Construction of a bacterial artificial chromosome library of Phytophthora infestans and transformation of clones into P. infestans.
A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.
Randall
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TA
Department of Plant Pathology, University of California at Riverside, Riverside, California 92521, USA.
Judelson
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HS
D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D015723:Gene Library; D009693:Nucleic Acid Hybridization; D010838:Phytophthora / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D014170:Transformation, Genetic
D004271:DNA, Fungal