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.
Appropriate populations for saturation mapping, high-resolution mapping, and genomic analysis of the Snn3-B1 and Snn3-D1 genes have been developed. For objective 1 as it relates to Snn3-B1, we developed two populations for saturation mapping. One was developed by crossing the wheat line BR34 with the Snn3-B1-containing line Sumai 3, and the other was developed by crossing a Chinese Spring-Triticum dicoccoides 5B disomic chromosome substitution line (CS-DIC 5B) with Sumai 3. Approximately 120 F2 progeny from each line will be evaluated for reaction to SnTox3 to map the Snn3-B1 gene, and microsatellite and EST-based markers will be surveyed to saturate the Snn3-B1 chromosomal region. The population that exhibits the highest frequency of recombination and marker polymorphism will be used for high-resolution mapping and subsequence genomic analysis.
The Snn3-D1 gene is found in the wheat relative Aegilops tauschii. Therefore, we crossed an Snn3-D1-containing Ae. tauschii line (TA2377) with an Ae. tauschii line that lacks Snn3-D1 (AL8/78) and generated a population of 120 F2 lines for saturation mapping. We also developed a synthetic hexaploid line by crossing TA2377 with the durum line Langdon (LDN2377syn), and crossed it to the hexaploid wheat line BR34 to develop a population of another 120 F2 lines with the Snn3-D1 gene segregating in a hexaploid background. Both populations will be evaluated for reaction to SnTox3 and with molecular markers to develop a saturated genetic linkage map of the Snn3-D1 region on chromosome 5D (objective 1). Both populations will be amplified for high-resolution mapping and subsequent genomic analysis of Snn3-D1.