Genetic studies and pathogen detection in plants using molecular methods require the isolation of DNA from a large number of samples in a short time span. A rapid and versatile protocol for extracting high-quality DNA from different plant species is described. This method yields from 1 to 2 mg of DNA per gram of tissue. The absorbance ratios (A260/A280) obtained ranged from 1.6 to 2.0. A minimal presence of contaminating metabolites (as polymerase chain reaction [PCR] inhibitors) in samples and a considerable savings in reagents are characteristics of this protocol, as well as the low cost of the analysis per sample. The quality of the DNA was suitable for PCR amplification.
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http://dx.doi.org/10.1385/MB:31:2:137 | DOI Listing |
PLoS One
December 2024
Institute of Genetics, Technische Universität Braunschweig, Braunschweig, Germany.
Diplodia sapinea (Fr.) Fuckel is a widespread fungal pathogen affecting conifers worldwide. Infections can lead to severe symptoms, such as shoot blight, canker, tree death, or blue stain in harvested wood, especially in Pinus species.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.
Yeast two-hybrid library screening enables the discovery of novel protein-protein interactions. Identifying cytosolic host proteins targeted by host-translocated Phytophthora effector proteins relies on the mRNA amount, quality, and composition used to prepare the yeast two-hybrid cDNA library. Here we describe the steps required for the preparation of a Pinus radiata cDNA library optimized for Phytophthora effector target screening in yeast.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
Horticultural Crops Disease and Pest Management Research Unit, United States Department of Agriculture-Agricultural Research Service, Corvallis, OR, USA.
Pathogens have evolved effector proteins to suppress host immunity and facilitate plant infections. RxLR effectors are small, secreted effector proteins with conserved RxLR and dEER amino acid motifs at the N terminus and highly variable C termini and are commonly found in oomycete species. We provide computational approaches to annotate RxLR candidate effector genes in a genome assembly in FASTA format with an available GFF file.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
Sanya Institute of Nanjing Agricultural University, Department of Plant Pathology, Nanjing Agricultural University, Nanjing, Jiangsu, China.
The establishment of reliable and efficient systems for genome editing in Phytophthora is very important for studying gene functions. Here, step-by-step methods for CRISPR/Cas9-based gene knockout and in situ complementation for Phytophthora sojae are presented. These steps include the sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement, complementation vector construction, P.
View Article and Find Full Text PDFMethods Mol Biol
December 2024
Department of Plant Breeding, Swedish University of Agricultural Sciences (SLU), Lomma, Sweden.
RNA silencing is a core cellular process that acts to defend the genome against potentially damaging genetic elements such as viruses and transposons. It has been extensively characterized in many eukaryotes and exploited as a tool for determining gene function through removing the activity of specific genes. It has also been used in Phytophthora species to reveal genes involved in different lifecycle stages.
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