6.16

GENETIC CHARACTERIZATION OF THE MAJOR RESISTANCE GENE CLUSTER IN LETTUCE (LACTUCA SATIVA)



DB CHIN, D SICARD, SS WOO, R ARROYO-GARCIA, O OCHOA and RW MICHELMORE
Department of Vegetable Crops, University of California, Davis, CA 95616

Background and objectives

Clustering of disease resistance genes is a recurring theme in many plant genomes. In lettuce, the majority of genes conferring resistance to lettuce downy mildew (Dm genes) cluster into three major linkage groups. To investigate the stability and evolution of these resistance specificities, we have been studying the genetic and physical organization of these clustered regions, with particular emphasis on the largest cluster which contains at least 10 downy mildew resistance genes, including Dm1 and Dm3. We have been examining the genetic changes underlying induced and spontaneous mutations in Dm specificities. Mutations were induced by both fast-neutron (FN) irradiation and ethyl methane sulfonate (EMS), while spontaneous mutations were examined in various genetic backgrounds. Extensive molecular analyses and complementation experiments have been conducted [1], [2]. Several approaches are being taken to investigate the evolutionary mechanisms generating resistance specificities. We have been investigating the genetic variation present in a variety of natural populations, cultivars and species. Both microsatellites and fingerprinting methods have been used to establish resistance gene haplotypes, and phylogenetic analysis of the data has been conducted. We have also been investigating patterns of recombination in the Dm1/3 region. Molecular analyses are coupled with phenotypic screens on informative recombinants and their progeny.

Results and conclusions

-Molecular analyses of induced mutations have traced loss of specificity to both large deletions and single point mutations. All losses of Dm activity studied mapped to single Dm genes.
-Over 11,000 S2 families have been screened and nine mutants identified--three at the Dm7 locus and six at the Dm3 locus. An additional 8,500 F1 individuals have been screened and 2 mutants identified--one at the Dm3 locus and one at the Dm1 locus. A majority of the Dm3 mutants show loss of several molecular markers, indicating deletions in the region.
-Phylogenetic analyses of a diversity of resistance gene haplotypes discovered in Lactuca species have shown that polymorphisms are ancient, predating the separation of species.
-Over 150 recombinants have been identified and a profile of their cross-over points assembled. High molecular weight DNA analyses are being used to investigate the possibility of unequal crossing-over and phenotypic analyses of progeny are underway.
-Current data indicate that unequal crossing-over is occurring but is not the primary evolutionary force generating new variation.

References

1. Okubara PA, Anderson PA, Ochoa OE, Michelmore RW, 1994. Genetics 137, 867-874.
2. Anderson PA, Okubara PA, Arroyo-Garcia R, Meyers BC, Michelmore RW, 1996. Mol. Gen. Genet. 251, 316-25.