Plant Genome IV Conference Plant Genome IV Conference
Citrus producers worldwide suffer huge losses from rapid tree decline and/or stem-pitting diseases caused by citrus tristeza virus (CTV). A single dominant resistance gene from Poncirus trifoliata, a sexually compatible related genus, confers immunity to the virus, but delicate introgression of the gene into elite cultivars using traditional approaches has been extremely difficult and disappointing. Efforts are being made to clone the resistance gene for subsequent transformation experiments. The gene region has been mapped with RAPD markers, and some of the most closely linked ones have been converted into SCAR markers. To facilitate gene isolation and cloning, a bacterial artificial chromosome library (BAC) is being constructed using the vector pBeloBAC11. The haploid genome size of citrus species is about 385 Mb, therefore a library of 15 000 clones with an average insert size of 125 Kb is expected to contain nearly five haploid genome equivalents, and thus to provide a 99% probability of representing any specific single-copy sequence. High molecular weight DNA has been isolated from an F1 intergeneric hybrid possessing the resistance gene, and vector preparation has been completed. Currently, fragment size selection, ligation, and bacterial transformation are being attempted.