RESUMEN
Heterobasidion annosum causes root and butt-rot in trees and is the most serious forest pathogen in the northern hemisphere. We developed a rapid and simple Agrobacterium-mediated method of gene delivery into H. annosum to be used in functional studies of candidate genes and for visualization of mycelial interactions. Heterobasidion annosum TC 32-1 was cocultivated at pH 5.6 and 20 degrees C in Hagems medium with Agrobacterium tumefaciens C58 carrying plasmids with hygromycin B resistance as the selectable marker and green fluorescent protein as a visual marker. We obtained 18 mitotically stable transformed isolates showing green fluorescence protein activity.
Asunto(s)
Agrobacterium tumefaciens/genética , Basidiomycota/genética , Transformación Genética , Agrobacterium tumefaciens/crecimiento & desarrollo , Basidiomycota/metabolismo , Transformación Genética/fisiologíaRESUMEN
Three different genes were sequenced from isolates of five plant-pathogenic Verticillium species, Verticillium albo-atrum, Verticillium dahliae, Verticillium longisporum, Verticillium nigrescens, and Verticillium tricorpus. The sequences covered parts of the mitochondrial cytochrome b gene ( cob), the mitochondrial small subunit rRNA gene ( rns) and the nuclear ITS2 region. When the sequences were combined, the five species clustered in five monophyletic groups, with V. nigrescens distantly related to the other species while V. tricorpus displayed a somewhat closer relationship to the three remaining species. V. albo-atrum, V. dahliae and V. longisporum were found to be very similar to each other, with V. albo-atrum and V. longisporum displaying the closest relationship. The species affiliation of V. longisporum is discussed.
Asunto(s)
ADN de Hongos/química , ADN Mitocondrial/química , Análisis de Secuencia de ADN , Verticillium/genética , Citocromos b/genética , ADN de Hongos/aislamiento & purificación , ADN Mitocondrial/aislamiento & purificación , ADN Ribosómico/química , ADN Ribosómico/aislamiento & purificación , ADN Espaciador Ribosómico/genética , Genes de ARNr , Datos de Secuencia Molecular , Filogenia , Verticillium/clasificación , Verticillium/aislamiento & purificaciónRESUMEN
In order to conduct studies concerning genetic variability of pollen beetles (Meligethes aeneus), a genotyping protocol was established. No genome information is available for pollen beetles so the amplified fragment length polymorphism (AFLP) technique was chosen since it does not depend on any prior sequence information of the samples and also is a sensitive and robust technique. However, several modifications were needed in order to adapt the method for analysis of pollen beetles. Basic modifications included (i) alterations of DNA purification, (ii) use of two six-cutter restriction enzymes, (iii) and modified PCR conditions. This protocol resulted in a favourable number of fragments of an appropriate size range for standard gel analysis by a DNA sequencer applicable to a single insect and even body parts enabling different assays to be conducted on a single specimen. Pollen beetles from different areas of Sweden were analysed to verify the reproducibility and efficacy of the protocol as well as for phenetic analysis. The high reproducibility of the modified AFLP protocol allows it to be used as a reliable tool for genotype analysis of pollen beetles.