Fine mapping of the FT1 locus for soybean flowering time using a residual heterozygous line derived from a recombinant inbred line

Fine-mapping of loci related to complex quantitative traits is essential for map-based cloning. A residual heterozygous line (RHL) of soybean (Glycine max) derived from a recombinant inbred line (RIL) was used for fine-mapping FT1, which is a major quantitative trait locus (QTL) responsible for soybean flowering time. The residual heterozygous line RHL1-156 was selected from the RILs that were derived from two distantly related varieties, Misuzudaizu and Moshidou Gong 503. The genome of RHL1-156 contains a heterozygous segment (approximately 17 cM) surrounding the FT1 locus but is homozygous in other regions, including three other loci affecting flowering time. A large segregating population of 1,006 individuals derived by selfing of RHL1-156 included two homozygous genotypes for the nearest marker of FT1 whose flowering time differed by 26 days. No continuous range of phenotypes was observed, in contrast to the F₂ population, suggesting that a single FT1 locus affected the flowering time in the RHL1-156 line. Linkage analysis revealed that the FT1 locus mapped as a single Mendelian factor between two tightly linked DNA markers, Satt365 and GM169, at distances of approximately 0.1 cM and 0.4 cM, respectively. Our results show that a RHL derived from RILs can be used to fine-map a QTL and that RHLs can be an efficient tool for a systematic fine-mapping of QTLs.     

Genetic characterization of Raffaelea quercivora isolates collected from areas of oak wilt in Japan

This study was undertaken to improve understanding of the phylogenetic position of pathogenic fungi implicated in the oak wilt in Japan. Sequences were obtained from three regions of partial nuclear ribosomal DNA of 25 isolates of Raffaelea quercivora including an ex-type strain, all of which were collected from seven areas of disease outbreak. All the isolates formed one clear clade with high bootstrap values, distinctly delimited from the closest species, R. montetyi. These results indicate that the R. quercivora is phylogenetically a well-defined taxon.     

Identification of the Augmin Complex in the Filamentous Fungus Aspergillus nidulans

Augmin is a protein complex that binds to spindle microtubules (MTs), recruits the potent MT nucleator, γ-tubulin, and thereby promotes the centrosome-independent MT generation within mitotic and meiotic spindles. Augmin is essential for acentrosomal spindle assembly, which is commonly observed during mitosis in plants and meiosis in female animals. In many animal somatic cells that possess centrosomes, the centrosome- and augmin-dependent mechanisms work cooperatively for efficient spindle assembly and cytokinesis. Yeasts have lost the augmin genes during evolution. It is hypothesized that their robust MT nucleation from the spindle pole body (SPB), the centrosome-equivalent structure in fungi, compensates for the lack of augmin. Intriguingly, however, a gene homologous to an augmin subunit (Aug6/AUGF) has been found in the genome of filamentous fungi, which has the SPB as a robust MT nucleation centre. Here, we aimed to clarify if the augmin complex is present in filamentous fungi and to identify its role in mitosis. By analysing the Aug6-like gene in the filamentous fungus Aspergillus nidulans, we found that it forms a large complex with several other proteins that share weak but significant homology to known augmin subunits. In A. nidulans, augmin was enriched at the SPB and also associated with spindle MTs during mitosis. However, the augmin gene disruptants did not exhibit growth defects under normal, checkpoint-deficient, or MT-destabilised conditions. Moreover, we obtained no evidence that A. nidulans augmin plays a role in γ-tubulin recruitment or in mitotic cell division. Our study uncovered the conservation of the augmin complex in the fungal species, and further suggests that augmin has several functions, besides mitotic spindle MT nucleation, that are yet to be identified.     

Tyrosine phosphorylation of pp185 by insulin receptor kinase in a cell-free system

Insulin treatment of rat H-35 hepatoma cells causes rapid tyrosine phosphorylation of a high molecular weight protein termed pp185 besides autophosphorylation of the beta-subunit of the insulin receptor (IR) in an intact cell system. To elucidate the molecular basis for tyrosine phosphorylation of pp185, cell-free phosphorylation of pp185 was performed using phosphotyrosine-containing proteins (PYPs) purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibody. After insulin treatment of cells, marked increases of tyrosine phosphorylation of pp185 and IR were observed compared to noninsulin-treated cells. Site-specific antibodies that specifically inactivate IR kinase inhibited tyrosine phosphorylation of pp185 as well as the beta-subunit of IR. PYPs purified from detergent-free cell extracts contained pp185 but little IR; tyrosine phosphorylation of pp185 did not occur. Addition of IR kinase purified from human placenta to these PYPs restored insulin-dependent tyrosine phosphorylation of pp185. These results suggest that tyrosine phosphorylation of pp185 is catalyzed directly by IR kinase in this cell-free system.