Development of EST-SSR markers and construction of a linkage map in faba bean (Vicia faba)

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a ‘Nubaria 2’ × ‘Misr 3’ F₂ mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F₂ plants were divided into three subpopulations according to the original F₁ plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated ‘Nubaria 2’ × ‘Misr 3’ map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.     

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis)

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.     

Reaction behavior of wood in an ionic liquid, 1-ethyl-3-methylimidazolium chloride

Reaction of Japanese beech (Fagus crenata) in an ionic liquid, 1-ethyl-3-methylimidazolium chloride ([C2mim][Cl]), which can dissolve cellulose, was investigated. Although both lignin and polysaccharides such as cellulose and hemicelluloses can be liquefied at a treatment temperature of around 100°C, the liquefaction of polysaccharides mainly occurs at the beginning of the treatment with [C2mim][Cl]. Cellulose crystallinity in the wood was gradually broken down as the treatment continued. The solubilized polymers were depolymerized to low molecular weight compounds. The results indicate that [C2mim][Cl] is an effective solvent and reagent for the liquefaction of wood components and subsequent depolymerization of them. Part of this report was presented at the 58th Annual Meeting of the Japan Wood Research Society, Tsukuba, April 2008     

Impacts of the nonlinear relationship between abundance and its index in a tuned virtual population analysis

The abundance index used in a tuned virtual population analysis (VPA) is usually assumed to be proportional to actual abundance. However, the actual abundance and abundance index do not always have a linear relationship. Such nonlinearity can cause biases in abundance estimates as well as retrospective biases arising from systematic differences in abundance estimates when more data are successively added. Severe retrospective biases can damage the reliability of stock assessments. In this study, we use an approach to estimate an additional parameter that controls the nonlinearity in a tuned VPA. A performance test using simulated data revealed that the tuned VPA was able to accurately estimate the nonlinearity parameter and thus yielded less biased abundance estimates and smaller retrospective biases. We also found that estimating the nonlinearity parameters was effective even under other model misspecification scenarios, such as disregarding historical increases in catchability and time-varying natural mortality. Moreover, we applied this approach to some Japanese fish stocks and evaluated its validity. We found that estimating the nonlinearity parameters in the tuned VPA enhances the reliability of fisheries stock assessments.     

Hypothalamic dopamine is required for salsolinol‐induced prolactin secretion in goats

The aim of the present study was to clarify the relationship between hypothalamic dopamine (DA) and salsolinol (SAL) for the secretion of prolactin (PRL) in goats. SAL or thyrotropin‐releasing hormone (TRH) was intravenously injected into female goats treated with or without the D₂ DA receptor antagonist haloperidol (Hal), which crosses the blood‐brain barrier, and the PRL‐releasing response to SAL was compared with that to TRH. PRL‐releasing responses to SAL, Hal, and Hal plus SAL were also examined after a pretreatment to augment central DA using carbidopa (Carbi) and L‐dopa. The PRL‐releasing response to Hal alone was greater than that to SAL or TRH alone. The PRL‐releasing response to Hal plus SAL was similar to that of Hal alone. In contrast, the PRL‐releasing response to Hal plus TRH was greater than that to TRH or Hal alone. The treatment with Carbi plus L‐dopa inhibited SAL‐ and Hal‐induced PRL secretion. The inhibition of the PRL‐releasing response to SAL disappeared when SAL was injected with Hal. These results indicate that the mechanisms underlying the SAL‐induced PRL response differ from those of TRH, and suggest that hypothalamic DA and its synthesis is associated in part with SAL‐induced PRL secretion in goats.     

日本对虾杆状核病毒性传染病诊断技术

本文提出了分别用暗视野显微技术和透射电子显微技术快速确诊日本对虾杆状核病毒性传染病的方法。在暗视野显微镜下观察到的血淋巴中的病毒粒子大小约为0.5μm;胃表层上皮被病毒感染的细胞核明显地呈白色均质小体,直径10～15μm,球形或椭圆形,坏死细胞被囊化为棕色小块(约20～50μm)。病虾血淋巴和胃经负染制样在透射电子显微镜下观察病毒颗粒大小为400×150nm;核衣壳大小为390×85nm。     

Ribotyping of EntomopathogenicVerticittium lecanii in Japan

To estimate the genetic diversity in 30 isolates ofVerticillium lecanii from aphids, whiteflies, mite and black pine in Japan, including two commercialized strains (Mycotal and Vertalec), DNA polymorphisms in ribosomal DNA of those isolates were analyzed using polymerase chain reaction (PCR). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the nuclear ribosomal RNA gene of each isolate were analyzed by PCR-RFLP (restriction fragment length polymorphism). The size of the PCR product from the ITS region was ~ 580 bp in 27 of the isolates. A 600 bp ITS product was detected in Mycotal and Vertalec. One Japanese isolate produced both the 580 bp and 600 bp products. Enzymatic digestion of the ITS region with Sau3A I,Msp I,Hae III andRsa I revealed RFLPs that consisted of eight haplotypes. Mycotal and Vertalec were specific haplotypes that differed from other isolates. The Japanese isolates had a complex relationship with the original host, but we identified several specific haplotypes common to an aphid origin. Ten distinct IGS haplotypes were detected in the IGS region, some of which were associated with aphid and whitefly origins. These results suggest that the haplotype of rDNA RFLP analysis can be used for studying genetic diversity inV. lecanii.     

Effects of VIP and GRF on the release of growth hormone in perifused bovine adenohypophysis

The effects of vasoactive intestinal polypeptide (VIP) and growth hormone releasing factor (GRF:hpGRF(1–29)-NH₂) on the release of growth hormone (GH) from anterior pituitaries from cows were examined by using an in vitro superfusion system. The pituitaries were excised randomly from cycling cows, dissected to obtain medial portions, and minced to obtain cubes with approximate dimensions of 1.5mm on a side. For each perifusion setup, 5 pieces of pituitary tissues were chambered and flushed with modified KRB solution saturated with 95% O₂-5% CO₂ at 38C. Perifusion with media containing 10⁻⁶ and 10⁻⁷M VIP for 30 min induced a significant release of GH during the treatments (P<0.05). VIP (10⁻⁸M) increased GH levels significantly (P<0.05), but to a minor degree. Perifusion with the media containing 10⁻⁶, 10⁻⁷ and 10⁻⁸M GRF for 30 min markedly increased the GH concentration and the effects continued up to 90 min after termination of the perifusion of the peptide (P<0.05, P<0.01). The GH releasing effects of GRF could be seen at doses as low as 10⁻¹¹M GRF (P<0.05, P<0.01).

These findings indicate that the GH releasing effect of VIP is less potent that that of GRF in cows.     

Sequencing of canine 5-hydroxytriptamine receptor (5-HTR) 1B, 2A, 2C genes and identification of polymorphisms in the 5-HTR1B gene

Polymorphisms of human genes encoding 5-hydroxytriptamine (serotonin) receptors (5-HTRs) are thought to be associated with psychiatric disorders and behavioral traits. In the present study, we searched for corresponding polymorphisms in the dog and compared allelic frequencies for the canine 5-HTR1B, 5-HTR2A, and 5-HTR2C genes among five canine breeds. The canine genes consisted of the following: 5-HTR1B, 1170 bp; 5-HTR2A, 1413 bp; and 5-HTR2C, 1377 bp. All of these genes were highly homologous with the human genes. We found six single nucleotide polymorphisms (SNPs) in the 5-HTR1B gene (G57A, A157C, G246A, C660G, T955C, and G1146C). Genotyping of the respective SNPs revealed that there were inter-breed variations in the genotypes and allelic frequencies for four out of the six identified SNPs, suggesting that further analyses of the polymorphisms of the 5-HTR1B gene would be useful in order to gain an understanding of the genetic background underlying the diversified behavioral traits among canine species.     

A direct time-resolved fluoroimmunoassay (TR-FIA) for measuring plasma estradiol-17beta concentrations in cattle

A direct Time-Resolved Fluoroimmunoassay (TR-FIA) system for measuring estradiol-17beta (E(2)) in bovine plasma was developed and evaluated. A 100 microl sample of bovine plasma was used for a TR-FIA without prior extraction and purification. The dose-response curves of reference standards ranged from 0.0625 to 10 pg/well. The minimum detectable concentration of this assay system was 0.625 pg/ml, and 19 pg/ml of E(2) caused a 50% reduction of maximum binding. The intra- and inter-assay coefficients of variation were 10.2 and 17.4%, respectively. The plasma E(2) concentrations measured by direct TR-FIA correlated closely with those measured after extraction (r=0.939). The results in the present study indicate that the TR-FIA reagent for E(2), designed for human research can also be utilized, with some modification, for direct assaying in bovine plasma. This assay type seems to fulfill the requirements for safety, sensitivity, specificity, reproducibility and practical convenience.