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Ear rot, caused by Fusarium verticillioides (FV), is a destructive disease of maize as it reduces grain yield and increases risks of mycotoxin production, thus endangering livestock. To identify genes differentially expressed during FV infection, four cDNA libraries were constructed for suppression subtractive hybridization using RNA isolated from bracts of an FV-resistant inbred maize line, Bt-1, as well as an FV-susceptible maize inbred line, Ye478. A total of 145 clones were obtained following reverse dot-blot hybridization, and these were sequenced from these libraries. Similarity analysis revealed that of these genes, 93 were unique, including 68 of known function, 24 of unknown function, and a single novel gene. Most genes of known function were predominantly involved in plant defense such as cell defense, transcription regulation, signal transduction, and metabolism. Expression profiles of eight representative genes, using semiquantitative reverse transcription-polymerase chain reaction, confirmed that differential gene regulation during FV infection was involved. These findings suggested that these differentially expressed genes might be involved in FV defense responses in maize. [ABSTRACT FROM AUTHOR]

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase ( SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers. [ABSTRACT FROM AUTHOR]

Tomato Yellow Leaf Curl virus (TYLCV-Eg) was isolated from whiteflies-infected tomato (Lycopersicon esculentum cv.Castle rock) plants growing in Nubaria and El-Behera Governorate. The infected plants exhibited systemic viral symptoms in the form of sever leaf curling, leaf crinkle with marginal yellowing, stem upright, twisted and stunted. TYLCV-Eg reacted positively with polyclonal antibodies specific to TYLCV using DAS-ELISA. It was transmitted by both syringe injection and whiteflies with transmission efficiency of about 80% and 100%, respectively. TYLCV-Eg isolate was transmitted to different species belonging to families Cucurbitaceae, Fabaceae, Solanaceae and Chenopodiaceae. TYLCV had TIP of 70ºC, DEP of 10-7 and LIV of about 6 days. Electron micrograph of the partially purified TYLCV revealed the presence of monomer and dimmer gemini particles with dimensions of 22 nm and 20 × 30 nm to 24 × 30 nm, respectively when negatively stained with uranyl acetate. Using degenerate oligonucleotide primers, the viral coat protein gene was amplified successfully by PCR, producing ∼ 500 bp fragment from tomato infected plants. The viral genome was detected by specific DNA probe using dot blot hybridization technique. Comparative nucleotide sequence analysis showed a similarity of 98% between TYLCV-Eg and other isolates. [ABSTRACT FROM AUTHOR]

Simple, reliable methods for identification of species are required for management of many species and lines in a plant gene bank. Species-specific probes were designed from published sequences of the ITS1 region in rDNA of 16 species in Brassica and its related genera, and used as probes for dot-blot hybridization with plant genomic DNA. All the probes detected species-specific signals at dot-blots of genomic DNAs of the 16 species in Brassica, Diplotaxis, Eruca, and Raphanus. Signals of the Brassica digenomic species in the U’s triangle, i.e., B. napus, B. juncea, and B. carinata, were detected by the probes of their parental monogenomic species, i.e., B. rapa, B. nigra, and B. oleracea. The probe for B. oleracea showed signals of B. balearica, B. cretica, B. incana, B. insularis, and B. macrocarpa, which have the C genome as B. oleracea. Eruca vesicaria DNA was detected by the probe for E. sativa, which has been classified as a subspecies of E. vescaria. DNA of leaf tissue extracted by an alkaline solution and seed DNA prepared by the NaI method can be used directly for dot-blotting. Misidentification of species was revealed in 20 accessions in the Tohoku University Brassica Seed Bank. These results indicate dot-blot hybridization to be a simple and efficient technique for identification of plant species in a gene bank. [ABSTRACT FROM AUTHOR]