DEVELOPMENT OF GENOME INTEGRATED PHYSICAL MAP; 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 glycine s i.) is conditioned by two loci: rhg1 on linkage group G and Rhg4 on linkage group A2. Using BSA (Bulked Segregant Analysis), AFLP and microsatellite markers, we constructed a high density genetic map for the interval carrying Rhg1 and Rhg4 (Meksem et al 1998). We have constructed a Forrest Bacterial Artificial Chromosome (BAC) library in the binary V41 vector (Meksem et al 1998). The library will provide 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. Using new technology for contig assembly by BAC fingerprinting (Tao and Zhang patent pending); we constructed about 950 Kb contig in the interval spanning Rhg1 and 450 Kb contig in the region around Rhg4. One clone, A109-4 (insert size of 127 Kb) contains the two 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. BAC fingerprinting and contig assembly has provided powerful tools to rapidly generate robust molecular physical maps. A physical map, integrated with the existing DNA marker genetic map, is under development for the entire soybean genome; This will provide a new strategy to clone genes known only by their phenotypes by gene golfing (Zhang and Wing 1997). Using the integrated physical map, DNA molecular markers up to 10 cM away from a gene of interest can be used to clone the target gene. Genes in regions where repeated sequences are rich can also be cloned with the integrated physical map, whereas it is difficult, if not impossible, to clone the genes in such regions by the currently used map-based cloning strategy. The development of an integrated physical map will provide a "highway" for isolation of large number of soybean genes. The methods developed will aid many other genetic and biological studies of plant and animal genomes.