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United States Department of Agriculture

Agricultural Research Service

Title: Construction of the first phage library of chickpea blight pathogen Ascochyta rabiei.

Authors
item White, David - WASHINGTON STATE UNIV
item Chen, Weidong

Submitted to: Inoculum
Publication Type: Abstract Only
Publication Acceptance Date: July 20, 2006
Publication Date: July 28, 2006
Citation: White, D., Chen, W. 2006. Construction of the first phage library of chickpea blight pathogen Ascochyta rabiei. Inoculum 57:39.

Technical Abstract: During studies on pathogenic determinants of Ascochyta rabiei, the causal agent of chickpea blight, we developed a library of insertional mutants of A. rabiei using Agrobacterium - mediated transformation. Non-pathogenic mutants were identified after screening more than 1000 transformants using pathogenicity bioassays. Because the genome sequence of A. rabiei is unavailable, a genomic library is needed in order to isolate the genes disrupted in the non-pathogenic mutants. This study was to generate a phage library of A. rabiei suitable for isolation of potential pathogenicity determinants. Genomic DNA fragments between 7,000 and 10,000 bps of ApoI digest from a pathotype II strain were ligated to EcoRI-digested Lambda ZAPII vector arms, packaged using Gigapack III extracts, and amplified in E. coli strain XL1-Blue. Ten randomly selected plaques were used to determine the average size of DNA insert. A phage library consisting of approximately 1.7 x 106 recombinants was constructed, with average insertion size of 6.5 Kb. A single round of amplification of the library was performed to produce a final titer of 1 x 1011 pfu/ml. Recombinant DNA can be rescued from the phage in the form of a plasmid. This represents the first phage library of A. rabiei.

Last Modified: 7/25/2014
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