ISHS Acta Horticulturae 461: International Symposium on Biotechnology of Tropical and Subtropical Species Part 2
A BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARY FOR CLONING A CITRUS TRISTEZA VIRUS-RESISTANCE GENE |
| Authors: | F.G. Gmitter Jr, E.S. Louzada, Z. Deng, S. Huang |
| Keywords: | Citrus, Poncirus, map-based cloning, disease resistance |
| Abstract:
A close relative of Citrus, Poncirus trifoliata L., is highly resistant to citrus tristeza virus (CTV). Resistance is controlled by a dominant allele at a single locus designated Ctv. Although this allele can be transmitted through hybridization for rootstock improvement, such an approach is impractical for scion cultivar improvement. An alternative strategy being pursued is positional cloning of the gene, for subsequent transformation of susceptible cultivars. A key component of this strategy is the development of a BAC library from which individual clones linked to or containing Ctv may be identified. High molecular weight (HMW) DNA has been isolated using nuclei prepared from leaf tissue, and embedded in agarose mini-plugs. Plasmid vector (pBeloBAC11) was isolated from E. coli, purified, digested with HindIII and dephosphorylated, prior to ligation with size- selected fragments (200–600 kb) from partial HindIII digests of HMW DNA separated on CHEF gels. After ligation, plasmids were electroporated into E. coli strain DH10B and grown on a selective medium. Experiments were conducted to optimize various steps of the protocol. Conclusions from some of these experiments are presented. | |
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