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Use of replicative genomic libraries to clone fungicide resistance alleles

Abstract : Telomere sequences have been found to enhance transformation frequencies of fungal plasmids. Such plasmids do not integrate into the genome; they may be replicated independently. We constructed two derivatives of the pFAC1 plasmid, which transform B. cinerea at high frequencies (1). Our plasmids pFACR5 and pFB2N both give rise to transformation frequencies 500-1.000 times higher than integrative plasmids. They are replicated under selective pressure but unstable without selection. We analyzed conformation and copy number of both plasmids in B. cinerea transformants. Integration of a fenhexamid resistance allele into the pFB2N vector induces fungicide resistance in the transformants showing that dominant alleles can be selected from replicative plasmids (2). Genomic libraries in the pFB2N vector were used to transform the B05.10 strain. Transformants analysis showed a ratio of recombinant plasmids and an average insert size comparable to the initial E. coli library. The replicative plasmids are found at roughly 1 copy/genome. After the construction of a 3-4X genome coverage B05.10 transformant pool, harboring the replicative library generated from a fenhexamid resistant strain, we screened the transformants for fenhexamid resistance. Results will be presented. Perspectives and limitations of the system will be discussed
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https://hal.inrae.fr/hal-02819504
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Submitted on : Saturday, June 6, 2020 - 5:01:21 PM
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3rd Botrytis genome workshop 2...
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  • HAL Id : hal-02819504, version 1
  • PRODINRA : 285387

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Catherine Lanen, Johann Confais, Christiane Auclair, Sabine, Helma Fillinger-David. Use of replicative genomic libraries to clone fungicide resistance alleles. 3rd Botrytis Genome Workshop, Sep 2008, Tenerife, Spain. ⟨hal-02819504⟩

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