ABSTRACT
We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.
Subject(s)
Arabidopsis/genetics , Cloning, Molecular/methods , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide , Alleles , Chromosome Mapping , DNA Primers , DNA, Plant/genetics , Genetic Markers , MutationABSTRACT
To identify components of the defense response that limit growth of a biotrophic fungal pathogen, we isolated Arabidopsis mutants with enhanced disease susceptibility to Erysiphe orontii. Our initial characterization focused on three mutants, eds14, eds15, and eds16. None of these is considerably more susceptible to a virulent strain of the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm). All three mutants develop a hypersensitive response when infiltrated with Psm expressing the avirulence gene avrRpt2, which activates resistance via the LZ-NBS/LRR resistance protein encoded by RPS2. The growth of Psm(avrRpt2), while somewhat greater in the mutants than in the wild type, is less than growth of the isogenic virulent strain. These results indicate that resistance mediated via LZ-NBS/LRR R genes is functional. Analysis of the growth of avirulent Peronospora parasitica strains showed that the resistance pathway utilized by TIR-NBS/LRR R genes is also operative in all three mutants. Surprisingly, only eds14 and eds16 were more susceptible to Erysiphe cichoracearum. Analysis of the expression profiles of PR-1, BGL2, PR-5 and PDF1.2 in eds14, eds15, and eds16 revealed differences from the wild type for all the lines. In contrast, these mutants were not significantly different from wild type in the deposition of callose at sites of E. orontii penetration. All three mutants have reduced levels of salicylic acid after infection. eds16 was mapped to the lower arm of chromosome I and found by complementation tests to be allelic to the salicylic acid-deficient mutant sid2.
