Dm. O'Sullivan et al., A maize bacterial artificial chromosome (BAC) library from the European flint inbred lined F2, THEOR A GEN, 103(2-3), 2001, pp. 425-432
We report here the construction and characterisation of a BAC library from
the maize flint inbred line F2, widely used in European maize breeding prog
rams. The library contains 86,858 clones with an average insert size of app
roximately 90 kb, giving approximately 3.2-times genome coverage. High-effi
ciency BAC cloning was achieved through the use of a single size selection
for the high-molecular-weight genomic DNA, and co-transformation of the lig
ation with yeast tRNA to optimise transformation efficiency. Characterisati
on of the library showed that less than 0.5% of the clones contained no ins
erts, while 5.52% of clones consisted of chloroplast DNA. The library was g
ridded onto 29 nylon filters in a double-spotted 8 x 8 array, and screened
by hybridisation with a number of single-copy and gene-family probes. A 3-d
imensional DNA pooling scheme was used to allow rapid PCR screening of the
library based on primer pairs from simple sequence repeat (SSR) and express
ed sequence tag (EST) markers. Positive clones were obtained in all hybridi
sation 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 charac
terised and found to form contigs covering most of this complex resistance
locus.