Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene

A soybean bacterial artificial chromosome (BAC) library, comprising approxi mately 45 000 clones, was constructed from high-molecular-weight nuclear DN A of cultivar Williams 82, which carries the Rps1-k gene for resistance aga inst Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random se quences including four markers linked to the Rps1 locus and 16 cDNAs. We id entified pools containing BACs for all sequences except for one cDNA. Addit ionally, when screened for possible contaminating BAC clones carrying chlor oplast genes, no sequences homologous to two barley chloroplast genes were found. The estimated average insert size of the BAC clones was about 105 kb . The library comprises about four genome equivalents of soybean DNA. There fore? this,gives a probability of 0.98 of finding a specific sequence from this library. This library should be a useful resource for the positional c loning of Rps1-k, and other soybean genes. We have also evaluated the feasi bility of an RFLP-based screening procedure for the isolation of BAC clones specific for markers that are members of repetitive sequence families, and are linked to the Rps1-k gene. We show that BAC clones isolated for two ge netically linked marker loci, Tgmr and TC1-2, are physically linked. Applic ation of this method in expediting the map-based cloning of a gene, especia lly from an organism, such as soybean, maize and wheat. with a complex geno me is discussed.