Detection of the EGFP sequence in breast muscle of 3‐year‐old chicken after transfection using sonoporation

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG‐EGFPac) and microbubbles were injected into the central disc of stage‐X blastoderm or the germinal crescent of stage‐4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP‐positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3‐year‐old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.     

Practical capability of a DNA pool‐based genome‐wide association study using BovineSNP50 array in a cattle population

Genome‐wide association mapping for complex traits in cattle populations is a powerful, but expensive, selection tool. The DNA pooling technique can potentially reduce the cost of genome‐wide association studies. However, in DNA pooling design, the additional variance generated by pooling‐specific errors must be taken into account. Therefore, this study aimed to investigate factors such as: (i) the accuracy of allele frequency estimation; (ii) the magnitude of errors in pooling construction and in the array; and (iii) the effect of the number of replicate arrays on P‐values estimated by a genome‐wide association study. Results showed that the Illumina correction method is the most effective method to correct the allele frequency estimation; pooling errors, especially array variance, should be taken into account in DNA pooling design; and the risk of a type I error can be reduced by using at least two replicate arrays. These results indicate the practical capability and cost‐effectiveness of pool‐based genome‐wide association studies using the BovineSNP50 array in a cattle population.     

草地景观视觉效果的定量评价研究--以日本北海道士幌新建草地为例

采用SD法(Semantic Differential Method)定量评价了居民对不同草地景观类型的主观印象.结果表明,评价者知识结构及经历的不同对评价结果有很大影响;草地景观的视觉效果由舒适性、协调性和空间性三评价轴所构成;草地内有林地的景观类型受到很高评价,今后在草地管理上为增加草地的视觉美学价值应重点研究草地内树林的配置结构及树种构成.     

Effect of CO2 enrichment on fruit growth and quality in Japanese pear (Pyrus serotina Reheder cv. Kosui)

Six year-old Japanese pear (Pyrus seratina Reheder cv. Kosui) trees grafted on P. serotina cv. Nihonyamanashi were grown in containers filled with Granite Regosol under glasshouse conditions. At different stages of fruit growth, pear trees were exposed to an elevated CO₂ concentration (130 Pa CO₂ ) along with a control (35 Pa CO₂). For one group of plants, CO₂ enrichment was applied for 79 d from 52 d after full bloom (DAB) to fruit maturity (long-term CO₂ enrichment) and for another group the same treatment was applied for 35 d from 96 DAB to fruit maturity (short-term CO₂ enrichment). The effects of the elevated CO₂ concentration on vegetative growth, mineral contents, and fruit production and quality were examined. Long-term CO₂ enrichment enhanced vegetative growth, without any significant effect on the mineral contents in either flower bud or fruit except for a remarkable increase in the K content. Long-term CO₂ enrichment increased the fruit size and fresh weight, but had no significant effect on the fruit quality. On the other hand, the short-term CO₂ enrichment did not induce any significant change in the fruit size but increased the fruit sugar concentration. Along with the reduction of the sorbitol concentration in fruit, the fructose and sucrose concentrations increased and these changes occurred earlier at elevated CO₂ than at ambient CO₂ concentrations. From these results, we concluded that the effect of CO₂ enrichment on fruit growth varies depending upon the growth stages of fruit: during the initial and fruitlet stages when fruit expansion occurs, CO₂ enrichment increases the fruit size, whereas, during maturation when fruit expansion has slowed down and sugar accumulation in fruit is active, it increases the fruit sugar concentration.     

Genetic and antigenic characterization of bovine viral diarrhea viruses isolated from cattle in Hokkaido,Japan

In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5′-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.     

Partitioning of photosynthates originating from bent shoots in the arching and high-rack culture systems of cut rose production

The arching and high-rack culture systems were developed and patented by Japanese rose growers. Both culture systems have bent canopies (lower bent shoots). In the arching culture system, shoots sprouting from the crown are harvested as cut flowers. However, the high-rack culture system also has a bent canopy originating from the mother stem (upper bent shoots) and flower stems sprout and is harvested at the top of each mother stem. Partitioning of photosynthates originating from bent shoots in arching and high-rack culture systems of rose production was investigated to elucidate how carbohydrates are re-allocated from the bent shoots in different culture systems of roses. At the flowering stage in both culture systems, 50–70% of ¹³C-photosynthates originated from bent shoots were exported to other parts within 72 h after ¹³CO₂ feeding to the bent shoot. In the arching culture system, photosynthates from lower bent shoots were partitioned mainly to the roots and crown. Similarly, in the high-rack culture system, between 71 and 86% of the exported carbon from the bent shoots were allocated to below the point of bending (roots + crown + mother stems) and only 9–28% was allocated to flowering shoots above the point of bending. In both culture systems, photosynthate translocation from the lower bent shoot directly to flowers was low. Accordingly, bent shoots in rose plants acted as a source of photosynthates, independent of culture system. The height of the bent shoots determined for a great deal in the re-allocation of the photosynthates, and provides a partial explanation for difference in production of cut roses.     

Characterization of ammonia-assimilating bacteria in a lagoon for wastewater from a paddock of dairy cattle

We investigated microorganisms that assimilated ammonia in lagoon treatment processes. Ammonia‐assimilating microorganisms were detected by nitrogen‐limited medium that contained ammonia as the sole nitrogen source. Numbers of ammonia‐assimilating aerobes (log CFU/g) were 3.4, 4.8, 5.0, 4.8 and 5.0 (log CFU/mL) on the culture plate incubated at 4°C, 10°C, 15°C, 20°C and 25°C, respectively. Many isolates used ammonia in high rates when they were purely cultivated in nitrogen‐limited medium added to sterilized lagoon extract. Many of them used ammonia even when they were cultivated in media containing viable microbial flora of the lagoon. Among them, enterobacteriaceae and Pseudomonas sp. were identified by analysis of 16S ribosomal DNA.