Physical isolation and sequence analysis of disease resistance gene analogs in soybean

Plant disease resistance is one of the most important control strategies against pathogens of agronomically important crops. The resistance response is triggered by the initial recognition of pathogen elicitors by plant resistance genes (R-genes) -encoded receptors. The majority of R-genes belong to a large gene family that encodes structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). This family can be divided into two classes based on the presence or absence of a short sequence with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes in the N-terminal domain of the proteins, called TiR. More than 19 NBS-LRR R-genes have been characterized and their conserved amino acid signatures are useful landmarks in the isolation of R-gene analogs (RGAs). Based on conserved motifs, a targeted polymerase chain reaction (PCR) approach was used to generate nearly 50 genomic soybean sequences with strong homology to cloned R-genes of the nonTIR-NBS-LRR (nonTIR) class. Multiple sequence alignments and phylogenetic analysis classified them into four main subclasses of RGAs within the nonTIR class. A representative clone from each of the four main classes was used as a restriction fragment length polymorphism (RFLP) probe for genetic mapping, bacterial artificial chromosome (BAC) library screening, fingerprinting and construction of RGA-containing BAC contigs. Twelve soybean nonTIR RGA loci, mapped within a 25 centimorgan region of linkage group F of soybean (LG-F), found to harbor more than 16 disease resistance loci by classical genetic studies. A majority of the genomic region encompassing the RGAs was physically isolated in eight contigs of BAC clones (together spanning 1 megabase or more of genomic sequence) that contained 16 RGA copies. Physical maps of the RGAs were constructed for each contig. Another RGA BAC contig was mapped to LG-K corresponding to a highly dissimilar RGA sequence. Phylogenetic and sequence analysis, together with genetic and physical mapping information, provided insights into the genome organization and possible evolution of this cluster of RGAs. Detailed information about these RGA clusters is currently being applied in positional cloning efforts to isolate economically important resistance genes against major pathogens of soybean.