Development of high molecular weight DNA methods in soybean for positional cloning of genes known only by phenotype

The purpose of this research was to develop high molecular weight DNA methods in soybean for map-based cloning of genes known only by their phenotype. Soybean will be a source of genes controlling nodulation and symbiotic nitrogen fixation. In particular, the work was started with the intention of cloning the nts gene of soybean, which is involved in autoregulation of nodule number in the symbiosis with Bradyrhizobium japonicum. Procedures for the preparation and analysis of high molecular weight DNA were used for physical mapping of a region in the soybean genome containing duplicated sequences using pulsed field gel electrophoresis. The duplicate soybean genome caused difficulties in relating bands on pulsed field gel Southerns to linkage groups on the genetic map, and these were resolved by using two-dimensional gel electrophoresis. An estimate of the relationship between physical and genetic distance in a cluster of markers was produced. 10% of a haploid soybean genome was cloned in yeast artificial chromosomes ranging in size from 50 to 900 kilobases, with an average of 175 kb. These clones were shown to be stable and useful for a variety of genome analysis applications, including rescue of end clones, cloning of repetitive sequences, and fluorescent in situ hybridization.