation of silt- and sand-size aggregates which are stable to sonic wave treatment, but are broken down into finer particles upon boiling with H₂ O₂.     

Aging of whiskey increases 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of Japanese whiskey after various aging periods in oak barrels was measured to evaluate the antioxidative effects of whiskey. The activity of the whiskey increased with the aging period with high correlation. The activity of various types of whiskey was measured and shown to be correlated to the potentiation of the GABAA receptor response measured in a previous paper. However, the fragrant compounds in the whiskey which potentiated the GABAA receptor response had low DPPH radical scavenging activity, while phenol derivatives had high radical scavenging activity. The whiskey was extracted by pentane. The aqueous part showed the scavenging activity, whereas the pentane part did not. Thus, both the DPPH radical scavenging activity and the potentiation of the GABAA receptor response increased during whiskey aging in oak barrels, but were due to different components. The whiskey protected the H2O2-induced death of E. coli more than ethanol at the same concentration as that of the whiskey. The changes that occurred in the whiskey during aging may be the reason aged whiskies are so highly valued.