Published in Probe Volume 5(2): April-June 1995
Guo-Liang Wang, and Pamela C. Ronald
Department of Plant Pathology
University of California at Davis
Davis, CA
One of the essentials for the success of map-based cloning and physical analysis of large chromosomal regions is the availability of libraries containing large inserts of genomic DNA. In recent years, yeast artificial chromosome (YAC) libraries have been constructed for human (Burke et al. 1987), mouse (Chartier et al. 1992), and plant species, such as Arabidopsis (Ward and Jen 1990, Grill and Somerville 1991), tomato (Martin et al. 1992), maize (Edwards et al. 1992), barley (Kleine et al. 1993) and japonica rice (Umehara et al. 1994).
These libraries have made valuable contributions to the production of physical maps of large regions and the isolation of many important genes (Arondel et al. 1992, Giraudat et al. 1992, Martin et al. 1994, Leyser et al. 1993). However, the large percentage of chimaeric ( Green et al. 1991, Libert et al. 1993, Umehara et al. 1994) and unstable (Neil et al. 1990, Dunford et al. 1993, Schmidt et al. 1994) YAC clones hashindered the usefulness of these libraries. Difficulties in isolation of YAC insert DNA and low transformation efficiency also affect YAC library construction.
Bacterial artificial chromosome (BAC) libraries overcome many of the disadvantages of YAC libraries (Shizuya et al. 1992). This system uses an F-factor-based vector and is capable ofmaintaining human genomic DNA fragments of > 300 kb. Compared with YAC cloning, DNA can be cloned with high efficiency, easily manipulated and stably maintained in E. coli (Shizuya et al. 1992). Recently, BAC libraries have been constructed for Arabidopsis, sorghum and rice (Wang et al., unpublished; Woo et al. 1994; Wang et al. 1995). All clones tested from the rice and sorghum BAC libraries hybridized to a single chromosomal location using fluorescence in situ hybridization (FISH) analysis (Jiang et al. 1995; Woo et al. 1994). These results indicate a very low level of chimerism in these libraries.
Plant BAC libraries have been used successfully in several collaborative efforts. For example, BAC clones linked to disease resistance genes have been isolated from the Arabidopsis and rice libraries (6x and 3x genome equivalents, respectively) (Wang et al., unpublished; Wang et al. 1995). In addition to their use for gene isolation within a species, BAC libraries from species with small genomes can be used to identify syntenous chromosomal regions in plant species with larger genomes. For example, compared with rice, other cereal crops such as maize, wheat, barley, and rye have a much higher percentage of repetitive sequences, yet the gene order is highly conserved along the chromosomes.
To test the usefulness of the BAC library in intergenomic gene cloning, we have isolated clones from the rice BAC library corresponding to a region of the barley genome containing the disease-resistance gene, Rpg1 (Kilian et al., unpublished). Single-copy end-probes of rice BAC clones can be used to jump or walk between genes in barley to avoid repetitive sequences. Finally, BAC clones can be mapped in plant chromosomes by FISH analysis(Jiang et al. 1995). This technique can be used for rapid determination of the chromosomal location of genomic clones or the physical distance between genetically mapped clones. Since BAC libraries can be constructed in a short time and analyzed more efficiently than can YAC libraries, the BAC system is likely to become an important tool for plant genome analysis.
The rice and Arabidopsis BAC libraries are available for distribution. Interested researchers should send requests to Pamela Ronald, pcronald@ucdavis.edu, phone: (916) 752-1654 0r fax (916) 752-5674.
This project was supported by grants from the Rockefeller Foundation and USDA (NRICGP, # 9300834).
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