POTENTIAL APPLICATIONS OF BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARIES IN ISOLATION OF DISEASE RESISTANCE GENES IN CITRUS

¹ University of Florida, Citrus Research & Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850

² Texas A&M University, Dept of Soil & Crop Sciences, Crop Biotechnology Center, College Station, TX 77843

Citrus is a perennial evergreen crop that suffers continuous attack from a wide spectrum of pathogens including the citrus tristeza virus (CTV) and citrus nematode (CN), among others. Improvement of disease resistance has been one of the top priorities in citrus research programs. Genetic engineering of resistance genes will be an effective approach to expedite this improvement process. Pyramiding advancements in several areas are making this approach a reality in citrus. One of these advancements is the construction and manipulation of bacterial artificial chromosomes (BAC) and BAC libraries, which make possible genome physical mapping, map-based gene cloning, etc. We have constructed one HindIII and one BamHI BAC library from an intergeneric hybrid between Citrus and its close relative Poncirus. The HindIII library consists of over 10,000 clones with an average insert size of 70 kb; the BamHI library consists of over 24,000 clones and has an average insert size of 110 kb. These two libraries have been used in map-based cloning of the CTV resistance gene and cloning of candidate resistance genes. More than 400 BAC clones that contain resistance gene candidate (RGC) sequences have been identified. Twenty six contigs have been constructed from 130 RGC-containing BACs. For map-based cloning of the CTV resistance gene, chromosome walking was initiated from five closely linked marker sites and extended across the gene region. The current BAC contig extends over 600 kb and appears to cover the resistance gene region. Putative resistance gene sequences have been identified within the CTV resistance BAC contig.