binary vector-based large insert library for Brassica napus and identification of clones linked to a fertility restorer locus for Ogura cytoplasmic male sterility (CMS)

We constructed and characterized a large DNA insert library for Brassica napus that would facilitate genome-related research and map-based cloning efforts in Brassica species. This library, consisting of 92 160 clones arrayed in 384-well microtiter dishes, was based on a conventional plant transformation vector (binary vector), and was constructed using a single ligation with transformation efficiency of over 5000 recombinants per microliter of ligation mixture. Every clone in this library contains an insert in the size range of 30-190 kb, facilitating both chromosome walking and plant transformation. Screening this library with three DNA markers (C2, F10, and CabR) that are linked to a fertility restorer locus for Ogura cytoplasmic male sterility (CMS) identified at least 17 positive clones for each probe. Among the 17 positive clones identified by C2, nine are linked to the restorer locus. Marker F10 identified 21 clones, of which only two are linked to the restorer locus. None of 68 clones identified by CabR is linked to the restorer locus. A stability test using two clones identified by the C2 marker indicated that large DNA inserts are stable in this conventional vector in both Escherichia coli and Agrobacterium.