PAG-IV Plant Genome IV Conference

Town & Country Conference Center, San Diego, CA, January, 1995.

P16
Cloning of Genes for Nematode (Heterodera schachtii) Resistance in Sugarbeet (Beta vulgaris L.)

M. Kleine(1), D. Cai(1), S. Kifle(1), H. Harloff(1), R. Klein-Lankhorst(2), N. Sandal(3), C. JUNG(1)
1. Institute of Crop Science and Plant Breeding, Christian Albrechts University of Kiel, Olshausenstrasse 40, D-24118 Kiel, Germany
2. DLO-Center for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen, The Netherlands
3. Laboratory of Gene Expression, University of Aarhus, Denmark

The beet cyst nematode (Heterodera schachtii Schm.) causes severe damage in sugar beet (Beta vulgaris L.) growing. Genes conferring dominant resistance against this pathogen are only available in wild beets of the Beta section IV (B. procumbens, B. webbiana and B. patellaris). After species hybridizations completely resistant monosomic addition lines were selected each carrying one wild beet chromosome. In the offspring of these addition lines numerous resistant introduction lines have been selected either carrying chromosome fragments derived from the original wild beet chromosome (2n=18+1) or translocations of the wild beet chromosome to a sugar beet chromosome (2n=18). A fine mapping around the resistance gene in sugar beet was not possible due to the lack of chromosome homology between wild beet and sugar beet chromosomes. Therefore, the smallest wild beet translocation was chosen for physically mapping and cloning the gene using a set of satellite DNA probes exclusively hybridizing with wild beet DNA. So far, nine YACs have been isolated from the translocation where the resistance gene is located on. With the help of the wild beet specific probes the YACs where arranged into three contigs of overlapping clones. Several cDNAs with homology to the YAC inserts have been isolated and characterized by sequence analysis, Northern analysis and by differential Southern hybridization with DNA from resistant and susceptible beets. A straightforward technique is used for the identification of the nematode resistance gene: The most promising cDNAs are subcloned into an A. rhizogenes plasmid vector under the transcriptional control of the 35S promotor. After transformation of susceptible sugar beet transgenic hairy roots can be directly tested for nematode resistance in vitro. After 4 weeks cysts are visible on transgenic roots lacking the resistance gene. However, the nematode is not able to complete its life cycle on roots carrying the resistance gene offering the possibility to identify the corresponding cDNA by lack of cyst formation.

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