1CSIRO Plant Industry, Canberra, ACT 2601, Australia; 2DNREM, Agriculture Victoria, Horsham, Australia; 3INRA BP29-35650 Le Rheu, France; 4ETS, Ciudad Universitaria, 28040 Madrid

Background and objectives
Cereal cyst nematode (CCN, causal agent Heterodera avenae ) causes severe losses to temperate cereals. In bread wheat, Triticum aestivum, the resistance genes, Cre1, Cre2, Cre3 ,Cre4 and CreX confer varying levels of resistance to different pathotypes of CCN found in Europe, Australia, West Asia and China. The Cre3 gene, originally identified in the diploid wild wheat Triticum tauschii, and transferred into cultivated wheat via a synthetic hexaploid, confers a high level of resistance to CCN pathotype Ha13. The Cre3 gene is inherited as a single dominant locus in T. tauschii and T. aestivum and is physically located on the most distal 0.06 fraction of the long arm of chromosome 2D. In a previous study we isolated a gene family predicted to encode a nucleotide binding site and leucine rich repeat (NBS-LRR) protein at the Cre3 locus [1], with similarities to other reported plant disease resistance genes [2]. Cosegregation between CCN phenotypes and the Cre3 NBS-LRR allelic variants have currently been observed in over 3000 lines from segregating families. We have explored the Cre3 NBS-LRR sequences as probes to: (i) investigate their relationship with the other Cre genes located in different parts of the wheat genome; (ii) characterise homologues associated with other Cre genes; and (iii) to utilise variant regions diagnostic for the different Cre genes in enhancing CCN resistance through gene pyramiding.

Results and conclusion
Three members of the NBS-LRR gene family have been identified at the Cre3 locus with sequence identities ranging from 82-87%. One of these genes is predicted to be functional based on the presence of an open reading frame and it also contains an additional 49 amino acids present as a direct repeat within the LRR. Frameshifts in the gene sequences were present in the other two members due to deletion and base substitution events. These sequences from the Cre3 locus hybridised to multiple fragments in bread wheat and were shown by genetic analysis to detect other homologues located at different loci. Analysis of appropriate genetic stocks for Cre1, Cre2 and CreX genes, which included near isogenic lines, segregating families and deletion lines revealed that the NBS-LRR sequences from the Cre3 locus detected diagnostic fragments specific for the Cre1, Cre2 and CreX loci. An extensive analysis on bread wheat selections made from over 5000 breeding lines segregating for the Cre1 gene as well as all commercial wheat cultivars carrying the Cre1 gene could be accurately predicted (100%) for its CCN resistance phenotype by using the Cre3 NBS-LRR gene sequences as probes to trace two diagnostic Cre1 associated homologues. DNA sequence analysis from one member of the NBS-LRR homologues at the Cre1 locus showed a sequence identity of 83% with the predicted functional gene at the Cre3 locus. For the purposes of a rapid DNA-based assay for CCN resistance, a PCR based selection has been developed for the Cre3 gene using the highly variable non coding region of the Cre3 NBS-LRR sequence. In addition to the selection efficiency, a major impact of the PCR based assay was is in reducing the long duration of three months, for the CCN bioassay, to one week with DNA based diagnostics. Thus combining Cre1 and Cre3 genes into a single cultivar for effective resistance has been made possible from the NBS-LRR gene family initially isolated from the Cre3 locus and currently forms the basis of CCN gene pyramiding in wheat breeding. The presence of closely related sequences to the NBS-LRR gene family from the Cre3 locus at other non homoeologous Cre loci in wheat, which includes the Cre1 as well as Cre2 and CreX genes suggests that the relationship of these CCN genes is not simply based on synteny.

1. Lagudah ES, Moullet O, Appels R, 1997. Genome 40, 659-665.
2. Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP, 1997. Science 276, 726-732.