Construction and characterization of a bacterial artificial chromosome (BAC) library for the A genome of wheat

A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the p arental lines used in the construction of a saturated genetic map. Leaves f rom this accession were used to isolate high-molecular-weight DNA from nucl ei. This DNA was partially digested with restriction enzyme Hind III, subje cted to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276 480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones wit h chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence repres ented in this library is higher than 99.6%. Clones were sorted in 720 384-w ell plates and blotted onto 15 high-density filters. High-density filters w ere screened with several single or low-copy clones and five positive BAC c lones were selected for further analysis. Since most of the T. monococcum B AC ends included repetitive sequences, a modification was introduced into t he classical end-isolation procedure to select low copy sequences for chrom osome walking.