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We report here the construction and characterisation of a BAC library from the maize flint inbred line F2, widely used in European maize breeding programs. The library contains 86,858 clones with an average insert size of approximately 90 kb, giving approximately 3.2-times genome coverage. High-efficiency BAC cloning was achieved through the use of a single size selection for the high-molecular-weight genomic DNA, and co-transformation of the ligation with yeast tRNA to optimise transformation efficiency. Characterisation of the library showed that less than 0.5% of the clones contained no inserts, while 5.52% of clones consisted of chloroplast DNA. The library was gridded onto 29 nylon filters in a double-spotted 8 × 8 array, and screened by hybridisation with a number of single-copy and gene-family probes. A 3-dimensional DNA pooling scheme was used to allow rapid PCR screening of the library based on primer pairs from simple sequence repeat (SSR) and expressed sequence tag (EST) markers. Positive clones were obtained in all hybridisation and PCR screens carried out so far. Six BAC clones, which hybridised to a portion of the cloned Rp1-D rust resistance gene, were further characterised and found to form contigs covering most of this complex resistance locus. [ABSTRACT FROM AUTHOR]