Construction and characterisation of a large DNA insert library from the D genome of wheat
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abstract
A large DNA fragment library consisting of 144 000 clones with an average insert size of 119 kb was constructed from nuclear DNA isolated from root and leaf tissue from Triticum tauschii (syn. Aegilops tauschii), the D-genome progenitor of wheat. The library was made in a binary vector that had previously been shown to stably maintain large inserts of foreign DNA in Escherichia coli. The use of root nuclei reduced considerably the proportion of the library containing clones derived from chloroplast DNA. Several experimental parameters were investigated and optimised, leading to a high cloning efficiency. Only three ligations were needed to construct the library which was estimated to be equivalent to 3.7 haploid genomes. The accuracy of this estimation was demonstrated by screening this library with three well-defined probes. One probe containing a glutenin gene sequence identified 5 clones covering at least 230 kb of the Glu-D1 locus and contained the two tightly linked high-molecular-weight glutenin genes Glu-D1x and -D1y. Each of the other two single-copy probes derived from the Cre3 cereal cyst nematode resistance gene locus hybridised with 4 clones containing gene sequences encoding nucleotide binding sites and a leucine-rich region. This is the first representative large-insert DNA library for wheat, and the results indicated that large molecules of wheat DNA can be efficiently cloned, stably maintained and manipulated in a bacterial system.
