Construction of a bacterial artificial chromosome (BAC) library for citrusand identification of BAC contigs containing resistance gene candidates

A BAC library was constructed from the genomic DNA of an intergeneric Citru s and Poncirus hybrid. The library consists of 24,576 clones with an averag e insert size of 115 kb, representing approximately seven haploid genome eq uivalents and is able to give a greater than 99% probability of isolating s ingle-copy citrus DNA sequences from this library. High-density colony hybr idization-based library screening was performed using DNA markers linked to the citrus tristeza virus (CTV) resistance gene and citrus disease resista nce gene candidate (RGC) sequences. Between four and eight clones were isol ated with each of the CTV resistance gene-linked markers, which agrees with the library's predicted genome coverage. Three hundred and twenty-two clon es were identified using 13 previously cloned citrus RGC sequences as probe s in library screening. One to four fragments in each BAC were shown to hyb ridize with RGC sequences. One hundred and nine of the RGC BAC clones were fingerprinted using a sequencing gel-based procedure. From the fingerprints , 25 contigs were assembled, each having a size of 120-250 kb and consistin g of 2-11 clones. These results indicate that the library is a useful resou rce for BAC contig construction and molecular isolation of disease resistan ce genes.