1a.Objectives (from AD-416):
1) Develop saturated molecular marker-based linkage maps of the genomic regions harboring the Snn3-B1 and Snn3-D1 loci;. 2)Develop high-resolution marker-based linkage maps of the Snn3-B1 and Snn3-D1 loci;. 3)Determine feasibility of cloning the Snn3-B1 and Snn3-D1 genes using a map-based approach; and. 4)Develop markers suitable for marker-assisted selection against the Snn3-B1 and Snn3-D1 genes.
1b.Approach (from AD-416):
Appropriate low-resolution mapping populations segregating for the Snn3-B1 and Snn3-D1 genes will be developed and phenotyped for reaction to the Stagonospora nodorum host-selective toxin SnTox3. The low-resolution populations will then be used to develop saturated genetic linkage maps of chromosome arms 5BS and 5DS, which are known to harbor the Snn3-B1 and Snn3-D1 genes, respectively. Sources of DNA-based markers for saturation mapping will include simple sequence repeats, expressed sequence tags identified based on available deletion mapping data and colinearity of the genomic regions with rice and Brachypodium, and other PCR-based markers. Markers flanking the target genes will be used to screen large segregating populations consisting of at least 3,000 individuals for high-resolution mapping. Plants harboring recombination events between the flanking markers will be further evaluated with markers that cosegregate with the Snn3 genes and scored for reaction to SnTox3. The most tightly flanking PCR-based markers will then be used to screen available BAC libraries to identify BAC clones and/or BAC contigs at the Snn3 loci. Physical to genetic distance ratios will be evaluated to determine the feasibility of cloning the Snn3 genes using a map-based approach. PCR-based markers tightly linked to the Snn3 genes will also be tested for their utility in marker-assisted selection schemes by evaluating genotypes of at least 100 wheat varieties and comparing the frequencies of marker alleles and response to SnTox3.
The generation of initial linkage maps of chromosome arm 5BS in the BR34 x Sumai 3 and CS-DIC 5B x Sumai 3 populations consisting of 19 and 20 markers each indicated that the BR34 x Sumai 3 population had significantly higher recombination frequencies than the CS-DIC 5B x Sumai 3 population. Therefore, the former should be most useful for fine mapping and cloning of the Snn3-B1 gene. Comparisons of the markers along the maps with orthologous genetic loci in the Brachypodium and rice genomes indicated that colinearity was highly conserved. These comparisons led to the identification of orthologous wheat gene sequences that were used to develop additional markers near the Snn3-B1 locus.
The two populations that were developed for mapping the Snn3-D1 gene were found to by highly polymorphic and 30 markers were used to assemble the initial genetic linkage map of chromosome arm 5DS. Colinearity of markers along 5DS was highly conserved with 5BS and also with Brachypodium and rice. These results provided further evidence that Snn3-B1 and Snn3-D1 are homoealleles and derived from a common ancestor. The development of high-resolution mapping populations for both Snn3-B1 and Snn3-D1 are in progress.