Construction and Characterization of a Bacterial Artificial Chromosome Library for the A-Genome of Cotton (G.arboreumL.)

A bacterial artificial chromosome (BAC) library for the A-genome of cotton has been constructed from the leaves ofG. arboreumL cv. Jianglinzhongmian. It is used as elite A-genome germplasm resources in the present cotton breeding program and has been used to build a genetic reference map of cotton. The BAC library consists of 123,648 clones stored in 322 384-well plates. Statistical analysis of a set of 103 randomly selected BAC clones indicated that each clone has an average insert length of 100.2 kb per plasmid, with a range of 30 to 190 kb. Theoretically, this represents 7.2 haploid genome equivalents based on an A-genome size of 1697 Mb. The BAC library has been arranged in column pools and superpools allowing screening with various PCR-based markers. In the future, the A-genome cotton BAC library will serve as both a giant gene resource and a valuable tool for map-based gene isolation, physical mapping and comparative genome analysis.

Stability of Barley stripe mosaic virus–Induced Gene Silencing in Barley

Virus-induced gene silencing (VIGS) can be used as a powerful tool for functional genomics studies in plants. With this approach, it is possible to target most genes and downregulate the messenger (m)RNA in a sequence-specific manner. Barley stripe mosaic virus (BSMV) is an established VIGS vector for barley and wheat; however, silencing using this vector is generally transient, with efficient silencing often being confined to the first two or three systemically infected leaves. To investigate this further, part of the barley Phytoene desaturase (PDS) gene was inserted into BSMV and the resulting photobleaching in infected barley plants was used as a reporter for silencing. In addition, downregulation of PDS mRNA was measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Using fragments of PDS ranging from 128 to 584 nucleotides in BSMV, we observed that insert length influenced stability but not efficiency of VIGS. Silencing was transient in most cases; however, the decrease in PDS mRNA levels measured by qRT-PCR began earlier and lasted longer than the photobleaching. Occasionally, silencing persisted and could be transmitted through seed as well as via mechanical inoculation, although large parts of the insert had been lost from the virus vector. The instability of the insert, observed consistently throughout our experiments, offers an explanation for the transient nature of silencing when using BSMV as a VIGS vector.

Reconstitution of V-Notched Charpy Impact Specimen Made of Short Insert

Reconstitution in this paper means to constitute the original size V-notched Charpy impact specimen, which is made of the irradiated insert cut out from broken piece and un-irradiated tabs welded to the insert. It is a promising technique to secure an adequate number of surveillance specimens for long-term operation of nuclear power plants. Every Japanese nuclear power plant has its own surveillance test program, and is operated considering its unique surveillance test results along with the general reduction tendency of fracture toughness. This practice should be continued and enhanced if possible, after the full use of originally installed specimens, because its fracture toughness is lower than before. Reconstitution of V-notched Charpy impact specimens to the original shape by using a short insert was studied. Charpy absorption energy is generally shifted by reconstitution, if the insert length is as short as 10 mm. Reconstitution with a short insert is necessary when the transverse property of the original specimen is required although only the longitudinal surveillance specimen is installed as in some early constructed reactor pressure vessels in Japan. This case is important when the reactor pressure vessel is suspected to be a so-called low upper shelf toughness reactor pressure vessel. The minimum required insert length to avoid affect on the specimen properties depends on the Charpy absorption energy of the insert and reconstitution weld condition. Correlation between Charpy absorption energy and plastic deformation size, and short time annealing properties of irradiated pressure vessel steels were investigated. A method to evaluate the minimum required insert length was proposed, which depends on the expected Charpy absorption energy and thermal transient during reconstitution. It was demonstrated that the reconstituted specimens of 10 mm-long irradiated inserts, whose upper shelf absorption energy was 69J and required insert length was 9.5mm, showed little shift of upper shelf absorption energy.