SOYBEAN GENE GOLFING; POSITIONAL CLONING OF THE CYST NEMATODE RESISTANCE LOCI IN SOYBEAN;

¹ Department of Plant Soil and General Agriculture, Southern Illinois University at Carbondale, Carbondale, IL 62901-4415 USA

² Department of Soil and Crop Sciences, Crop Biotechnology Center, Texas A & M University, College Station, TX 77843-2123 USA

³ Department of Agronomy, University of Missouri, Columbia MO 65211 USA

Inheritance of field resistance in soybean [Glycine max (L.) Merr. In"Forrest" cultivar (Peking source) to soybean cyst nematode (SCN) race 3(Heterodera glycines i.) is conditioned by two loci: rhg1 on linkage group G and Rhg4 on linkage group A2. Bulked segregant analysis, AFLP's and microsatellite were used to select molecular markers closely linked to rhg1 and Rhg4. We have screened 1024 (EcoRI/MseI) primer combinations against two pools of DNA (a resistant and a susceptible pool). This revealed about 10,000 AFLP polymorphic bands, 20 of which map to G in coupling with rhg1 and 9 of which map to A2 in coupling with Rhg4. Two AFLP markers place rhg1 within a 1 cM interval and three AFLP markers place Rhg4 within a 0.5 cM interval. We have constructed a Forrest Bacterial Artificial Chromosome (BAC) library in the Binary V41 vector. The library provides clones for physical mapping of the soybean genome and for chromosome walking or landing. Candidate clones containing target genes can be directly used to transform plants for genetic complementation tests via Agrobacterium-mediated methods. We are developing new techniques for BAC contig assembly based on BAC fingerprinting (Tao and Zhang). We isolated a contiguous 950 Kb region from overlapping BAC DNAs spaning the 5 cM interval carrying rhg1 (and rfs1) and a 450 Kb region in the 3 cM interval around Rhg4. Within the 950 kb region on one clone, A109-4 (insert size of 127 Kb) contains the two AFLP markers flanking either side of rhg1 in Forrest. Sequencing of subclones of A109-4 has identified candidate resistance genes. Transformation with candidate genes from the A109-4 clone is in process. A physical map integrated with the developed DNA marker genetic maps will provide a new strategy to clone genes known only by their phenotypes by gene golfing (Zhang and Wing 1997). The development of the soybean integrated physical map will provide a "highway" for isolation of large number of genes. Methods developed will aid many other genetic and biological studies of plant and animal genomes. Currently, the integrated physical map of the soybean is under development by a multi-disciplinary group using the BAC fingerprinting and contigs assembly technologies.