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Bulked Segregant Analysis Using the GoldenGate Assay to Locate the Rpp3 Locus that Confers Resistance to Soybean Rust in Soybean

^a Soybean Genomics and Improvement Laboratory, U.S. Dep. of Agriculture-Agricultural Research Service, Beltsville, MD 20705
^b Crop Genetics and Production Research Unit, U.S. Dep. of Agriculture-Agricultural Research Service, Stoneville, MS 38776
^c Foreign Disease-Weed Science Research Unit (FDWSRU), U.S. Dep. of Agriculture-Agricultural Research Service, Ft. Detrick, MD 21702
^d Dep. of Plant Science and Landscape Architecture, Univ. of Maryland, College Park, MD 20742

View larger version (13K): [in this window] [in a new window]   Figure 1. The clustering of a typical GoldenGate assay result that was considered a positive hit for bulked segregant analysis where the three susceptible bulks clustered with the susceptible genotype Williams 82. The normalized R (y axis) is the normalized sum of intensities of the two channels (Cy3 and Cy5) and normalized theta (x axis) is [(2/)Tan–1 (Cy5/Cy3)] where a normalized theta value nearest 0 is a homozygote for allele A and a theta value nearest 1 is homozygous for allele B (Fan et al., 2006).

View larger version (14K): [in this window] [in a new window]   Figure 2. Genetic linkage maps of the Rpp3 region of soybean linkage group C2. Cumulative cM distances are in parenthesis next to the marker name. The Rpp3 resistance allele confers a reddish-brown lesion response to the P. pachyrhizi isolate IN73–1. a) Genetic map generated using the Kosambi's mapping function from 110 F2:3 lines of Williams 82 x PI 462312. b) Soybean consensus genetic map of the same SSR markers on linkage group C2 as reported by Choi et al. (2007).

View larger version (28K): [in this window] [in a new window]   Figure 3. Diagram displaying the gene annotation for the region in the Soybean Genome Project, DoE Joint Genome Institute whole soybean 7x genome sequence scaffold-60 sequence between nucleotides 1,077,000 and 1,977,000. The SSR markers Satt460 and Sat_263 enclosing the Rpp3 resistance locus are at either end of the genomic sequence depicted. BLASTX alignments with high sequence similarity to the NCBI non-redundant (nr) protein sequence database with an expected value < E-20 were marked and grouped based on similar protein descriptions. The group labeled LRR Receptor-like Kinase includes gene descriptions for S-locus, carbohydrate-binding, lectin, LRR transmembrane, and receptor-like kinases. The group labeled TIR-NBS-LRR was separated from the other LRR protein kinases because the sequence description for these proteins included the phrase "disease resistant LRR kinase." The group labeled retrotransponson includes sequence descriptions of polyprotein, pol protein, retroelement, retrotransposon, retrotransposable, reverse transcriptase, retro-virus related, RT-like, integrase, RNA-directed DNA polymerase, transposase, and transposon. The STS group is the positions of sequence tagged sites used for haplotyping Williams 82, PI 462312, and PI 506764.