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Genomic Polymorphism Identifies a Subtilisin-Like Protease near the Rhg4 Locus in Soybean

^a Dep. of Crop and Soil Environmental Science, 276 Poole Agricultural Center, Clemson Univ., Clemson, SC 29634-0359
^b Agronomy College, Shanxi Agricultural Univ., Taigu, Shanxi Province 030801, P.R. China
^c USDA-ARS-PSI, Soybean Genomics & Improvement Lab., Bldg. 006, Room 118, BARC-West, 10300 Baltimore Blvd., Beltsville, MD 20705-2350

View larger version (72K): [in a new window]   Fig. 1. Southern hybridization analysis with PI 437654 BAC clone 15G19. Genomic DNA from susceptible germplasms Essex (1), Faribault (2), and Williams 82 (3), and resistant germplasms PI 209332 (4) and PI 437654 (5) digested with BamHI was hybridized with a random-prime generated probe from a PI 437654 BAC clone associated with the Rhg4 resistance locus. The 7.5-kb polymorphic fragment is indicated by the arrow.

View larger version (66K): [in a new window]   Fig. 2. Southern hybridization analysis confirming isolation of the DNA fragment producing the specific genomic polymorphism. Genomic DNA from soybean germplasms Essex (1), Faribault (2), Williams 82 (3), PI 209332 (4), and PI 437654 (5) digested with BamHI was hybridized with a PCR generated probe from an 8-kb BamHI fragment of a PI 437654 BAC clone associated with the Rhg4 resistance locus.

View larger version (55K): [in a new window]   Fig. 3. Structure and sequence analysis of the GmSUB1 gene. (A) Schematic diagram of GmSUB1. Open boxes represent exon sequences. Shaded boxes represent repeated sequences in the promoter region. (B) Alignment of the deduced amino acid sequence of GmSUB1 with AIR3 of Arabidopsis. Identical residues are shaded in black. The predicted cleavage sites for the signal peptide (open triangle) and propeptide domain (shaded triangle) are indicated. Conserved residues forming the catalytic triad and substrate binding site are indicated with an asterisk (*). GenBank accession number AY277949.

View larger version (45K): [in a new window]   Fig. 4. Southern hybridization analysis to determine GmSUB1 copy number in soybean. Genomic DNA from Williams 82 (1, 3, and 5) and PI 437654 (2, 4, and 6) digested with BamHI (1 and 2), DraI (3 and 4), and EcoRV (5 and 6) were hybridized with a GmSUB1 gene-specific probe (A). Differences in hybridization intensity with GmSUB1 probe were normalized to 26S rRNA content to determine differences in gene copy number (B).

View larger version (70K): [in a new window]   Fig. 5. GmSUB1 expression in soybean. (A) Relative GmSUB1 mRNA levels in uninfected roots, SCN infected roots, and leaves of PI 437654 (resistant) and Williams 82 (susceptible) were determined by quantitative real-time PCR using gene-specific primers and normalizing to 18S rRNA values. Quantitation is based on CT value as described in Materials and Methods. (B) Representative data set from quantitative real-time PCR analysis with GmSUB1 and 18S gene-specific primers. Inset panel shows standard curve graph for GmSUB1 (top) and 18S (bottom).