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Objective: To analyze the T-DNA integration pattern in the genome of grey mold Botrytis cinerea., Methods: T-DNA (Transfer DNA) inserted mutant library of Botrytis cinerea was created by Agrobactirium tumfacience mediated transformation. By using TAIL-PCR (Thermal asymmetric interlaced polymerase chain reaction), we amplified and cloned the chromosomal regions flanking T-DNA insertions. The obtained T-DNA flanking sequences were subjected to alignment with standard T-DNA border sequence for identification and analysis of integration., Results: Up to 69% T-DNA inserted at noncoding regions and 30% inserted at exons. Recombination including deletion or addition of bases in T-DNA region was observed. The right borders of the T-DNA were frequently truncated, and by contrast the left borders were less prone to degradation and appeared to have been inserted in a relatively integrated manner. Extra sequence additions also occurred in T-DNA integration sites., Conclusion: Analysis of T-DNA integration pattern in B. cinerea genome will stimulate the functional genomics study of this fungus.

Objective: Construction of Botrytis cinerea T-DNA insertion mutant library through Agrobacterium-mediated transformation and for further understanding of the molecular mechanisms underlying the pathogenicity of Botrytis cinerea., Methods: Agrobacterium containing the binary vector pCMBIA1390 was used for transformation of Botrytis cinerea. Hygromycin resistant transformants were screened out and subjected to biological and morphological observation, Detached tomato leaves were used for pathogenicity assay. The flanking sequence of T-DNA inserted in mutant genome was cloned and analyzed by using TAIL-PCR., Results: A variety of mutants were obtained and important phenotypes including reduction in growth rate, loss or reduction in conidiation and loss of pathogenicity were screened out. The T-DNA flanking sequence of one of the mutants was successfully amplified with TAIL-PCR., Conclusion: Agrobacterium-mediated transformation system of Botrytis cinerea was established and a T-DNA insertion mutants library was constructed. TAIL-PCR was an effective method for isolating the flanking sequence of T-DNA inserted in genome.